WO2022033163A1 - Molecular marker related to aging and healthy aging and application thereof in improving healthy aging - Google Patents

Abstract

The present invention belongs to the field of biotechnology and relates in particular to a molecular marker related to aging and healthy aging and an application thereof in improving healthy aging. Individuals who practice Tai Chi were used as an experimental group. In carrying out protein mass spectrometry comparison between normal young individuals and elderly individuals, the FABP4 protein was screened, and the expression thereof in the blood of the elderly individuals increased significantly compared with that in the young individuals. However, said trend was significantly reversed in elderly individuals who practice Tai Chi. The knockdown of FABP4 gene expression or the inhibition of FABP4 activity in aged mice significantly improves cognitive function and inhibits aging and related inflammation indicators. It is suggested that the FABP4 gene has the effect of promoting aging and can be used as a molecular target for improving healthy aging, thereby providing a novel target and novel strategy for delaying aging.

Description

A molecular marker related to aging and healthy aging and its application in improving healthy aging technical field

The invention belongs to the field of biotechnology, and in particular relates to a molecular marker related to aging and healthy aging and its application in improving healthy aging.

Background technique

The world has ushered in the era of "global aging", and my country has entered an aging society and is in a stage of deepening aging. It is estimated that by 2020, the population of the elderly over 60 years old in China will increase to about 255 million, accounting for about 17.8% of the total population, and the social burden brought by aging is increasing. Aging is closely associated with numerous age-related diseases, including Alzheimer's disease, Parkinson's disease, stroke, peripheral neuropathy, macular degeneration, cataracts, presbycusis, type 2 diabetes, osteoporosis, osteoarthritis, arterial Atherosclerosis, benign prostatic hyperplasia and various cancers.

Aging is a complex process in which the function of the body's organs continues to decline under the combined action of genetic and environmental factors, and it is one of the basic characteristics of life. At present, theories on the mechanism of aging include: oxidative stress theory, decreased genome stability, shortening of telomeres, epigenetic changes, dysregulation of protein homeostasis, etc. These theories generally support that aging is a programmed and can be intervened biological process. With the prolongation of human life expectancy and the advent of an aging society, the risk of Alzheimer's disease, cancer, diabetes, cardiovascular and cerebrovascular diseases, etc. in the elderly group has also greatly increased. Extending lifespan does not make much sense if it does not improve the quality of life of the elderly population. Scientists believe that prolonging lifespan should be premised on delaying aging and maintaining a certain quality of life. How to delay aging-related functional degradation, prevent and treat senile diseases has become an urgent and significant scientific problem.

In recent years, many aging studies have attempted to explore aging-related molecular markers macroscopically through proteomics technology. Dunston et al identified significant changes in ferroportin, complement C3b and transthyretin expression by comparing the proteomes of young and old. Miura et al. selected long-lived populations and found that haptoglobin, microglobulin and clusterin are aging-related protein factors. Thambisetty et al. found that the expression level of apolipoprotein ApoE is closely related to Alzheimer's disease in the elderly. However, these studies generally have insufficient detection depth, cannot accurately quantify low-abundance proteins, and cannot reflect the broad-spectrum regulation of protein factors in the blood circulation by the aging process, and there is a lack of repeated verification between the findings of each study. Therefore, further proteomic studies are needed, especially for accurate quantification of low-abundance proteins.

How to maintain the health of the aging population to the greatest extent, intervene in aging-related diseases and delay the occurrence of aging is a huge challenge for the medical system, sports system and even the whole society. The concept of "exercise is good medicine" advocated and promoted by European and American scholars in recent years reflects the broad recognition of modern medicine for the view that "exercise promotes health". Taijiquan has become the common choice of health experts all over the world. It is a traditional sports event in my country with thousands of years of development history and plays an important role in promoting human health. Studies have shown that Tai Chi exercises can significantly improve various human diseases such as Parkinson's disease, coronary atherosclerosis, diabetes, fibromyoma, and arthritis. Practice has proved that the elderly often participate in Tai Chi exercise, not only can prevent disease and fitness, but also delay the aging process. In this regard, Tai Chi exercise provides an excellent model for studying the molecular mechanisms of aging and aging-related diseases.

