Application of polypeptide in preparation of diabetic nephropathy relieving agent

Abstract

The invention discloses application of polypeptide in preparation of a diabetic nephropathy alleviator, and belongs to the technical field of medicines. The cordyceps sinensis beta-tubulin polypeptide is synthesized by analyzing the structure of the cordyceps sinensis beta-tubulin and selecting amino acid peptide fragments from the 84th site to the 107th site. The apoptosis of the glomerular mesangial cells is induced by using 50 mmol / L of glucose, and meanwhile, the apoptosis of the glomerular mesangial cells induced by high glucose can be relieved by adding the cordyceps sinensis beta-tubulin polypeptide. A treatment test of the cordyceps sinensis beta-tubulin polypeptide on diabetic nephropathy model mice shows that the body mass and fasting blood-glucose levels of the mice in the cordyceps sinensis beta-tubulin polypeptide group are remarkably reduced, the 24-hour urine protein content of the mice is remarkably reduced, and the kidney injury of the mice in the cordyceps sinensis beta-tubulin polypeptide group is remarkably relieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a polypeptide in preparation of a diabetic nephropathy relieving agent. BACKGROUND

[0002] Diabetic nephropathy is one of the most common and most serious microvascular complications of diabetes, which refers to chronic kidney disease caused by diabetes. Its essence is that long-term high blood sugar damages the microvessels and filtration system (glomerulus) of the kidney, leading to decreased kidney filtration function, and eventually possibly developing into kidney failure (uremia). Diabetic nephropathy has become the leading cause of chronic kidney disease and end-stage renal disease (uremia). Long-term high blood sugar can generate a series of biochemical reactions to generate advanced glycation end products, activate harmful pathways, and damage the endothelial cells of glomerular capillaries. In the early stage of diabetes, the glomerulus will be in a "three-high" state of "high perfusion, high filtration, and high pressure". This long-term high load work will accelerate the sclerosis and damage of the glomerulus like an overused sieve. The damage will trigger an inflammatory response, eventually leading to the replacement of normal kidney tissue by useless fibrous scar tissue, and permanent loss of kidney function. More and more studies have pointed out that mesangial cells (MCs) play a core role in maintaining glomerular homeostasis and promoting fibrosis. In the development process of diabetic nephropathy, MCs undergo significant changes, leading to a large amount of matrix production, which is a hallmark feature of diabetic glomerulopathy.

[0003] Currently, the treatment methods for diabetic nephropathy mainly include drug therapy, diet therapy, blood glucose control, etc. However, these methods have certain limitations. With the in-depth study of the pathogenesis of diabetic nephropathy, new treatment targets are emerging. Cordyceps sinensis, as a precious traditional Chinese medicine, has certain application and research support in the management of diabetic nephropathy. The polypeptide substances contained in Cordyceps sinensis may assist in relieving the development process of diabetic nephropathy through multiple mechanisms. Therefore, developing new Cordyceps sinensis polypeptide drugs and preparing diabetic nephropathy relieving agents can provide new drug options for patients. SUMMARY

[0004] The purpose of the present application is to provide application of a polypeptide in preparation of a diabetic nephropathy relieving agent, which belongs to the technical field of medicine.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions. Firstly, the present application provides application of a polypeptide in preparation of a diabetic nephropathy relieving agent, wherein the polypeptide is a Cordyceps sinensis fungus beta-tubulin polypeptide.

[0006] Further, the amino acid sequence of the Cordyceps sinensis fungus beta-tubulin polypeptide is SEQ ID NO. 1.

[0007] Furthermore, the Cordyceps sinensis β-tubulin polypeptide can be used to alleviate high glucose-induced apoptosis of glomerular mesangial cells.

[0008] Furthermore, the Cordyceps sinensis β-tubulin polypeptide can reduce body weight in patients with diabetic nephropathy.

[0009] Furthermore, the Cordyceps sinensis β-tubulin polypeptide can reduce fasting blood glucose levels in diabetic nephropathy.

[0010] Furthermore, the Cordyceps sinensis β-tubulin polypeptide can reduce kidney damage under diabetic nephropathy conditions.

[0011] Secondly, the present invention provides a polypeptide for reducing high glucose-induced apoptosis of glomerular mesangial cells, wherein the polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.1.

[0012] Thirdly, the present invention provides a polypeptide for reducing body weight under diabetic nephropathy conditions, wherein the polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.1.

[0013] Fourth, the present invention provides a polypeptide for reducing fasting blood glucose levels under diabetic nephropathy conditions, wherein the polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.1.

[0014] Fifth, the present invention also provides a polypeptide for reducing kidney damage under diabetic nephropathy conditions, wherein the polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.1.

