Inhibitor in diabetic nephropathy medicine, application of inhibitor and medicine

Abstract

The invention provides an inhibitor in a diabetic nephropathy drug, application of the inhibitor and the drug, and belongs to the field of pharmaceutical preparations, the inhibitor is an HDAC8 inhibitor, and administration of the HDAC8 inhibitor improves pathological structure changes of renal tubules caused by diabetes and inhibits the fibrosis process of the renal tubules at the same time; glycogen deposition is improved to a certain degree after administration, then renal tubular epithelial cell apoptosis is relieved, the treatment effect is achieved, and a new medicine and a new treatment mode are provided for treatment of diabetic nephropathy.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to an inhibitor in a diabetic nephropathy drug, and also relates to the application of the inhibitor and a drug containing the inhibitor. Background Art

[0002] Diabetes mellitus (DM) is a common metabolic disease, and hyperglycemia is its main clinical feature. Chronic hyperglycemia can induce multiple tissue damage, especially microvascular tissue. Diabetic nephropathy (DKD) is the main microvascular complication of DM and the main cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) worldwide. The occurrence of DKD can further increase the incidence and mortality of cardiovascular diseases complicated by DM, and is also one of the main reasons for the shortened life expectancy of DM patients. The early pathological manifestations of DKD are glomerular hypertrophy, basement membrane thickening, and extracellular matrix deposition in the mesangial region. In the later stage, it develops into glomerular sclerosis and tubulointerstitial damage, and finally develops into irreversible renal failure.

[0003] Traditional treatment strategies for DKD include regulating the angiotensin-aldosterone system, lowering blood sugar levels and reducing weight, such as the invention patent with patent number: CN201510596981.5. However, even if blood sugar and blood pressure are effectively controlled, 30%-40% of DM patients will eventually develop DKD. It can be seen that the existing treatment methods cannot effectively reduce the risk of death from DKD, and the lack of early effective intervention measures has become a bottleneck problem in the treatment of DKD. Therefore, it is of great significance to find a treatment method to delay the occurrence and development of DKD.

[0004] Histone deacetylase (HDACs) is a class of proteases and also an important class of cytokines, playing a key role in many biological processes, such as cell cycle regulation, apoptosis regulation, DNA repair and tumor-related regulation. Based on structural and functional homology, the 18 members of the eukaryotic HDACs family can be divided into four categories, among which class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8) are widely studied. Summary of the invention

[0005] Based on the above problems in the prior art, the present invention provides an inhibitor in a diabetic nephropathy drug, wherein the inhibitor is an HDAC8 inhibitor.

[0006] Preferably, the HDAC8 inhibitor includes one or more of an inhibitor that inhibits HDAC8 activity and an inhibitor that inhibits HDAC8 synthesis.

[0007] Preferably, the HDAC8 inhibitor is PCI-34051.

[0008] Preferably, the molecular structure of PCI-34051 is:

[0009]

[0010] The present invention also discloses a use of the inhibitor in the diabetic nephropathy drug as described above in preparing a therapeutic drug for diabetic nephropathy.

[0011] The present invention also discloses a use of the inhibitor in the diabetic nephropathy drug as described above in preparing a therapeutic kit for diabetic nephropathy.

[0012] The present invention also provides a drug for treating diabetic nephropathy, which comprises the inhibitor as described above.

[0013] Preferably, the drug is in solid powder dosage form.

[0014] Preferably, the inhibitor in the diabetic nephropathy drug is the only active ingredient of the drug.

[0015] The beneficial effects of the present invention are: the administration of HDAC8 inhibitors improves the pathological structural changes of the renal tubules caused by diabetes, and at the same time inhibits the fibrosis process of the renal tubules; glycogen deposition is improved to a certain extent after administration, and then the apoptosis of renal tubular epithelial cells is alleviated, thereby achieving a therapeutic effect, and providing a new drug and treatment method for the treatment of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the results of apoptosis rate detection in three groups of HK-2 cells: NG, HG, and HG+PCI-34051.