The clinical trial research of Taijiquan intervention is becoming more and more internationalized. Following the publication of Taijiquan intervention in PD treatment in the New England Journal, the research of Taijiquan in the field of rehabilitation and prevention by foreign researchers has also developed rapidly. In the rehabilitation study of the disease, Yeh et al. conducted three Taijiquan rehabilitation studies in patients with chronic heart failure within 10 years; Salmoirago-Blotcher et al. found that 2 times/week Taijiquan exercise for 12 weeks significantly improved coronary heart disease. Disease recovery; Pan et al. conducted a study of 120 people and found that 3-month Tai Chi exercise significantly promoted the recovery of pulmonary respiratory function in patients with non-small cell lung cancer after surgery. These studies suggest that Taijiquan exercise has obvious effects of delaying aging and preventing aging-related diseases. It is an ideal model for studying aging mechanisms and can make up for the disadvantage that prospective studies cannot be carried out in the field of clinical aging.

Lipids are essential components of many biological processes and are critical to the pathogenesis of many common diseases. Fatty acid binding proteins (FABPs) are a class of small, highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands and regulate lipid flux, transport, signaling, and metabolism. Intracellular and extracellular interactions with proteins enable the function and diversity of lipids. FABPs were originally described as intracellular proteins that can affect lipid flux, metabolism, and signaling within cells. As the functions of this protein family are further elucidated, it is clear that they are important mediators of metabolic and inflammatory processes both locally and systemically, and are therefore potential therapeutic targets. FABPs are lipid carriers involved in secretion, blood sucking, and intracellular fatty acid delivery to subcellular organelles such as mitochondria and peroxisomes, and are involved in glucose metabolism and lipid oxidation.

Fatty acid binding protein FABP4 (also known as Adipocyte-FABP, A-FABP, and aP2), contains 132 amino acids and has a molecular mass of 14719Da. FABP4 is a fatty acid-binding protein found in adipocytes and is a lipid transporter in adipocytes; it has a beta-barrel structure that accommodates hydrophobic ligands, naturally occurring long-chain fatty acids and synthetic Hydrophobic ligands can be accepted therein. Long-chain fatty acids and retinoic acid are provided to cognate receptors in the nucleus by binding them. FABP4 acts as a transporter of endogenous fatty acids from the cell surface to various sites of fatty acid storage and metabolism.

FABP4 is secreted by adipocytes and its levels are higher in obese people. In humans, high circulating FABP4 levels are associated with obesity, metabolic disease and cardiac dysfunction. FABP4 regulates metabolic and inflammatory pathways, and inhibition of FABP4 ameliorates type 2 diabetes, atherosclerosis, and immunometabolic disease in mouse models. Genetic deficiency and small-molecule-mediated inhibition of FABP4 can effectively improve glucose homeostasis and reduce atherosclerosis in mouse models. The circulating form of FABP4 has a key hormonal function in systemic metabolism, it is highly expressed in adipocytes and macrophages, and has been implicated in the development of insulin resistance, metabolic syndrome and atherosclerosis. Furthermore, this protein acts as a cytoplasmic shuttle protein for ligand activation of the nuclear receptor PPAR to activate its downstream transcriptional targets involved in cell differentiation, apoptosis, and anti-inflammatory responses. The FABP4 inhibitor BMS309403 is a selectively targeted FABP4 inhibitor. Its Ki value is less than 2nM. BMS309403 is an aromatic biphenylazo compound that competes with fatty acids for the highly specific A-FABP binding pocket. BMS309403 has been shown to reduce monocyte chemoattractant protein-1 (MCP-1) secretion by THP-1 macrophages. BMS309403 is effective in preventing severe atherosclerosis and type 2 diabetes.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the present invention provides a molecular marker related to aging and healthy aging and its application, aiming to solve some problems in the prior art or at least alleviate some of the problems in the prior art.

