Application of ACSS2 protein in diabetic nephropathy and gene medicine

Abstract

The present application relates to the technical field of gene drug therapy, and in particular to an application of ACSS2 protein in diabetic nephropathy and a gene drug; the application includes: using ACSS2 protein with expression levels lower than normal as a biomarker for diabetic nephropathy; the gene drug includes overexpressed ACSS2 protein and / or an adeno-associated virus vector and an adeno-associated virus for overexpressing a target gene of the ACSS2 protein; based on a large number of experimental findings, ACSS2 protein may be a potential therapeutic target for diabetic nephropathy. Based on the characteristics of this discovery, it is clarified that the kidneys can be directly protected by reversing the gene expression imbalance caused by the disease. Therefore, overexpression of ACSS2 protein mediated by adeno-associated virus can achieve improvement of diabetic nephropathy through gene therapy, thereby filling the gap in the existing technology for the application of ACSS2 protein in diabetic nephropathy.

Description

Technical Field

[0001] The present application relates to the technical field of gene drug therapy, and in particular to an application of ACSS2 protein in diabetic nephropathy and gene drugs. Background Art

[0002] Diabetes is a chronic metabolic disease that poses a serious threat to human health, with an increasing incidence each year. Furthermore, the complications resulting from diabetes across various systems also place a significant burden on socioeconomic and social development. Among these complications, diabetic kidney disease (DKD) is the primary microvascular complication of diabetes, characterized by proteinuria and progressive renal function decline. Because the pathogenesis of DKD remains unclear and specific prevention and treatment options are currently lacking, identifying and identifying precise therapeutic targets for diabetic kidney disease is a pressing task. Although blood sugar can be controlled to inhibit the development of DKD through nutritional restriction, smoking cessation, exercise and weight loss, most patients have poor compliance with nutritional restriction, smoking cessation, exercise and weight loss. Therefore, relying solely on these methods cannot effectively control blood sugar and improve the progression of diabetic kidney disease. Although SGLT2 inhibitors, DPP-4 inhibitors and insulin can be used to assist in weight loss, patients who use Western medicine oral medications and insulin injections also have the defect of poor compliance, and these methods are also prone to hypoglycemia, which can induce serious conditions such as myocardial infarction and stroke.

[0003] In recent years, the field of gene therapy has gradually attracted attention. Adeno-associated virus (AAV), as the main gene vector for the treatment of various human diseases, has achieved preclinical and clinical success in mediated gene replacement, gene silencing and gene editing, making gene therapy a new treatment model. Currently, the AAV vectors commonly used in kidney research are AAV2, AAV6, AAV8 and AAV9, and AAV9 is currently the most effective serotype for direct injection transduction of adult mouse kidneys. Previous studies have found that AAV9-mediated changes in gene expression in the kidney have significant potential in chronic tubular fibrosis, but there is currently a lack of AAV9-mediated targets to achieve the improvement of diabetic kidney disease through gene therapy; therefore, how to provide AAV9-mediated targets to improve diabetic kidney disease through gene therapy is a technical problem that urgently needs to be solved.

[0004] Acetyl-CoA synthetase 2 (ACSS2) is a key member of the ACSS family, catalyzing the conversion of acetate into acetyl-CoA, a crucial intermediate metabolite in energy metabolism. ACSS2 has been shown to inhibit lipid synthesis in vivo, and studies have also shown that insulin can positively regulate ACSS2 expression. ACSS2 is not only a key enzyme in energy metabolism but also the rate-limiting enzyme for a variety of protein post-translational modification substrates. It catalyzes the synthesis of the corresponding coenzyme A from butyrate, crotonic acid, and lactate, and influences protein post-translational modification, implicated in a variety of diseases, including cancer, alcoholic fatty liver disease, and HIV / AIDS. However, no studies have yet reported on the role of ACSS2 in diabetic nephropathy. Summary of the Invention

[0005] The present application provides an application of ACSS2 protein in diabetic nephropathy and a gene drug to fill the gap in the application of ACSS2 in diabetic nephropathy in the prior art.

[0006] In a first aspect, the present application provides an application of ACSS2 protein in protecting diabetic nephropathy, the application comprising:

[0007] ACSS2 protein expression levels below normal were used as a biomarker for diabetic kidney disease.

