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WO2025176039A1 - USE OF IL-1α INHIBITOR IN PREPARING MEDICAMENT FOR TREATING LEUKEMIA THERAPY-INDUCED CARDIAC INJURY - Google Patents

USE OF IL-1α INHIBITOR IN PREPARING MEDICAMENT FOR TREATING LEUKEMIA THERAPY-INDUCED CARDIAC INJURY

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Publication number
WO2025176039A1
WO2025176039A1 PCT/CN2025/076776 CN2025076776W WO2025176039A1 WO 2025176039 A1 WO2025176039 A1 WO 2025176039A1 CN 2025076776 W CN2025076776 W CN 2025076776W WO 2025176039 A1 WO2025176039 A1 WO 2025176039A1
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Prior art keywords
leukemia
cardiac
chemotherapy
mice
specific embodiment
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PCT/CN2025/076776
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French (fr)
Chinese (zh)
Inventor
张�浩
刘一为
周星亮
沈熠
冯蓓
颜艺
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Shanghai Children's Medical Center School Of Medicine Shanghai Jiao Tong University
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Shanghai Children's Medical Center School Of Medicine Shanghai Jiao Tong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the use of an IL-1 ⁇ inhibitor in the preparation of a medicine for treating heart damage induced by leukemia treatment.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • a recent clinical study showed that among childhood cancers, leukemia survivors face a significantly higher long-term disease burden than survivors of other cancer types, primarily due to cardiovascular disease. 1
  • the incidence of heart disease in children with AML is significantly higher than in those with ALL, primarily manifesting as cardiomyopathy and heart failure.
  • one of the technical solutions provided by the present invention is: use of an IL-1 ⁇ (i.e., interleukin 1 ⁇ ) inhibitor in the preparation of a drug for treating leukemia-induced cardiac damage.
  • an IL-1 ⁇ i.e., interleukin 1 ⁇
  • the immunotherapy is CAR-T therapy.
  • the leukemia treatment results in necrosis of the leukemia cells.
  • the chemotherapy drug is an anthracycline chemotherapy drug.
  • the anthracycline chemotherapy drug is daunorubicin.
  • the anti-IL-1 ⁇ antibody is a monoclonal antibody.
  • the anti-IL-1 ⁇ antibody is a neutralizing antibody.
  • the immunotherapy is CAR-T therapy.
  • the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and/or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.
  • Figure 1 shows the construction of cardiac-specific Il1r1 knockout mice.
  • the control group consisted of IL1R1 fl/fl cells, while the experimental group consisted of IL1R1 cKO cells.
  • the right side shows the protein expression of IL1R1 relative to actin.
  • IL1R1 interleukin-1 receptor type 1; Actin: actin.
  • Statistical differences between the two groups were calculated using a two-sample t-test ( * P ⁇ 0.05).
  • Figure 6D shows the evaluation of myocardial ATP content in the two groups of mice.
  • FIG8C shows a pseudo-time series analysis of all cardiomyocytes.
  • Figure 9C shows the cardiac PET-CT examination of the two groups of mice.
  • TF tumor-free
  • DNR daunorubicin
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular short-axis contraction fraction
  • FTHA 6-thio-heptadecanoic acid
  • FDG deoxyglucose
  • ATP adenosine triphosphate
  • FIG10A-FIG10C are cardiac transcriptomic analyses of AML mice after DNR chemotherapy.
  • Figure 10A shows the KEGG pathway enrichment analysis of differentially expressed genes in cardiac transcriptomics results after chemotherapy.
  • the cytokine and cytokine interaction pathways were significantly enriched and highlighted in red.
  • Figure 10B shows the biological response pathway analysis of cardiac transcriptomics results after chemotherapy, with PPAR signaling significantly downregulated and highlighted in blue.
  • Figure 10C is a protein interaction network analysis of differentially expressed genes in the PPAR pathway, showing that IL-1 signaling and NF-KappaB signaling closely interact with the key metabolic protein PPARGC1A.
  • TF tumor-free
  • AML acute myeloid leukemia
  • DNR daunorubicin. Differences between samples were calculated using analysis of variance followed by the Tukey-Kramer test (NS: not significant; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001).
  • Figures 12A-12C show that AML cells undergo necrosis and release IL-1 ⁇ after DNR chemotherapy.
  • FIG12A and FIG12B are flow cytometric measurements of the ratio of live cells to necrotic cells in primary AML cell lines after 24 h of intervention with different DNR concentration gradients.
  • Figure 12C shows the detection of IL-1 ⁇ content in the cell culture supernatant after 24 hours of intervention of AML primary cell line with 0.5 ⁇ M DNR.
  • IL-1 ⁇ interleukin 1 ⁇
  • DNR daunorubicin
  • 13A-13D show the changes in cardiac metabolism and function in normal tumor-free mice after administration of IL-1 ⁇ .
  • FIG13C shows the cardiac PET-CT examination of the two groups of mice.
  • FIG13D shows the detection of myocardial ATP content in two groups of mice.
  • Figure 14A- Figure 14C show that cardiac-specific Il1r1 knockout improves cardiac metabolism and function in mice after chemotherapy.
  • Figure 14A is a schematic diagram of the experiment, in which DNR was used to intervene in AML mice with cardiomyocyte-specific knockout of IL1R1 (IL1R1cKO group) and control AML mice (IL1R1 fl/fl group).
  • AML acute myeloid leukemia
  • DNR daunorubicin
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular fractional contraction
  • ATP adenosine triphosphate
  • Figure 15 shows the correlation analysis between the increase rate of plasma IL-1 ⁇ and the decrease rate of LVEF in patients with acute myeloid leukemia before and after chemotherapy.
  • LVEF left ventricular ejection fraction.
  • Figure 16A- Figure 16C show that knockdown of Il1a in AML cells improves cardiac metabolism and function in mice after chemotherapy.
  • FIG 16A is a schematic diagram of the experiment, in which DNR intervention was performed on AML models constructed using control (scramble group) and IL-1 ⁇ knockdown (shIL-1 ⁇ group) cells.
  • FIG16B shows cardiac ultrasound examination of the two groups of mice.
  • WT wild type
  • AML acute myeloid leukemia
  • DNR daunorubicin
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular fractional contraction
  • ATP adenosine triphosphate
  • Figure 18 shows immunofluorescence and immunoblotting of myocardial tissues in AML patients treated with DNR chemotherapy and normal myocardium.
  • (Top) Immunofluorescence analysis of IL-1 ⁇ in myocardium after chemotherapy (patient) and normal myocardium (control); gray indicates ACTIN2, green indicates IL-1 ⁇ , and blue indicates DAPI; scale bar: 50 ⁇ m;
  • (Bottom) Immunoblotting analysis of the NF- ⁇ B signaling pathway in myocardium after chemotherapy and normal myocardium.
  • PGC-1 ⁇ peroxisome proliferator-activated receptor ⁇ coactivator 1- ⁇
  • p65 transcription factor p65
  • p-p65 phosphorylated transcription factor p65
  • Actin actin.
  • Figure 19 shows that an NF- ⁇ B signaling antagonist improves cardiac metabolism and function in mice after chemotherapy.
  • PDTC group DNR intervention in AML mice treated with PDTC (PDTC group) or untreated (control group);
  • PDTC group PDTC group
  • control group untreated (control group)
  • PDTC group PDTC group
  • Control group untreated (control group)
  • PDTC group Cardiac ultrasound examination of the two groups of mice
  • Bottom Myocardial ATP content in the two groups of mice.
  • Figure 20 shows changes in cardiac PGC-1 ⁇ expression in AML mice after DNR chemotherapy.
  • (Top) Detection of cardiac PGC-1 ⁇ protein expression in AML mice after chemotherapy.
  • (Right) Detection of PGC-1 ⁇ protein expression relative to Actin protein.
  • (Bottom) Detection of cardiac PGC-1 ⁇ transcript levels in AML mice after chemotherapy.
  • AML acute myeloid leukemia
  • DNR daunorubicin
  • PGC-1 ⁇ peroxisome proliferator-activated receptor gamma coactivator 1- ⁇
  • Actin actin.
  • Statistical differences between the two groups were calculated using a two-sample t-test ( ** P ⁇ 0.01).
  • 21A-21D show the expression of myocardial PGC-1 ⁇ in children with AML after DNR chemotherapy.
  • FIG21A is a UMAP image showing that PGC-1 ⁇ is mainly expressed in cardiomyocytes in cardiac tissue.
  • FIG21B is a pseudo-time series analysis showing the cell fate of different cardiomyocyte subpopulations.
  • FIG21C shows the expression levels of PGC-1 ⁇ in different cardiomyocyte subsets.
  • FIG21D is a pseudo-time series analysis showing the relationship between different cardiomyocyte fates and PGC-1 ⁇ expression levels.
  • Figure 22 shows a dual-luciferase reporter assay. Overexpression of p65 significantly inhibited PGC-1 ⁇ transcription. TSS: transcription start site; LUC: firefly luciferase; RLU: relative fluorescence intensity; PGC-1 ⁇ : peroxisome proliferator-activated receptor ⁇ coactivator 1- ⁇ ; p65: transcription factor p65 ( *** P ⁇ 0.001).
  • FIG. 23 shows that cardiac PGC-1 ⁇ overexpression improves cardiac metabolism and function in mice after chemotherapy.
  • WT wild type
  • AAV9 adeno-associated virus type 9
  • NC negative control
  • PGC-1 ⁇ peroxisome proliferator-activated receptor ⁇ coactivator 1- ⁇
  • AML acute myeloid leukemia
  • DNR daunorubicin
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular short-axis contraction fraction
  • ATP adenosine triphosphate
  • FIG24A and FIG24B show that IL-1 ⁇ neutralizing antibodies improve cardiac metabolism and function in mice after chemotherapy.
  • Figure 24B shows the percentage of residual GFP+ tumor cells in the bone marrow of the two groups of mice on day 4 after chemotherapy.
  • Figure 25 illustrates the mechanism by which chemotherapy leads to cardiac metabolic disorders and functional impairment.
  • tumor cell necrosis releases IL-1 ⁇ , which acts on the cardiac IL1R1 receptor, activating the NF-KappaB signaling pathway and inhibiting PGC-1 ⁇ expression, leading to cardiac metabolic disorders and functional abnormalities.
  • B-ALL acute lymphoblastic B-cell leukemia
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular short-axis contraction fraction
  • ATP adenosine triphosphate
  • the heart is an organ with a high energy demand, beating over 100,000 times daily, pumping 10 tons of blood throughout the body and consuming 6 kilograms of ATP.
  • the heart In the fasting state, the heart relies on fatty acids for 70% of its energy supply, making energy conversion a core metabolic process.
  • cardiac energy metabolism undergoes remodeling, shifting from a primary fatty acid to glucose energy supply, accompanied by decreased cardiac ATP synthesis and functional impairment.
  • Numerous studies have demonstrated a close link between impaired cardiac energy metabolism and cardiac damage. For example, a recent study found that energy imbalance induced by insulin resistance during adolescence directly leads to cardiac damage and dysfunction.
  • 9 is the post-chemotherapy cardiotoxicity in children with leukemia related to abnormal energy metabolism? Can improving cardiac energy metabolism mitigate chemotherapy-induced cardiovascular damage? These questions remain unclear and require further exploration.
  • Example 2 Tumor cell-derived IL-1 ⁇ induces cardiac metabolic and functional disorders
  • the cardiac energy metabolism disorder after chemotherapy may not be a direct effect of chemotherapy drugs, but a comprehensive result of the interaction between chemotherapy drugs and leukemia cells.
  • the inventors performed transcriptomic sequencing on the heart tissue of leukemia mice after chemotherapy. The results showed that the cytokine and cytokine receptor interaction pathway was significantly enriched in the myocardial tissue after chemotherapy ( Figure 10A- Figure 10C). This result suggests that chemotherapy may cause certain tumor-derived cytokines to be released into the blood, leading to energy metabolism disorders in the heart.
  • Luminex multifactorial detection technology to perform high-throughput screening of cytokines that change in the plasma of leukemia mice and normal tumor-free mice after chemotherapy.
  • the results showed that among all the cytokines tested, nine cytokines were significantly elevated after chemotherapy in leukemia mice. Because tumor-specific cytokines released by tumor cells need to follow a pattern of increase in leukemia mice after chemotherapy but not in healthy tumor-free mice, the inventors validated each of these nine cytokines individually.
  • the results showed that only interleukin-1 ⁇ (IL-1 ⁇ ) met this trend ( Figures 11A-11D).
  • the IL-1 family is divided into two subtypes: IL-1 ⁇ and IL-1 ⁇ .
  • IL-1 ⁇ is currently considered a damage-associated model molecule, constitutively expressed in both blood and non-blood cells. After cell necrosis, IL-1 ⁇ is released into the intercellular space or blood circulation to exert its biological functions locally or systemically. 16 The inventors' previous in vitro experiments showed that DNR can induce necrosis in leukemia cells, and the necrosis rate increases in a dose-dependent manner ( Figures 12A and 12B).
  • the inventors also found that the protein expression of IL-1 ⁇ in the leukemia cell culture supernatant was significantly increased after the addition of DNR ( Figure 12C). Therefore, the IL-1 ⁇ released by leukemia cell necrosis caused by chemotherapy drugs may be a potential cause of cardiac metabolic disorders and cardiac function damage.
  • the inventors used systemic IL-1 ⁇ knockout mice to construct a leukemia model.
  • the inventors knocked down the IL-1 ⁇ gene in primary leukemia cells using short hairpin RNA (shRNA) technology, and then constructed a leukemia model using the IL-1 ⁇ knockdown cell line.
  • shRNA short hairpin RNA
  • PDTC an inhibitor of the NF-KappaB signaling pathway
  • Example 4 PGC-1 ⁇ reduction is the core of IL-1 ⁇ -mediated cardiometabolic disorders
  • PGC-1 ⁇ is a transcriptional coactivator that binds to multiple transcription factors and plays a crucial role in processes such as fatty acid oxidation, glycolysis, and mitochondrial biogenesis. 19 In recent years, PGC-1 ⁇ has been found to be closely linked to cardiac energy metabolism. Studies have shown that decreased PGC-1 ⁇ expression inhibits cardiac fatty acid metabolism and further induces cardiac dysfunction. 20 Studies have also shown that activation of the NF-kappaB signaling pathway can downregulate cellular PGC-1 ⁇ expression, ultimately altering cellular metabolic patterns. 21
  • IL-1 ⁇ neutralizing antibodies have translational value and broad application prospects for improving cardiac damage in treated patients.
  • IL-1 ⁇ plays a key role in both leukemia development and cardiac damage, and blocking the IL-1 ⁇ -mediated cardiac NF-kappaB/PGC-1 ⁇ axis may reduce the potential risk of cardiotoxicity.

