Application of Protein Acetylation Modification in the Diagnosis and Treatment of Diabetic Nephropathy

Abstract

The present invention belongs to the fields of biomedicine and molecular biology, and relates to the application of protein acetylation in the diagnosis and treatment of diabetic nephropathy. The present invention demonstrates that acetylation of the TFAM K76 site is differentially expressed in diabetic nephropathy, demonstrating its beneficial diagnostic and prognostic effects. Furthermore, the present invention has found that regulating the TFAM K76 site, particularly inhibiting acetylation of the TFAM K76 site, can alleviate high-glucose-induced inflammation and fibrosis in renal tubular epithelial cells and renal tissue. This approach can be used to treat diabetic nephropathy and further screen for small molecule compounds that mitigate kidney damage in diabetic nephropathy mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and relates to the application of acetylation modification of proteins in the diagnosis and treatment of diabetic nephropathy. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes. Approximately 30-40% of diabetic patients eventually develop DKD, making it the leading cause of end-stage renal disease (ESRD). Characteristic pathological changes include glomerular basement membrane thickening, mesangial matrix expansion, podocyte injury, and tubulointerstitial fibrosis. Clinical manifestations include proteinuria, progressive renal function decline, and ultimately renal failure. While current treatments (such as blood sugar and blood pressure control and RAS inhibitors) can slow progression, some patients still cannot avoid disease progression, necessitating the urgent need for therapeutic strategies targeting novel pathological mechanisms.

[0004] The core pathophysiological mechanisms of diabetic nephropathy currently include the following aspects, such as (1) metabolic abnormalities and oxidative stress: sustained hyperglycemia causes mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) through the polyol pathway, hexosamine pathway, and accumulation of advanced glycation end products (AGEs), leading to oxidative damage to glomerular endothelial cells and podocytes, and destroying the integrity of the filtration barrier. (2) Activation of inflammatory signals: hyperglycemia activates macrophage infiltration through pathways such as NF-κB and NLRP3 inflammasome, releasing proinflammatory factors such as IL-6 and TNF-α, promoting glomerular sclerosis and interstitial fibrosis. (3) Fibrosis process driver: overexpression of transforming growth factor-β1 (TGF-β1) induces epithelial-mesenchymal transition (EMT), stimulating excessive deposition of extracellular matrix (ECM) (such as collagen IV and fibronectin), ultimately leading to structural remodeling and functional loss of the nephron. (4) Imbalance of epigenetic regulation: Recent studies have found that abnormal acetylation modification of histones / non-histones plays a key role in the progression of diabetic nephropathy by regulating the chromatin accessibility of inflammation- and fibrosis-related genes, and has become an emerging therapeutic target.

[0005] Protein acetylation is a crucial post-translational modification, primarily through the covalent attachment of acetyl groups to proteins (particularly lysine residues), regulating protein stability, subcellular localization, enzymatic activity, and interactions. This process is dynamically regulated by acetyltransferases (HATs) and deacetylases (HDACs / Sirtuins), and is closely linked to gene expression, metabolic regulation, and inflammatory responses. Abnormal protein acetylation can increase inflammation and oxidative stress in DKD. For example, hyperglycemia inhibits sirtuin-1 activity, increasing acetylation levels of NF-κB, p65, and STAT3, leading to increased release of proinflammatory cytokines and oxidative stress, and contributing to glomerular podocyte damage and proteinuria. Mitochondrial dysfunction is a key feature of DKD, and acetylation affects energy metabolism by regulating metabolic pathways such as oxidative phosphorylation and the tricarboxylic acid (TCA) cycle. Literature reports indicate that mitochondrial protein acetylation is significantly increased in the kidneys of patients with DKD, leading to decreased metabolic adaptability. Furthermore, acetylation modifies the progression of DKD fibrosis. For example, abnormally elevated histone acetylation (such as H3K18Ac and H4K8Ac) in DKD promotes the expression of profibrotic genes (such as TGF-β and CTGF) and accelerates tubulointerstitial fibrosis. Furthermore, hyperglycemia-induced changes in histone acetylation can persist over the long term (metabolic memory), driving DKD progression even after glycemic control. Therefore, targeting protein acetylation is an important strategy for slowing DKD progression.

