CN117982520A - A composition and its application - Google Patents

Abstract

The invention relates to a new application of a natural medicine, in particular to a composition and application thereof, namely application of the pharmaceutical composition in preparing medicines for improving or preventing diabetic nephropathy and kidney fibrosis. The cyclocarya paliurus active ingredient can cooperatively balance TGF-beta ₁/BMP 7 pathway through combined application, so that extracellular matrix accumulation of a renal tubule is blocked, the progress of renal fibrosis of diabetic nephropathy is inhibited, and the effect is better than that of a positive medicine dapagliflozin group.

Description

A composition and its application

Technical Field

The present invention relates to natural medicines, in particular to a composition and application thereof, namely, application of asiatic acid and astragaloside, active ingredients of Cyclocarya paliurus, in improving renal fibrosis in diabetic nephropathy by balancing TGF- _β1 /BMP7.

Background technique

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes. The latest epidemiological results show that there are currently 537 million adults aged 20-79 suffering from diabetes. This accounts for 10.5% of the global population in this age group. It is estimated that by 2030, the total number will rise to 643 million (11.3%). The incidence of diabetic nephropathy among diabetic patients worldwide is about 30-40%. At present, DN patients have a high probability of developing end-stage renal disease (ESRD) due to progressive renal fibrosis in clinical practice. Once ESRD develops, patients can only use long-term dialysis or kidney transplantation for treatment, which imposes a heavy burden on their living standards and socioeconomic pressures.

DN progression occurs in several stages. Early changes in the kidneys of diabetic patients include significant glomerular hyperfiltration and hypertrophy, followed by impaired glomerular filtration barrier, increased urinary albumin excretion, mesangial matrix accumulation, hypertrophy, nodular glomerulosclerosis, and tubulointerstitial fibrosis, which are key to the progression of ESRD and renal failure. Renal fibrosis plays a key role in the pathogenesis of DN, and the current clinical treatment of diabetic nephropathy is generally angiotensin enzyme inhibitor antihypertensive drugs such as captopril, or metformin and other hypoglycemic drugs, these drugs can not effectively improve or block the development of renal fibrosis, the treatment of diabetic nephropathy has obvious limitations. Therefore, the development of drugs with significant effects on renal fibrosis is a feasible treatment strategy to improve diabetic nephropathy.

Asiatic acid (AA) is a pentacyclic triterpenoid compound with an ursane skeleton isolated from Centella asiatica of the Umbelliferae family. Since the discovery of the ability of asiatic acid to treat skin trauma in 1971, a large number of studies have been conducted on it and it has been found that asiatic acid also has a variety of pharmacological activities such as antibacterial, anti-tumor, anti-inflammatory, liver protection, improvement of neurocognitive disorders, and hypoglycemic. In particular, its anti-tumor activity has been widely studied. It has been found that asiatic acid can effectively inhibit the proliferation of various cancer cells including liver cancer, breast cancer, human tongue squamous cell carcinoma, ovarian cancer, and melanoma.

Astragalus glycoside is a natural flavonoid compound widely found in medicinal plants. It has anti-inflammatory, antioxidant, cardiotonic, analgesic, antibacterial, anti-allergic, and anti-hepatotoxic effects. It can also enhance the body's resistance, stimulate the production of interferon, anti-arrhythmia, dilate blood vessels, and protect the myocardium.

Therefore, more effective treatment strategies are urgently needed to prevent the occurrence and development of DN. Different from the single target of synthetic small molecule drugs, traditional Chinese medicine and natural products have the advantages of multi-target and low toxicity, and have multi-pathway synergistic effects on the complex lesions of metabolic diseases, which may be a better choice to alleviate the progression of DN.

Summary of the invention

In order to solve the problem that the current clinical treatment of renal fibrosis in patients with diabetic nephropathy is ineffective, the present invention provides an active ingredient of Cyclocarya paliurus for use in combination with Asiatic Acid (AA) and Astragalin (AG) for preparing a drug for improving or preventing renal fibrosis in diabetic nephropathy.

In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: The present invention discloses a new use of Cyclocarya paliurus, and the present invention first finds that the active ingredients of Cyclocarya paliurus have a synergistic effect on the combination of asiatic acid and astragalus glycosides in improving renal fibrosis in diabetic nephropathy. Among them, astragalus glycosides have not been reported in the literature on renal fibrosis in nephropathy.

