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WO2018032513A1 - Application d'un astragaloside dans la prévention et le traitement d'une néphropathie diabétique de type 2 - Google Patents

Application d'un astragaloside dans la prévention et le traitement d'une néphropathie diabétique de type 2 Download PDF

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Publication number
WO2018032513A1
WO2018032513A1 PCT/CN2016/096096 CN2016096096W WO2018032513A1 WO 2018032513 A1 WO2018032513 A1 WO 2018032513A1 CN 2016096096 W CN2016096096 W CN 2016096096W WO 2018032513 A1 WO2018032513 A1 WO 2018032513A1
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astragaloside
effect
mice
shows
group
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Chinese (zh)
Inventor
李顺民
易铁钢
孙惠力
韩鹏勋
邵牧民
王文静
宋高峰
余学问
戈娜
王太芬
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Shenzhen Traditional Chinese Medicine Hospital
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Shenzhen Traditional Chinese Medicine Hospital
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to a novel application of astragaloside IV, and more particularly to the use of astragaloside IV in the preparation of a medicament for the prevention and treatment of type 2 diabetic nephropathy.
  • Diabetic nephropathy is an important complication of diabetes and gradually becomes the main cause of chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • the dysfunction of Akt and its related signaling pathways plays an important role in the progression of DN.
  • Astragaloside IV is an effective monomeric compound extracted from the traditional Chinese medicine Astragalus membranaceus, which can enhance the body's immunity and improve the body's disease resistance.
  • the invention patent CN 1569884A discloses a method for preparing astragaloside IV and application thereof in preparing medicine for preventing and treating diabetic nephropathy, and constructing a rat model of type 1 diabetic nephropathy by intraperitoneal injection of high-dose streptozotocin (STZ).
  • STZ high-dose streptozotocin
  • STZ high-dose streptozotocin
  • the invention is based on the research of astragaloside IV for type 2 DN, and finds that astragaloside IV has a strong preventive and therapeutic effect on type 2 DN, and the object of the present invention is to provide a drug for preventing and treating type 2 diabetic nephropathy in the preparation of a drug for preventing and treating type 2 diabetic nephropathy.
  • the present invention provides the following technical solutions:
  • the present invention has the following beneficial effects: the present invention aims to investigate the protective effect of astragaloside IV on type 2 DN and its mechanism of action, and the results show that astragaloside IV can reduce urinary albumin excretion in type 2 diabetes db/db mice. Rate, improve glomerular and renal tubular pathological damage, reduce urine NAG, NGAL and TGF- ⁇ 1 excretion, astragaloside can also inhibit the activation of Akt/mTOR, NF ⁇ B, Erk1/2 signaling pathway, and does not show obvious Hepatotoxicity.
  • astragaloside IV has a certain protective effect on type 2 DN, and its mechanism of action is related to the inhibition of Akt/mTOR, NF ⁇ B and Erk1/2 signaling pathways.
  • Figure 1 shows the effect of astragaloside on urinary albumin excretion rate in mice.
  • Figure 1 (a) shows the effect of astragaloside on urinary albumin excretion rate in mice
  • Figure 1 (b) shows astragaloside IV. results Effect on creatinine clearance in mice; wherein, compared with the wild type group, * P ⁇ 0.05, *** P ⁇ 0.001; compared with db / db group, # P ⁇ 0.05, ### P ⁇ 0.001;
  • Figure 2 shows the effect of astragaloside on the metabolic index of mice.
  • Figure 2(a) shows the effect of astragaloside on blood glucose in mice
  • Figure 2(b) shows the effect of astragaloside on the glycosylated hemoglobin in mice.
  • Fig. 2(c) shows the effect of astragaloside on urine glucose in mice
  • Fig. 2(d) shows the effect of astragaloside on serum insulin in mice; among them, **P ⁇ 0.01 compared with the wild type group. ***P ⁇ 0.001;
  • Figure 3 shows the effect of astragaloside on the physiological indexes of mice.
  • Figure 3 (a) shows the effect of astragaloside on the body weight of mice
  • Figure 3 (b) shows the effect of astragaloside on the kidney weight of mice. Among them, compared with the wild type group, ***P ⁇ 0.001;
  • Figure 4 shows the effect of astragaloside on glomerular injury in mice.