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Abstract

Provided are use of an IL-1α inhibitor in preparing a medicament for treating leukemia therapy-induced cardiac injury and use of an IL1R1 antagonist in preparing a medicament for treating leukemia therapy-induced cardiac injury. The present disclosure can effectively reduce cardiovascular complications and the occurrence of long-term cardiovascular adverse events in the period of leukemia treatments, thereby helping alleviate the cardiac metabolic dysfunction and cardiac dysfunction after treatment.

Description

IL-1α的抑制剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用Application of IL-1α inhibitors in the preparation of drugs for treating leukemia-induced cardiac damage

本申请要求申请日为2024/2/20的中国专利申请2024101907113的优先权。本申请引用上述中国专利申请的全文。This application claims the benefit of Chinese patent application No. 2024101907113, filed on February 20, 2024. The entire text of the aforementioned Chinese patent application is incorporated herein by reference.

技术领域Technical Field

本发明涉及生物医学领域，具体涉及IL-1α的抑制剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用。The present invention relates to the field of biomedicine, and in particular to the use of an IL-1α inhibitor in the preparation of a medicine for treating heart damage induced by leukemia treatment.

背景技术Background Art

肿瘤疾病与心血管疾病是严重威胁人类健康的两大类疾病。白血病占儿童恶性肿瘤患者的三分之一，是最常见的儿童恶性肿瘤。有数据显示：随着新治疗手段的出现与治疗方案的优化，儿童急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)和急性髓系白血病(acute myelocytic leukemia，AML)的五年总体生存率分别达到90％与78.2％，从而使得白血病幸存者基数不断扩大。最新的一项临床研究表明，儿童肿瘤疾病中白血病幸存者的长期疾病负担远高于其他类型肿瘤幸存者，且主要以心血管疾病负担为主1。其中AML患儿的心脏疾病发生率显著高于ALL，主要表现为心肌病与心力衰竭2。目前普遍认为，白血病幸存患儿的心脏疾病死亡风险主要来源于肿瘤化疗。例如，上世纪70年代就有研究报道认为儿童白血病化疗方案中的核心药物蒽环类化疗药具有明确的剂量依赖性心脏毒性3。一项针对儿童AML幸存者的研究表明，12％的幸存者在5年内会发生心功能障碍，其中约70％的心功能障碍在围化疗期出现，尤为重要的是其早期是否出现治疗相关心脏毒性决定了其远期生存率的高低4因此早期识别化疗诱导的心脏毒性并积极干预将有可能降低其心血管疾病的负担5。Cancer and cardiovascular disease are two major diseases that pose a serious threat to human health. Leukemia accounts for one-third of childhood malignant tumors and is the most common childhood malignancy. Data show that with the emergence of new treatments and optimized regimens, the five-year overall survival rates for childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have reached 90% and 78.2%, respectively, leading to a growing base of leukemia survivors. A recent clinical study showed that among childhood cancers, leukemia survivors face a significantly higher long-term disease burden than survivors of other cancer types, primarily due to cardiovascular disease. 1 The incidence of heart disease in children with AML is significantly higher than in those with ALL, primarily manifesting as cardiomyopathy and heart failure. 2 It is generally believed that the risk of cardiac mortality in leukemia survivors is primarily due to chemotherapy. For example, studies dating back to the 1970s reported that anthracyclines, a core drug in childhood leukemia chemotherapy regimens, exhibit dose-dependent cardiotoxicity. 3 A study of childhood AML survivors showed that 12% of survivors will develop cardiac dysfunction within 5 years, of which approximately 70% develop during the peri-chemotherapy period. Importantly, the presence of treatment-related cardiotoxicity early on determines long-term survival. 4 Therefore, early identification of chemotherapy-induced cardiotoxicity and active intervention may reduce the burden of cardiovascular disease. 5

能量代谢是心脏最重要的生理活动，许多研究发现能量代谢异常是心脏损伤的重要原因。Neubauer等率先提出心衰的心脏是“燃料耗竭的机器”的概念6，但是化疗药是否会通过干扰心肌能量代谢进而损伤心脏目前尚不十分清楚。另一方面，以往肿瘤心脏病学领域的研究主要采用健康小鼠观察长期大剂量多疗程化疗后药物对于心脏的直接损害，例如：Wallace等发现蒽环类药物会通过促进线粒体呼吸链中活性氧的生成破坏心肌细胞6，Zhang等发现蒽环类药物可以通过抑制拓扑异构酶Ⅱβ的活性造成心脏的损伤7。然而，临床上发明人发现白血病患儿在短期诱导化疗阶段即可出现化疗药相关心脏损伤，此外以往的研究忽略了化疗后肿瘤微环境改变对心脏的间接毒性作用，忽视了肿瘤、化疗药物、心脏三者之间可能存在的交互关系。这些问题促使发明人思考是否存在尚未探明的化疗相关心脏损伤机制，继而通过精准化干预，减轻白血病患儿化疗后的心血管疾病负担。Energy metabolism is the heart's most important physiological activity, and numerous studies have found that abnormal energy metabolism is a major cause of cardiac damage. Neubauer et al. pioneered the concept that the heart in heart failure is a "fuel-depleted machine." 6 However, whether chemotherapy drugs can damage the heart by interfering with myocardial energy metabolism remains unclear. Previous studies in the field of cardiology oncology have primarily used healthy mice to investigate direct cardiac damage after long-term, high-dose, multi-course chemotherapy. For example, Wallace et al. found that anthracyclines damage cardiomyocytes by promoting the production of reactive oxygen species in the mitochondrial respiratory chain. 6 Zhang et al. found that anthracyclines can cause cardiac damage by inhibiting topoisomerase IIβ activity. 7 However, in clinical practice, the inventors have discovered that children with leukemia can develop chemotherapy-related cardiac damage even during the short-term induction chemotherapy phase. Furthermore, previous studies have overlooked the indirect toxic effects of altered tumor microenvironment post-chemotherapy on the heart, and have overlooked the potential interactions between tumors, chemotherapy drugs, and the heart. These questions prompted the inventors to explore the potential mechanisms of chemotherapy-related cardiac damage, and to explore targeted interventions to alleviate the cardiovascular burden of chemotherapy-induced leukemia in children.

发明内容Summary of the Invention

为了解决现有技术中缺乏一种精准、有效地改善白血病治疗导致的心脏损伤，例如心脏代谢障碍和心功能异常的方法的缺陷，本发明提供了IL-1α的抑制剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用。In order to address the deficiency in the prior art of lacking a method for accurately and effectively improving cardiac damage caused by leukemia treatment, such as cardiac metabolic disorders and abnormal cardiac function, the present invention provides the use of IL-1α inhibitors in the preparation of drugs for treating leukemia treatment-induced cardiac damage.

为解决上述技术问题，本发明提供的技术方案之一为：IL-1α(即白介素1α，Interleukin1α)的抑制剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用。To solve the above technical problems, one of the technical solutions provided by the present invention is: use of an IL-1α (i.e., interleukin 1α) inhibitor in the preparation of a drug for treating leukemia-induced cardiac damage.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the invention, the leukemia treatment results in necrosis of the leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug.

在本发明的具体实施方案中，所述蒽环类化疗药物为柔红霉素。In a specific embodiment of the present invention, the anthracycline chemotherapy drug is daunorubicin.

在本发明的具体实施方案中，所述IL-1α的抑制剂为抗IL-1α的抗体。In a specific embodiment of the present invention, the IL-1α inhibitor is an anti-IL-1α antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体为单克隆抗体。In a specific embodiment of the present invention, the anti-IL-1α antibody is a monoclonal antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体为中和抗体。In a specific embodiment of the present invention, the anti-IL-1α antibody is a neutralizing antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体购自R&D Systems、货号为AB-400-NA。In a specific embodiment of the present invention, the anti-IL-1α antibody was purchased from R&D Systems with the product number AB-400-NA.

在本发明的具体实施方案中，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

为解决上述技术问题，本发明提供的技术方案之二为：购自R&D Systems、货号为AB-400-NA的抗体在制备治疗白血病治疗诱导的心脏损伤的药物中的应用。To solve the above technical problems, the second technical solution provided by the present invention is: the use of an antibody purchased from R&D Systems with the product number AB-400-NA in the preparation of a drug for treating heart damage induced by leukemia treatment.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；和/或，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and/or, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the invention, the leukemia treatment results in necrosis of the leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，优选柔红霉素；和/或，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and/or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

为解决上述技术问题，本发明提供的技术方案之三为：购自R&D Systems、货号为AB-400-NA的抗体在制备通过抑制IL-1α对白血病治疗诱导的心脏损伤进行治疗的药物中的应用。To solve the above technical problems, the third technical solution provided by the present invention is: the use of an antibody purchased from R&D Systems with the product number AB-400-NA in the preparation of a drug for treating heart damage induced by leukemia treatment by inhibiting IL-1α.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；和/或，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and/or, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the invention, the leukemia treatment results in necrosis of the leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，优选柔红霉素；和/或，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and/or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

为解决上述技术问题，本发明提供的技术方案之四为：IL1R1(即白细胞介素-1受体1型，Interleukin-1 receptor type I)的拮抗剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用。To solve the above technical problems, the fourth technical solution provided by the present invention is: use of an antagonist of IL1R1 (i.e., interleukin-1 receptor type 1, Interleukin-1 receptor type I) in the preparation of a drug for treating heart damage induced by leukemia treatment.

本发明已经证明了心脏IL1R1受体的敲除可以达到心脏功能保护作用(实施例2)，本领域技术人员能够据此合理预期IL1R1的拮抗剂可以治疗白血病诱导的心脏损伤。The present invention has demonstrated that knockout of the cardiac IL1R1 receptor can protect cardiac function (Example 2). Based on this, those skilled in the art can reasonably anticipate that IL1R1 antagonists can treat leukemia-induced cardiac damage.

在本发明的具体实施方案中，所述IL1R1的拮抗剂为抗IL1R1的药物。In a specific embodiment of the present invention, the antagonist of IL1R1 is an anti-IL1R1 drug.

在本发明的具体实施方案中，所述IL1R1的拮抗剂为IL1R1的竞争性抑制药物或者封闭药物。In a specific embodiment of the present invention, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1.

在本发明的具体实施方案中，所述竞争性抑制药物为Anakinra(阿那白滞素，一种重组、非糖基化形式的人白细胞介素-1受体拮抗剂(IL-1Ra)，CAS号：143090-92-0)。In a specific embodiment of the present invention, the competitive inhibitory drug is Anakinra (Anakinra, a recombinant, non-glycosylated form of human interleukin-1 receptor antagonist (IL-1Ra), CAS No.: 143090-92-0).

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，例如柔红霉素；和/或，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy. In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; and/or the leukemia treatment causes leukemia cell necrosis.

在本发明的具体实施方案中，所述白血病为急性髓系白血病或急性淋巴细胞白血病；和/或，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia; and/or the cardiac injury is selected from one or more of: cardiac metabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述急性髓系白血病为MLL-AF9融合蛋白引起的；和/或，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and/or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

为解决上述技术问题，本发明提供的技术方案之五为：一种治疗白血病治疗诱导的心脏损伤的方法，所述方法包括向有需要的患者施用治疗有效量的IL-1α的抑制剂。To solve the above technical problems, the fifth technical solution provided by the present invention is: a method for treating leukemia-induced cardiac damage, the method comprising administering a therapeutically effective amount of an IL-1α inhibitor to a patient in need.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy. In a specific embodiment of the present invention, the leukemia treatment causes necrosis of leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug.

在本发明的具体实施方案中，所述蒽环类化疗药物为柔红霉素。In a specific embodiment of the present invention, the anthracycline chemotherapy drug is daunorubicin.

在本发明的具体实施方案中，所述IL-1α的抑制剂为抗IL-1α的抗体。In a specific embodiment of the present invention, the IL-1α inhibitor is an anti-IL-1α antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体为单克隆抗体。In a specific embodiment of the present invention, the anti-IL-1α antibody is a monoclonal antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体为中和抗体。In a specific embodiment of the present invention, the anti-IL-1α antibody is a neutralizing antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体购自R&D Systems、货号为AB-400-NA。In a specific embodiment of the present invention, the anti-IL-1α antibody was purchased from R&D Systems with the product number AB-400-NA.

在本发明的具体实施方案中，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

在本发明的具体实施方案中，所述患者为儿童。In a specific embodiment of the invention, the patient is a child.