[0017] Figure 2 The graph shows the expression of BAX and Bcl-2 in three groups of HK-2 cells: NG, HG, and HG+PCI-34051.

[0018] Figure 3 This is a graph showing the expression of HDAC8 in the kidneys of three groups of mice: db / m, db / db, and db / db+PCI-34051;

[0019] Figure 4 This is a graph showing the changes in various biochemical indicators of the three groups of mice: db / m, db / db, and db / db+PCI-34051.

[0020] A is the body weight of the three groups of mice; B is fasting blood glucose (FBG); C is serum creatinine (Serum creatinine); D is blood ureanitrogen (BUN); and E is urine microalbumin (MALB). ## p<0.01vs db / m; *p<0.05vs db / db; **p<0.01vs db / db.

[0021] Figure 5 The hematoxylin and eosin of the renal tubules of the three groups of db / m, db / db and db / db+PCI-34051 mice

[0022] (H&E) staining, MASSON staining and Sirius Red staining results;

[0023] Figure 6 This is the PAS staining result of renal tubules of three groups of mice: db / m, db / db, and db / db+PCI-34051;

[0024] Figure 7 These are the results of immunohistofluorescence staining of tubular apoptosis-related proteins BAX and BCL-2 in three groups of mice: db / m, db / db, and db / db+PCI-34051.

[0025] in Figure 1-3 Figures 5-7 are flow cytometry detection images or staining experiment result images. The colors in the images are part of the results. Color images are used to clearly and accurately display the results. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1: An inhibitor in a diabetic nephropathy drug.

[0028] An inhibitor in a diabetic nephropathy drug, wherein the inhibitor is an HDAC8 inhibitor, including an inhibitor that inhibits HDAC8 activity or an inhibitor that inhibits HDAC8 synthesis; the HDAC8 inhibitor is preferably PCI-34051, and the molecular structure is:

[0029]

[0030] The inhibitor in the above-mentioned diabetic nephropathy drug can be used in the preparation of a therapeutic drug for diabetic nephropathy, and can also be used in the preparation of a therapeutic kit for diabetic nephropathy.

[0031] Embodiment 2: A drug for treating diabetic nephropathy.

[0032] A drug for treating diabetic nephropathy, comprising the inhibitor in the drug for treating diabetic nephropathy as described in Example 1, wherein the drug is in a solid powder dosage form, and the inhibitor in the drug for treating diabetic nephropathy is the only active ingredient of the drug.

[0033] Next, we will study the role of HDAC8 inhibitors in DKD from the cellular and animal levels.

[0034] At the cellular level, the apoptosis rates of HK-2 cells in the three groups of NG, HG, and HG+PCI-34051 were first detected by a kit. The results showed that HDAC8 inhibitors could inhibit the apoptosis level of HK-2 cells under high glucose conditions. Then, the expression of apoptotic proteins BAX and Bcl-2 in HK-2 cells was detected by immunofluorescence. The results showed that the administration of PCI-34051 could downregulate the apoptosis level of HK-2 cells cultured in HG.

[0035] At the animal level, immunohistofluorescence staining was performed on the kidney tissues of three groups of mice, db / m, db / db, and db / db+PCI-34051. The results showed that HDAC8 was upregulated in the renal parenchyma of db / db mice, and its expression was downregulated after administration of the HDAC8 inhibitor (PCI-34051). Subsequently, the pathological changes of the renal tubules of each group of mice were observed by H&E staining, MASSON staining, and Sirius Red staining. The experimental results showed that the administration of PCI-34051 improved the pathological structural changes of the renal tubules caused by diabetes and inhibited the fibrosis process of the renal tubules. Then, PAS staining was used to explore the renal tubular glycogen deposition of each group of mice. The results showed that the renal tubular glycogen deposition of db / db mice was improved to a certain extent after administration. Finally, the staining results of apoptosis-related proteins BCL-2 and BAX showed that PCI-34051 alleviated the apoptosis of renal tubular epithelial cells in db / db mice. The detailed research process is as follows:

[0036] Research example 1: Apoptosis rate of HK-2 cells in three groups: NG, HG and HG+PCI-34051.