  • Figure 4(a) shows the effect of astragaloside on glomerular vasospasm in mice
  • Figure 4(b) shows the effect of astragaloside on small
  • Figure 4(c) is the effect of astragaloside on the thickness of mouse glomerular basement membrane
  • Figure 4(d) is the effect of astragaloside on the width of mouse foot.
  • Figure 5 shows the effect of astragaloside on the ratio of mesangial matrix and fibronectin in mice.
  • Figure 5 (a) shows the effect of astragaloside on the ratio of mesangial matrix in mice.
  • (b) The effect of astragaloside on the level of fibronectin in mice, and
  • Figure 5(c) shows the effect of astragaloside on the expression of fibronectin in mouse kidney by immunoblotting.
  • Figure 6 shows the effect of astragaloside on renal tubular injury in mice.
  • Figure 6 (a) shows the effect of astragaloside on urine NAG in mice
  • Figure 6 (b) shows the effect of astragaloside on mouse urine NGAL.
  • Figure 6 (c) is the effect of astragaloside on the urine TGF- ⁇ 1 in mice
  • Figure 6 (d) is the effect of astragaloside on the proximal renal tubular area of mice
  • Figure 6 (e) The effect of astragaloside on the lumen area of the proximal renal tubules in mice
  • Fig. 6(f) shows the effect of astragaloside on the proximal tubule wall area of mice
  • FIG. 6(g) is the jaundice The effect of glycoside on the thickness of renal tubular basement membrane in mice
  • Fig. 6(h) shows the effect of PAS staining and electron microscopy on the effect of astragaloside on the renal tubules of mice; among them, compared with the wild type group, **P ⁇ 0.01, *** P ⁇ 0.001; compared with db / db group, # P ⁇ 0.05, ## P ⁇ 0.01;
  • Figure 7 shows the effect of astragaloside on the renal Akt and its related signaling pathways in mice.
  • Figure 7 (a) shows the effect of astragaloside on Akt and its related signaling pathway proteins in immunoblotting experiments
  • Figure 7 (b) The effect of astragaloside on p-Akt in mice
  • Figure 7(c) shows the effect of astragaloside on p-mTOR in mice
  • Figure 7(d) shows the effect of astragaloside on mouse p-NF-
  • Figure 7(e) is the effect of astragaloside on p-Erk1/2 in mice; among them, compared with the wild type group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001; compared with db / db group, # P ⁇ 0.05, ## P ⁇ 0.01;
  • Figure 8 shows the effect of astragaloside on liver function in mice
  • Figure 8 (a) shows the effect of astragaloside on serum.
  • Fig. 8(b) shows the effect of astragaloside on serum aspartate aminotransferase (AST) in mice; **P ⁇ 0.01 compared with the wild type group.
  • DN is mainly characterized by glomerular hyperfiltration and microalbuminuria.
  • the pathological changes are mainly glomerular, tubular basement membrane thickening and mesangial expansion.
  • the onset of DN is still unclear and the treatment is scarce.
  • Akt and its related signaling pathways (mTOR, NF ⁇ B and Erk1/2) play important roles in cell growth and proliferation.
  • Akt signaling pathway dysfunction also plays an important role in human cancer, diabetes, cardiovascular and neurological diseases.
  • Recent studies have shown that Akt overactivation is closely related to DN progression, and statin, troglitazone, rapamycin or sirolimus can protect kidneys from DN by inhibiting the overactivation of Akt and its related signaling pathways.
  • Astragaloside IV is the main active ingredient of Astragalus.
  • AS-IV belongs to saponin compound and has a molecular weight of 784.97kDa. It has anti-inflammatory, anti-viral and anti-cancer effects.
  • AS-IV is mainly used in the heart, liver and nervous system, and its mechanism of action is related to the inhibition of Akt and its related signaling pathways.
  • Existing studies have shown that AS-IV has a certain renal protective effect on type 1 DN mice.
  • type 2 DN there is no research report on type 2 DN, and the type 1 DN animal model used and the type 2 DN model used in the present invention are not reported. There is an essential difference in the pathogenesis.
  • the present invention aims to observe the protective effect of AS-IV on type 2 DN nephropathy and its effect on Akt-related signaling pathways.