为解决上述技术问题，本发明提供的技术方案之六为：一种治疗白血病治疗诱导的心脏损伤的方法，所述方法包括向有需要的患者施用治疗有效量的购自R&D Systems、货号为AB-400-NA的抗体。To solve the above technical problems, the sixth technical solution provided by the present invention is: a method for treating cardiac damage induced by leukemia treatment, the method comprising administering a therapeutically effective amount of an antibody purchased from R&D Systems with the product number AB-400-NA to a patient in need.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；和/或，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and/or, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the invention, the leukemia treatment results in necrosis of the leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，优选柔红霉素；和/或，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and/or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

在本发明的具体实施方案中，所述患者为儿童。In a specific embodiment of the invention, the patient is a child.

为解决上述技术问题，本发明提供的技术方案之七为：一种治疗白血病治疗诱导的心脏损伤的方法，所述方法包括向有需要的患者施用治疗有效量的IL1R1的拮抗剂。To solve the above technical problems, the seventh technical solution provided by the present invention is: a method for treating leukemia-induced cardiac damage, comprising administering a therapeutically effective amount of an IL1R1 antagonist to a patient in need.

在本发明的具体实施方案中，所述IL1R1的拮抗剂为抗IL1R1的药物。In a specific embodiment of the present invention, the antagonist of IL1R1 is an anti-IL1R1 drug.

在本发明的具体实施方案中，所述IL1R1的拮抗剂为IL1R1的竞争性抑制药物或者封闭药物。In a specific embodiment of the present invention, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1.

在本发明的具体实施方案中，所述竞争性抑制药物为Anakinra。In a specific embodiment of the present invention, the competitive inhibitory drug is Anakinra.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，例如柔红霉素；和/或，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy. In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; and/or the leukemia treatment causes leukemia cell necrosis.

在本发明的具体实施方案中，所述白血病为急性髓系白血病或急性淋巴细胞白血病；和/或，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia; and/or the cardiac injury is selected from one or more of: cardiac metabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述急性髓系白血病为MLL-AF9融合蛋白引起的；和/或，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and/or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

在本发明的具体实施方案中，所述患者为儿童。In a specific embodiment of the invention, the patient is a child.

如本发明所用，术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示，与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强正常生理功能的量。As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Additionally, the term "therapeutically effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

为解决上述技术问题，本发明提供的技术方案之八为：一种IL-1α的抑制剂，其用于治疗白血病治疗诱导的心脏损伤。To solve the above technical problems, the eighth technical solution provided by the present invention is: an IL-1α inhibitor for treating heart damage induced by leukemia treatment.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the invention, the leukemia treatment results in necrosis of the leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug.

在本发明的具体实施方案中，所述蒽环类化疗药物为柔红霉素。In a specific embodiment of the present invention, the anthracycline chemotherapy drug is daunorubicin.

在本发明的具体实施方案中，所述IL-1α的抑制剂为抗IL-1α的抗体。In a specific embodiment of the present invention, the IL-1α inhibitor is an anti-IL-1α antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体为单克隆抗体。In a specific embodiment of the present invention, the anti-IL-1α antibody is a monoclonal antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体为中和抗体。In a specific embodiment of the present invention, the anti-IL-1α antibody is a neutralizing antibody.

在本发明的具体实施方案中，所述抗IL-1α的抗体购自R&D Systems、货号为AB-400-NA。In a specific embodiment of the present invention, the anti-IL-1α antibody was purchased from R&D Systems with the product number AB-400-NA.

在本发明的具体实施方案中，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

为解决上述技术问题，本发明提供的技术方案之九为：购自R&D Systems、货号为AB-400-NA的抗体，其用于治疗白血病治疗诱导的心脏损伤。To solve the above technical problems, the ninth technical solution provided by the present invention is: an antibody purchased from R&D Systems with the product number AB-400-NA, which is used to treat heart damage induced by leukemia treatment.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；和/或，所述白血病为急性髓系白血病或急性淋巴细胞白血病。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and/or, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the invention, the leukemia treatment results in necrosis of the leukemia cells.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，优选柔红霉素；和/或，所述急性髓系白血病为MLL-AF9融合蛋白引起的。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and/or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

在本发明的具体实施方案中，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiometabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

为解决上述技术问题，本发明提供的技术方案之十为：一种IL1R1的拮抗剂，其用于治疗白血病治疗诱导的心脏损伤。To solve the above technical problems, the tenth technical solution provided by the present invention is: an antagonist of IL1R1, which is used to treat heart damage induced by leukemia treatment.

在本发明的具体实施方案中，所述IL1R1的拮抗剂为抗IL1R1的药物。In a specific embodiment of the present invention, the antagonist of IL1R1 is an anti-IL1R1 drug.

在本发明的具体实施方案中，所述IL1R1的拮抗剂为IL1R1的竞争性抑制药物或者封闭药物。In a specific embodiment of the present invention, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1.

在本发明的具体实施方案中，所述竞争性抑制药物为Anakinra。In a specific embodiment of the present invention, the competitive inhibitory drug is Anakinra.

在本发明的具体实施方案中，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种。In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

在本发明的具体实施方案中，所述免疫治疗为CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

在本发明的具体实施方案中，所述免疫治疗为CD19 CAR-T治疗。In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

在本发明的具体实施方案中，所述化疗的药物为蒽环类化疗药物，例如柔红霉素；和/或，所述白血病治疗导致白血病细胞坏死。In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; and/or the leukemia treatment causes leukemia cell necrosis.

在本发明的具体实施方案中，所述白血病为急性髓系白血病或急性淋巴细胞白血病；和/或，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种。In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia; and/or the cardiac injury is selected from one or more of: cardiac metabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure.

在本发明的具体实施方案中，所述急性淋巴细胞白血病为急性淋系T细胞白血病或急性淋系B细胞白血病。In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

在本发明的具体实施方案中，所述急性髓系白血病为MLL-AF9融合蛋白引起的；和/或，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and/or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are commercially available.

本发明的积极进步效果在于：The positive progress effect of the present invention is:

本发明率先提出白血病治疗(例如化疗或免疫治疗)引起的肿瘤源性细胞因子IL-1α的释放是导致心脏能量代谢重塑和功能异常的关键，强调药物与肿瘤的相互作用在白血病治疗药物(例如化疗药或CAR-T)的心脏毒性中扮演了重要角色。本发明提供的IL-1α的抑制剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用将有效降低白血病治疗期间的心血管并发症，减少远期心血管不良事件的发生率，有助于改善治疗后的心脏代谢障碍和心功能异常，将有可能显著改善白血病患者的远期预后，使众多白血病患者获益，从而获得明显的社会和经济效应。The present invention is the first to propose that the release of tumor-derived cytokine IL-1α caused by leukemia treatment (such as chemotherapy or immunotherapy) is the key to causing cardiac energy metabolism remodeling and functional abnormalities, emphasizing that the interaction between drugs and tumors plays an important role in the cardiotoxicity of leukemia treatment drugs (such as chemotherapy drugs or CAR-T). The use of the IL-1α inhibitor provided by the present invention in the preparation of a drug for treating leukemia treatment-induced cardiac damage will effectively reduce cardiovascular complications during leukemia treatment, reduce the incidence of long-term adverse cardiovascular events, and help improve cardiac metabolic disorders and cardiac function abnormalities after treatment. It is likely to significantly improve the long-term prognosis of leukemia patients, benefiting many leukemia patients, thereby achieving significant social and economic effects.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为心脏特异性Il1r1敲除小鼠构建。(上)构建示意图；(下)免疫印迹实验验证IL1R1敲除效果，对照组为IL1R1 fl/fl，实验组为IL1R1 cKO，右侧为IL1R1相对于Actin的蛋白表达量。IL1R1：白细胞介素-1受体1型；Actin：肌动蛋白。两组样本之间的统计学差异使用两独立样本t检验进行计算(*P<0.05)。Figure 1 shows the construction of cardiac-specific Il1r1 knockout mice. (Top) Schematic diagram of the construction; (Bottom) Western blot analysis to verify the effect of IL1R1 knockout. The control group consisted of IL1R1 fl/fl cells, while the experimental group consisted of IL1R1 cKO cells. The right side shows the protein expression of IL1R1 relative to actin. IL1R1: interleukin-1 receptor type 1; Actin: actin. Statistical differences between the two groups were calculated using a two-sample t-test ( * P < 0.05).

图2为Il1a稳定敲降的AML细胞构建。(上)构建示意图；(下)RT-PCR验证敲除效果，对照组使用scramble序列，实验组使用shIL-1α序列，右侧为Il1a相对于Gapdh的mRNA表达量。两组样本之间的统计学差异使用两独立样本t检验进行计算(***P<0.001)。Figure 2 shows the construction of AML cells with stable Il1a knockdown. (Top) Schematic diagram of the construction; (Bottom) RT-PCR verification of knockdown efficacy. The control group used a scramble sequence, and the experimental group used a shIL-1α sequence. The right side shows the mRNA expression levels of Il1a relative to Gapdh. Statistical differences between the two groups were calculated using a two-sample t-test ( *** P < 0.001).

图3为心肌特异性过表达PGC-1α效果验证。对照组为AAV9-NC，实验组为AAV9-PGC1α，右侧为PGC-1α相对于Actin的蛋白表达量。NC：阴性对照，PGC-1α：过氧化物酶体增殖体激活受体γ辅激活因子1-α，Actin：肌动蛋白。两组样本之间的统计学差异使用两独立样本t检验进行计算(**P<0.01)。Figure 3 demonstrates the efficacy of myocardial-specific overexpression of PGC-1α. The control group received AAV9-NC, while the experimental group received AAV9-PGC1α. The protein expression levels of PGC-1α relative to actin are shown on the right. NC: negative control; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-α; Actin: actin. Statistical differences between the two groups were calculated using a two-sample t-test ( ** P<0.01).

图4为p65过表达质粒和PGC-1α启动子荧光报告质粒。FIG4 shows the p65 overexpression plasmid and the PGC-1α promoter fluorescence reporter plasmid.

图5为MLL-AF9 AML小鼠模型的构建示意图。AML：急性髓系白血病。Figure 5 is a schematic diagram of the construction of the MLL-AF9 AML mouse model. AML: acute myeloid leukemia.

图6A-图6D为AML小鼠使用DNR化疗后的心脏代谢和功能变化。Figure 6A-Figure 6D show the changes in cardiac metabolism and function in AML mice after DNR chemotherapy.

图6A为AML小鼠分为两组，一组接受DNR化疗(AML+DNR组)，一组使用PBS作为对照(AML组)。Figure 6A shows that AML mice were divided into two groups, one group received DNR chemotherapy (AML+DNR group), and the other group received PBS as a control (AML group).

图6B为两组小鼠的心脏超声检测。FIG6B shows cardiac ultrasound examination of the two groups of mice.

图6C为两组小鼠的心脏PET-CT检测，氟18标记的FTHA与FDG探针信号强弱分别反映了心脏对脂肪酸和葡萄糖的利用水平。Figure 6C shows the cardiac PET-CT examination of the two groups of mice. The signal strengths of the fluorine-18 labeled FTHA and FDG probes reflect the levels of fatty acid and glucose utilization by the heart, respectively.

图6D为两组小鼠的心肌ATP含量评估。Figure 6D shows the evaluation of myocardial ATP content in the two groups of mice.

AML：急性髓系白血病；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；FDG：脱氧葡萄糖；FTHA：6-硫-十七烷酸；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(*P<0.05；**P<0.01***P<0.001)。AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional contraction; FDG: deoxyglucose; FTHA: 6-thio-heptadecanoic acid; ATP: adenosine triphosphate; the statistical differences between the two groups were calculated using the two-sample t-test ( * P <0.05; ** P < 0.01 *** P < 0.001).

图7为白血病小鼠化疗后心脏脂代谢关键酶表达下降。AML：急性髓系白血病；DNR：柔红霉素；CD36：白细胞分化抗原36；CPT1：肉毒碱棕榈酰基转移酶1；LCAD：长链酰基辅酶A脱氢酶；MCAD：中链酰基辅酶A脱氢酶；Actin：肌动蛋白；两组样本之间的统计学差异使用两独立样本t检验进行计算(*P<0.05；**P<0.01；***P<0.001)。Figure 7 shows decreased expression of key enzymes of cardiac lipid metabolism in leukemic mice after chemotherapy. AML: acute myeloid leukemia; DNR: daunorubicin; CD36: leukocyte differentiation antigen 36; CPT1: carnitine palmitoyltransferase 1; LCAD: long-chain acyl-CoA dehydrogenase; MCAD: medium-chain acyl-CoA dehydrogenase; Actin: actin. Statistical differences between the two groups were calculated using a two-sample t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

图8A-图8D为AML患儿使用DNR化疗后心肌与正常心肌的单细胞测序分析。Figures 8A-8D show single-cell sequencing analysis of the myocardium of AML children after DNR chemotherapy and normal myocardium.

图8A为UMAP图展示心脏细胞聚类分布。FIG8A is a UMAP image showing the cluster distribution of cardiac cells.

图8B为正常心肌和化疗后心肌细胞的scMetabolism代谢评分；糖代谢通路使用红色进行了突出显示，脂肪酸代谢通路使用蓝色进行了突出显示。Figure 8B shows the scMetabolism metabolic scores of normal myocardium and myocardial cells after chemotherapy; the carbohydrate metabolism pathway is highlighted in red, and the fatty acid metabolism pathway is highlighted in blue.

图8C为对所有心肌细胞进行拟时序分析。FIG8C shows a pseudo-time series analysis of all cardiomyocytes.

图8D为对所有心肌细胞进行拟时序分支点分析。FIG8D shows a pseudo-sequential branch point analysis of all cardiomyocytes.