[0037] Reagents used:

[0038] AnnexinV-mCherry apoptosis detection kit was purchased from Bio-Technology Co., Ltd.;

[0039] Cell-based culture:

[0040] Human renal tubular epithelial cells (HK-2 cells) were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; HK-2 cells were cultured in a 37°C, 5% CO2 incubator using DMEM BASIC medium containing 10% FBS.

[0041] Cell model construction:

[0042] In order to simulate the internal environment of DKD renal tubules, the following cell model was constructed using glucose. The cells were divided into three groups, including

[0043] NG group: HK-2 cells cultured in 5.56mmol / l normal DMEM medium;

[0044] HG group: HK-2 cells cultured in 5.56mmol / L DMEM medium (where the sugar concentration was 60mmol / L);

[0045] HG+PCI-34051 group: HK-2 cells cultured in 5.56 mmol / L DMEM medium (wherein the sugar concentration was 60 mmol / L) were co-cultured with 10 μM PCI-34051 for 48 h.

[0046] Specific experimental steps:

[0047] (1) After the HK-2 cell model was successfully established, the cells were digested and collected using EDTA-free trypsin.

[0048] (2) Wash the cells with PBS, count them, and collect 1-5x10 5 cells.

[0049] (3) Add 500 μl of Binding Buffer and gently blow to form a single-cell suspension.

[0050] (4) Add 5 μl of AnnexinV-EGFP and mix well, then add 5 μl of Propidium Iodide and mix well.

[0051] (5) Incubate at room temperature, away from light, for 10 minutes.

[0052] (6) Perform flow cytometric observation and detection within 1 hour.

[0053] The experimental results are as follows Figure 1 As shown, compared with the NG group, the apoptosis rate of the HG group was significantly increased, and the apoptosis rate was decreased after the administration of the inhibitor PCI-34051.

[0054] Conclusion: PCI-34051 administration can down-regulate the apoptosis rate of HK-2 cells cultured in HG.

[0055] Study 2: Changes in the expression of BCL-2 and BAX proteins in the three groups of HK-2 cells: NG, HG, and HG+PCI-34051.

[0056] Reagents used:

[0057] DAPI staining solution, anti-fluorescence quenching sealing solution, Western primary antibody diluent, Western secondary antibody diluent, BCL-2 and BAX antibodies were all purchased from Proteintech, USA; pepsin was purchased from Beyotime Biotechnology Co., Ltd.; immunohistochemistry reagents were purchased from Beijing Zhongshan Jinqiao Co., Ltd.; goat anti-rabbit near-infrared fluorescent secondary antibody was purchased from Xuzhou Micro-Comed Bioengineering Co., Ltd.

[0058] The basic cell culture was the same as in Research Example 1.

[0059] The construction of the cell model was the same as that of Research Example 1.

[0060] Immunohistofluorescence was used to detect the protein expression level of HDAC8 in mouse kidney tissue samples. Specific experimental steps:

[0061] (1) HK-2 cells were cultured on glass coverslips for 48 hours. When the cells grew to 80-90%, they were removed and rinsed three times with PBS for three minutes each time. They were fixed with paraformaldehyde at room temperature for 10 minutes, rinsed with PBS, penetrated with 0.1% Triton solution for 15 minutes, and rinsed with PBS for 3 minutes. 2% BSA was added to block the cells, and the cells were blocked at room temperature for 30 minutes. Excess liquid on the tissue was carefully wiped off with qualitative filter paper.

[0062] (2) Primary antibody application: Antibodies (BCL-2, 1:200; BAX, 1:200) were diluted and added dropwise to the kidney tissue and kept in a refrigerator at 4°C overnight.

[0063] (3) The next day, the tissue sections were placed in a 37°C oven to warm up for 30 min and washed with PBS for 3 min, for a total of 3 washes.

