WO2018032513A1

WO2018032513A1 - Application of astragaloside in preventing and treating type 2 diabetic nephropathy

Info

Publication number: WO2018032513A1
Application number: PCT/CN2016/096096
Authority: WO
Inventors: 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
Original assignee: 深圳市中医院
Priority date: 2016-08-19
Filing date: 2016-08-19
Publication date: 2018-02-22

Abstract

An application of an astragaloside for preparing a pharmaceutical product for preventing and treating type 2 diabetic nephropathy. The astragaloside can reduce urine albumin excretion of a db/db mouse with type 2 diabetes, mitigate a pathological injury of a glomerulus and renal tubule, and reduce urine excretion of NAG, NGAL and TGF-β1. The astragaloside can also suppress an activation of an Akt/mTOR, NFκB, and Erk1/2 signal pathway, and does not exhibit significant hepatotoxicity.

Description

Application of astragaloside IV in prevention and treatment of type 2 diabetic nephropathy

Technical field

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.

Background technique

Diabetic nephropathy (DN) is an important complication of diabetes and gradually becomes the main cause of chronic kidney disease (CKD). The dysfunction of Akt and its related signaling pathways (mTOR, NFκB and Erk1/2) plays an important role in the progression of DN.

Astragaloside IV (AS-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). The therapeutic experiment of astragaloside IV by intragastric administration proves that astragaloside has a certain protective effect on type 1 DN. However, there is no research report on type 2 DN. Type 1 diabetic nephropathy is insulin-dependent diabetes mellitus, and type 2 diabetic nephropathy is non-insulin-dependent diabetes mellitus, which is essentially different in pathogenesis, diagnosis, and treatment strategies.

Summary of the invention

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. Applications.

In order to achieve the above object, the present invention provides the following technical solutions:

1. To study the effect of AS-IV treatment on urinary albumin excretion rate in db/db mice;

2. To study the effects of AS-IV treatment on physiological and metabolic indexes of db/db mice;

3. To study the effect of AS-IV treatment on glomerular injury in db/db mice;

4. To study the effect of AS-IV treatment on the improvement of renal tubular injury in db/db mice;

5. To study the effects of AS-IV treatment on Akt and its related signaling pathways in db/db mice;

6. To study the effect of AS-IV treatment on liver function of db/db mice;

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-β1 excretion, astragaloside can also inhibit the activation of Akt/mTOR, NFκB, Erk1/2 signaling pathway, and does not show obvious Hepatotoxicity. According to the above studies, it can be concluded that astragaloside IV has a certain protective effect on type 2 DN, and its mechanism of action is related to the inhibition of Akt/mTOR, NFκB and Erk1/2 signaling pathways. The use of the existing preparation process to prepare astragaloside IV into an oral administration dosage form, an injection administration dosage form, a mucosal administration dosage form or a transdermal administration dosage form, or a tablet, a capsule, a granule, an oral solution, a patch Or a gelling agent that can be used to prevent and treat type 2 diabetic nephropathy.

DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:

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, and 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, and 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, and 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, and 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, and Figure 4(b) shows the effect of astragaloside on small The effect of glomerular vasospasm volume on mouse, Figure 4(c) is the effect of astragaloside on the thickness of mouse glomerular basement membrane, and Figure 4(d) is the effect of astragaloside on the width of mouse foot. As a result, Fig. 4(e) shows the results of PAS staining and electron microscopy showing the effect of astragaloside on glomeruli in mice; among them, **P<0.01, ***P<0.001 compared with the wild type group; Comparison of db groups, ^# P<0.05, ^### P<0.001;

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 5 (d) Immunohistochemical staining showed the effect of astragaloside on the expression of fibronectin in mice; *P<0.05, ***P<0.001 compared with the wild type group;

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, and Figure 6 (b) shows the effect of astragaloside on mouse urine NGAL. The results of the effect, Figure 6 (c) is the effect of astragaloside on the urine TGF-β1 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, and 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, and Figure 7(d) shows the effect of astragaloside on mouse p-NF- The effect of κB p65, 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, and Figure 8 (a) shows the effect of astragaloside on serum. Results of alanine aminotransferase (ALT), Fig. 8(b) shows the effect of astragaloside on serum aspartate aminotransferase (AST) in mice; **P<0.01 compared with the wild type group.

detailed description

The embodiments of the present invention will be specifically described below with reference to the accompanying drawings.

Early 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, NFκB 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. It 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. However, 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.

First, the experimental method

1. 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. Experimental 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.

2. Physiological and metabolic parameters: 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-β-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.

3. 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.

4, light microscopy: paraffin section (4 microns thick) using PAS staining method to evaluate the pathological damage of glomeruli and renal tubules. Image processing analysis was performed using the 4.10 version of NIS-Elements image processing software (Nikon Corporation, Tokyo, Japan). Each slice was measured for 40-50 glomerular tuft area (GTA), 20-30 glomerular system. Membrane substrate area, 80-100 renal tubules and tubular lumen area (length to short axis ratio less than 1.5). The glomerular tuft volume (GTV) is measured by the method invented by Weibel. This measurement requires only the average of the random cross-sectional area of the glomerulus and is calculated according to the following formula: V _G =Area ^1.5 ×β/K, where _VG is the glomerular volume, β=1.38, and K (distribution coefficient) is set to 1.10. The tubular wall area is equal to the area of the renal tubule minus the area of the tubular lumen.