其中在非适应态心肌细胞中表达下调的与脂肪酸代谢相关的基因名称使用蓝色进行了突出显示，表达上调的与糖代谢相关的基因名称用红色进行了突出显示。The names of genes related to fatty acid metabolism that are downregulated in non-adapted cardiomyocytes are highlighted in blue, and the names of genes related to glucose metabolism that are upregulated are highlighted in red.

图9A-图9D为正常无瘤小鼠使用DNR化疗后的心脏代谢和功能变化。Figure 9A-Figure 9D show the changes in cardiac metabolism and function in normal tumor-free mice after DNR chemotherapy.

图9A为健康无瘤小鼠分为两组，一组接受DNR化疗(TF+DNR组)，一组使用PBS(TF组)作为对照。Figure 9A shows that healthy tumor-free mice were divided into two groups, one group received DNR chemotherapy (TF+DNR group), and the other group received PBS (TF group) as a control.

图9B为两组小鼠的心脏超声检测。FIG9B shows cardiac ultrasound examination of the two groups of mice.

图9C为两组小鼠的心脏PET-CT检测。Figure 9C shows the cardiac PET-CT examination of the two groups of mice.

图9D为两组小鼠心肌ATP含量检测。FIG9D shows the detection of myocardial ATP content in two groups of mice.

TF：无瘤；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；FTHA：6-硫-十七烷酸；FDG：脱氧葡萄糖；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(NS：无显著性差异)。TF: tumor-free; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; FTHA: 6-thio-heptadecanoic acid; FDG: deoxyglucose; ATP: adenosine triphosphate; the statistical differences between the two groups were calculated using the two-sample t-test (NS: not significant).

图10A-图10C为AML小鼠使用DNR化疗后的心脏转录组学分析。FIG10A-FIG10C are cardiac transcriptomic analyses of AML mice after DNR chemotherapy.

图10A为化疗后心脏转录组学结果的差异基因KEGG通路富集分析，细胞因子与细胞因子互作通路显著富集并用红色进行了突出显示。Figure 10A shows the KEGG pathway enrichment analysis of differentially expressed genes in cardiac transcriptomics results after chemotherapy. The cytokine and cytokine interaction pathways were significantly enriched and highlighted in red.

图10B为化疗后心脏转录组学结果的生物反应路径分析，PPAR信号显著下调并用蓝色进行了突出显示。Figure 10B shows the biological response pathway analysis of cardiac transcriptomics results after chemotherapy, with PPAR signaling significantly downregulated and highlighted in blue.

图10C为PPAR通路差异基因的蛋白互作网络分析，显示IL-1信号，NF-KappaB信号与代谢关键蛋白PPARGC1A紧密互作。Figure 10C is a protein interaction network analysis of differentially expressed genes in the PPAR pathway, showing that IL-1 signaling and NF-KappaB signaling closely interact with the key metabolic protein PPARGC1A.

图11A-图11D为AML小鼠使用DNR化疗后血浆多因子检测，方框指示IL-1α。Figures 11A-11D show the detection of multiple factors in plasma of AML mice after DNR chemotherapy, and the boxes indicate IL-1α.

TF：无瘤；AML：急性髓系白血病；DNR：柔红霉素。样本之间的差异使用方差分析事后Tukey-Kramer检验进行计算(NS：无显著性差异；*P<0.05；**P<0.01；***P<0.001)。TF: tumor-free; AML: acute myeloid leukemia; DNR: daunorubicin. Differences between samples were calculated using analysis of variance followed by the Tukey-Kramer test (NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

图12A-图12C为DNR化疗后AML细胞发生坏死释放IL-1α。Figures 12A-12C show that AML cells undergo necrosis and release IL-1α after DNR chemotherapy.

图12A和图12B为流式细胞学检测AML原代细胞系在不同DNR浓度梯度干预24h后的活细胞与坏死细胞比例。FIG12A and FIG12B are flow cytometric measurements of the ratio of live cells to necrotic cells in primary AML cell lines after 24 h of intervention with different DNR concentration gradients.

图12C为0.5μM DNR干预AML原代细胞系24h后细胞培养上清中IL-1α的含量检测。Figure 12C shows the detection of IL-1α content in the cell culture supernatant after 24 hours of intervention of AML primary cell line with 0.5 μM DNR.

IL-1α：白细胞介素1α；DNR：柔红霉素；两组样本之间的统计学差异使用两独立样本t检验进行计算，两组以上样本之间的差异使用方差分析事后Tukey-Kramer检验进行计算(*P<0.05；**P<0.01；***P<0.001)。IL-1α: interleukin 1α; DNR: daunorubicin; the statistical differences between two groups of samples were calculated using the two-independent sample t-test, and the differences between more than two groups of samples were calculated using the post-hoc Tukey-Kramer test for analysis of variance (*P<0.05; **P<0.01; ***P<0.001).

图13A-图13D为正常无瘤小鼠使用IL-1α后的心脏代谢和功能变化。13A-13D show the changes in cardiac metabolism and function in normal tumor-free mice after administration of IL-1α.

图13A为健康无瘤小鼠分为两组，一组接受IL-1α(TF+IL-1α组)，一组使用PBS作为对照(TF组)。FIG13A shows healthy tumor-free mice divided into two groups, one group receiving IL-1α (TF+IL-1α group) and the other group receiving PBS as a control (TF group).

图13B为两组小鼠的心脏超声检测。FIG13B shows cardiac ultrasound examination of the two groups of mice.

图13C为两组小鼠的心脏PET-CT检测。FIG13C shows the cardiac PET-CT examination of the two groups of mice.

图13D为两组小鼠心肌ATP含量检测。FIG13D shows the detection of myocardial ATP content in two groups of mice.

TF：无瘤；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；FTHA：6-硫-十七烷酸；FDG：脱氧葡萄糖；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(*P<0.05；**P<0.01***P<0.001)。TF: tumor-free; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; FTHA: 6-thio-heptadecanoic acid; FDG: deoxyglucose; ATP: adenosine triphosphate; the statistical differences between the two groups were calculated using the two-sample t-test ( * P <0.05; ** P < 0.01 *** P < 0.001).

图14A-图14C为心脏特异性Il1r1敲除改善化疗后小鼠心脏代谢和功能。Figure 14A-Figure 14C show that cardiac-specific Il1r1 knockout improves cardiac metabolism and function in mice after chemotherapy.

图14A为实验示意图，DNR干预心肌细胞特异性敲除IL1R1的AML小鼠(IL1R1cKO组)与对照AML小鼠(IL1R1 fl/fl组)。Figure 14A is a schematic diagram of the experiment, in which DNR was used to intervene in AML mice with cardiomyocyte-specific knockout of IL1R1 (IL1R1cKO group) and control AML mice (IL1R1 fl/fl group).

图14B为两组小鼠的心脏超声检测。FIG14B shows cardiac ultrasound examination of the two groups of mice.

图14C为两组小鼠心肌ATP含量检测。FIG14C shows the detection of myocardial ATP content in two groups of mice.

AML：急性髓系白血病；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(***P<0.001)。AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional contraction; ATP: adenosine triphosphate; The statistical differences between the two groups were calculated using the two-independent-sample t-test (***P<0.001).

图15为急性髓细胞性白血病患者化疗前后血浆IL-1α升高率与LVEF降低率的相关性分析。LVEF：左心室射血分数。Figure 15 shows the correlation analysis between the increase rate of plasma IL-1α and the decrease rate of LVEF in patients with acute myeloid leukemia before and after chemotherapy. LVEF: left ventricular ejection fraction.

图16A-图16C为AML细胞Il1a敲降改善化疗后小鼠心脏代谢和功能。Figure 16A-Figure 16C show that knockdown of Il1a in AML cells improves cardiac metabolism and function in mice after chemotherapy.

图16A为实验示意图，DNR干预使用对照(scramble组)与敲降IL-1α(shIL-1α组)细胞构建的AML模型。Figure 16A is a schematic diagram of the experiment, in which DNR intervention was performed on AML models constructed using control (scramble group) and IL-1α knockdown (shIL-1α group) cells.

图16B为两组小鼠的心脏超声检测。FIG16B shows cardiac ultrasound examination of the two groups of mice.

图16C为两组小鼠心肌ATP含量检测。FIG16C shows the detection of myocardial ATP content in two groups of mice.

WT：野生型；AML：急性髓系白血病；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(***P<0.001)。WT: wild type; AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional contraction; ATP: adenosine triphosphate; the statistical differences between the two groups were calculated using the two-sample t-test ( *** P < 0.001).

图17为正常无瘤小鼠和AML小鼠使用DNR化疗后心脏NF-KappaB信号通路的检测。(上)正常无瘤小鼠化疗后的心脏NF-KappaB通路免疫印迹检测；(下)AML小鼠化疗后心脏NF-KappaB通路免疫印迹检测。TF：无瘤；AML：急性髓系白血病；DNR：柔红霉素；p-p65：磷酸化转录因子p65；p65：转录因子p65；IκBα：核因子κB抑制蛋白；Actin：肌动蛋白。两组样本之间的统计学差异使用两独立样本t检验进行计算(NS：无显著性差异；*P<0.05；**P<0.01)。Figure 17 shows the detection of the cardiac NF-κB signaling pathway in normal tumor-free mice and AML mice after chemotherapy with DNR. (Top) Immunoblotting of the cardiac NF-κB pathway in normal tumor-free mice after chemotherapy; (Bottom) Immunoblotting of the cardiac NF-κB pathway in AML mice after chemotherapy. TF: tumor-free; AML: acute myeloid leukemia; DNR: daunorubicin; p-p65: phosphorylated transcription factor p65; p65: transcription factor p65; IκBα: inhibitor of nuclear factor κB; Actin: actin. Statistical differences between the two groups were calculated using a two-sample independent t-test (NS: not significant; * P <0.05; ** P < 0.01).

图18为AML患儿使用DNR化疗后心肌与正常心肌的免疫荧光和免疫印迹检测。(上)化疗后心肌(Patient)和正常心肌(Control)的IL-1α免疫荧光检测；灰色指示ACTIN2，绿色指示IL-1α，蓝色指示DAPI；标尺：50μm；(下)化疗后心肌和正常心肌的NF-KappaB信号通路免疫印迹检测。PGC-1α：过氧化物酶体增殖受体γ辅激活因子1-α；p65：转录因子p65；p-p65：磷酸化转录因子p65；Actin：肌动蛋白。Figure 18 shows immunofluorescence and immunoblotting of myocardial tissues in AML patients treated with DNR chemotherapy and normal myocardium. (Top) Immunofluorescence analysis of IL-1α in myocardium after chemotherapy (patient) and normal myocardium (control); gray indicates ACTIN2, green indicates IL-1α, and blue indicates DAPI; scale bar: 50 μm; (Bottom) Immunoblotting analysis of the NF-κB signaling pathway in myocardium after chemotherapy and normal myocardium. PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1-α; p65: transcription factor p65; p-p65: phosphorylated transcription factor p65; Actin: actin.

图19为NF-KappaB信号拮抗剂改善化疗后小鼠心脏代谢和功能。(上)实验示意图，DNR干预PDTC处理(PDTC组)或非处理(control组)的AML小鼠；(中)两组小鼠的心脏超声检测；(下)两组小鼠心肌ATP含量检测。AML：急性髓系白血病；DNR：柔红霉素；PDTC：吡咯烷二硫代氨基甲酸铵；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(**P<0.01；***P<0.001)。Figure 19 shows that an NF-κB signaling antagonist improves cardiac metabolism and function in mice after chemotherapy. (Top) Experimental schematic: DNR intervention in AML mice treated with PDTC (PDTC group) or untreated (control group); (Middle) Cardiac ultrasound examination of the two groups of mice; (Bottom) Myocardial ATP content in the two groups of mice. AML: acute myeloid leukemia; DNR: daunorubicin; PDTC: ammonium pyrrolidine dithiocarbamate; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; ATP: adenosine triphosphate; Statistical differences between the two groups were calculated using a two-sample t-test (**P < 0.01; ***P < 0.001).

图20为AML小鼠使用DNR化疗后心脏PGC-1α的表达改变。(上)AML小鼠化疗后心脏PGC-1α蛋白表达检测，右侧为PGC-1α相对于Actin的蛋白表达量；(下)AML小鼠化疗后心脏Pgc-1α转录水平检测。AML：急性髓系白血病；DNR：柔红霉素；PGC-1α：过氧化物酶体增殖受体γ辅激活因子1-α；Actin：肌动蛋白。两组样本之间的统计学差异使用两独立样本t检验进行计算(**P<0.01)。Figure 20 shows changes in cardiac PGC-1α expression in AML mice after DNR chemotherapy. (Top) Detection of cardiac PGC-1α protein expression in AML mice after chemotherapy. (Right) Detection of PGC-1α protein expression relative to Actin protein. (Bottom) Detection of cardiac PGC-1α transcript levels in AML mice after chemotherapy. AML: acute myeloid leukemia; DNR: daunorubicin; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-α; Actin: actin. Statistical differences between the two groups were calculated using a two-sample t-test ( ** P < 0.01).

图21A-图21D为AML患儿使用DNR化疗后心肌PGC-1α的表达。21A-21D show the expression of myocardial PGC-1α in children with AML after DNR chemotherapy.

图21A为UMAP图展示PGC-1α主要在心脏组织中的心肌细胞表达。FIG21A is a UMAP image showing that PGC-1α is mainly expressed in cardiomyocytes in cardiac tissue.

图21B为拟时序分析显示不同心肌细胞亚群的细胞命运。FIG21B is a pseudo-time series analysis showing the cell fate of different cardiomyocyte subpopulations.

图21C为不同心肌细胞亚群的PGC-1α表达量。FIG21C shows the expression levels of PGC-1α in different cardiomyocyte subsets.

图21D为拟时序分析显示不同心肌细胞命运与PGC-1α表达量水平的关系。FIG21D is a pseudo-time series analysis showing the relationship between different cardiomyocyte fates and PGC-1α expression levels.