[0008] Optionally, the expression level of the ACSS2 protein that is lower than the normal expression level is lower than 50% or more of the normal ACSS2 protein expression level.

[0009] Optionally, the application further includes:

[0010] Overexpressed ACSS2 protein is used as an inhibitory drug for diabetic nephropathy-related diseases.

[0011] Optionally, the expression level of the overexpressed ACSS2 protein is 1 times or more higher than the expression level of the normal ACSS2 protein.

[0012] Optionally, the diabetic nephropathy-related diseases include at least one of the following:

[0013] Diabetic nephropathy, renal tissue fibrosis in diabetic nephropathy, and renal inflammation in diabetic nephropathy.

[0014] In a second aspect, the present application provides an adeno-associated virus, which includes an adeno-associated virus vector and a target gene contained in the adeno-associated virus vector, wherein the target gene is used to overexpress ACSS2 protein.

[0015] Optionally, the adeno-associated viral vector comprises at least one of the following:

[0016] AAV2, AAV6, AAV8 and AAV9.

[0017] Optionally, the adeno-associated viral vector includes AAV9.

[0018] In a third aspect, the present application provides an application of an adeno-associated virus vector, which includes using the adeno-associated virus described in the second aspect to prepare a therapeutic drug for diabetic nephropathy.

[0019] In a fourth aspect, the present application provides a gene drug for treating or inhibiting diabetic nephropathy, wherein the gene drug comprises overexpressed ACSS2 protein and / or the adeno-associated virus described in the second aspect.

[0020] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0021] The examples of the present application provide an application of an ACSS2 protein in the protection of diabetic nephropathy. Based on a large number of experimental findings, in the in vivo high-glucose microenvironment formed by diabetic nephropathy, the high-glucose environment can induce a downregulation of the expression level of the ACSS2 protein in kidney tissue, thereby promoting the occurrence and development of diabetic nephropathy. This suggests that the ACSS2 protein may be a potential therapeutic target for diabetic nephropathy. Based on the characteristics of this discovery, it is clarified that it can directly protect the kidneys by reversing the gene expression imbalance caused by the disease. Therefore, adeno-associated virus-mediated overexpression of the ACSS2 protein can be used to improve diabetic nephropathy through gene therapy, thereby filling the gap in the existing technology for the application of the ACSS2 protein in diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 Schematic diagram of the immunohistochemical staining for detecting the expression of ACSS2 protein in kidney tissue of patients with diabetic nephropathy and the quantitative results provided in the examples of the present application;

[0025] Figure 2 This is a graph showing the results of the Elisa assay provided in the examples of this application for detecting the serum ACSS2 expression level in patients with diabetic kidney disease;

[0026] Figure 3 Schematic diagram of the results of Western Blot detection of ACSS2 protein expression level and relative expression amount of ACSS2 protein in kidney tissue of diabetic nephropathy mice provided in the examples of the present application;

[0027] Figure 4 Schematic diagram of the results of Western Blot detection of ACSS2 protein expression level and relative expression of ACSS2 protein in MEC cells induced by high glucose provided in the examples of the present application;

[0028] Figure 5 A map of the adeno-associated virus vector overexpressing ACSS2 provided in the examples of this application;

[0029] Figure 6 Schematic diagram of the experimental strategy flow for overexpressing ACSS2 protein in mice using AAV9-ACSS2 provided in the examples of the present application;

[0030] Figure 7 This is a graph showing the results of immunohistofluorescence detection of the expression of ACSS2 and GFP-AAV9 in mouse kidneys after injection of AAV9-ACSS2, as provided in the examples of the present application;

[0031] Figure 8 This is a graph showing the results of Western Blot analysis of ACSS2 protein expression in mouse kidneys after injection of AAV9-Con and AAV9-ACSS2, as provided in the examples of this application;

[0032] Figure 9 This is a graph showing the results of immunohistofluorescence detection of ACSS2 protein expression levels in mouse kidneys after injection of AAV9-Con and AAV9-ACSS2, as provided in the examples of this application;

[0033] Figure 10 A graph comparing the effects of AAV9-Con and AAV9-ACSS2 injections on mouse renal function provided in the examples of this application;