The present invention is realized by a molecular marker related to aging and healthy aging, wherein the molecular marker is the expression product of FABP4 gene and/or FABP4 gene.

Application of the above-mentioned molecular markers related to aging and healthy aging in the preparation of reagents and/or kits for detecting and/or treating animal aging indicators or aging-related diseases.

Further, the aging index includes at least one of animal body weight and pancreatic islet cell disease.

Application of the above-mentioned molecular markers related to aging and healthy aging in the preparation of reagents and/or kits for detecting and/or treating cognitive function in animals.

Further, the cognitive function includes animal learning and/or memory ability.

Application of the above-mentioned molecular markers related to aging and healthy aging in the preparation of reagents and/or kits for detecting and/or treating inflammatory conditions in animals.

Application of the above-mentioned molecular markers related to aging and healthy aging in the preparation of reagents and/or kits for detecting and/or treating animal liver function.

Further, the reagents and/or kits comprise inhibitors of FABP4.

Further, the inhibitor of FABP4 is any one of siRNA of FABP4 gene, RNA interference vector of FABP4 gene, antibody of FABP4 and other inhibitors that can inhibit the expression of FABP4.

Application of the above-mentioned molecular markers related to aging and healthy aging in the preparation of cell models and/or animal models for screening diagnostic and/or therapeutic drugs related to aging.

To sum up, the advantages and positive effects of the present invention are:

1. The present invention discovers a new function of the FABP4 gene, that is, the role of the FABP4 gene in accelerating aging-related diseases.

2. Based on the function of FABP4 in accelerated aging-related diseases, provide a target for the drugs of aging diseases.

3. The inhibitors of FABP4 can be used to prepare medicines for protecting liver function and treating aging-related diseases.

The invention closely follows the national health project strategic plan, and is based on cross-sectional studies of elderly controls and Taijiquan exercisers, combined with animal experiments to study the molecular mechanism of aging, especially the regulatory mechanism affecting metabolism in the aging process. Explore new mechanisms of aging from the molecular level, and provide new molecular targets for clinical prevention and treatment of aging. It not only provides scientific evidence for Tai Chi to prevent aging, but also has great significance for mankind to work together to deal with the common threats and challenges of aging and aging-related diseases .

The present invention analyzes the elderly who have practiced Tai Chi for more than 3 years, as well as control elderly and control young people of the same age in the preliminary research process. The physical examination data shows that Tai Chi exercise significantly improves the declining cerebrovascular function and cardiac contraction of the elderly. and diastolic function and bone mineral density. The protein spectrum classification method was used to identify the secreted proteins in the blood; the protein molecules whose abundance changed during the aging process and were reversed in the Taijiquan group were screened. Through the study of plasma proteomics, we screened a number of Significantly differentially expressed proteins associated with aging. Among them, the protein FABP4 was significantly low-expressed in young people and significantly high-expressed in the elderly group, while the expression of FABP4 in the elderly group who practiced Taijiquan tended to be low in young people. These results suggest that FABP4 may play an important role in reversing the development of senescence.

The invention takes the FABP4 gene knockout mice as the experimental objects, and by constructing an aging model, the results show that compared with the wild-type C57 mice, the FABP4 gene knockout mice, the cognitive function of the old mice is improved, and the aging and inflammatory indicators are inhibited . This suggests that the FABP4 gene has a role in promoting aging, and provides a theoretical basis and clinical basis for the role of FABP4 in the study of new targets and strategies for anti-aging.