A use of a pharmaceutical composition in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy, characterized in that the pharmaceutical composition comprises asiatic acid and astragaloside.

The application is characterized in that the pharmaceutical composition is composed of asiatic acid, astragalus glycoside and pharmaceutically acceptable excipients.

A Cyclocarya paliurus ethanol extract, characterized in that it is prepared by the following method:

Take 49.59 kg of dried Cyclocarya paliurus leaves, crush them, add 80% ethanol at a solid-liquid ratio of 1:6, and extract them by immersion method for 3 times, each time for 7 days. After the extracts are combined, they are concentrated under reduced pressure until there is no alcohol taste, so as to obtain Cyclocarya paliurus alcohol extract.

The Cyclocarya paliurus ethanol extract is characterized in that its active ingredients are asiatic acid and astragaloside.

The Cyclocarya paliurus ethanol extract is characterized in that the mass ratio of the asiatic acid to astragaloside is 1:1.

The application of the Cyclocarya paliurus ethanol extract in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy.

Beneficial effects:

1. The present invention verifies that the ethanol extract of Cyclocarya paliurus (whose active ingredients are asiatic acid and astragaloside) and a mixture of asiatic acid and astragaloside monomer compounds can synergistically balance the TGF-β/BMP7 pathway through combined use, block the accumulation of extracellular matrix in renal tubular cells, and inhibit the progression of renal fibrosis in diabetic nephropathy, and the effect is better than that of the positive drug dapagliflozin group.

2. The active ingredients of Cyclocarya paliurus, Centella asiatica and Astragalus glycosides, are used in combination to prepare drugs for improving or preventing renal fibrosis in diabetic nephropathy.

3. As a new food resource in my country, Cyclocarya paliurus has a long history of application and is highly safe.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: HPLC profiles of Cyclocarya paliurus total ethanol extract (CPE), AA and AG.

Figure 2: Changes in body weight (A) and blood glucose (B) in mice with diabetic nephropathy induced by AA and AG intervention.

Figure 3: A is the change of urinary albumin in diabetic nephropathy mice after AA and AG intervention; B is the change of urea nitrogen; C is the change of creatinine.

Figure 4: Immunohistochemical results of AA and AG on the morphological changes of kidney tissue in diabetic mice.

Figure 5: Schematic diagram of Masson staining of collagen deposition and fibronectin immunohistochemistry of AA and AG on the kidneys of diabetic mice.

Figure 6: The change in the survival ratio of renal tubular epithelial cells induced by advanced glycation end products (AGEs) by AA and AG in Example 2, wherein A is AA, B is AG, and C is AA:AG=1:1.

Figure 7: Schematic diagram of the ELISA results of AA and AG on fibronectin of renal tubular epithelial cells induced by advanced glycation end products (AGEs) in Example 2, wherein A is AA, B is AG, C is AA:AG=1:1, D is Fa, E is Log (Fa/Fu), F is CI, and G is Log (CI).

Figure 8: Western-blot diagram of cell protein blotting method-immunoblotting experiment (Western-blot) in Example 2; A is a protein blotting diagram; B is a protein grayscale integral diagram.

Figure 9: Western-blotting of mouse kidney tissue in Example 3. A is an extracellular matrix protein imprint; B is an extracellular matrix protein grayscale integral map; C is a TGF-β ₁ /BMP7/Smads pathway protein imprint; DF are TGF-β ₁ /BMP7/Smads pathway protein grayscale integral maps, specifically D is a BMP7, TGF-β ₁ grayscale integral map, E is a Smad1, Smad3 and Smad4 grayscale integral map, and F is a TIMP1 and TIMP2 grayscale integral map.

Figure 10: Co-inmunoprecipitation Western-blotting of the kidney tissue of mice in Example 3. A is a co-immunoprecipitation Western-blotting image; B is a co-immunoprecipitation positive control Western-blotting image; C is a grayscale integral image of Smad4 protein binding, D is a grayscale integral image of Smad4 binding Smad3 protein, and E is a grayscale integral image of Smad4 binding Smad1 protein.

Detailed ways

The essential contents of the present invention are described in detail below with reference to the accompanying drawings and examples. However, the present invention is not limited to the following embodiments. The experimental methods in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are available from public commercial channels unless otherwise specified.