[0034] Figure 11 The results of HE, Masson, PAS, and Sirius red staining analysis of mouse kidney tissue pathological morphology, glycogen deposition, and fibrosis levels provided in the examples of this application;

[0035] Figure 12 This is a graph showing the results of electron microscopy analysis of the basement membrane and foot processes of mouse kidney tissue provided in the examples of this application;

[0036] Figure 13This is a graph showing the results of immunohistochemical staining analysis of the protein expression levels of inflammatory factors IL-1β and IL-6 and fibrosis indicator TGF-β in mouse kidney tissue provided in the examples of this application;

[0037] Figure 14 The results of Western Blot analysis of the protein levels of inflammatory factors IL-6 and IL-18 and fibrosis factor TGF-β in mouse kidney tissue after injection of AAV9-Con and AAV9-ACSS2 are provided in the examples of this application;

[0038] Figure 15 This is a graph showing the results of cell immunofluorescence detection of ACSS2 expression in MEC cells overexpressing ACSS2 and stimulated with high glucose, as provided in the examples of this application;

[0039] Figure 16 This is a graph showing the results of Western Blot analysis of ACSS2 expression in MEC cells overexpressing ACSS2 and stimulated with high glucose, as provided in the examples of this application;

[0040] Figure 17 The results of Western Blot detection of the protein expression levels of inflammatory factors IL-6, IL-18 and fibrosis factor TGF-β in MEC cells overexpressing ACSS2 and stimulated with high glucose are provided in the examples of this application. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0042] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0043] like Figure 1 As shown, the present application embodiment provides an application of ACSS2 protein in protecting diabetic nephropathy, and the application includes:

[0044] ACSS2 protein expression levels below normal were used as a biomarker for diabetic kidney disease.

[0045] In some optional embodiments, the expression level of the ACSS2 protein that is lower than the normal expression level is lower than 50% or more of the normal ACSS2 protein expression level.

[0046] In the examples of the present application, by comparing ACSS2 proteins with expression levels lower than normal and ACSS2 proteins with normal expression levels, the specific expression levels of ACSS2 proteins with expression levels lower than normal can be clarified, thereby clarifying the specific expression levels of ACSS2 proteins as biomarkers for diabetic nephropathy, thereby facilitating improving the accuracy of the biomarkers.

[0047] In some optional implementations, the application further includes:

[0048] Overexpressed ACSS2 protein is used as an inhibitory drug for diabetic nephropathy-related diseases.

[0049] In the examples of the present application, based on the principle that ACSS2 protein with expression levels below normal can serve as a biomarker for diabetic nephropathy, experiments have found that reversing the expression imbalance caused by diabetic nephropathy can directly protect the kidneys. Therefore, mediating overexpression of ACSS2 protein may be one of the protective measures for diabetic nephropathy-related diseases. This discovery fills the gap in targets that can be used by adeno-associated viruses in gene therapy and provides a basic principle for subsequent overexpression of ACSS2 protein in diabetic nephropathy mediated by adeno-associated virus vectors.

[0050] It should be noted that diabetic nephropathy-related diseases refer to complications caused by high glucose expression in diabetic nephropathy, such as renal tissue fibrosis and renal inflammation.

[0051] In some optional embodiments, the expression level of the overexpressed ACSS2 protein is 1 times or more higher than the normal ACSS2 protein expression level.

[0052] In the examples of the present application, by refining the specific expression level of the overexpressed ACSS2 protein, the specific expression level of the ACSS2 protein in the inhibitory drug for diabetic nephropathy can be clarified, which facilitates improving the inhibitory effect of the inhibitory drug on diabetic nephropathy-related diseases, thereby playing a protective role on diabetic kidneys.

[0053] In some optional embodiments, the diabetic nephropathy-related diseases include at least one of the following:

[0054] Diabetic nephropathy, renal tissue fibrosis in diabetic nephropathy, and renal inflammation in diabetic nephropathy.

[0055] In the examples of this application, the specific components of diabetic nephropathy-related diseases are clarified, which can cover the significant characteristics of diabetic nephropathy-related diseases, thereby further clarifying that the expressed ACSS2 protein can also treat and / or inhibit renal tissue fibrosis and renal inflammation in diabetic nephropathy.