Therefore, FABP4 gene can be used as a drug target to construct in vitro cell models or animal models of FABP4 gene knockout and overexpression for screening prevention, remission and/or treatment and aging-related drugs; FABP4 gene can also be used in gene therapy. Design and prepare drugs and/or biological reagents to prevent, alleviate and/or treat aging diseases, and achieve the purpose of preventing, alleviating and/or treating aging diseases through genetic engineering technology. For example, with FABP4 as the target gene, siRNA, shRNA, and AAV-shRNA that can interfere with the expression of FABP4 are designed. After chemical synthesis, they are injected into the human body to silence the FABP4 gene through RNA interference to treat aging-related diseases; Design small molecule compound inhibitors with FABP4 as the target, use the in vitro cell model or animal model of FABP4 gene overexpression, and find the molecules that can specifically inhibit FABP4 through screening, so as to provide a new treatment for liver ischemic diseases sex molecules.

The present invention relates to the regulation effect of FABP4 on aging, and will have great significance for exploring the mechanism research and drug development related to aging.

Description of drawings

Fig. 1 is the proteomic screening result in embodiment 2;

Fig. 2 is the detection result to mouse cognitive function in embodiment 4;

Fig. 3 is the detection to aging and inflammation index in embodiment 5;

Fig. 4 is the experimental result of metabolic cage in embodiment 6;

Fig. 5 is the liver fat evaluation result in embodiment 7;

6 is a graph showing the improvement results of the FABP4 inhibitor BMS309403 in Example 8 on the improvement of physiological indicators in aging mice.

detailed description

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels unless otherwise specified. The specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention.

The proteins or fragments thereof contemplated in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants) using recombinant techniques.

The present invention discloses a molecular marker related to aging and healthy aging and its application in improving healthy aging, as shown in the following embodiments. Experimental animals involved in the present invention: wild-type mice (WT, purchased from Beijing Huafukang Biotechnology Co., Ltd.), 8-10 weeks old, weighing 24g-27g, and the background is male C57BL/6 strain, adenovirus AAV9 -shNeg and adenovirus AAV9-shFabp4 construction. Referring to the Fabp4 sequence of mice, Hanheng Biotechnology (Shanghai) Co., Ltd. used pHBAAV-U6-MCS-CMV-EGFP plasmid vector to construct a eukaryotic expression plasmid containing shFabp4 fragment, wherein the Forward primer of shFABP4 was rGrGrCrCrArArGrCrCrCrArArCrArUrGrArUrCrArUrCrAGT, SEQ ID NO.1, Reverse primer is rArCrUrGrArUrGrArUrCrArUrGrUrUrGrGrGrCrUrUrGrGrCrCrArU, SEQ ID NO.2. The plasmid was packaged as adeno-associated virus AAV9-shFabp4, while the empty pHBAAV-U6-MCS-CMV-EGFP plasmid was packaged as adeno-associated virus AAV9-shNeg as a control. Mice were injected with AAV9 virus via tail vein when they were 16 months old: control group adenovirus AAV9-Neg and FABP4 knockdown group adenovirus AAV9-shFabp4, the dose of virus was 5.5×10 ¹¹ particles per mouse injection.

Breeding environment: All experimental mice were bred in the SPF-level experimental animal center of the People's Hospital of Wuhan University. The special feed for mice was provided by the Animal Center of the Chinese Academy of Military Medical Sciences. Breeding conditions: room temperature between 22-24 ℃, humidity between 40-70%, alternating light and dark lighting time for 12h, free water and food intake.

The number of FABP4 (human) involved in the present invention in the NCBI database: gene ID is 2167, nucleotide sequence number NM_001442.3, protein sequence NP_001433.1. The number of FABP4 (mouse) in the NCBI database: the gene ID is 11770, the nucleotide sequence number is NM_024406.3, and the protein sequence is NP_077717.1 (the mouse sequence was used when constructing the adenovirus).

Example 1 The improvement effect of Taijiquan exercise on various indicators of the human body

Participants: 22-25-year-old youth control group, 60-69-year-old elderly control group, and 60-69-year-old people who have practiced Taijiquan for more than 3 years were selected as the youth control group, the elderly control group and the Taijiquan group, respectively.