Asiatic acid and astragalus glycosides can synergistically improve renal fibrosis and reduce extracellular matrix accumulation in diabetic nephropathy.

In order to find out the mechanism by which asiatic acid and astragalus glycosides can improve renal fibrosis in diabetic nephropathy, in vitro cell and in vivo animal experiments were carried out, and the expression of TGF-β ₁ /BMP7 pathway proteins, a key pathway of fibrosis, was detected. Western Blotting and immunoprecipitation experiments were used to verify that asiatic acid and astragalus glycosides can improve renal fibrosis in diabetic nephropathy by synergistically balancing the TGF-β ₁ /BMP7 pathway.

In a preferred embodiment of the present invention, the experimental animals are SPF-grade C57BL/6J mice, and the kidney cells are renal tubular epithelial cells HK-2. The experimental results of the specific implementation method show that at the animal level, asiatic acid and astragalus glycosides can synergistically improve the urine albumin, creatinine, and urea nitrogen levels of C57BL/6J mice stimulated by streptozotocin (STZ), and restore the imbalance of the TGF- _β1 /BMP7 pathway, thereby reducing the accumulation of extracellular matrix and improving renal fibrosis in diabetic nephropathy. At the cellular level, asiatic acid and astragalus glycosides can synergistically improve the accumulation of extracellular matrix in renal tubular cells induced by AGEs, thereby achieving the purpose of improving renal fibrosis in diabetic nephropathy.

Example 1 Asiatic acid and astragaloside can synergistically improve renal function damage in diabetic nephropathy mice

A total of 75 SPF male C57BL/6J mice weighing 21±3.2g were selected. After adaptive feeding for 1 week, they were fasted for 12h. STZ was dissolved in 0.1mmol·L ^-1 citric acid buffer (pH=4.2) and injected intraperitoneally into 60 random mice at a dose of 55mg·kg ^-1 ·d ^-1 (10mL·kg ^-1 ·d ^-1) for 5 consecutive days. The remaining 15 mice were injected with citric acid buffer at the same dose. Blood glucose levels were measured one week later, and the blood glucose levels were all greater than 16.7mmol·L ^-1 . Then the 60 mice were randomly divided into 5 groups, with 15 mice in each group, namely model group (STZ group), asiatic acid group (AA group), astragaloside group (AG group), asiatic acid and astragaloside combined application group (AA+AG group), Cyclocarya paliurus ethanol extract group (CPE group), and positive drug dapagliflozin group (DAPA group). Each group was intragastrically administered with CMC-Na solution, 40mg·kg ^-1 ·d ^-1 asiatic acid, 40mg·kg ^-1 ·d ^-1 astragaloside, 40mg·kg ^-1 ·d ^-1 asiatic acid and 40mg·kg ^-1 ·d ^-1 astragaloside mixture, 240mg·kg ^-1 ·d ^-1 Cyclocarya paliurus ethanol extract, 5mg·kg ^-1 ·d ^-1 dapagliflozin, the intervention lasted for 18 weeks, body weight was measured once a week, fasting blood sugar was measured every three weeks, after the experiment, the mice were killed, serum was collected, blood sugar, urine albumin, creatinine, urea nitrogen levels were detected. Kidney tissue was taken, and HE staining was performed to observe the morphological changes of kidney tissue.

Kidney tissues of mice in each group were obtained, fixed with 4% paraformaldehyde fixative for 48 h, then embedded in paraffin, cut into 5 μm thick sections, subjected to Masson staining and fibronectin immunohistochemical staining, and then observed and photographed under an optical upright microscope.

Results: It was found that AA+AG can play a synergistic role in protecting the kidneys. The results of the effects on body weight and blood sugar are shown in Figure 2. AA+AG can synergistically reduce the body weight of diabetic nephropathy mouse models, and AG can improve the blood sugar of diabetic nephropathy mouse models, while AA cannot. The CPE group has a better hypoglycemic effect than AA+AG. As shown in Figure 1, the ethanol extract group of Cyperus paliurus contains other ingredients in addition to AA and AG, and its dosage is also high (240 mg·kg-1·d-1); the results of the effects on urine albumin, urea nitrogen, and creatinine are shown in Figure 3. AA+AG can synergistically improve the renal function damage of diabetic nephropathy mouse models. The effects on the morphological changes of mouse kidney tissue are shown in Figure 4. AA+AG can synergistically improve tubular atrophy (blue arrows) and cell shedding (red arrows) as well as glomerular sclerosis (black arrows). The effects on the morphological changes of mouse kidney tissue are shown in Figure 5. AA+AG can synergistically improve the collagen deposition and fibronectin deposition in the kidneys of diabetic nephropathy mice.