图22为双荧光素报告酶实验。p65过表达显著抑制了PGC-1α的转录。TSS：转录起始位点；LUC：萤火虫酶；RLU：相对荧光强度；PGC-1α：过氧化物酶体增殖受体γ辅激活因子1-α；p65：转录因子p65(***P<0.001)。Figure 22 shows a dual-luciferase reporter assay. Overexpression of p65 significantly inhibited PGC-1α transcription. TSS: transcription start site; LUC: firefly luciferase; RLU: relative fluorescence intensity; PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1-α; p65: transcription factor p65 ( *** P < 0.001).

图23为心脏PGC-1α过表达改善化疗后小鼠心脏代谢和功能。(上)实验示意图，DNR干预心脏过表达PGC-1α的AML小鼠(AAV9-PGC1α组)与对照AML小鼠(AAV9-NC组)；(中)两组小鼠的心脏超声检测；(下)两组小鼠心肌ATP含量检测。WT：野生型；AAV9：腺相关病毒9型；NC：阴性对照；PGC-1α：过氧化物酶体增殖受体γ辅激活因子1-α；AML：急性髓系白血病；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(*P<0.05；***P<0.001)。Figure 23 shows that cardiac PGC-1α overexpression improves cardiac metabolism and function in mice after chemotherapy. (Top) Experimental schematic: DNR intervention in AML mice with cardiac PGC-1α overexpression (AAV9-PGC1α group) and control AML mice (AAV9-NC group); (Middle) Cardiac ultrasound examination of the two groups of mice; (Bottom) Myocardial ATP content in the two groups of mice. WT: wild type; AAV9: adeno-associated virus type 9; NC: negative control; PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1-α; AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; ATP: adenosine triphosphate; Statistical differences between the two groups were calculated using a two-sample t-test ( * P <0.05; *** P < 0.001).

图24A和图24B为IL-1α中和抗体改善化疗后小鼠心脏代谢和功能。FIG24A and FIG24B show that IL-1α neutralizing antibodies improve cardiac metabolism and function in mice after chemotherapy.

图24A(上)实验示意图，DNR干预IL-1α中和抗体处理(IL-1αAb组)或同型对照抗体处理(IgG组)的AML小鼠；(中)两组小鼠的心脏超声检测；(下)两组小鼠心肌ATP含量检测。Figure 24A (top) Schematic diagram of the experiment, DNR intervention in AML mice treated with IL-1α neutralizing antibody (IL-1αAb group) or isotype control antibody (IgG group); (middle) cardiac ultrasound detection of the two groups of mice; (bottom) detection of myocardial ATP content in the two groups of mice.

图24B为两组小鼠化疗结束后第4天骨髓残余GFP+肿瘤细胞占比。Figure 24B shows the percentage of residual GFP+ tumor cells in the bone marrow of the two groups of mice on day 4 after chemotherapy.

AML：急性髓系白血病；DNR：柔红霉素；Ab：抗体；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；BM：骨髓；GFP：绿色荧光蛋白；两组样本之间的统计学差异使用两独立样本t检验进行计算(NS：无显著性差异；*P<0.05；**P<0.01)。AML: acute myeloid leukemia; DNR: daunorubicin; Ab: antibody; LVEF: left ventricular ejection fraction; LVFS: left ventricular fraction; ATP: adenosine triphosphate; BM: bone marrow; GFP: green fluorescent protein; The statistical differences between the two groups were calculated using the two-sample t-test (NS: not significant; * P <0.05; ** P < 0.01).

图25为化疗导致心脏代谢紊乱和功能损伤的机制图。化疗后肿瘤细胞坏死释放IL-1α，作用于心脏IL1R1受体后激活NF-KappaB信号通路并抑制PGC-1α的表达，进而导致心脏代谢紊乱和功能异常。Figure 25 illustrates the mechanism by which chemotherapy leads to cardiac metabolic disorders and functional impairment. Following chemotherapy, tumor cell necrosis releases IL-1α, which acts on the cardiac IL1R1 receptor, activating the NF-KappaB signaling pathway and inhibiting PGC-1α expression, leading to cardiac metabolic disorders and functional abnormalities.

图26为IL-1α中和抗体改善T-ALL小鼠化疗后心脏代谢和功能。(左)实验示意图，DNR干预IL-1α中和抗体处理(IL-1αAb组)或同型对照抗体处理(IgG组)的T-ALL小鼠；(中)两组小鼠的心脏超声检测；(右)两组小鼠心肌ATP含量检测。T-ALL：急性淋系T细胞白血病；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(**P<0.01；***P<0.001)。Figure 26 shows that IL-1α neutralizing antibodies improve cardiac metabolism and function in T-ALL mice after chemotherapy. (Left) Experimental schematic: DNR intervention in T-ALL mice treated with IL-1α neutralizing antibodies (IL-1αAb group) or isotype control antibodies (IgG group); (Middle) Cardiac ultrasound examination of the two groups of mice; (Right) Myocardial ATP content in the two groups of mice. T-ALL: acute lymphoblastic T-cell leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; ATP: adenosine triphosphate; Statistical differences between the two groups were calculated using a two-sample t-test (**P < 0.01; ***P < 0.001).

图27为IL-1α中和抗体改善B-ALL小鼠CAR-T治疗后心脏代谢和功能。(左)实验示意图，CD19 CAR-T细胞干预IL-1α中和抗体处理(IL-1αAb组)或同型对照抗体处理(IgG组)的B-ALL小鼠；(中)两组小鼠的心脏超声检测；(右)两组小鼠心肌ATP含量检测。B-ALL：急性淋系B细胞白血病；CAR-T：嵌合抗原受体T细胞免疫疗法；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(*P<0.05；**P<0.01)。Figure 27 shows that IL-1α neutralizing antibodies improve cardiac metabolism and function in B-ALL mice after CAR-T therapy. (Left) Schematic diagram of the experiment: CD19 CAR-T cells intervene in B-ALL mice treated with IL-1α neutralizing antibodies (IL-1αAb group) or isotype control antibodies (IgG group); (Middle) Cardiac ultrasound examination of the two groups of mice; (Right) Myocardial ATP content in the two groups of mice. B-ALL: acute lymphoblastic B-cell leukemia; CAR-T: chimeric antigen receptor T cell immunotherapy; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; ATP: adenosine triphosphate; Statistical differences between the two groups were calculated using a two-sample t-test (*P < 0.05; **P < 0.01).

图28为Anakinra改善小鼠化疗后心脏代谢和功能。(左)实验示意图，DNR干预Anakinra处理(Anakinra组)或非处理(control组)的AML小鼠；(中)两组小鼠的心脏超声检测；(右)两组小鼠心肌ATP含量检测。AML：急性髓系白血病；DNR：柔红霉素；LVEF：左心室射血分数；LVFS：左心室短轴收缩率；ATP：腺嘌呤核苷三磷酸；两组样本之间的统计学差异使用两独立样本t检验进行计算(***P<0.001)。Figure 28 shows that Anakinra improves cardiac metabolism and function in mice after chemotherapy. (Left) Schematic diagram of the experiment: DNR intervention in AML mice treated with Anakinra (Anakinra group) or not treated with Anakinra (control group); (Middle) Cardiac ultrasound examination of the two groups of mice; (Right) Myocardial ATP content in the two groups of mice. AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional contraction; ATP: adenosine triphosphate; Statistical differences between the two groups were calculated using a two-sample t-test (***P < 0.001).

具体实施方式DETAILED DESCRIPTION

下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

实验方法Experimental methods

小鼠心脏功能评估Mouse cardiac function assessment

富士Vevo3100小动物超声，可以实现对心脏的高分辨率成像，本发明利用超声对白血病小鼠化疗前后的心脏功能进行了评估，明确了白血病化疗引起的心脏功能变化。更精确的心脏功能数据通过9.4T的Bruker BioSpin小动物磁共振获取，该设备配套有小鼠专用心脏线圈和心脏门控装置，可实现小鼠心功能的准确评估。The Fujifilm Vevo3100 small animal ultrasound system enables high-resolution imaging of the heart. This study used ultrasound to assess cardiac function in leukemia mice before and after chemotherapy, clarifying the changes in cardiac function caused by leukemia chemotherapy. More precise cardiac function data was acquired using a 9.4T Bruker BioSpin small animal magnetic resonance imaging system, equipped with a mouse-specific cardiac coil and cardiac gating device, enabling accurate assessment of mouse cardiac function.

心脏同位素示踪代谢流检测Cardiac isotope tracer metabolic flux detection

心脏同位素示踪代谢流检测能准确反应糖脂代谢底物在心脏能量代谢中的具体流向与通量，本发明使用sigma公司的C13标记葡萄糖和棕榈酸，依据Motoaki Sano等报道的方法(J Mol Cell Cardiol.2015May；82:116-24.)，选择了1mg/g的C13-葡萄糖注射量，依据Kim等报道的BSA加热溶解法(Sci Rep.2017；7:4335.)，并根据小鼠的耐受情况选择了0.5mg/g的C13-棕榈酸注射量，并在注射后15分钟取下小鼠心脏进行后续质谱分析。Cardiac isotope tracer metabolic flux detection can accurately reflect the specific flow direction and flux of glycolipid metabolic substrates in cardiac energy metabolism. The present invention uses C13-labeled glucose and palmitic acid from Sigma. According to the method reported by Motoaki Sano et al. (J Mol Cell Cardiol. 2015 May; 82: 116-24.), a C13-glucose injection dose of 1 mg/g was selected. According to the BSA heating dissolution method reported by Kim et al. (Sci Rep. 2017; 7: 4335.), a C13-palmitic acid injection dose of 0.5 mg/g was selected based on the tolerance of mice. The mouse heart was removed 15 minutes after injection for subsequent mass spectrometry analysis.

预备例Preparatory Example

白血病小鼠治疗模型的构建Construction of a leukemia mouse therapeutic model

AML小鼠模型构建：AML mouse model construction:

本发明使用的白血病小鼠化疗模型基于慢病毒包装MLL-AF9质粒转染正常小鼠的原代髓系干/祖细胞，然后通过流式分选技术筛选GFP+的白血病细胞，并进一步将其移植至接受辐照处理后髓系细胞被清除的小鼠中扩增，从而构建MLL-AF9 AML小鼠。该模型造模保留了受体小鼠的免疫功能，可模拟儿童白血病发病的病理生理过程，是血液肿瘤领域目前公认的AML模型。接受白血病细胞骨髓移植的小鼠，约20-30天后会出现脾大、乏力等白血病症状。The leukemia mouse chemotherapy model used in this study involves transfecting primary myeloid stem/progenitor cells from normal mice with a lentiviral-packaged MLL-AF9 plasmid. GFP+ leukemia cells are then screened for growth using flow cytometry and transplanted into irradiated mice in which myeloid cells have been eliminated for expansion, thereby creating the MLL-AF9 AML mouse model. This model preserves the immune function of the recipient mice and mimics the pathophysiological process of childhood leukemia, making it a recognized AML model in the field of hematologic malignancies. Mice that receive leukemia cell bone marrow transplants develop leukemia symptoms such as splenomegaly and fatigue approximately 20-30 days after transplantation.

T-ALL小鼠模型构建(参考文献：PD-1signalling defines and protects leukaemic stemcells from T cell receptor-induced cell death in T cell acute lymphoblastic leukaemia.DOI:10.1038/s41556-022-01050-3)：Construction of T-ALL mouse model (reference: PD-1 signaling defines and protects leukaemic stem cells from T cell receptor-induced cell death in T cell acute lymphoblastic leukaemia. DOI: 10.1038/s41556-022-01050-3):

使用的T-ALL小鼠模型基于慢病毒包装NOTCH1质粒转染正常小鼠的原代髓系干/祖细胞，然后通过流式分选技术筛选GFP+的白血病细胞，并进一步将其移植至接受辐照处理后的小鼠中扩增，从而构建T-ALL小鼠。NOTCH1的过度激活突变出现在超过60％的人类T-ALL中。The T-ALL mouse model used is based on transfection of primary myeloid stem/progenitor cells from normal mice with a lentiviral NOTCH1 plasmid. GFP+ leukemic cells are then selected by flow cytometry and expanded by transplantation into irradiated mice. Overactivating mutations in NOTCH1 occur in over 60% of human T-ALL.

B-ALL小鼠模型构建(参考文献：Ink4a and Arf are crucial factors in the determination of the cell of origin and the therapeutic sensitivity of Myc-induced mouse lymphoid tumor.DOI:10.1038/onc.2011.462)：Construction of B-ALL mouse model (reference: Ink4a and Arf are crucial factors in the determination of the cell of origin and the therapeutic sensitivity of Myc-induced mouse lymphoid tumor. DOI: 10.1038/onc.2011.462):

使用的B-ALL小鼠模型基于慢病毒包装N-Myc质粒转染正常小鼠的原代造血干细胞，然后通过流式分选技术筛选GFP+的白血病细胞，并进一步将其移植至接受辐照处理后的小鼠中扩增，从而构建B-ALL小鼠。The B-ALL mouse model used is based on transfection of primary hematopoietic stem cells of normal mice with lentiviral-packaged N-Myc plasmid, followed by flow cytometry screening of GFP+ leukemia cells, which are further transplanted into irradiated mice for expansion, thereby constructing B-ALL mice.

本发明使用的化疗方案基于临床白血病患儿在诱导化疗阶段的蒽环类药物剂量，采取连续两天给予5mg/kg的DNR进行化疗。The chemotherapy regimen used in the present invention is based on the anthracycline dosage of clinical leukemia children in the induction chemotherapy stage, and adopts the method of administering 5 mg/kg of DNR for two consecutive days for chemotherapy.