[0006] Mitochondrial transcription factor A (TFAM) is a key DNA-binding protein encoded by a nuclear gene and translocated to the mitochondrial matrix. It plays a central role in maintaining mitochondrial genome (mtDNA) stability and regulating mitochondrial biogenesis. Its structure contains two high-mobility group (HMG-box) domains, which bind specifically to mtDNA to form a nucleic acid-protein complex. TFAM is directly involved in mtDNA transcription and replication and regulates the synthesis of subunits of mitochondrially encoded respiratory chain complexes (such as Complexes I, III, and IV). Studies have reported that TFAM undergoes multiple post-translational modifications, including phosphorylation, ubiquitination, and acetylation. Acetylation of TFAM is a key modulator of its function, significantly reducing its binding affinity for mitochondrial DNA (mtDNA). Furthermore, by inhibiting TFAM's interaction with mtDNA, acetylation reduces the expression of genes critical for mitochondrial biogenesis, leading to a decrease in mtDNA copy number. In models of vascular dementia (VaD), dysregulation of the SIRT3 / TFAM pathway leads to overacetylation of TFAM, which increases mitochondrial damage. Restoring its deacetylation improves mitochondrial function and alleviates the disease. In acute kidney injury (AKI), upregulation of TFAM acetylation mediated by GCN5L1 is associated with mitochondrial damage. Knockdown of GCN5L1 reduces TFAM acetylation and alleviates renal pathological changes. Accumulating evidence indicates that TFAM acetylation plays an important role in regulating mtDNA function and mitochondrial biogenesis. However, no studies have reported on TFAM acetylation in DKD, and its abnormal expression and biological functions in DKD progression remain largely unclear. Summary of the Invention

[0007] The present invention found that (1) acetylation modification of the TFAM K76 site was significantly increased in DKD patients and mice and high glucose-induced HK-2 cells; (2) acetylation of the TFAMK76 site promoted inflammation and mitochondrial damage in high glucose-induced HK-2 cells; (3) acetylation of the TFAMK76 site promoted renal damage and mitochondrial damage in DKD mice; (4) small molecule compounds targeting acetylation of the TFAMK76 site were screened; (5) small molecule compounds could reduce inflammation and mitochondrial damage in HK-2 cells induced by high glucose; (6) small molecule compounds could reduce renal damage and mitochondrial damage in DKD mice. Based on the above research results, the present invention provides the application of protein acetylation modification in the diagnosis and treatment of diabetic nephropathy.

[0008] Specifically, the technical solutions of the present invention are as follows:

[0009] The first aspect of the present invention provides the use of any one of the following a1) to a4) in the preparation of a medicament for diagnosing diabetic nephropathy:

[0010] a1) Acetylation modification of TFAM K76 site in the renal tissue of the subjects;

[0011] a2) a nucleic acid encoding an acetyltransferase that modifies the acetylation of TFAM K76;

[0012] a3) Reagents for detecting TFAM K76 acetylation expression;

[0013] a4) Reagents for detecting the expression of acetyltransferases encoding TFAM K76 acetylation modifications.

[0014] Through research, the present invention found that the acetylation level of the TFAM K76 site is increased in DKD tissues and cell lines, clarifying that TFAM K76 acetylation is a marker for the diagnosis and prognosis of DKD, and can thus serve as an important key target for its treatment. Targeting TFAM K76 acetylation can slow the progression of DKD.

[0015] The subject of the present invention is a human (especially a diabetic patient) or a non-human mammal, and the non-human mammal is a rat, mouse, guinea pig, rabbit, dog, monkey, orangutan, etc.

[0016] The reagent for detecting the expression of acetylated modification of TFAM K76 site in the present invention can be a reagent based on immunoblotting, a reagent based on mass spectrometry, a reagent based on enzyme-linked immunosorbent assay, and the like.

[0017] The second aspect of the present invention provides the use of an active ingredient in at least one of the following b1) to b4):

[0018] b1) Preparation of products for reducing inflammation and / or fibrosis of renal tubular epithelial cells;

[0019] b2) preparing products for reducing mitochondrial damage in renal tubular epithelial cells;

[0020] b3) preparing products for screening to slow the progression of diabetic nephropathy;

[0021] b4) Preparation of medicaments for treating diabetic nephropathy;

[0022] The active ingredients include at least the following c1) or c2):

[0023] c1) Substances that reduce or inhibit the expression level of TFAM K76 acetylation modification;

[0024] c2) A substance that reduces or inhibits the expression level of a nucleic acid encoding an acetyltransferase that modifies TFAM K76 acetylation.

[0025] Wherein, in b3), the diabetic nephropathy is a disease caused by diabetes and characterized by lesions of kidney tissue (including renal tubular epithelial cells).

[0026] The product of the present invention can be a medicine or an experimental reagent. The experimental reagent can be used in scientific research, such as constructing a diabetic nephropathy model.