[0064] (4) Add Dylight 594 or Dylight 488 (1:200) dropwise, incubate at room temperature in the dark for 1 h, wash with PBS for 3 min, and wash three times in total.

[0065] (5) Stain with DAPI for 15 min, wash with PBS buffer for 3 min, and wash three times in total.

[0066] (6) Apply a drop of fluorescence quencher and seal the slide.

[0067] The experimental results are as follows Figure 2 As shown, compared with the NG group, the BAX expression of HK-2 cells in the HG group was significantly upregulated, and its expression level decreased after PCI-34051 administration; the trend of Bcl-2 was opposite to that of BAX, that is, PCI-34051 administration blocked the HG-induced downregulation of Bcl-2 expression.

[0068] Conclusion: PCI-34051 administration inhibits HG-induced apoptosis in HK-2 cells.

[0069] Research example three: Expression of HDAC8 in the renal parenchyma of three groups of mice: db / m, db / db, and db / db+PCI-34051.

[0070] Reagents used:

[0071] DAPI staining solution, anti-fluorescence quenching sealing solution, Western primary antibody diluent, Western secondary antibody diluent, and HDAC8 antibody were purchased from Proteintech, USA; pepsin was purchased from Biotech Biotechnology Co., Ltd.; immunohistochemistry reagents were purchased from Beijing Zhongshan Jinqiao Co., Ltd.; goat anti-rabbit near-infrared fluorescent secondary antibody was purchased from Xuzhou Micro-Comed Bioengineering Co., Ltd.

[0072] Animal model construction:

[0073] 11-week-old db / m mice and 11-week-old db / db mice were selected as research subjects, and the experimental groups were db / m group, db / db group and db / db+PCI-34051 group (n=6). After one week of adaptive feeding, the mice in the db / m group and db / db group were not injected, and the mice in the db / db+PCI-34051 group were intraperitoneally injected with PCI-34051 (10 mg / kg). The preparation method of the injection drug was as follows: taking 10 mL of the drug as an example, 1 mL of 25.0 mg / mL clear DMSO stock solution was added to 400 μL PEG300 and mixed evenly; 500 μL Tween-80 was added to the above system and mixed evenly; then 4.5 mL of normal saline was added to make the volume 10 mL, and finally 10 mg PCI-34051 was added to prepare the final drug, and the drug was administered according to the weight of the mouse, and the dosage standard was 10 mg / kg.

[0074] The drug was administered for four consecutive weeks, and the weight, fasting blood glucose (FBG), blood urea nitrogen (BUN), serum creatinine, and urine microalbumin (MALB) and other biochemical indicators of the mice were measured and recorded. After four weeks of administration, the mice were killed, and the kidney tissues of the mice were removed and placed in paraformaldehyde fixative for later use. This experiment has passed the ethical review of the Animal Center of Xuzhou Medical University.

[0075] Immunohistofluorescence detection of HDAC8 protein expression in mouse kidney tissue samples:

[0076] (1) Kidney tissue was removed from the paraformaldehyde fixative and dehydrated and dewaxed using an automatic dehydrator. The steps were as follows: soak in 75% ethanol for 1 hour, soak in 80% ethanol for 1 hour, soak in 90% ethanol for 1 hour, soak in 95% ethanol for 1 hour, soak in 100% ethanol for 1 hour, soak in xylene for 1 hour, then soak in xylene for another 1 hour, soak in paraffin for 1 hour, then soak in paraffin for another 1 hour. After the operation, the tissue was embedded in paraffin, cooled on ice, and sliced ​​(thickness: 4 μm) for later use.

[0077] (2) Add 0.4% pepsin to the kidney tissue wax block, repair at 37°C for 30 min, and wash with PBS for 3 min, for a total of 3 washes.

[0078] (3) Reagent I (endogenous peroxidase blocker) was then added to the kidney tissue wax block, reacted at room temperature for 10 min, and washed with PBS for 3 min, for a total of 3 washes.