本发明使用的CAR-T治疗的方案(参考文献：Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus.DOI:10.1038/s41423-020-0472-1)：The CAR-T therapy regimen used in the present invention (reference: Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus. DOI: 10.1038/s41423-020-0472-1):

1.嵌合抗原受体的组成：1D3 anti-CD19抗体的轻链和重链的可变区作为scFv片段，以mCD8作为跨膜区，以m4-1BB和mCD3ζ信号作为胞质区。将上述序列合成后连接到带有IRES-EGFP荧光信号的慢病毒载体中。1. Chimeric Antigen Receptor Composition: The variable regions of the light and heavy chains of the 1D3 anti-CD19 antibody were used as scFv fragments, with mCD8 as the transmembrane region and m4-1BB and mCD3ζ signaling as the cytoplasmic region. These sequences were synthesized and ligated into a lentiviral vector carrying an IRES-EGFP fluorescent signal.

2.慢病毒感染T细胞后，获得CD19 CAR-T细胞。2. After lentivirus infects T cells, CD19 CAR-T cells are obtained.

3.连续两天给予1E6/只的CAR-T细胞治疗(B-ALL小鼠经过清淋处理)。3. 1E6 CAR-T cells were administered to each mouse for two consecutive days (B-ALL mice were cleared of lymphocytes).

心脏特异性Il1r1敲除小鼠和全身性Il1a敲除小鼠的构建Construction of cardiac-specific Il1r1 knockout mice and systemic Il1a knockout mice

Il1r1-Flox小鼠购自上海南方模式生物科技股份有限公司，该品系小鼠在Il1r1基因的3-4外显子区域插入了flox位点，然后通过与Myh6-CreERT2转基因小鼠交配构建了Il1r1flox/flox；Myh6-CreERT2小鼠。通过使用100mg/kg他莫昔芬干预5天，等待3周后，免疫印迹检测显示小鼠心肌组织IL1R1蛋白表达量明显降低，从而证实该基因工程小鼠品系的有效性(图1)。全身性Il1a敲除小鼠购自上海南方模式生物科技股份有限公司，该品系小鼠敲除了Il1a基因5-6外显子区域，且基因型已通过测序验证。Il1r1-Flox mice were purchased from Shanghai Model Organisms Science Co., Ltd. This strain contains a flox site inserted into exons 3-4 of the Il1r1 gene. Il1r1 flox/flox;Myh6-CreERT2 mice were then mated with Myh6-CreERT2 transgenic mice to create Il1r1 flox/flox ;Myh6-CreERT2 mice. Treatment with 100 mg/kg tamoxifen for 5 days, followed by a 3-week wait, revealed a significant decrease in IL1R1 protein expression in myocardial tissue by immunoblotting, confirming the effectiveness of this genetically engineered mouse strain (Figure 1). Systemic Il1a knockout mice were purchased from Shanghai Model Organisms Science Co., Ltd. This strain contains a knockout site in exons 5-6 of the Il1a gene, and their genotype was verified by sequencing.

Il1a敲低的AML细胞构建Il1a knockdown AML cell construction

利用分子克隆相关技术改造sigma公司商品化MISSION shRNA质粒，增加红色荧光蛋白基因序列(RFP)以及靶向小鼠Il1a的shRNA序列，然后将质粒进行慢病毒包装后转染原代AML细胞，最后流式分选出GFP+RFP+的细胞。通过RT-PCR实验验证了GFP+RFP+的AML细胞中Il1a的表达显著降低，证明成功构建了Il1a稳定敲低的AML细胞系(图2)。Using molecular cloning techniques, Sigma's commercially available MISSION shRNA plasmid was modified to include a red fluorescent protein (RFP) gene sequence and a shRNA targeting mouse Il1a. The plasmid was then packaged into lentiviral vectors and transfected into primary AML cells. Finally, GFP+RFP+ cells were flow-sorted. RT-PCR confirmed that Il1a expression was significantly reduced in these GFP+RFP+ AML cells, demonstrating the successful establishment of an AML cell line with stable Il1a knockdown (Figure 2).

AAV9-PGC-1α病毒构建AAV9-PGC-1α virus construction

携带心肌特异性启动子的AAV9-PGC-1α病毒购自上海汉恒生物科技有限公司。在本发明中，申请者采用尾静脉注射病毒的方式，病毒数量按照文献报道选取4*1013vg/kg的剂量注射(Gene Ther.2011Jan；18(1):43-52)。野生型小鼠注射病毒3周后，免疫印迹检测证实其心脏PGC-1α的表达显著上升(图3)。AAV9-PGC-1α virus carrying a myocardial-specific promoter was purchased from Shanghai Hanheng Biotechnology Co., Ltd. In the present invention, the applicant used tail vein injection of the virus, using a dose of 4 × 10 13 vg/kg as reported in the literature (Gene Ther. 2011 Jan; 18(1): 43-52). Three weeks after virus injection, immunoblotting confirmed a significant increase in cardiac PGC-1α expression in wild-type mice (Figure 3).

p65过表达质粒和PGC-1α启动子荧光报告质粒构建Construction of p65 overexpression plasmid and PGC-1α promoter fluorescence reporter plasmid

本发明将扩增的人p65全长编码序列(NM_021975)克隆到CV702载体中，同时已将覆盖1100bp片段(从-1000bp到+100bp)的人PGC-1α(NM_013261)启动子序列克隆到GV238荧光素酶载体中(图4)。The present invention cloned the amplified full-length human p65 coding sequence (NM_021975) into the CV702 vector, and simultaneously cloned the human PGC-1α (NM_013261) promoter sequence covering a 1100 bp fragment (from -1000 bp to +100 bp) into the GV238 luciferase vector ( FIG4 ).

实施例1白血病化疗诱导心脏能量代谢紊乱与心脏损伤Example 1: Leukemia chemotherapy induces cardiac energy metabolism disorder and heart damage

心脏是一个能量需求旺盛的器官，每天搏动10万余次，向全身输送10吨血液，消耗6公斤ATP6。在禁食状态下，心脏70％的能量依靠脂肪酸供应，因此能量转换是心脏最为核心的代谢过程。心衰的心脏能量代谢会发生重塑，从脂肪酸供能为主转变为葡萄糖供能，伴随心脏ATP的合成下降和功能障碍8。众多研究已证实心脏能量代谢障碍和心脏损伤之间存在密切联系，例如在近期的一项研究中，申请者发现青春期胰岛素抵抗诱导的能量失衡将直接导致心脏损伤和功能异常9。然而，白血病患儿在化疗后出现的心脏毒性是否与能量代谢异常有关？能否通过改善心脏能量代谢进而减轻化疗导致的心血管系统损伤？这些问题尚不清楚，有待进一步探索。The heart is an organ with a high energy demand, beating over 100,000 times daily, pumping 10 tons of blood throughout the body and consuming 6 kilograms of ATP. 6 In the fasting state, the heart relies on fatty acids for 70% of its energy supply, making energy conversion a core metabolic process. In heart failure, cardiac energy metabolism undergoes remodeling, shifting from a primary fatty acid to glucose energy supply, accompanied by decreased cardiac ATP synthesis and functional impairment. 8 Numerous studies have demonstrated a close link between impaired cardiac energy metabolism and cardiac damage. For example, a recent study found that energy imbalance induced by insulin resistance during adolescence directly leads to cardiac damage and dysfunction. 9 However, is the post-chemotherapy cardiotoxicity in children with leukemia related to abnormal energy metabolism? Can improving cardiac energy metabolism mitigate chemotherapy-induced cardiovascular damage? These questions remain unclear and require further exploration.

目前普遍认为儿童白血病以基因融合为主要发病机制10，MLL-AF9融合蛋白在25％的儿童初发AML中存在11，携带此融合蛋白的AML小鼠模型被广泛应用于儿童血液肿瘤研究领域。发明人通过逆转录病毒在小鼠髓系细胞中转染了MLL-AF9融合基因，构建了急性髓系白血病(MLL-AF9 AML)小鼠荷瘤模型(图5)。然后发明人在模型鼠出现白血病相关体征后将其分为两组，一组参照临床AML短期诱导化疗方案使用蒽环类化疗药物柔红霉素(DNR)进行干预(连续两天给予5mg/kg的DNR)，另一组则使用磷酸盐缓冲液(PBS)作为对照(图6A)。进一步发明人使用小动物心脏超声，检测了小鼠化疗后左室射血分数(LVEF)和左室短轴收缩率(LVFS)的变化情况。结果显示，相比于对照组，白血病小鼠在短期诱导化疗后心功能出现了明显的下降(图6B)。然后，发明人应用PET-CT评估了白血病小鼠化疗后的心脏能量代谢变化。结果表明，相比于对照组，白血病小鼠化疗后心脏对脂肪酸摄取降低，对葡萄糖的摄取增强(图6C)。进一步，发明人检测了化疗后白血病小鼠心肌组织的ATP含量，结果显示化疗后心脏ATP含量出现明显下降(图6D)。最后，发明人使用免疫印迹评估了脂代谢相关蛋白的表达，结果显示化疗后心脏脂代谢关键酶表达出现显著降低(图7)。这些结果证明白血病小鼠化疗后出现了脂肪酸代谢的障碍，伴随心脏功能的下降。为了进一步在人类标本中验证发明人的结论，发明人收集了在使用DNR化疗后出现心功能异常的AML患儿的心肌活检标本以及因为脑死亡而捐献的儿童正常心肌标本，并进行了单细胞转录组分析。结果同样显示，化疗后患儿心肌脂肪酸代谢下降，葡萄糖代谢增强(图8A-图8D)。综上，发明人认为：心脏能量代谢重塑与化疗引起的心脏损伤和心功能降低密切相关，脂肪酸代谢受损是其心功能障碍的重要原因。It is currently generally believed that gene fusion is the primary pathogenesis of childhood leukemia.10 The MLL-AF9 fusion protein is present in 25% of newly diagnosed childhood AML.11 AML mouse models carrying this fusion protein are widely used in pediatric hematological tumor research. The inventors used retroviral transfection of the MLL-AF9 fusion gene into mouse myeloid cells to construct a mouse model of acute myeloid leukemia (MLL-AF9 AML) (Figure 5). After the model mice developed leukemia-related signs, the inventors divided them into two groups. One group received the anthracycline chemotherapy drug daunorubicin (DNR) (5 mg/kg DNR for two consecutive days) according to the clinical AML short-term induction chemotherapy regimen, while the other group received phosphate-buffered saline (PBS) as a control (Figure 6A). The inventors further used small animal cardiac ultrasound to examine changes in left ventricular ejection fraction (LVEF) and left ventricular fractional contraction (LVFS) in the mice after chemotherapy. The results showed that compared with the control group, the leukemic mice showed a significant decline in cardiac function after short-term induction chemotherapy (Figure 6B). Then, the inventors used PET-CT to evaluate the changes in cardiac energy metabolism in leukemia mice after chemotherapy. The results showed that compared with the control group, the heart of leukemia mice had reduced fatty acid uptake and enhanced glucose uptake after chemotherapy (Figure 6C). Furthermore, the inventors detected the ATP content in the myocardial tissue of leukemia mice after chemotherapy, and the results showed that the ATP content in the heart decreased significantly after chemotherapy (Figure 6D). Finally, the inventors used immunoblotting to evaluate the expression of lipid metabolism-related proteins, and the results showed that the expression of key enzymes of cardiac lipid metabolism was significantly reduced after chemotherapy (Figure 7). These results demonstrate that leukemia mice had an impaired fatty acid metabolism after chemotherapy, accompanied by a decline in cardiac function. In order to further verify the inventors' conclusions in human specimens, the inventors collected myocardial biopsy specimens from children with AML who developed abnormal cardiac function after using DNR chemotherapy and normal myocardial specimens from children donated because of brain death, and performed single-cell transcriptome analysis. The results also showed that myocardial fatty acid metabolism in children decreased and glucose metabolism increased after chemotherapy (Figures 8A-8D). In summary, the inventors believe that cardiac energy metabolism remodeling is closely related to chemotherapy-induced heart damage and decreased cardiac function, and impaired fatty acid metabolism is an important cause of its cardiac dysfunction.

实施例2肿瘤细胞来源的IL-1α引起心脏代谢与功能障碍Example 2 Tumor cell-derived IL-1α induces cardiac metabolic and functional disorders

是什么因素推动了白血病小鼠化疗后心脏能量代谢的改变？有研究表明，体外灌注蒽环类药物的心脏会降低肉碱含量从而影响心脏代谢12；被批准用于治疗糖尿病等代谢性疾病的药物，如：二甲双胍(MET)和恩格列净(EMPA)可以预防蒽环类药物的心脏毒性13。另一方面，脑膜炎球菌性疾病引发的炎症因子释放，如干扰素γ(IFN-γ)等，也被报道可能会导致心脏损伤14。那么究竟是化疗药物本身会造成小鼠的心脏能量代谢障碍？还是化疗引起肿瘤细胞破坏后释放的某些毒性细胞因子导致心脏能量代谢异常？What factors drive changes in cardiac energy metabolism in leukemia mice after chemotherapy? Studies have shown that ex vivo perfusion of hearts with anthracyclines reduces carnitine levels, thereby affecting cardiac metabolism. 12 Drugs approved for the treatment of metabolic diseases such as diabetes, such as metformin (MET) and empagliflozin (EMPA), can prevent anthracycline-induced cardiotoxicity. 13 On the other hand, the release of inflammatory factors, such as interferon gamma (IFN-γ), triggered by meningococcal disease has also been reported to cause cardiac damage. 14 So, is it the chemotherapy drugs themselves that cause the impairment of cardiac energy metabolism in mice? Or is it certain toxic cytokines released after chemotherapy-induced tumor cell destruction that lead to abnormal cardiac energy metabolism?