[0056] Based on a general inventive concept, an embodiment of the present application provides an adeno-associated virus, which includes an adeno-associated virus vector and a target gene contained in the adeno-associated virus vector, wherein the target gene is used to overexpress ACSS2 protein.

[0057] The adeno-associated virus is realized based on the application of the above-mentioned ACSS2 protein in the protection of diabetic nephropathy. The specific raw materials for the application of the ACSS2 protein in the protection of diabetic nephropathy can be referred to the above-mentioned embodiments. Since the adeno-associated virus adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0058] It should be noted that the target gene can adopt a nucleotide sequence known in the prior art that can express ACSS2 protein.

[0059] In some optional embodiments, the adeno-associated viral vector comprises at least one of the following:

[0060] AAV2, AAV6, AAV8 and AAV9.

[0061] In some optional embodiments, the adeno-associated viral vector comprises AAV9.

[0062] In the examples of the present application, by refining the type of adeno-associated viral vector, since AAV9 is currently the most effective serotype for direct injection transduction of adult mouse kidneys, and AAV9-mediated gene expression changes in the kidneys have obvious potential in chronic tubular fibrosis, it is clear that the adeno-associated viral vector is AAV9, and AAV9-mediated overexpression of ACSS2 protein can improve diabetic nephropathy, thereby filling the gap in AAV9-mediated available targets for improving diabetic nephropathy through gene therapy.

[0063] Based on a general inventive concept, an embodiment of the present application provides an application of an adeno-associated virus vector, which includes using the adeno-associated virus to prepare a therapeutic drug for diabetic nephropathy.

[0064] The application of this adeno-associated virus vector is based on the application of the above-mentioned ACSS2 protein in the protection of diabetic nephropathy. The specific principles of the application of the ACSS2 protein in the protection of diabetic nephropathy can be referred to the above-mentioned embodiments. Since the application of this adeno-associated virus vector adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0065] Based on a general inventive concept, an embodiment of the present application provides a gene drug for treating or inhibiting diabetic nephropathy, wherein the gene drug comprises overexpressed ACSS2 protein and / or the adeno-associated virus described in the second aspect.

[0066] This gene drug is based on the application of the above-mentioned ACSS2 protein in the protection of diabetic nephropathy. The specific principles of the application of the ACSS2 protein in the protection of diabetic nephropathy can be referred to the above-mentioned embodiments. Since this gene drug adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0067] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples where specific conditions are not specified are generally measured according to industry standards. If there are no corresponding industry standards, then the methods are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0068] Example 1

[0069] With the approval of the hospital ethics committee and based on the patients' informed consent, patients who were hospitalized in the Affiliated Hospital of Southwest Medical University from January 2021 to December 2021 and required renal puncture biopsy due to their condition and were clinically diagnosed with DKD were randomly selected.

[0070] Diagnostic criteria: T2DM is diagnosed according to the diagnostic criteria proposed by WHO in 1999; DKD is diagnosed based on random urine microalbumin / urine creatinine (ACR) > 30 mg / gcr.

[0071] Volunteers who underwent health checkups at the Affiliated Hospital of Southwest Medical University during the same period were randomly selected and peripheral blood was collected as normal controls (NC). Paraffin sections of the collected clinical kidney specimens were prepared and immunohistochemistry was performed (the results are shown in Figure 2). Figure 1 As shown), serum samples were tested by Elisa (the results were as shown Figure 2 ). Figure 1 and Figure 2 The results showed that compared with normal controls, the expression of ACSS2 protein in the renal tissue of patients with diabetic nephropathy was significantly reduced, and the corresponding serum ACSS2 level was also reduced.

[0072] Based on the above results, 10 7-week-old SPF C57BKSdb / db male mice and 5 wild-type male mice from the same littermate were selected and purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The standard feed used in the experiment was purchased from Chengdu Dashuo Company.