Example 2 AA+AG can synergistically improve the accumulation of extracellular matrix in renal tubular epithelial cells

The human renal tubular epithelial cells (HK-2) were divided into 5 groups, each group had three parallel wells, and 5*10 ³ cells were inoculated into 96-well plates in each well, and cultured with DMEM high-glucose medium containing 10% fetal bovine serum in a 37°C incubator with 5% CO ₂ ; after inoculation, the three groups of cells were treated as follows: Group 1 was a blank group, Group 2 was a high-glucose group, and the AGEs concentration was 800 mg/mL, Groups 3 and 4 were AA group (20 μM) and AG group (20 μM), respectively, and the AGEs concentration was 800 mg/mL, and Group 5 was a combined application group of AA and AG (AA+AG=10 μM+10 μM), and all five groups were treated for 72 hours. After the above five groups of cells were treated as described above, the culture medium was discarded, thiazolyl blue (MTT) was added, and DMSO was added 4 hours later to perform the MTT experiment, and the absorbance at 490 nm was measured by an enzyme marker to calculate the cell viability, and the results are shown in Figure 6. At the same time, HK-2 cells were inoculated into a 48-well plate at a density of 10*10 ⁴ cells per well. After the above 4 groups of cells were treated, the culture medium was discarded, and the cell samples were scraped and washed with PBS for 3 times. After that, the cells were repeatedly frozen and thawed for 3 times at -80°C for lysis and detected by ELISA. The protein was quantified by the BCA method and the results were normalized. The results are shown in Figure 7.

Results: The synergistic effect index CI in Figures 6 and 7, especially Figures 7F-G, indicated that AA+AG produced a synergistic effect, which improved AGEs-induced extracellular matrix accumulation in renal tubular epithelial cells.

After sampling, the above five groups of cells were added with RIPA lysis buffer containing protease inhibitors, ground with a tissue grinder, lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the supernatant protein samples were taken. The protein concentration was determined by the BCA method, and Western Blotting experiments were performed. The protein samples were separated using 8% SDS-PAGE and transferred to nitrocellulose (NC) membranes. At room temperature, the membrane was placed in 5% BSA configured in TBST buffer for 1 hour. Then, the membrane was incubated with the first antibody extracellular matrix protein at 4°C overnight. The next day, the membrane was incubated with the relevant second antibody at room temperature for 1 hour, and the protein expression was detected by luminescence of the ECL solution, where GAPDH was used as the normalized internal reference protein.

Results: Figure 8 shows the ratio of extracellular matrix proteins to internal reference proteins in Western Blotting animals. Fibronectin, Laminin 1, Collagen I and Collagen IV were significantly increased in the AGEs group, while the AA group, AG group and AA+AG group all had a significant decrease, and the combined effect was better than AA and AG. The above shows that the combination of AA and AG has a synergistic improvement effect on the accumulation of extracellular matrix in diabetic renal tubular cells.

Example 3 Synergistic balancing effect of AA+AG on TGF-β ₁ /BMP7/Smads pathway

Example 1 The kidney tissue was washed and chopped, 20 mg of tissue was weighed and added to the RIPA lysis solution containing protease inhibitors, and after grinding with a tissue grinder, it was lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the protein sample of the supernatant was taken. The protein concentration was determined by the BCA method, and a Western Blotting experiment was performed. The protein sample was separated using 8% SDS-PAGE and transferred to a nitrocellulose (NC) membrane. At room temperature, the membrane was placed in 5% BSA configured in TBST buffer and blocked for 1 hour. Then, the membrane was incubated with the first antibody extracellular matrix protein and TGF-β ₁ /BMP7/Smads pathway protein at 4 ° C overnight. The next day, the membrane was incubated with the relevant second antibody at room temperature for 1 hour, and the expression of the protein was detected by luminescence of the ECL solution, wherein GAPDH was used as a normalized internal reference protein.