[0079] (4) Add 2% BSA to block the kidney tissue and incubate at room temperature for 30 min. Use qualitative filter paper to carefully wipe off excess liquid on the tissue.

[0080] (5) Primary antibody application: The diluted antibody (HDAC8, 1:200) was added dropwise to the kidney tissue and stored in a 4°C refrigerator overnight.

[0081] (6) The next day, the tissue sections were placed in a 37°C oven to warm up for 30 min and washed with PBS for 3 min, for a total of 3 washes.

[0082] (7) Add Dylight 594 or Dylight 488 (1:200), incubate at room temperature in the dark for 1 h, wash with PBS for 3 min, and wash three times in total.

[0083] (8) Stain with DAPI for 5 min, wash with PBS buffer for 3 min, and wash three times in total.

[0084] (9) Apply a drop of fluorescence quencher and seal the slide.

[0085] The experimental results are as follows Figure 3 As shown, the expression of HDAC8 in the renal tubules of db / db mice was significantly upregulated compared with that in the db / m group, and its expression level was decreased after administration of PCI-34051.

[0086] Conclusion: PCI-34051 can down-regulate the protein expression of HDAC8 in the renal tubules of db / db mice.

[0087] Research Example 4: Testing the body weight, FBG and renal function-related indicators of three groups of mice: db / m, db / db and db / db+PCI-34051.

[0088] Reagents used

[0089] Blood glucose meter was purchased from Tianjin Jiuan Medical Electronics Co., Ltd., Cr and BUN test kits were purchased from Nanjing Jiancheng Bioengineering Institute; MALB test kit was purchased from Shanghai Lanpai Biotechnology Co., Ltd.

[0090] The animal model construction was the same as that in Study 3.

[0091] Specific experimental operation steps: perform operations and calculations according to the instructions of the kit.

[0092] The experimental results are as follows Figure 4 As shown: Compared with db / db mice, the body weight and FBG of db / db mice decreased after PCI-34051 administration, and renal function indicators such as creatinine, urea nitrogen and trace urine protein decreased.

[0093] Conclusion: PCI-34051 can downregulate the body weight and FBG of db / db mice, and improve the renal function of db / db mice to a certain extent.

[0094] Study Example 5: Changes in renal pathological morphology and degree of renal fibrosis in the three groups of mice, db / m, db / db, and db / db+PCI-34051, after administration.

[0095] Reagents used:

[0096] Hematoxylin and eosin (H&E) staining kit, MASSON staining kit and Sirius Red staining kit were purchased from Beijing Solebow Technology Co., Ltd.

[0097] The animal model construction was the same as that in Study 3.

[0098] Specific experimental steps for hematoxylin and eosin staining experiment:

[0099] (1) Conventional dewaxing to water, the tissue is removed from the paraformaldehyde fixative and dehydrated and dewaxed in an automatic dehydrator. The operation steps are as follows: 75% ethanol 1h → 80% ethanol 1h → 90% ethanol 1h → 95% ethanol 1h → 100% ethanol 1h → xylene 1h → xylene 1h → paraffin 1h → paraffin 1h. After the operation, the tissue is embedded in paraffin, placed on ice for cooling, and then sliced ​​(thickness: 4μm) for later use.

[0100] (2) Add hematoxylin solution to the tissue wax block for 5 minutes and gently rinse it with running water.

[0101] (3) Add hydrochloric acid ethanol differentiation solution to the tissue wax block for 30 seconds and rinse with double distilled water for 10 minutes.

[0102] (4) Add eosin staining solution to the tissue wax block for 1 min, wash briefly with double distilled water, and dehydrate quickly.

[0103] (5) Wash with 75% ethanol, 80% ethanol, 90% ethanol and 100% ethanol for 1 min each; clear with xylene twice, 1 min each; seal with neutral gum and observe under a microscope.