[0073] All animal experiments were performed under the following conditions: room temperature of 23°C ± 1°C, relative humidity of 60% ± 10%, and alternating 12-h light-dark cycles in individually ventilated IVC cages. For AAV9-mediated expression of ACSS2 in mouse kidneys, a plasmid with the sequence pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV-MCS-3flag was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. (Guangzhou, China). The map is shown in Figure 2. Figure 5 As shown, according to Figure 6 As shown in the process, 100 μL of AAV9-ACSS2-GFP (1E+13 GC / mL) or negative control Vector (1E+13 GC / mL) was injected through the tail vein of db / db and homologous littermate control mice to obtain ACSS2 kidney overexpression diabetic mouse models. The transfection results were shown in Figure 2. Figure 7 、 8 , 9 , indicating that the transfection was successful. After that, each group was given a normal diet for 12 weeks. After 12 weeks, the mice were anesthetized with sodium pentobarbital, killed, and samples were collected.

[0074] Mouse mesangial cells (MECs) were cultured in DMEM medium containing 10% fetal bovine serum and supplemented with 1% penicillin-streptomycin. MEC cells were cultured at 37°C with 5% CO2 until 60% to 70% confluency and exposed to solvent control or high glucose for 48 h. Mouse ACSS2 overexpression plasmid (GFP-ACSS2) and vector plasmid (GFP) were purchased from Chengdu Bowen Biotechnology Co., Ltd. and transfected into MEC cells using Lipofectamine 3000 (Invitrogen). The transfection results are shown in Figure 2. Figure 15 、 16 As shown, indicating that the transfection was successful.

[0075] Example 2

[0076] Based on the mouse samples treated in Example 1, subsequent treatment was performed:

[0077] Mouse urine microalbumin / urine creatinine (ACR), blood creatinine detection:

[0078] After 12 weeks of intervention, clean urine was collected from mice and urine microalbumin / creatinine was detected using Siemens automatic biochemical analyzer. A creatinine (Cr) kit was purchased from Nanjing Jiancheng Bioengineering Institute to detect serum creatinine levels. The test results were as follows: Figure 10 shown.

[0079] Example 3

[0080] Based on the mouse samples treated in Example 1, subsequent treatment was performed:

[0081] Western Blot experiment:

[0082] Kidney tissue: 50 mg + 1000 μL RRIPA lysis buffer (containing 1% PMSF) (P0013B, Shanghai Beyotime Biotechnology Co., Ltd.) was used for tissue homogenization, and the mixture was centrifuged at 12000 g for 10 min. The supernatant was collected and added with 5X loading buffer, and heated at 100°C for 10 min to denature the protein.

[0083] MEC cells: Remove the treated cells, wash them three times with pre-chilled PBS, and then lyse the cells with RIPA lysis buffer (containing 1% PMSF) for 20 minutes. Centrifuge at 12,000 g for 10 minutes, collect the supernatant, add 5X gel loading buffer, and heat at 100°C for 10 minutes to denature the protein.

[0084] After electrophoresis on 12% SDS-PAGE gel, the membrane was transferred to PVDF membrane and blocked with 5% BSA at room temperature for 1 hour. The primary antibodies were incubated with shaking in a refrigerator at 4°C overnight. The primary antibody catalog numbers were: ACSS2 (SC-398559, Santa Cruz, USA), IL-1β (AF7209, Beyotime, China), IL-18 (AF7266, Beyotime, China), IL-6 (AF0201, Beyotime, China), and TGF-β (AF0297, Beyotime, China).

[0085] The next day, the membrane was washed three times with PBST and then the secondary antibody of the corresponding species was replaced. The membrane was incubated at room temperature for 1 hour, washed again with PBST, and developed. The membrane was placed in developer (Millipore Corporation), then dried and exposed in a luminescence detection machine. The band intensity was quantified using ImageJ software.

[0086] The results of Western Blot analysis of kidney tissues of mice in the AAV9-ACSS2 control group are as follows: Figure 8 The results of Western Blot detection of MEC cells are shown in Figure 16 As shown in Figure 2, the expression levels of IL-18, IL-6, and TGF-β proteins in MEC cells were Figure 17 shown.

[0087] Depend on Figure 3 and Figure 4 It can be seen that the expression level of ACSS2 protein was significantly downregulated in the kidney tissue of diabetic nephropathy mice and high glucose-induced MEC cells.

[0088] Depend on Figure 8 、 Figure 16 and Figure 17 It can be seen that overexpression of ACSS2 can significantly improve DKD inflammation-fibrosis damage.