1. Cerebrovascular function test

The cerebrovascular function detector (GT-3000) was used to analyze the hemodynamics of bilateral common carotid arteries in the subjects. The pressure sensing probe collects information related to cerebral vascular elasticity, and obtains related indicators such as peripheral resistance and pulse wave velocity. Detection indicators: average flow rate (Qmean), average flow rate (Vmean), maximum flow rate (Vmax), minimum flow rate (Vmin), pulse wave velocity (Wv), characteristic impedance (Zcv), expandability (ID), peripheral resistance (Rv) ), dynamic resistance (DR), critical pressure level (Cp), diastolic pressure and critical pressure difference (Dp), etc.

2. Color Doppler Ultrasound Detection of Cardiac Function

The digital color ultrasound diagnostic system (SONOLINE Premier, SIEMENS) was used to test the cardiac function of the subjects. Measurement. Detection indicators: Ascending aortic diameter (AAOD), pulmonary artery diameter (MPAD), left atrial diameter (LAD), left atrial diameter (LVD), right atrial diameter (RAD), right atrial diameter (RVD) , End-diastolic ventricular septal thickness (IVSD), left ventricular posterior wall thickness (LVPWD), left ventricular ejection fraction (LVEF), aortic valve stroke volume (AVSV), mitral valve E peak velocity (MVDV: E ), mitral valve A peak velocity (MVDV: A), pulmonary valve stroke volume (PVSV).

3. Bone density test

Bone mineral density (BMD) of anteroposterior lumbar spine (L1-4) of all subjects was measured by dual-energy X-ray absorptiometry (Unigamma M, Ican scientific laboratories). Bone mineral density is an important indicator of bone strength, expressed in grams per square centimeter (g/cm ² ), which is an absolute value. In clinical use of bone mineral density values, due to the different absolute values of different bone density detectors, Usually the T value is used to judge whether the bone density is normal.

The physical indicators of the above groups were tested, and the results are shown in Table 1. Through the comparative analysis between the Taijiquan elderly group and the normal elderly group, it was revealed that Taijiquan exercise significantly improved the cerebrovascular function, cardiac function, and bone mineral density of the elderly.

Table 1. Effects of Tai Chi exercise on cerebrovascular function, cardiac systolic and diastolic function, and bone mineral density in the elderly.

^# , P value is calculated by comparing the elderly group and the Tai Chi elderly group.

Example 2 Proteomic Screening

The plasma samples of the young control group, the elderly control group and the Taijiquan group were randomly divided into three mixed samples according to gender, each group of mixed samples contained samples from 15 individuals, and two 10-label, 36-level tandem mass spectrometry labels (Tandem Mass Tag, TMT) protein profile identification, and screened the protein molecular markers whose abundance changed during the aging process and the change trend of the Tai Chi group was reversed.

The results are shown in Figure 1: A. ELISA detection of IGF-1 expression in serum. B. ELISA detection of FABP4 expression in serum. Protein profiling reveals that Tai Chi exercise reverses the elevation of circulating FABP4 in the elderly.

Example 3 Construction of FABP4 transgenic mice

Establishment of a natural aging mouse model: 8-week-old C57BL/6 wild-type male mice were purchased, and when they were raised to 14 months, adenovirus AAV9-shNeg and adenovirus AAV9-shFabp4 were injected to construct Fabp4 knockdown mice and its control mice. Mice were cultured to 20 months of age to establish a natural aging mouse model. When the mice were 20 months old, the relevant indicators were detected.

Experimental grouping: 20 senescent mice transfected with adenovirus AAV9-shNeg at 20 months, 20 senile mice transfected with adenovirus AAV9-shFabp4 at 20 months, and 20 8-week-old young mice with C57BL/6 wild type.

Example 4 Detection of Cognitive Function in Mice

Morris water maze (MWM) experiment is an experiment in which experimental animals are forced to swim and learn to find platforms hidden in water. The Morris water maze is mainly used to test the sense of spatial position and direction (spatial orientation) of experimental animals. evaluation of learning and memory ability. By detecting the indicators Island latency, Island entries, Island time, speed, etc., the effect of Fabp4 on learning and memory in mice was clarified.