  • Animal model Eight-week-old male wild type mice (wild type) and db/db mice (BKS.Cg-Dock7 m +/+Lepr db /JNju) were purchased from the Model Animal Research Center of Nanjing University. Animal research experiments are carried out in strict accordance with the guidelines and regulations of animal ethics of Guangzhou University of Traditional Chinese Medicine. The experimental animals were controlled to freely ingest and drink at a constant room temperature of 20 ⁇ 1 ° C, 12 hours light and 12 hours dark cycle.
  • mice were randomly assigned to the following groups (8-10 per group): normal control mice were fed conventional food (wild type mice as normal group, ie, wild type group in the chart of the present invention), db/db mice were fed Conventional food (db/db group), db/db mice were fed AS-IV-added food (db/db+AS-IV group).
  • AS-IV was purchased from Chengdu ConBon Biotechnology Co., Ltd. (China) and added to the food of mice at a standard rate of 1 g/kg. The above experimental treatment lasted for 12 weeks.
  • mice The body weight of the mice was weighed every two weeks, and the blood glucose of each group was measured using a blood glucose meter (Roche, Basel, Switzerland), and urine was collected using a metabolic cage (Tenibus, Italy). . After 12 weeks of treatment, the mice were sacrificed and blood and kidney tissue samples were taken. The content of glycated hemoglobin (HbA 1C ) was measured using an Ultra2 glycated hemoglobin analyzer. Biochemical indicators of urine and blood were measured using Roche's automatic biochemical analyzer, including urinary creatinine, glucose, NAG (urinary N-acetyl- ⁇ -glucosidase), serum creatinine, ALT, and AST. Creatinine clearance (Ccr) was calculated by urine creatinine x urine volume x 1000 / serum creatinine / 1440 and expressed in microliters per minute.
  • HbA 1C glycated hemoglobin
  • Biochemical indicators of urine and blood were measured using Roche's automatic biochemical analyzer
  • Tissue preparation Immediately after the mice were sacrificed, the kidneys were removed, weighed, rinsed in phosphate buffer, and a certain amount of kidney tissue was cut along the longitudinal section and fixed with 10% formalin, and according to histopathology and immunological group. The method of research is carried out. One cubic millimeter of renal cortical tissue was fixed in a 2.5% glutaraldehyde solution, followed by treatment with 1% citric acid and analysis under an electron microscope. The remaining kidney tissue was immediately frozen in liquid nitrogen and stored at -80 °C for subsequent experimental studies.
  • the tubular wall area is equal to the area of the renal tubule minus the area of the tubular lumen.
  • HRP-polymer-conjugated anti-mouse/rabbit lgG secondary antibody (Fuzhou Maixin Biotechnology Development Co., Ltd.) was added for 15 minutes at room temperature, using diamino
  • the benzidine hydrochloride solution was used as a display reagent to measure the peroxidase activity.
  • ELISA ELISA test according to the manufacturer's instructions to detect serum insulin (Merck, Germany), urinary albumin (Bethyl, USA), urine TGF- ⁇ 1 (Daktronics, Shenzhen, China) and urine NGAL (American R&Dsystem) The content.
  • p-Akt (#4060) antibody, p-mTOR (#5536) antibody, p-NF- ⁇ B p65 (#3033) antibody and p-Erk1/2 (#4370) antibody were purchased from CST Company, USA; ⁇ -actin Antibody (A2228) was purchased from Sigma, USA; fibronectin antibody (ab2413) was purchased from Abcam, UK.
  • Measurement data are expressed as mean ⁇ standard deviation. Statistical differences between the two groups of samples were analyzed using independent sample t-test. Comparisons between groups of samples were performed using one-way ANOVA, and statistical analysis was performed using SPSS 16.0 statistical software. A statistically significant difference was considered when P ⁇ 0.05.
  • AS-IV can reduce the excretion rate of albuminuria in db/db mice, and does not improve the glomerular hyperfiltration.
  • Figure 1 shows the effect of astragaloside on urinary albumin excretion rate in mice.
  • Figure 1 (a) shows the effect of astragaloside on urinary albumin excretion rate in mice
  • Figure 1 (b) shows astragaloside IV.
  • the effect of creatinine clearance on mice was compared with that of the wild type group.
  • the urinary albumin of the db/db group increased significantly and gradually increased.
  • the glomerular filtration rate of db/db mice was significantly increased. Raise.