[0089] Example 4

[0090] Based on the samples processed in Example 1 and Example 3, subsequent processing is performed:

[0091] 1. Masson and HE staining of kidney tissue:

[0092] (1) Sectioning: Keep the tissue wax block in a low-temperature cooling state and slice it at a thickness of 4 μm, trying to ensure the integrity of the tissue section. Spread the cut kidney tissue slices flat in a preheated 37°C water tank to expand them as much as possible. Use a slide to remove the kidney tissue slices to avoid mixing with impurities and other tissue fragments. After the slices are taken, place the kidney tissue slices in a 37°C constant temperature oven and bake them overnight for later use.

[0093] (2) Dewaxing: Place the kidney tissue sections on a slide rack and soak them in xylene in the following order: xylene I (pure) 10 min → xylene II (pure) 10 min → xylene III (pure) 10 min → xylene IV (pure) 10 min for dewaxing.

[0094] (3) Hydration: Place the kidney tissue sections on a slide rack and soak them in gradient ethanol: 100% ethanol for 10 min → 95% ethanol for 10 min → 90% ethanol for 10 min → 80% ethanol for 10 min.

[0095] (4) Rinse thoroughly with distilled water and set aside.

[0096] (5) Staining: HE staining: Place the slide in hematoxylin solution for 5 minutes → rinse with tap water; 1% hydrochloric acid alcohol for 30 seconds → rinse with tap water → stain the cytoplasm with eosin for 10 minutes → rinse with tap water; dehydrate with gradient ethanol; Masson staining: Soak the above slide in double distilled water for 1 minute, drop R1 stain solution on the slide for 1 minute, then discard and rinse for 30 seconds, drop R2 stain solution on the slide for 30 seconds, then discard and rinse for 30 seconds, R3 stain solution for 8 minutes, then discard, and R4 stain solution for 5 minutes, then discard and rinse with anhydrous ethanol.

[0097] (6) Then, dry the tissue sections, seal the sections, read the sections under a microscope, and take photos.

[0098] The steps of PAS staining include:

[0099] (1) Sectioning, dewaxing, and hydration are consistent with the HE and Masson procedures.

[0100] (2) Place in oxidant for 5 minutes and rinse with tap water.

[0101] (3) Soak in distilled water for 2 minutes, repeat 3 times

[0102] (4) Soak in Schiff Reagent for 15 minutes and rinse with tap water.

[0103] (5) Hematoxylin staining stains the nucleus for 2 minutes.

[0104] (6) Differentiate in acidic differentiation solution for 2s to 5s, rinse with tap water, and use double-distilled water to turn blue.

[0105] (7) Dehydrate with conventional gradient ethanol, make transparent with xylene, and seal with neutral gum.

[0106] (8) After drying, observe under a microscope and take photos.

[0107] The steps of the Sirius red staining experiment include:

[0108] (1) Sectioning, dewaxing, and hydration are consistent with the HE and Masson procedures.

[0109] (2) Prepare iron hematoxylin staining solution just before use, add it to the tissue and stain for 5 to 10 minutes, then wash with distilled water for 10 to 20 seconds to remove excess staining solution.

[0110] (3) Wash with tap water for 5 to 10 minutes.

[0111] (4) Add Sirius red staining solution to the mouse kidney tissue and stain for 15 to 30 minutes, then shake off the staining solution.

[0112] (5) Wash with tap water for 5 to 10 minutes.

[0113] (6) Dehydrate using gradient alcohol concentrations and make transparent using xylene.

[0114] (7) Allow to dry, seal the slides, read the slides under a microscope, and take photos.

[0115] The steps of transmission electron microscopy include:

[0116] (1) Fixation: Kidney tissue was fixed with 3% glutaraldehyde and then with 1% osmium tetroxide.

[0117] (2) Dehydration: Dehydration step by step using concentration gradient acetone.

[0118] (3) Infiltration and embedding: dehydrating agent and Epon812 embedding agent, the volume ratios are 3:1, 1:1, and 1:3, respectively, and finally Ep812 embedding.

[0119] (4) Ultrathin sectioning: Prepare ultrathin sections of approximately 70 nm using a microtome, spread the sections, and then place them on a copper mesh.

[0120] (5) Staining: Stain with uranyl acetate for 15 minutes, then stain with lead citrate for 2 minutes.