1. Acquired training

(1) Put the mouse head into the water with its head facing the pool wall, and randomly choose one of the four starting positions of east, west, south and north. Record the time(s) for the animal to find the underwater platform. In the first few training sessions, if this time exceeds 90 s, guide the animal to the platform. Let the animal stay on the platform for 15 s.

(2) Remove the animal and dry it. If necessary, the animals (especially rats) were baked under a 150W incandescent lamp for 5 minutes and returned to their cages. Each animal was trained 4 times a day, with an interval of 15-20 min between the two training sessions, and continued training for 5 days.

2. Probe training

The day after the last acquired training, the platform was removed and the 60s exploration training began. The animal was placed in the water from the opposite side of the original platform quadrant. The time spent in the target quadrant (the quadrant where the platform was originally placed) and the number of times the animal entered the quadrant were recorded as indicators of spatial memory.

3. Contrast training

The working memory of the animals was measured. On the second day after the exploratory training, the 4-day alignment training began. The platform is placed in the opposite quadrant of the original platform in the same way as for acquisition training. Train 4 times a day. The time to find the platform and the swimming distance and swimming speed were recorded each time.

4. Alignment detection training

The second day of the last alignment training. The approach is similar to the profiling training described above. The time spent in the target quadrant (the area where the platform was located for the second time) and the number of times the animal entered the area were recorded within 60 s.

The results are shown in Figure 2: A. Time required for mice to find the target area on day 5; B. Number of times they entered the target area; C. Time required for mice to find the target area on day 5; D. Stay in the target area time. E. Swimming speed. Experiments show that knockdown of Fabp4 can improve cognitive function.

The water maze experiment is divided into two stages, 1-5 days is the stage of learning and memory ability, as shown in Figure 2A, it is the time it takes for mice to find the target area in these 5 days, the learning ability and memory ability of aging mice Both were weaker than young mice, and slightly improved after Fabp4 knockout. The BE in Figure 2 reflects the results of the 6th day detection period. Compared with the young mice, the number of times the aged mice entered the target area and the time they stayed in the target area decreased, the time to find the target area was prolonged, and the swimming speed slowed down , while knocking out Fabp4 improved the above parameters, but only the number of times entering the target region was statistically significant.

Example 5 Detection of aging and inflammation indicators

RNA extraction: Add 1 ml of Trizol solution to the collected cardiomyocytes, pipet repeatedly on ice until the cardiomyocytes are fully lysed, let stand for 5 minutes, add 0.2 ml of chloroform, shake vigorously for 15-30 seconds, let stand for 2-3 minutes, Centrifuge at 12000rpm x 15min at 4°C. Pipette the aqueous layer into a new EP tube, add 0.5ml of isopropanol, mix the liquid in the tube gently, let stand for 10 minutes at room temperature, and centrifuge at 12000rpm x 10min at 4°C. Discard the supernatant, add 1 ml of pre-cooled 75% ethanol to the pellet, resuspend the pellet and wash thoroughly, and centrifuge at 12000rpm x 5min at 4°C. The supernatant was discarded, air-dried, and an appropriate amount of DEPC water was added to solubilize the RNA at 65°C. Take 2ul RNA for agarose gel electrophoresis to check whether the RNA is extracted successfully, and store the remaining RNA at -80°C when not in use.

Real-time quantitative PCR (RT-PCR): reverse transcription to cDNA using the Thermo Fisher Scientific reverse transcription kit. The reverse transcribed cDNA was diluted five times with ddH2O, and the mRNA level was detected by SYBR ^TM Green reagent from Thermo Fisher Scientific. The specific reaction system is shown in Table 2.

Table 2 Real-time fluorescence quantitative PCR reaction system

Reaction conditions:

95°C for 10min; 95°C for 10sec, 60°C for 10sec, 72°C for 10sec, 40cycles, 40°C for 30sec, draw a dissolution curve, and analyze the final data with 2 ^-ΔΔCt .