[0015] The beneficial effects of this invention are as follows: This invention analyzes the structure of Cordyceps sinensis β-tubulin and selects the amino acid peptide from positions 84-107 to synthesize Cordyceps sinensis β-tubulin polypeptide. Inducing glomerular mesangial cell apoptosis with 50 mmol / L glucose, the addition of Cordyceps sinensis β-tubulin polypeptide alleviates high glucose-induced glomerular mesangial cell apoptosis. Treatment trials of Cordyceps sinensis β-tubulin polypeptide in diabetic nephropathy model mice showed that the mice in the Cordyceps sinensis β-tubulin polypeptide group had significantly reduced body weight and fasting blood glucose levels, significantly reduced 24-hour urinary protein content, and significantly reduced kidney damage. The application of Cordyceps sinensis β-tubulin polypeptide in the preparation of diabetic nephropathy allergens provides a new direction for the development of drugs for the treatment of diabetic nephropathy patients. Attached Figure Description

[0016] Figure 1 Mass spectrometry detection of β-tubulin polypeptide synthesis in Cordyceps sinensis; Figure 2 Figure showing the effect of Cordyceps sinensis β-tubulin polypeptide on cell viability; Figure 3 This is a diagram of a glucose-induced apoptosis assay. Figure 4 A diagram illustrating the effect of Cordyceps sinensis β-tubulin polypeptide on inhibiting high glucose-induced apoptosis in glomerular mesangial cells; Figure 5 Figure showing the effect of Cordyceps sinensis β-tubulin polypeptide on body weight of model mice; Figure 6 Figure showing the effect of Cordyceps sinensis β-tubulin polypeptide on fasting blood glucose in diabetic model mice; Figure 7 The figure shows the effect of Cordyceps sinensis β-tubulin peptide on renal tubular interstitial injury indicators in diabetic model mice. Detailed Implementation

[0017] The following is a more detailed description of the present invention, illustrated by examples. It should be understood that these examples are merely illustrative of the invention and are intended to explain the principles and functions of the invention, and are not intended to limit the scope of protection of the invention.

[0018] Example 1: Synthesis of Cordyceps sinensis β-tubulin polypeptide The GenBank accession number for Cordyceps sinensis β-tubulin is AGG17824.1. Structural analysis revealed that the active region of beta-tubulin, specifically the peptide segment containing amino acids 84-107, was selected. Its amino acid sequence is SEQ ID NO. 1. This peptide was synthesized by Nanjing GenScript Biotech Co., Ltd. Figure 1 This is the mass spectrometry detection result of the synthesized peptide.

[0019] Example 2: Assay of Cordyceps sinensis β-tubulin peptide inhibiting high glucose-induced glomerular mesangial cell apoptosis. (1) Cell culture of SV40 MES-13 mouse glomerular mesangial cells: The cells were cultured in DMEM (low glucose) medium containing 10% fetal bovine serum at 37 ℃ in a 5% carbon dioxide incubator. The medium was changed once a day. When the cell density reached 90%, the cells were digested with 0.25% trypsin and passaged.

[0020] (2) Effect of Cordyceps sinensis β-tubulin peptide on cell viability: The control group was cultured in DMEM (low glucose) medium containing 10% fetal bovine serum, and the culture medium was supplemented with 30, 60, 120, 240, 480 and 960 μg / L Cordyceps sinensis β-tubulin peptide, respectively. After 72 h of drug intervention, CCK-8 reagent was added and incubated in an incubator for 2 h. The absorbance (A) value was detected at 450 nm wavelength using an ELISA reader. Each group was set up with 3 replicates. from Figure 2 The results showed that, compared with the control group, SV40 MES-13 cells treated with 30–960 μg / L Cordyceps sinensis β-tubulin peptide for 72 h showed no significant effect on cell viability when the concentration of Cordyceps sinensis β-tubulin peptide was measured by the CCK-8 assay.

[0021] (3) Flow cytometry detection of apoptosis: SV40 MES-13 cells were cultured for 72 h in medium containing 30 and 50 mmol / L glucose, respectively, while a control group was added to low-glucose medium. After discarding the medium, the cells were washed with PBS, digested with trypsin, centrifuged, resuspended in binding buffer, and then 5 μL of Annexin V-kFluor647 and 5 μL of propidium iodide (PI) were added. After mixing, the cells were incubated at room temperature in the dark and analyzed by flow cytometry. Control group, model group, and Cordyceps sinensis β-tubulin peptide (60, 120, 240 μg / L) groups were set up. The control group was added to low-glucose medium, the model group was added to medium containing 50 mmol / L glucose, and each drug group was added to medium containing 50 mmol / L glucose and the corresponding drug. After 72 h of intervention, apoptosis was detected by flow cytometry.