为了排除白血病细胞的影响因素，发明人首先参照之前实验的诱导化疗方案在无白血病的正常小鼠中化疗，观察化疗药对小鼠心脏能量代谢和心脏功能的直接作用。发明人将健康无瘤小鼠分成两组，其中一组使用DNR化疗，一组使用PBS作为对照，然后进行心脏超声与PET-CT的检测(图9A)。有趣的是，发明人发现健康无瘤小鼠在化疗后心脏代谢和功能并未发生明显改变(图9B-图9C)，心脏ATP的含量也未出现显著下降(图9D)。因此化疗后的心脏能量代谢紊乱可能不是化疗药物的直接作用，而是化疗药与白血病细胞相互作用后的综合结果。进一步，发明人对白血病小鼠化疗后的心脏组织进行了转录组学测序，结果提示化疗后心肌组织中细胞因子与细胞因子受体互作通路显著富集(图10A-图10C)，这一结果提示：或许是化疗引起某种肿瘤源性的细胞因子释放入血导致了心脏的能量代谢障碍。In order to exclude the influencing factors of leukemia cells, the inventors first referred to the induction chemotherapy regimen of the previous experiment to perform chemotherapy in normal mice without leukemia, and observed the direct effects of chemotherapy drugs on the cardiac energy metabolism and cardiac function of mice. The inventors divided healthy tumor-free mice into two groups, one of which used DNR chemotherapy and the other used PBS as a control, and then performed cardiac ultrasound and PET-CT detection (Figure 9A). Interestingly, the inventors found that the cardiac metabolism and function of healthy tumor-free mice did not change significantly after chemotherapy (Figure 9B-Figure 9C), and the content of cardiac ATP did not show a significant decrease (Figure 9D). Therefore, the cardiac energy metabolism disorder after chemotherapy may not be a direct effect of chemotherapy drugs, but a comprehensive result of the interaction between chemotherapy drugs and leukemia cells. Further, the inventors performed transcriptomic sequencing on the heart tissue of leukemia mice after chemotherapy. The results showed that the cytokine and cytokine receptor interaction pathway was significantly enriched in the myocardial tissue after chemotherapy (Figure 10A-Figure 10C). This result suggests that chemotherapy may cause certain tumor-derived cytokines to be released into the blood, leading to energy metabolism disorders in the heart.

基于以上猜想，发明人利用Luminex多因子检测技术，高通量筛选了白血病小鼠和正常无瘤小鼠化疗后血浆中发生变化的细胞因子。结果显示在所有检测的细胞因子中，有9种细胞因子在白血病小鼠化疗后显著升高。由于肿瘤细胞释放的特异性细胞因子需要符合在白血病小鼠化疗后升高而在健康无瘤小鼠化疗后无明显升高的变化模式，因此发明人对这9种细胞因子进行了逐一验证，结果显示只有白细胞介素1α(IL-1α)符合这一趋势特征(图11A-图11D)。IL-1家族分为IL-1α和IL-1β两个亚型。在心血管疾病研究领域，目前已有许多研究报道了IL-1β对于心肌细胞的影响15，但是对于IL-1α的研究主要集中于其对心脏血管内皮细胞和成纤维细胞的作用，其对心肌细胞直接影响的相关研究较少。目前认为，IL-1α是一种损伤相关模式分子，在血液或非血液细胞中组成性表达。在细胞坏死后，IL-1α释放进入细胞间隙或血液循环在局部或全身发挥其生物功能16。发明人前期的体外实验表明，DNR能引起白血病细胞坏死，且其坏死比例呈剂量依赖性升高(图12A和图12B)。此外发明人还发现，在DNR加入后白血病细胞培养上清中IL-1α的蛋白表达显著升高(图12C)。因此，化疗药物引起白血病细胞坏死释放的IL-1α可能是造成心脏代谢障碍和心功能损伤的潜在原因。Based on the above hypothesis, the inventors used Luminex multifactorial detection technology to perform high-throughput screening of cytokines that change in the plasma of leukemia mice and normal tumor-free mice after chemotherapy. The results showed that among all the cytokines tested, nine cytokines were significantly elevated after chemotherapy in leukemia mice. Because tumor-specific cytokines released by tumor cells need to follow a pattern of increase in leukemia mice after chemotherapy but not in healthy tumor-free mice, the inventors validated each of these nine cytokines individually. The results showed that only interleukin-1α (IL-1α) met this trend (Figures 11A-11D). The IL-1 family is divided into two subtypes: IL-1α and IL-1β. In the field of cardiovascular disease research, many studies have reported the effects of IL-1β on cardiomyocytes. However, research on IL-1α has primarily focused on its effects on cardiac endothelial cells and fibroblasts, with limited studies on its direct effects on cardiomyocytes. IL-1α is currently considered a damage-associated model molecule, constitutively expressed in both blood and non-blood cells. After cell necrosis, IL-1α is released into the intercellular space or blood circulation to exert its biological functions locally or systemically. 16 The inventors' previous in vitro experiments showed that DNR can induce necrosis in leukemia cells, and the necrosis rate increases in a dose-dependent manner (Figures 12A and 12B). In addition, the inventors also found that the protein expression of IL-1α in the leukemia cell culture supernatant was significantly increased after the addition of DNR (Figure 12C). Therefore, the IL-1α released by leukemia cell necrosis caused by chemotherapy drugs may be a potential cause of cardiac metabolic disorders and cardiac function damage.

为了验证IL-1α在心脏代谢与功能中的作用，发明人在无白血病小鼠中直接注射IL-1α重组蛋白，发现IL-1α对心脏有直接的损伤作用(图13A-图13D)。进一步发明人使用心脏特异性IL-1α受体(IL1R1)敲除小鼠构建白血病模型，发现心脏IL-1α受体敲除保护了白血病小鼠化疗后心脏代谢和功能。以上两个实验从正反两个方面证实了IL-1α在化疗后心脏损伤中的关键作用(图14A-图14C)。同时，发明人采集了白血病患儿化疗后的血液样本，对其血浆IL-1α的浓度与化疗前后的心脏功能进行评估，证明患儿IL-1α的升高与心功能的下降具有相关性(图15)。In order to verify the role of IL-1α in cardiac metabolism and function, the inventors directly injected IL-1α recombinant protein into leukemia-free mice and found that IL-1α had a direct damaging effect on the heart (Figure 13A-Figure 13D). The inventors further used cardiac-specific IL-1α receptor (IL1R1) knockout mice to construct a leukemia model and found that cardiac IL-1α receptor knockout protected the cardiac metabolism and function of leukemia mice after chemotherapy. The above two experiments confirmed the key role of IL-1α in cardiac damage after chemotherapy from both positive and negative aspects (Figure 14A-Figure 14C). At the same time, the inventors collected blood samples from children with leukemia after chemotherapy, evaluated their plasma IL-1α concentration and cardiac function before and after chemotherapy, and proved that the increase in IL-1α in children was correlated with the decline in cardiac function (Figure 15).

为了验证IL-1α的来源，发明人使用全身性IL-1α敲除小鼠构建白血病模型，同时发明人通过短发卡RNA(shRNA)技术敲低白血病原代细胞中的IL-1α基因，然后再用IL-1α敲低的细胞系构建白血病模型，最后通过检测两种模型化疗后血浆IL-1α的含量变化，明确化疗后的血浆IL-1α升高来源于肿瘤细胞的破坏和释放(图16A-图16C)。To verify the source of IL-1α, the inventors used systemic IL-1α knockout mice to construct a leukemia model. At the same time, the inventors knocked down the IL-1α gene in primary leukemia cells using short hairpin RNA (shRNA) technology, and then constructed a leukemia model using the IL-1α knockdown cell line. Finally, by detecting the changes in plasma IL-1α levels in the two models after chemotherapy, it was confirmed that the increase in plasma IL-1α after chemotherapy was derived from the destruction and release of tumor cells (Figures 16A-16C).

实施例3 NF-KappaB信号通路介导了IL-1α引起的心脏损伤Example 3 NF-KappaB signaling pathway mediates IL-1α-induced cardiac damage

那么化疗后血浆IL-1α水平的升高如何引起心脏代谢紊乱？已有的研究表明IL-1α的受体IL1R1激活后可进一步激活下游NF-KappaB信号通路，进而调控基因的表达17。目前普遍认为NF-KappaB信号是许多炎症细胞因子发挥生物学功能的共同通路，在心肌梗死、心衰的进展中扮演了重要角色。NF-KappaB蛋白家族以异源二聚体复合物的形式存在，其中p50/p65是心脏中最主要的NF-KappaB复合物。p65亚基是一种转录因子，其在发生磷酸化后可进入细胞核对下游基因的转录进行调控18。近年来的研究表明，p65在细胞的多种代谢基因调节中发挥了重要作用。So how does the increase in plasma IL-1α levels after chemotherapy cause cardiac metabolic disorders? Existing studies have shown that activation of the IL-1α receptor IL1R1 can further activate the downstream NF-KappaB signaling pathway, thereby regulating gene expression17 . It is generally believed that NF-KappaB signaling is a common pathway for many inflammatory cytokines to exert their biological functions and plays an important role in the progression of myocardial infarction and heart failure. The NF-KappaB protein family exists in the form of a heterodimeric complex, of which p50/p65 is the most important NF-KappaB complex in the heart. The p65 subunit is a transcription factor that, after phosphorylation, can enter the cell nucleus to regulate the transcription of downstream genes18 . Recent studies have shown that p65 plays an important role in the regulation of various metabolic genes in cells.

转录组学结果提示白血病小鼠化疗后心脏NF-KappaB信号通路显著富集(图10A)。进一步，发明人通过免疫印迹检测对白血病小鼠化疗后心脏的NF-KappaB信号通路的激活水平进行了初步验证，结果显示心脏中p65磷酸化水平提高，NF-KappaB抑制蛋白IkBα的表达水平降低。然而，在健康无瘤小鼠化疗后心脏NF-KappaB信号通路的激活水平并未出现显著变化(图17)。此外，发明人前期使用白血病患儿化疗后的心肌组织进行了免疫荧光和免疫印迹检测，结果显示化疗后心肌组织间隙出现IL-1α富集伴随NF-KappaB信号通路激活水平的上升(图18)。因此发明人推测化疗引起血浆IL-1α的升高可能激活了心肌细胞的NF-KappaB信号通路，进而影响了下游代谢基因的表达。The transcriptomic results suggest that the NF-KappaB signaling pathway in the heart of leukemia mice is significantly enriched after chemotherapy (Figure 10A). Furthermore, the inventors preliminarily verified the activation level of the NF-KappaB signaling pathway in the heart of leukemia mice after chemotherapy by immunoblotting. The results showed that the level of p65 phosphorylation in the heart increased, and the expression level of the NF-KappaB inhibitory protein IkBα decreased. However, there was no significant change in the activation level of the NF-KappaB signaling pathway in the heart of healthy tumor-free mice after chemotherapy (Figure 17). In addition, the inventors previously performed immunofluorescence and immunoblotting tests on myocardial tissue after chemotherapy in leukemia children. The results showed that IL-1α was enriched in the myocardial tissue gap after chemotherapy, accompanied by an increase in the activation level of the NF-KappaB signaling pathway (Figure 18). Therefore, the inventors speculated that the increase in plasma IL-1α caused by chemotherapy may have activated the NF-KappaB signaling pathway in myocardial cells, thereby affecting the expression of downstream metabolic genes.

为了验证这一猜想，发明人使用NF-KappaB信号通路抑制剂PDTC对白血病小鼠进行干预，PDTC干预组与对照组相比，化疗后心脏代谢和功能显著改善(图19)。To verify this hypothesis, the inventors used PDTC, an inhibitor of the NF-KappaB signaling pathway, to intervene in leukemia mice. Compared with the control group, the cardiac metabolism and function of the PDTC intervention group were significantly improved after chemotherapy ( FIG19 ).

实施例4 PGC-1α降低是IL-1α介导心脏代谢紊乱的核心Example 4 PGC-1α reduction is the core of IL-1α-mediated cardiometabolic disorders

PGC-1α是一种转录共激活因子，能与多种转录因子结合，在脂肪酸氧化、糖酵解、线粒体生物合成等过程中发挥重要作用19。近年来，PGC-1α被发现与心脏的能量代谢密切相关，已有研究发现PGC-1α的表达下降会抑制心脏的脂肪酸代谢，并进一步诱发心脏功能异常20。已有研究表明NF-KappaB信号通路的激活可以下调细胞PGC-1α的表达，最终使细胞代谢模式发生改变21。PGC-1α is a transcriptional coactivator that binds to multiple transcription factors and plays a crucial role in processes such as fatty acid oxidation, glycolysis, and mitochondrial biogenesis. 19 In recent years, PGC-1α has been found to be closely linked to cardiac energy metabolism. Studies have shown that decreased PGC-1α expression inhibits cardiac fatty acid metabolism and further induces cardiac dysfunction. 20 Studies have also shown that activation of the NF-kappaB signaling pathway can downregulate cellular PGC-1α expression, ultimately altering cellular metabolic patterns. 21

在前期研究中，发明人使用生物反应路径分析系统(Ingenuity Pathway Analysis)对白血病小鼠化疗后的心脏转录组学数据进行了通路富集，结果提示与代谢紧密相关的过氧化物酶体增殖物激活受体(PPAR)信号通路显著下调(图10B)。进一步，发明人对PPAR通路中的差异基因进行了蛋白互作网络分析，在中枢基因中发现PGC-1α与p65存在互作关系(图10C)。然后，发明人通过RT-PCR、免疫印迹检测了白血病小鼠化疗后心脏PGC-1α在转录与翻译水平的表达改变。结果显示白血病小鼠化疗后心脏PGC-1α的表达显著下调(图20)。此外，发明人前期的单细胞转录组学结果显示白血病患儿化疗后心肌细胞的PGC-1α表达水平下降，且其表达水平的高低决定了化疗后心肌细胞的不同命运(图21A-图21D)。由此发明人推测：化疗后心肌NF-KappaB信号通路的激活可能通过抑制PGC-1α的表达造成心脏能量代谢障碍和功能损伤。In previous studies, the inventors used the Ingenuity Pathway Analysis system to perform pathway enrichment on the cardiac transcriptomic data of leukemia mice after chemotherapy. The results showed that the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which is closely related to metabolism, was significantly downregulated (Figure 10B). Furthermore, the inventors performed a protein interaction network analysis on the differentially expressed genes in the PPAR pathway and found that PGC-1α and p65 interacted with each other among the central genes (Figure 10C). Then, the inventors detected the expression changes of PGC-1α at the transcriptional and translational levels in the hearts of leukemia mice after chemotherapy by RT-PCR and immunoblotting. The results showed that the expression of PGC-1α in the hearts of leukemia mice was significantly downregulated after chemotherapy (Figure 20). In addition, the inventors' previous single-cell transcriptomics results showed that the expression level of PGC-1α in cardiomyocytes of leukemia children decreased after chemotherapy, and its expression level determined the different fates of cardiomyocytes after chemotherapy (Figure 21A-Figure 21D). Therefore, the inventors speculate that the activation of the myocardial NF-KappaB signaling pathway after chemotherapy may cause cardiac energy metabolism disorders and functional damage by inhibiting the expression of PGC-1α.