[0104] Specific experimental steps of Masson staining:

[0105] (1) Dewaxing of paraffin tissue sections;

[0106] (2) Stain with reagent A for 5-10 minutes;

[0107] (3) Differentiate with reagent B for 5-15 seconds and wash with water;

[0108] (4) Reagent C turns blue for 3-5 minutes, then rinse with distilled water for 1 minute;

[0109] (5) After staining with reagent D for 5-10 minutes, wash with reagent E for 1 minute;

[0110] (6) Wash with reagent F for 2 minutes;

[0111] (7) Wash with reagent E for 1 min;

[0112] (8) Reagent G was dripped for 1 min;

[0113] (9) Wash with reagent F for 1 min.

[0114] Specific experimental steps for Sirius Red staining:

[0115] (1) Conventional dewaxing to water;

[0116] (2) Staining with Weigert iron hematoxylin solution for 10-20 min;

[0117] (3) Differentiation in acidic differentiation medium for 5 seconds;

[0118] (4) Rinse with tap water for 5-10 minutes and then with distilled water once;

[0119] (5) Stain with Sirius red solution for 1 hour;

[0120] (6) Rinse with running water to remove the surface staining liquid;

[0121] (7) Conventional dehydration to make it transparent, and then sealing with neutral gum.

[0122] The experimental results are as follows Figure 5 As shown, H&E staining results showed that compared with the db / m group mice, the renal tubular epithelial cells of db / db mice often had severe vacuoles, tubular expansion, and thickening of the glomerular basement membrane. After drug administration, the above pathological changes were improved to a certain extent;

[0123] The results of MASSON staining and Sirius Red staining showed that compared with the db / m group mice, the degree of renal tubular fibrosis in db / db mice was significantly aggravated, while after the administration of PCI-34051, the degree of fibrosis was alleviated to a certain extent.

[0124] Conclusion: PCI-34051 can improve the renal pathological morphological changes and reduce the degree of tubular fibrosis in db / db mice.

[0125] Study Example 6: Changes in renal glycogen deposition in the three groups of mice, db / m, db / db, and db / db+PCI-34051, after administration of medication.

[0126] Reagents used:

[0127] The PAS staining kit was purchased from Beijing Solebow Technology Co., Ltd.

[0128] Glycogen staining (PAS) specific experimental staining steps:

[0129] (1) Use 10% formalin for routine fixation, routine dehydration and embedding;

[0130] (2) Paraffin sections were dewaxed and placed in distilled water, while frozen sections were placed directly in distilled water;

[0131] (3) Rinse with tap water for 2-3 minutes, then rinse twice with distilled water;

[0132] (4) Place the sample in Schiff staining solution and place it in a dark place at room temperature for 10-20 minutes;

[0133] (5) Rinse with tap water for 10 minutes;

[0134] (6) The sample was placed in hematoxylin staining solution and the cell nucleus was stained for 1-2 min;

[0135] (7) Rinse with tap water for 10 minutes;

[0136] (8) Differentiation in acidic differentiation solution for 2-5 seconds;

[0137] (9) Rinse with tap water for 10-15 minutes to turn it back to blue;

[0138] (10) conventional ethanol dehydration step by step;

[0139] (11) Xylene is transparent and neutral gum is used for sealing.

[0140] The experimental results are as follows Figure 6 As shown, compared with the db / m group mice, the deposition of glycogen in the renal tubules of db / db mice was significant, and administration of PCI-34051 alleviated the deposition of glycogen in the renal tubules of mice.

[0141] Conclusion: PCI-34051 can alleviate renal tubular glycogen deposition in db / db mice.

[0142] Study Example 7: Changes in protein expression of BCL-2 and BAX in the renal tubules of the three groups of mice, db / m, db / db, and db / db+PCI-34051, after administration.