FIG9 shows the ratio of extracellular matrix proteins and TGF-β ₁ /BMP7/Smads pathway proteins to internal reference proteins in Western Blotting animals. Western Bolting results show that AA and AG can significantly improve the accumulation of extracellular matrix in the kidneys of DN mice, whether used alone or in combination. The effect of CPE on renal fibrosis in DN mice is not much different from that of AA and AG combined, indicating that AA and AG are the main synergistic effectors of CPE in treating renal fibrosis in DN mice. The expression of TGF-β ₁ in DN mice was significantly increased, and the combination of AA and AG or single use can reduce the expression of TGF-β ₁ , but the inhibitory effect of AA+AG combination is significantly better than that of single use. AG can significantly increase the expression of BMP7, while AA can inhibit the expression of BMP7 by exciting Smad7. The combination of AA+AG can simultaneously increase the expression of Smad7 and BMP7, thereby playing a better role in reducing TGF-β _1. The stimulating effect of CPE on BMP7 and Smad7 is basically the same as that of AA+AG combined, while DAPA has basically no stimulating effect on them. The expression of Smad1 and Smad3 was basically unaffected by drug administration, while the expression of Smad4 was reduced in DN mice, indicating that there was a significant imbalance in the balance of the TGF family in DN mice, and drug administration could restore this balance.

The kidney tissue in Example 1 was washed and chopped, 20 mg of tissue was weighed and added to RIPA lysis buffer containing protease inhibitors, ground with a tissue grinder, lysed on ice for 30 min, centrifuged at 12,000 rpm for 15 min, and the protein sample of the supernatant was taken. The protein concentration was determined by the BCA method, normalized to a uniform concentration, and protein quantification was performed using the EnhancedBCAProteinAssay Kit. An equal amount of protein (1 mg) was taken and immunoprecipitated using the rProtein A/G Magnetic IP/Co-IP Kit, mouse Smad1, Smad3 and Smad4. After denaturation, rabbit Smad1, Smad3 and Smad4 were used for Western Bolting detection.

Figure 10 shows the binding of Smad2/3, Smad1/5/8 and Smad4 pathway proteins in animal kidney tissue by co-inmunoprecipitation Western Blotting. The results show that the binding of Smad3 and Smad4 in DN mice increased significantly, while drug administration significantly reduced their binding, and AA had a better inhibitory effect on their binding than AG, and the combined use was better than AA alone. The binding rate of Smad1 and Smad4 in the kidney of DN mice decreased, and the administration of AG significantly increased their binding, while AA could not, and the effect of the combined group was slightly lower than that of the AG alone group. The above results indicate that the combination of AA and AG can synergistically restore the balance of the TGF-β ₁ /BMP7/Smads pathway.

Obviously, the purpose of the above embodiments is to clearly and specifically illustrate the content of the present invention, and it is not to limit the implementation mode. For those skilled in the art to which the present invention belongs, other different forms of changes can be made on the basis of the content of the present invention. The present invention does not need to and cannot exhaustively list all the implementation modes. Therefore, the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (7)

1. Use of a pharmaceutical composition in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy, characterized in that the pharmaceutical composition comprises asiatic acid and astragaloside. 2. The use according to claim 1, characterized in that the mass ratio of asiatic acid to astragaloside is 1:1. 3. The use according to claim 1, characterized in that the pharmaceutical composition is composed of asiatic acid, astragaloside and pharmaceutically acceptable excipients. 4. A Cyclocarya paliurus ethanol extract, characterized in that it is prepared by the following steps: 49.59 kg of dried Cyclocarya paliurus leaves were taken, crushed, added with 80% ethanol at a solid-liquid ratio of 1:6, extracted three times by immersion method, and the extracts were combined and concentrated under reduced pressure until there was no alcohol taste, thereby obtaining Cyclocarya paliurus alcohol extract. 5. The Cyclocarya paliurus ethanol extract according to claim 4, characterized in that the active ingredients thereof are asiatic acid and astragaloside. 6. The Cyclocarya paliurus ethanol extract according to claim 5, characterized in that the mass ratio of asiatic acid to astragaloside is 1:1. 7. Use of the Cyclocarya paliurus ethanol extract according to any one of claims 4 to 6 in the preparation of a drug for improving or preventing renal fibrosis in diabetic nephropathy.