  • the albumin excretion rate of the db/db+AS-IV group was significantly reduced, and the effect was maintained until 12 weeks. However, after 12 weeks of intervention, there was no significant decrease in glomerular filtration rate.
  • Table 1 shows the metabolic characteristics of each group of mice. As shown in Table 1, compared with the wild type group, the db/db group showed polydipsia and polyuria symptoms at 8 weeks, AS-IV intervention. After these symptoms were significantly improved; Figure 2 is the effect of astragaloside on the metabolic index of mice, Figure 2 (a) is the effect of astragaloside on blood glucose in mice, and Figure 2 (b) is the effect of astragaloside on mice The effect of glycated hemoglobin, Figure 2 (c) is the effect of astragaloside on urine glucose in mice, and Figure 2 (d) is the effect of astragaloside on serum insulin in mice; 0, 2, 4, 6, At 8, 10, and 12 weeks, blood glucose was significantly increased in db/db mice, and glycated hemoglobin was also significantly elevated at 12 weeks. Urine sugar and serum insulin levels also increased significantly at 12 weeks. AS-IV treatment had no significant effect on blood glucose, glycosylated hemoglobin, urine glucose, and serum insulin.
  • Figure 3 shows the effect of astragaloside on the physiological indexes of mice.
  • Figure 3 (a) shows the effect of astragaloside on the body weight of mice
  • Figure 3 (b) shows the effect of astragaloside on the kidney weight of mice.
  • body weight and kidney weight of the db/db group were significantly increased.
  • AS-IV treatment reduced body weight and kidney weight, but did not show statistical difference.
  • AS-IV can improve glomerular injury
  • Figure 4 shows the effect of astragaloside on glomerular injury in mice.
  • Figure 4(a) shows the effect of astragaloside on glomerular vasospasm in mice
  • Figure 4(b) shows the effect of astragaloside on small
  • Figure 4(c) is the effect of astragaloside on the thickness of mouse glomerular basement membrane
  • Figure 4(d) is the effect of astragaloside on the width of mouse foot.
  • Figure 5 shows the effect of astragaloside on the ratio of mesangial matrix and fibronectin in mice.
  • Figure 5 (a) shows the effect of astragaloside on the ratio of mesangial matrix in mice.
  • (b) The effect of astragaloside on the level of fibronectin in mouse kidney tissue
  • Figure 5(c) shows the effect of astragaloside on the expression of fibronectin in mouse kidney tissue by immunoblotting
  • Figure 5(d) Immunohistochemical staining showed the effect of astragaloside on the expression of fibronectin in mice; compared with the wild type group, the ratio of mesangial matrix and fibronectin in db/db group increased significantly, AS-IV intervention These changes can be reduced but do not show statistical differences.
  • Figure 6 shows the effect of astragaloside on renal tubular injury in mice.
  • Figure 6 (a) shows the effect of astragaloside on urine NAG in mice
  • Figure 6 (b) shows the effect of astragaloside on mouse urine NGAL.
  • Figure 6 (c) is the effect of astragaloside on the urine TGF- ⁇ 1 in mice
  • Figure 6 (d) is the effect of astragaloside on the proximal renal tubular area of mice
  • Figure 6 (e) The effect of astragaloside on the lumen area of the proximal renal tubules in mice
  • Fig. 6(f) shows the effect of astragaloside on the proximal tubule wall area of mice
  • FIG. 6(g) is the jaundice The effect of glycoside on the thickness of renal tubular basement membrane in mice
  • Fig. 6(h) shows the effect of PAS staining and electron microscope on the effect of astragaloside on mouse renal tubules; at 12 weeks, compared with the wild type group, db/db group NAG, NGAL, and TGF- ⁇ 1 excretion were significantly increased, the proximal renal tubules, lumen and wall area increased, and the renal tubule basement membrane was significantly thickened.
  • AS-IV treatment can significantly reduce NAG, NGAL, TGF- ⁇ 1 excretion, reduce renal tubular, lumen and wall area, and reduce basement membrane thickness.
  • AS-IV inhibits the activation of p-Akt (Ser473), p-mTOR (Ser2448), p-NF- ⁇ B p65 (Ser536) and p-Erk1/2 (Thr202/Tyr204)
  • Figure 7 shows the effect of astragaloside on the renal Akt and its related signaling pathways in mice.