发明人通过双荧光素酶报告实验证明p65能结合PGC-1α基因的启动子区域从而抑制PGC-1α的转录(图22)。使用携带包含心肌特异性启动子和PGC-1α基因编码序列的9型腺相关病毒(AAV9-PGC-1α)特异性过表达白血病小鼠心脏的PGC-1α，纠正了化疗后的心脏能量代谢紊乱和功能异常，进而证明PGC-1α在化疗相关性心功能障碍中的核心作用(图23)。The inventors used a dual-luciferase reporter assay to demonstrate that p65 can bind to the promoter region of the PGC-1α gene, thereby inhibiting PGC-1α transcription (Figure 22). Using adeno-associated virus type 9 (AAV9-PGC-1α) carrying a myocardial-specific promoter and the PGC-1α gene coding sequence to specifically overexpress PGC-1α in the hearts of leukemia mice, they corrected the cardiac energy metabolism disorder and functional abnormalities after chemotherapy, further demonstrating the central role of PGC-1α in chemotherapy-related cardiac dysfunction (Figure 23).

实施例5 IL-1α中和抗体减轻白血病治疗引起的心脏损伤与代谢紊乱Example 5 IL-1α neutralizing antibodies alleviate cardiac damage and metabolic disorders caused by leukemia treatment

明确白血病治疗(化疗或免疫治疗等)引起心脏损伤的机制后，发明人认为IL-1α中和抗体改善治疗患者的心脏损伤具有转化价值与广阔的应用前景。IL-1α在白血病发生与心脏损伤中均扮演了重要的角色，阻断IL-1α介导的心脏NF-KappaB/PGC-1α轴可能降低潜在的心脏毒性风险。After clarifying the mechanism by which leukemia treatment (chemotherapy or immunotherapy, etc.) causes cardiac damage, the inventors believe that IL-1α neutralizing antibodies have translational value and broad application prospects for improving cardiac damage in treated patients. IL-1α plays a key role in both leukemia development and cardiac damage, and blocking the IL-1α-mediated cardiac NF-kappaB/PGC-1α axis may reduce the potential risk of cardiotoxicity.

本发明发现，使用10μg/只剂量的IL-1α中和抗体(Mouse IL-1alpha/IL-1F1 antib ody，货号：AB-400-NA，供应商：R&D Systems，商品网址：https://www.rndsystems.co m/cn/products/mouse-il-1alpha-il-1f1-antibody_ab-400-na#product-details)能减轻白血病小鼠(AML、T-ALL或B-ALL)治疗后的心脏代谢与功能损伤，且不影响治疗效果(图24A和图24B、图26和图27)。The present invention found that the use of a 10 μg/mouse dose of IL-1α neutralizing antibody (Mouse IL-1alpha/IL-1F1 antib ody, catalog number: AB-400-NA, supplier: R&D Systems, product website: https://www.rndsystems.com/cn/products/mouse-il-1alpha-il-1f1-antibody_ab-400-na#product-details) can reduce cardiac metabolic and functional damage in leukemia mice (AML, T-ALL or B-ALL) after treatment without affecting the therapeutic effect ( Figures 24A and 24B, Figures 26 and 27 ).

实施例6 IL1R1的拮抗剂减轻白血病治疗引起的心脏损伤与代谢紊乱Example 6 IL1R1 antagonists alleviate cardiac damage and metabolic disorders induced by leukemia treatment

本发明发现，使用30mg/kg/d×5d剂量的IL1R1的拮抗剂(Anakinra，阿那白滞素，CAS号：143090-92-0)能减轻白血病小鼠(AML小鼠)治疗后的心脏代谢与功能损伤，且不影响治疗效果(图28)。The present invention found that the use of an IL1R1 antagonist (Anakinra, CAS No.: 143090-92-0) at a dose of 30 mg/kg/d×5 days can alleviate cardiac metabolic and functional damage in leukemia mice (AML mice) after treatment without affecting the therapeutic effect ( Figure 28 ).

小结summary

“肿瘤心脏病学”概念的提出使得抗肿瘤治疗引起的心脏损伤并发症越发受到关注。然而目前很多研究聚焦于化疗药物直接引起的心脏损伤，却忽视了肿瘤与化疗药物之间的相互作用，以及肿瘤负荷水平可能造成化疗后心脏损伤的差异。本发明发现：肿瘤细胞、化疗药物、心脏损伤之间存在紧密的关联，化疗通过诱导肿瘤细胞坏死释放IL-1α，其作用于心肌IL1R1受体后激活NF-KappaB信号通路，进而抑制PGC-1α的表达并最终导致心脏能量代谢紊乱和心脏功能异常(图25)。The introduction of the concept of "oncological cardiology" has made the complications of cardiac damage caused by anti-tumor treatment more and more concerned. However, many studies currently focus on cardiac damage directly caused by chemotherapy drugs, but ignore the interaction between tumors and chemotherapy drugs, and the differences in tumor load levels that may cause cardiac damage after chemotherapy. The present invention found that there is a close relationship between tumor cells, chemotherapy drugs, and cardiac damage. Chemotherapy releases IL-1α by inducing tumor cell necrosis, which acts on the myocardial IL1R1 receptor and activates the NF-KappaB signaling pathway, thereby inhibiting the expression of PGC-1α and ultimately leading to cardiac energy metabolism disorders and abnormal cardiac function (Figure 25).

儿童白血病幸存者基数逐年增加，预期寿命不断延长，随之带来的长期心血管负担持续加重，化疗引发的心血管健康问题严重影响了其生存质量。目前尚缺乏针对白血病化疗相关心脏损伤的有效保护策略，早期识别白血病化疗导致的心脏病理生理改变并积极干预将有效降低白血病患儿化疗后发生心血管系统疾病的风险。针对儿童独特的肿瘤疾病谱与生理特点制定个体化的围化疗期心血管保护策略迫在眉睫，这将有效降低化疗期间的心血管并发症，减少远期心血管不良事件的发生率，使众多白血病患儿获益，从而获得明显的社会和经济效应。The number of childhood leukemia survivors is increasing year by year, and life expectancy is continuously increasing. The resulting long-term cardiovascular burden continues to increase, and cardiovascular health problems caused by chemotherapy seriously affect their quality of life. Currently, there is a lack of effective protective strategies for leukemia chemotherapy-related cardiac damage. Early identification of cardiac pathological and physiological changes caused by leukemia chemotherapy and active intervention will effectively reduce the risk of cardiovascular diseases in children with leukemia after chemotherapy. It is urgent to develop personalized peri-chemotherapy cardiovascular protection strategies based on the unique spectrum of tumor diseases and physiological characteristics of children. This will effectively reduce cardiovascular complications during chemotherapy and reduce the incidence of long-term adverse cardiovascular events, benefiting many children with leukemia and thus achieving significant social and economic effects.

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Claims (10)

IL-1α的抑制剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用。Use of an IL-1α inhibitor in the preparation of a drug for treating leukemia-induced cardiac damage. 如权利要求1所述的应用，其特征在于，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；The use according to claim 1, wherein the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; 优选地，所述白血病治疗满足选自以下条件的一种或多种：Preferably, the leukemia treatment satisfies one or more of the following conditions: 所述化疗的药物为蒽环类化疗药物，例如柔红霉素；The chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; 所述白血病治疗导致白血病细胞坏死；和，The leukemia treatment results in necrosis of leukemia cells; and, 所述免疫治疗为CAR-T治疗，例如CD19 CAR-T治疗。The immunotherapy is CAR-T therapy, such as CD19 CAR-T therapy. 如权利要求1所述的应用，其特征在于，所述白血病为急性髓系白血病或急性淋巴细胞白血病，例如急性淋系T细胞白血病或急性淋系B细胞白血病；和/或，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种；The use according to claim 1, characterized in that the leukemia is acute myeloid leukemia or acute lymphocytic leukemia, such as acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia; and/or the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure; 优选地，所述急性髓系白血病为MLL-AF9融合蛋白引起的；和/或，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。Preferably, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and/or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism. 如权利要求1所述的应用，其特征在于，所述IL-1α的抑制剂为抗IL-1α的抗体；The use according to claim 1, wherein the IL-1α inhibitor is an anti-IL-1α antibody; 较佳地，所述抗IL-1α的抗体为单克隆抗体和/或中和抗体；Preferably, the anti-IL-1α antibody is a monoclonal antibody and/or a neutralizing antibody; 更佳地，所述抗IL-1α的抗体购自R&D Systems、货号为AB-400-NA。More preferably, the anti-IL-1α antibody is purchased from R&D Systems with the product number AB-400-NA. 购自R&D Systems、货号为AB-400-NA的抗体在制备治疗白血病治疗诱导的心脏损伤的药物中的应用；The use of an antibody purchased from R&D Systems, catalog number AB-400-NA, in the preparation of a drug for the treatment of leukemia-induced cardiac damage; 较佳地，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；和/或，所述白血病为急性髓系白血病或急性淋巴细胞白血病，例如急性淋系T细胞白血病或急性淋系B细胞白血病；Preferably, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and/or, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia, such as acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia; 更佳地，所述白血病治疗导致白血病细胞坏死；和/或，所述免疫治疗为CAR-T治疗，例如CD19 CAR-T治疗。More preferably, the leukemia treatment causes necrosis of leukemia cells; and/or, the immunotherapy is CAR-T therapy, such as CD19 CAR-T therapy. 如权利要求5所述的应用，其特征在于，所述化疗的药物为蒽环类化疗药物，优选柔红霉素；和/或，所述急性髓系白血病为MLL-AF9融合蛋白引起的。The use according to claim 5, characterized in that the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and/or the acute myeloid leukemia is caused by MLL-AF9 fusion protein. 如权利要求5所述的应用，其特征在于，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种；The use according to claim 5, wherein the cardiac injury is selected from one or more of: cardiac metabolic disorders, abnormal cardiac function, cardiomyopathy and heart failure; 优选地，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。Preferably, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism. IL1R1的拮抗剂在制备治疗白血病治疗诱导的心脏损伤的药物中的应用；Use of an antagonist of IL1R1 in the preparation of a medicament for treating cardiac damage induced by leukemia treatment; 较佳地，所述IL1R1的拮抗剂为IL1R1的竞争性抑制药物或者封闭药物；Preferably, the IL1R1 antagonist is a competitive inhibitory drug or a blocking drug of IL1R1; 更佳地，所述竞争性抑制药物为Anakinra。More preferably, the competitive inhibitory drug is Anakinra. 如权利要求8所述的应用，其特征在于，所述白血病治疗的方式选自：化疗、放疗、分子靶向治疗和免疫治疗中的一种或多种；The use according to claim 8, wherein the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; 优选地，所述白血病治疗满足选自以下条件的一种或多种：Preferably, the leukemia treatment satisfies one or more of the following conditions: 所述化疗的药物为蒽环类化疗药物，例如柔红霉素；The chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; 所述白血病治疗导致白血病细胞坏死；和，The leukemia treatment results in necrosis of leukemia cells; and, 所述免疫治疗为CAR-T治疗，例如CD19 CAR-T治疗。The immunotherapy is CAR-T therapy, such as CD19 CAR-T therapy. 如权利要求8所述的应用，其特征在于，所述白血病为急性髓系白血病或急性淋巴细胞白血病，例如急性淋系T细胞白血病或急性淋系B细胞白血病；和/或，所述心脏损伤选自：心脏代谢障碍、心功能异常、心肌病和心力衰竭中的一种或多种；The use according to claim 8, characterized in that the leukemia is acute myeloid leukemia or acute lymphocytic leukemia, such as acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia; and/or the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure; 优选地，所述急性髓系白血病为MLL-AF9融合蛋白引起的；和/或，所述心脏代谢障碍为心肌脂肪酸代谢下降，葡萄糖代谢增强。Preferably, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and/or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.
PCT/CN2025/076776 2024-02-20 2025-02-11 USE OF IL-1α INHIBITOR IN PREPARING MEDICAMENT FOR TREATING LEUKEMIA THERAPY-INDUCED CARDIAC INJURY Pending WO2025176039A1 (en)

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