[0143] Reagents used:

[0144] DAPI staining solution, anti-fluorescence quenching sealing solution, Western primary antibody diluent, Western secondary antibody diluent, BCL-2, BAX antibodies were purchased from Proteintech, USA, pepsin was purchased from Biyuntian Biotechnology Co., Ltd., immunohistochemistry reagents were purchased from Beijing Zhongshan Jinqiao Co., Ltd., and goat anti-rabbit near-infrared fluorescent secondary antibody was purchased from Xuzhou Micro-Comand Bioengineering Co., Ltd.

[0145] Immunohistofluorescence was used to detect the expression of BAX and Bcl-2 proteins in mouse kidney tissue samples. Specific experimental steps:

[0146] (1) Kidney tissue was removed from paraformaldehyde fixative and dehydrated and dewaxed using an automatic dehydrator. The operation steps were as follows: 75% ethanol for 1 hour → 80% ethanol for 1 hour → 90% ethanol for 1 hour → 95% ethanol for 1 hour → 100% ethanol for 1 hour → xylene for 1 hour → xylene for 1 hour → paraffin for 1 hour → paraffin for 1 hour. After the operation, the tissue was embedded in paraffin, cooled on ice, and then sliced ​​(thickness: 4 μm) for later use.

[0147] (2) Add 0.4% pepsin and repair at 37°C for 30 min. Wash with PBS for 3 min, for a total of 3 times.

[0148] (3) Add reagent I (endogenous peroxidase blocker) to the kidney tissue wax block, react at room temperature for 10 min, and wash with PBS for 3 min, for a total of 3 washes.

[0149] (4) Add 2% BSA to block the kidney tissue at room temperature for 30 min, and carefully wipe off the excess liquid on the tissue with qualitative filter paper.

[0150] (5) Primary antibody application: Antibodies (BCL-2, 1:200; BAX, 1:200) were diluted and added dropwise to the kidney tissue and stored in a refrigerator at 4°C overnight.

[0151] (6) The next day, the tissue sections were placed in a 37°C oven to warm up for 30 min and washed with PBS for 3 min, for a total of 3 washes.

[0152] (7) Add Dylight 594 or Dylight 488 (1:200), incubate at room temperature in the dark for 1 h, wash with PBS for 3 min, and wash three times in total.

[0153] (8) Stain with DAPI for 5 min, wash with PBS buffer for 3 min, and wash three times in total.

[0154] (9) Apply a drop of fluorescence quencher and seal the slide.

[0155] The experimental results are as follows Figure 7 As shown, compared with the db / m group mice, BAX was significantly upregulated and Bcl-2 was significantly downregulated in the renal tubules of db / db mice; after administration of PCI-34051, the expression of BAX decreased, while the expression of Bcl-2 was the opposite.

[0156] Conclusion: PCI-34051 administration alleviates renal tubular apoptosis in db / db mice.

[0157] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. An inhibitor in a diabetic nephropathy drug, characterized in that: The inhibitor is an HDAC8 inhibitor.

2. The inhibitor in a diabetic nephropathy drug according to claim 1, characterized in that: The HDAC8 inhibitor includes one or a mixture of inhibitors that inhibit HDAC8 activity and inhibitors that inhibit HDAC8 synthesis.

3. The inhibitor in a diabetic nephropathy drug according to claim 1, characterized in that: The HDAC8 inhibitor is PCI-34051.

4. The inhibitor in a diabetic nephropathy drug according to claim 3, characterized in that: The molecular structure of PCI-34051 is:

5. Use of the inhibitor in the diabetic nephropathy drug according to any one of claims 1 to 4 in the preparation of a therapeutic drug for diabetic nephropathy.

6. Use of the inhibitor in the diabetic nephropathy drug according to any one of claims 1 to 4 in the preparation of a therapeutic kit for diabetic nephropathy.

7. A drug for treating diabetic nephropathy, characterized in that: It comprises the inhibitor according to any one of claims 1 to 4.

8. The drug for treating diabetic nephropathy according to claim 7, characterized in that: The drug is in solid powder dosage form.

9. The drug for treating diabetic nephropathy according to claim 7, characterized in that: The inhibitor in the diabetic nephropathy drug is the only active ingredient of the drug.

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