[0121] (6) Electron microscopy observation: A transmission electron microscope was used to capture images of the copper mesh and observe the lesions.

[0122] The results of Masson, HE, PAS and Sirius red staining of kidney tissue are as follows Figure 11 and Figure 12 As shown, the results showed that overexpression of ACSS2 protein can significantly improve the renal pathological morphology caused by diabetic nephropathy.

[0123] Example 5

[0124] Based on the samples processed in Example 1 and Example 3, combined with the method of Example 4, subsequent processing is performed:

[0125] 1. The immunohistochemical staining steps include:

[0126] (1) The dewaxing and hydration procedures for kidney tissue sections were the same as those for Masson staining.

[0127] (2) A ready-to-use high-efficiency immunohistochemistry secondary antibody kit (abs957, Aibixin, China) was added with 100 μL of solution A for 10 min to block endogenous peroxidase and reduce nonspecific binding to reduce background staining, and the samples were washed with PBS for 5 min three times.

[0128] (3) Add primary antibody (concentration 1:200), incubate at room temperature for 40 min, and wash with PBS for 5 min, three times.

[0129] (4) Add 100 μL of solution C and incubate for 10 min. Wash with PBS for 5 min, three times.

[0130] (5) Add 100 μL of solution D and incubate for 10 min, protecting from light. Wash with PBS for 5 min, three times.

[0131] (6) Prepare fresh substrate solution: According to the number of slides, prepare fresh substrate solution according to the volume ratio (F solution: E solution = 3:100), add appropriate amount of fresh substrate solution, incubate for 5 minutes, and rinse with deionized water.

[0132] (7) Restaining: After washing with PBS, stain with hematoxylin for 2 minutes and rinse repeatedly with tap water; differentiate with 1% hydrochloric acid alcohol for 2 seconds to 3 seconds, saturated lithium carbonate solution for 1 minute, and rinse repeatedly with tap water; dehydrate with gradient alcohol (mass concentration is 80% → 85% → 90% → 95% → 100%, each time for 2 minutes).

[0133] (8) Seal the slides and take photos: Carefully seal the slides with neutral gum, observe under a microscope and take photos.

[0134] Immunohistochemical staining results of kidney tissue sections Figure 13As shown, this indicates that overexpression of ACSS2 protein can significantly improve the activation of inflammatory-fibrotic factors IL-1β, IL-6, and TGF-β caused by diabetic kidney disease.

[0135] In summary, the examples of the present application provide an application of an ACSS2 protein in diabetic nephropathy. Experiments have found that ACSS2 expression is downregulated in diabetic nephropathy, which suggests that ACSS2 may be a potential therapeutic target for diabetic nephropathy. Therefore, it can directly protect the kidneys by reversing the gene expression imbalance caused by the disease. Therefore, combined with adeno-associated virus AAV9-mediated ACSS2 overexpression, it is possible to improve diabetic nephropathy through gene therapy, thereby filling the gap in AAV9-mediated targets that can be used to improve diabetic nephropathy through gene therapy.

[0136] At the same time, the experimental results provided in the examples of this application demonstrate that AAV-mediated ACSS2 renal heterologous overexpression may be a new therapeutic approach to improve diabetic nephropathy, and may also be one of the effective means of treating diabetic nephropathy.

[0137] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a hard limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numbers within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0138] In this application, unless otherwise specified, the directional words used, such as "upper" and "lower", refer specifically to the directions of the drawings in the accompanying drawings. In addition, in the description of the present application specification, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" refers to one or more, and "plurality" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple.

[0139] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. An application of an adeno-associated virus vector, characterized in that: The application includes using an adeno-associated virus to prepare a therapeutic drug for diabetic nephropathy; the adeno-associated virus includes an adeno-associated virus vector and a target gene contained in the adeno-associated virus vector, and the target gene is used to overexpress the ACSS2 protein; the expression level of the overexpressed ACSS2 protein is 1 times or more higher than the normal ACSS2 protein expression level.

2. The use according to claim 1, characterized in that The adeno-associated viral vector comprises at least one of the following: AAV2, AAV6, AAV8 and AAV9.

3. The use according to claim 2, characterized in that The adeno-associated virus vector includes AAV9.

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