  • Figure 7 (a) shows the effect of astragaloside on Akt and its related signaling pathway proteins in immunoblotting experiments
  • Figure 7 (b) The effect of astragaloside on p-Akt in mice
  • Figure 7(c) shows the effect of astragaloside on p-mTOR in mice
  • Figure 7(d) shows the effect of astragaloside on mouse p-NF-
  • Figure 7(e) is the effect of astragaloside on p-Erk1/2 in mice; at 12 weeks, db/db mouse kidney cortex p-Akt (Ser473), p-mTOR (Ser2448 ), p-NF- ⁇ B p65 (Ser536) and p-Erk1/2
  • the content of (Thr202/Tyr204) was significantly increased, and AS-IV intervention significantly reduced these protein levels.
  • Figure 8 shows the effect of astragaloside on liver function in mice.
  • Figure 8(a) shows the effect of astragaloside on ALT
  • Figure 8(b) shows the effect of astragaloside on AST in mice; at 12 weeks
  • the blood biochemistry of db/db mice showed that ALT and AST were significantly increased, and there was no significant difference between db/db+AS-IV group and db/db group.
  • astragaloside as the main active ingredient of Chinese medicine Astragalus membranaceus can reduce the urinary albumin excretion rate of type 2 diabetes db/db mice, improve the pathological damage of glomeruli and renal tubules, reduce urine NAG, NGAL and Excretion of TGF- ⁇ 1. Astragaloside can also inhibit the activation of Akt/mTOR, NF ⁇ B and Erk1/2 signaling pathways, and does not show significant hepatotoxicity.
  • astragaloside IV has a certain protective effect on type 2 DN, and its mechanism of action is related to inhibition of Akt and its related signaling pathway.

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Abstract

L'invention concerne une application d'un astragaloside pour préparer un produit pharmaceutique pour prévenir et traiter une néphropathie diabétique de type 2. L'astragaloside peut réduire l'excrétion urinaire d'albumine d'une souris db/db atteinte de diabète de type 2, atténuer une lésion pathologique d'un glomérule et d'un tubule rénal et réduire l'excrétion urinaire de NAG, de NGAL et de TGF-ß1. L'astragaloside peut également supprimer une activation d'une voie de signalisation Akt/mTOR, NFκB et Erk1/2 et ne présente pas d'hépatotoxicité significative.
PCT/CN2016/096096 2016-08-19 2016-08-19 Application d'un astragaloside dans la prévention et le traitement d'une néphropathie diabétique de type 2 Ceased WO2018032513A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220260558A1 (en) * 2019-01-25 2022-08-18 Provigate Inc. Antigen measuring method and measuring apparatus
CN119235846A (zh) * 2024-10-16 2025-01-03 山西医科大学 Gpr55拮抗剂cid16020046在糖尿病肾病治疗中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1569884A (zh) * 2004-04-29 2005-01-26 南京医科大学 黄芪甲苷的制法及在制备防治糖尿病肾病药物中的应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1569884A (zh) * 2004-04-29 2005-01-26 南京医科大学 黄芪甲苷的制法及在制备防治糖尿病肾病药物中的应用

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Title
GUI, DINGKUN ET AL.: "Renal protective effects and mechanisms of astragaloside IV", CHINESE JOURNAL OF KIDNEY DISEASE INVESTIGATION, vol. 2, no. 4, 31 August 2013 (2013-08-31), pages 204 - 206, ISSN: 2095-3216 *
LI, ZHONG ET AL.: "Effect of astragaloside through the TGF-beta / Smad signaling pathway on the kidneys of diabetic rats with nephropathy", GUANGDONG MEDICAL JOURNAL, vol. 37, no. 11, 30 June 2016 (2016-06-30), pages 1623 - 1628, ISSN: 1001-9448 *
ZHANG, TINGJI: "Clinical Research Progress of Astragalus Injection in Treating Diabetic Nephropathy", THE 10TH NATIONAL INTEGRATIVE MEDICINE NEPHROLOGY SYMPOSIUM ON PROCEEDINGS, 11 June 2009 (2009-06-11), pages 910 - 913 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220260558A1 (en) * 2019-01-25 2022-08-18 Provigate Inc. Antigen measuring method and measuring apparatus
CN119235846A (zh) * 2024-10-16 2025-01-03 山西医科大学 Gpr55拮抗剂cid16020046在糖尿病肾病治疗中的应用

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