The total RNA was extracted from the liver tissues of the mice in each group. After reverse transcription, RT-PCR was used to detect the mRNA expression levels of the aging-related factor p21, as well as the expression levels of Interleukin-6 (IL-6) and Interleukin-1beta (IL-1β). The mRNA expression level was based on the mRNA level of 18S rRNA. The primer sequences are shown in the table below:

  Forward Primer Reverse Primer P21 CCTGGTGATGTCCGACCTG, SEQ ID NO.3 CCATGAGCGCATCGCAATC,SEQ ID NO.4 IL-1β CCGTGGACCTTCCAGGATGA, SEQ ID NO.5 GGGAACGTCACACACCAGCA, SEQ ID NO.6 IL-6 AGTTGCCTTCTTGGGACTGA, SEQ ID NO.7 TCCACGATTTCCCAGAGAAC, SEQ ID NO. 8 18S AGGGTTCGATTCCGGAGAGG, SEQ ID NO.9 CAACTTTAATATACGCTATTGG, SEQ ID NO.10

The results of the determination of the senescence of liver tissue cells are shown in Figure 3, A. The senescence index p21. B. Inflammatory factor indicators IL-6 and IL-1β. The experimental results show that knocking down Fabp4 can improve the levels of aging indicators and inflammatory factors. This part of the experiment detected the aging marker p21 and the inflammatory markers IL-6 and IL-1β in young mice (Young), old control mice (Old-shNeg) and old Fabp4 knockdown mice (Old-shFabp4). mRNA expression levels. RT-PCR detection showed that the aging status and inflammation level of the three indicators FABP4 knockdown aged mice were significantly improved compared with the control aged mice, and there was no statistical difference compared with young mice, which further suggested that FABP4 is closely related to the related to aging. These results suggest that inhibition of FABP4 expression can improve liver tissue aging.

Example 6 Metabolic cage experiment

Computers are used to monitor the diet, gas metabolism, and activities of mice in real time for one or several days, and comprehensively analyze the possible abnormal phenotypes in their metabolism. Using the Oxymax/CLAMS animal metabolism system (Columbus, USA), the mice were kept in metabolic cages after standard gas calibration for the accuracy of gas detection, avoiding human interference, and calculated by measuring changes in gases (oxygen and carbon dioxide). The respiration entropy and heat production of the mice were detected in real time, and the changes in the food intake of the mice were detected in real time, and the real-time activity of the mice was calculated by the number of infrared rays passing through.

Detection indicators include: animal water and food consumption, solid and liquid excrement quality, oxygen consumption (VO2 consumption), carbon dioxide consumption (CO2 consumption), carbon dioxide production (CO2 production), heat generation (Heat production), activity (Activity), etc.

The results are shown in Figure 4, A and B. Graphs of hourly oxygen consumption versus carbon dioxide production. C and D. Histograms of total nocturnal and daytime oxygen consumption and total carbon dioxide production in mice. E and F. 24-hour calorie production and activity times in mice. The results indicated that Fabp4 knockdown could increase the basal metabolic rate in aging mice.

In Figure 4, A and B are the curves of the total amount of oxygen consumption and carbon dioxide production at night and during the day. Compared with the young mice, the oxygen consumption and carbon dioxide production of the old mice were significantly decreased. The oxygen consumption and carbon dioxide production of aging mice with FABP4 increased, mainly at night when the mice were active. In Figure 4, C-F are the calorie generation and activity times in 24 hours. The FABP4 knockout aging mice increased calorie production and activity times, which was consistent with the changes in oxygen consumption.

Example 7 Evaluation of liver fat

In order to show the fat in the tissue, Oil Red O is often used for staining. Oil Red O is a fat-soluble dye, highly soluble in fat, and can specifically color neutral fats such as triglycerides in tissues. Tissue samples were processed by cryosection and nuclei were stained by Hoechst33342. The fat droplets inside the hepatocytes are red and the nuclei are blue. The degree of hepatic steatosis was evaluated by the size of the red area. After taking pictures, use ImageJ image processing software to analyze the proportion of senescent cells.