[0022] from Figure 3 The results showed that treating cells with culture media containing 30 and 50 mmol / L glucose for 72 h, respectively, and then detecting cell apoptosis by flow cytometry, both 30 and 50 mmol / L glucose significantly induced apoptosis in SV40 MES-13 cells, with 50 mmol / L glucose inducing apoptosis more effectively.

[0023] from Figure 4 The results showed that when glomerular mesangial cells were induced to apoptosis by 50 mmol / L glucose and treated with different concentrations of Cordyceps sinensis β-tubulin peptide (60, 120, 240 μg / L) for 72 h, the apoptosis rate of each treatment group was significantly reduced compared with the model group, indicating that Cordyceps sinensis β-tubulin peptide can alleviate high glucose-induced apoptosis of glomerular mesangial cells.

[0024] Example 3: Treatment trial of Cordyceps sinensis β-tubulin polypeptide on diabetic nephropathy model mice. (1) Animal grouping and administration: SPF-grade 6-week-old male diabetic nephropathy models C57BL / KsJ db / db mice and db / m mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. After 4 weeks of feeding, db / db mice were randomly divided into model group and Cordyceps sinensis β-tubulin polypeptide groups of 60, 120, and 240 μg / kg according to body weight, with 10 mice in each group. Ten db / m mice were used as the control group. Each drug group was administered the corresponding drug by gavage, while the control group and model group were administered an equal volume of distilled water (5 mL / kg) by gavage once a day for 4 consecutive weeks.

[0025] (2) Biochemical index detection: The body weight of mice in each group was measured weekly. Mice were fasted but allowed free access to water. Blood was collected from the inner canthus of the eye, and fasting blood glucose levels were measured using a blood glucose meter. After the administration of the drug, the mice were placed in metabolic cages and allowed free access to food and water. 24-hour urine was collected, and 24-hour urine protein levels were measured according to the kit instructions.

[0026] from Figure 5 , Figure 6 The results showed that, compared with the model group, the body weight and fasting blood glucose levels of mice in the Cordyceps sinensis β-tubulin polypeptide group were significantly reduced, and the 24-hour urinary protein content of mice was significantly reduced.

[0027] (3) Observation of pathological changes in kidney tissue: Kidney tissue from mice in each group was fixed with 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, cut into 3 μm thick sections, stained with hematoxylin and eosin (HE), and observed under a microscope. Ten fields of view were randomly selected to semi-quantitatively score the indicators of glomerular mesangial dilation and tubulointerstitial injury. Scoring criteria: presence of apoptosis, necrosis, or reduced villi in the renal tubular epithelium (1 point), presence of dilation in the renal tubular lumen (1 point), presence of casts or inflammatory infiltration in the renal interstitium (1 point), presence of congestion in the renal interstitium or glomerular capillary congestion (1 point). 1 point was added for each item, with a total score of 5 points.

[0028] HE staining was performed on kidney tissues from mice in each group, and glomerular sclerosis and renal tubular interstitial collagen deposition were observed under a light microscope. Compared with the control group, the model group mice showed significant dilation of the renal mesangial area and renal tubules, and severe extracellular matrix deposition. Compared with the model group, the Cordyceps sinensis β-tubulin polypeptide group showed significant relief of ECM deposition, interstitial damage, and dilation of the membranous area and renal tubules. Semi-quantitative scoring was performed on indicators of glomerular mesangial dilation and renal tubular interstitial damage. Figure 7 The results showed that the kidney damage in mice treated with Cordyceps sinensis β-tubulin polypeptide was significantly reduced.

Claims

1. The application of a polypeptide in the preparation of a diabetic nephropathy relief agent, characterized in that, The polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the Cordyceps sinensis β-tubulin polypeptide is SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The Cordyceps sinensis β-tubulin polypeptide can be used to reduce high glucose-induced apoptosis of glomerular mesangial cells.

3. The application according to claim 1, characterized in that, The Cordyceps sinensis β-tubulin polypeptide can reduce body weight in diabetic nephropathy.

4. The application according to claim 1, characterized in that, The Cordyceps sinensis β-tubulin polypeptide can reduce fasting blood glucose levels in diabetic nephropathy.

5. The application according to claim 1, characterized in that, The Cordyceps sinensis β-tubulin polypeptide can reduce kidney damage under diabetic nephropathy conditions.

6. A polypeptide for alleviating high glucose-induced apoptosis in glomerular mesangial cells, characterized in that, The polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.

1.

7. A polypeptide for reducing body weight in patients with diabetic nephropathy, characterized in that, The polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.

1.

8. A polypeptide for reducing fasting blood glucose levels in diabetic nephropathy, characterized in that, The polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.

1.

9. A polypeptide for reducing kidney damage in diabetic nephropathy, characterized in that, The polypeptide is Cordyceps sinensis β-tubulin polypeptide, and the amino acid sequence of the polypeptide is SEQ ID NO.1.

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