5. Immunohistochemistry: paraffin sections of kidney tissue (4 micron thick), dewaxed, hydrated, and the sections were placed in boiled 10 mM sodium citrate buffer (pH 6) for antigenic repair for 20 minutes, and then cooled to room temperature. After treatment with 3% hydrogen peroxide for 10 minutes, the sections were blocked with goat serum for 30 minutes, followed by addition of fibronectin primary antibody overnight at 4 °C (ab2413, 1:200, Abcam, Cambridge, UK). After rinsing the buffer, 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.

6. ELISA: ELISA test according to the manufacturer's instructions to detect serum insulin (Merck, Germany), urinary albumin (Bethyl, USA), urine TGF-β1 (Daktronics, Shenzhen, China) and urine NGAL (American R&Dsystem) The content.

7. Western blot: The frozen renal cortex tissue was homogenized in lysis buffer, the total protein concentration was balanced, and then separated by SDS-PAGE gel electrophoresis, and the protein was transferred to the PVDF membrane to PVDF. The membrane was placed in TBST buffer containing 0.5 g/l skim milk powder, and reacted at room temperature for 1 hour to block a non-specific site on the membrane, then a primary antibody was added, and the mixture was incubated at 4 ° C overnight. After washing, using ChemiDoc ^TM MP imaging system (Bio-Rad Corporation USA) protein bands were detected and analyzed, the results of using β-actin as an internal control for comparison. p-Akt (#4060) antibody, p-mTOR (#5536) antibody, p-NF-κB p65 (#3033) antibody and p-Erk1/2 (#4370) antibody were purchased from CST Company, USA; β-actin Antibody (A2228) was purchased from Sigma, USA; fibronectin antibody (ab2413) was purchased from Abcam, UK.

8, statistical analysis

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.

Second, the results

1. 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, and 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. At 12 weeks, the glomerular filtration rate of db/db mice was significantly increased. Raise. After 8 weeks of AS-IV intervention, 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.

2. Changes in physiological and metabolic indicators

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.

Table 1 metabolic characteristics (n=8 per group)

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 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, and Figure 3 (b) shows the effect of astragaloside on the kidney weight of mice. Compared with the wild type group, the 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.

3, 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, and Figure 4(b) shows the effect of astragaloside on small The effect of glomerular vasospasm volume on mouse, Figure 4(c) is the effect of astragaloside on the thickness of mouse glomerular basement membrane, and Figure 4(d) is the effect of astragaloside on the width of mouse foot. As a result, Fig. 4(e) shows the effect of astragaloside on the glomerulus of mice by PAS staining and electron microscopy; at 12 weeks, the area and volume of glomerular vasospasm in db/db mice increased, glomerular base The membrane is thickened and the foot is broadly variable. AS-IV intervention can improve this Some changes. PAS staining and electron microscopy showed the characteristics of each group.

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, and 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.

4, AS-IV improves renal tubular injury

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, and Figure 6 (b) shows the effect of astragaloside on mouse urine NGAL. The results of the effect, Figure 6 (c) is the effect of astragaloside on the urine TGF-β1 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, and 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-β1 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-β1 excretion, reduce renal tubular, lumen and wall area, and reduce basement membrane thickness.

5. AS-IV inhibits the activation of p-Akt (Ser473), p-mTOR (Ser2448), p-NF-κB 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, and Figure 7(d) shows the effect of astragaloside on mouse p-NF- The effect of κB p65, 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-κB p65 (Ser536) and p-Erk1/2 The content of (Thr202/Tyr204) was significantly increased, and AS-IV intervention significantly reduced these protein levels.

6. No significant hepatotoxicity was observed after 12 weeks of AS-IV intervention.

Figure 8 shows the effect of astragaloside on liver function in mice. Figure 8(a) shows the effect of astragaloside on ALT, and 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.

The above experimental results show that 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-β1. Astragaloside can also inhibit the activation of Akt/mTOR, NFκB and Erk1/2 signaling pathways, and does not show significant hepatotoxicity. In conclusion, 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.

The embodiments of the present invention have been described above with reference to the drawings, but the present invention is not limited to the specific embodiments described above, and the specific embodiments described above are merely illustrative and not restrictive, and those skilled in the art In the light of the present invention, many forms may be made without departing from the spirit and scope of the invention as claimed.

Claims

The application of astragaloside IV in the preparation of a medicament for preventing and treating type 2 diabetic nephropathy.
The use according to claim 1, wherein the drug is an oral administration form, an injection administration form, a mucosal administration form or a transdermal administration form.
The use according to claim 1, wherein the drug is a tablet, a capsule, a granule, an oral solution, a patch or a gel.