The results are shown in Figure 5, young mice (Young), old control mice (Old-shNeg) and old Fabp4 knockdown mice (Old-shFabp4). The results of oil red O staining in liver tissue showed that there were very few lipid droplets in the liver tissue of young mice, and there were a large number of lipid droplets in the liver tissue of the aging control group, but they were not yet vacuolated, and some mice knocked down Fabp4. few. The results indicate that Fabp4 knockdown can improve the fat level of aging mice.

Example 8 The effect of FABP4 inhibitor BMS309403 on the improvement of physiological indicators in aging mice

BMS309403 (APExBIO, Boston, MA; Catalog No. B7794; Cas No. 300657-03-8; molecular formula C31H26N2O3) is a specific inhibitor of FABP4. FABP4 was inhibited by gavage with BMS309403.

There are three groups of mice: 8-week-old young mice group, aging mice control group (CMC-Na solvent group), and aging mice FABP4 inhibitor group (BMS30940345); 10 mice in each group.

Male mice with C57BL/6J background were gavaged at 15 mg/kg/d twice a week, starting from 20 weeks old to 22 weeks (males) and 22.5 weeks (females). Spleen samples were collected for morphological examination.

The results are shown in Figure 6, grouped into: young mice (Young), old control mice (Old Control) and old Fabp4 inhibitor BMS309403 (Old BMS309403).

After gavage with the FABP4 inhibitor BMS309403, it slightly improved the body weight of mice (Figure 6A). By staining with Oil Red O, it could be observed that BMS309403 significantly improved the heart, brown fat, and liver of mice (Figure 6A). Figure 6B). Figure 6C shows the results of HE staining of islet cells after intragastric administration of the FABP4 inhibitor BMS309403: under the microscope, the islet cells of the young mouse group (Young) were basically normal, and the cell structure was neat; while the islet cells of the old control group (Old Control) were disordered, and There are necrotic foci of different sizes and irregular shapes. The structure of islet cells in the necrotic foci is blurred, disordered, and severed. Compared with normal islet cells, the islet cell nucleus has typical necrotic changes, and nuclear pyknosis can be seen. This phenomenon was significantly improved in the FABP4 inhibitor BMS309403 group (Fig. 6C). The results show that the FABP4 inhibitor BMS309403 can improve the physiological indicators of aging mice.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

A molecular marker related to aging and healthy aging, characterized in that: the molecular marker is the expression product of FABP4 gene and/or FABP4 gene. The application of an aging and healthy aging-related molecular marker according to claim 1 in the preparation of a reagent and/or kit for detecting and/or treating an animal aging index or an aging-related disease. The application according to claim 2, wherein the aging index comprises at least one of animal body weight and pancreatic islet cell disease. The application of an aging and healthy aging-related molecular marker according to claim 1 in the preparation of reagents and/or kits for detecting and/or treating animal cognitive function. The application according to claim 4, wherein the cognitive function includes animal learning and/or memory ability. The application of an aging and healthy aging-related molecular marker according to claim 1 in the preparation of a reagent and/or kit for detecting and/or treating inflammation in animals. The application of an aging and healthy aging-related molecular marker according to claim 1 in the preparation of a reagent and/or kit for detecting and/or treating animal liver tissue aging and hepatic steatosis. The use according to any one of claims 2-7, wherein the reagent and/or the kit comprises an inhibitor of FABP4. The application according to claim 8, wherein the inhibitor of FABP4 is any one of siRNA of FABP4 gene, RNA interference vector of FABP4 gene, antibody of FABP4 and other inhibitors capable of inhibiting the expression of FABP4. The application of an aging and healthy aging-related molecular marker according to claim 1 in the preparation of a diagnostic and/or therapeutic drug for screening aging-related.

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