[0027] The substance for reducing or inhibiting the expression level of TFAM K76 site acetylation modification of the present invention can be a compound for reducing or inhibiting the expression level of TFAM K76 site acetylation modification. Specifically, the molecular formula of the compound is C 21 H 25 N3O2S, CAS number 353261-40-2, has the following chemical structure:

[0028] .

[0029] In some embodiments, the drug is a pharmaceutical composition, further comprising pharmaceutical excipients.

[0030] The pharmaceutical excipients may be carriers, excipients, etc. commonly used in pharmacy. Moreover, according to conventional methods, the preparations may be prepared into dosage forms such as granules, powders, sprays, tablets, capsules, suspensions, emulsions, syrups, etc., in the form of oral preparations, external preparations, suppositories, and sterile injection solutions.

[0031] The non-pharmaceutical active ingredients such as carriers and excipients that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards. The excipients include binders, fillers, disintegrants, preservatives, lubricants, etc.

[0032] In some embodiments, the carriers and excipients include but are not limited to alumina, serum protein, lecithin, maltitol, starch, gum arabic, sucrose, lactose, alginate, glucose, sorbitol, mannitol, magnesium stearate, mineral oil, xylitol, erythritol, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, phosphate, water, etc.

[0033] The medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the corresponding tissue by systemic intravenous delivery or local injection. Alternatively, it can be administered intravenously, percutaneously, intranasally, through the mucosa or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on various factors to a great extent, such as the target cell, biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0034] In some embodiments, the pharmaceutical composition can be administered to humans and non-human mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, orangutans, and the like.

[0035] A third aspect of the present invention provides a method for treating diabetic nephropathy, comprising administering to a subject a substance that reduces acetylation of the TFAM K76 site.

[0036] Beneficial technical effects of one or more of the above technical solutions or implementation plans of the present invention are:

[0037] The subject of the present invention is a human (especially a diabetic patient) or a non-human mammal, and the non-human mammal is a rat, mouse, guinea pig, rabbit, dog, monkey, orangutan, etc.

[0038] This invention reports for the first time the differential expression of acetylation of the TFAM K76 site in diabetic nephropathy, indicating that it has good diagnostic and prognostic effects on diabetic nephropathy. Studies have also found that regulating the TFAM K76 site, especially inhibiting the acetylation modification of the TFAM K76 site, can reduce high-glucose-induced inflammation and fibrosis in renal tubular epithelial cells and renal tissues, and can be used to treat diabetic nephropathy. At the same time, small molecule compounds that specifically target the TFAM K76 site are screened out. The screened small molecule compounds can significantly reduce high-glucose-induced inflammation and fibrosis in renal tubular epithelial cells and renal tissues, and can slow down kidney damage in mice with diabetic nephropathy.

[0039] In summary, the present invention provides a new mechanism for the development and progression of diabetic nephropathy and a promising treatment strategy for patients with diabetic nephropathy, and therefore has great potential for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0041] Figure 1 The immunoprecipitation in Example 1 of the present invention verified that the acetylation level of renal tubular epithelial cells increased under high glucose induction.

[0042] Figure 2 The immunofluorescence result in Example 1 of the present invention verified that the acetylation level of TFAM K76 in renal tubular epithelial cells increased under high glucose induction.

[0043] Figure 3 The immunohistochemical results in Result 1 of Example 1 of the present invention confirm that the acetylation level of TFAM K76 in the kidney tissue of diabetic patients is increased.

[0044] Figure 4 Result 1 of Example 1 of the present invention shows that the acetylation level of TFAM K76 in the kidney tissue of diabetic mice is increased by immunohistochemistry.

[0045] Figure 5 The Western blot technique in Example 1 of the present invention detected an increase in the acetylation level of TFAMK76 in the kidney tissue of diabetic mice.

[0046] Figure 6 The Western blot technique in Example 2 of the present invention was used to detect the levels of TFAM K76 acetylation promoting inflammation and fibrosis in renal tubular epithelial cells.

[0047] Figure 7 The PCR technology in Example 2 of the present invention verified that TFAM K76 acetylation in renal tubular epithelial cells increased TGF-β levels.

[0048] Figure 8 The Western blot technique in Example 2 of the present invention detected that TFAM K76 acetylation in renal tubular epithelial cells reduced mitochondrial biogenesis.

[0049] Figure 9 The PCR technology in Example 2 of the present invention verifies that TFAM K76 acetylation in renal tubular epithelial cells inhibits mitochondrial DNA replication.

[0050] Figure 10 The Western blot technique in Example 3 of the present invention detected that TFAM K76 acetylation increased kidney damage in DKD mice.

[0051] Figure 11 The PCR technology in Example 3 of the present invention verifies that TFAM K76 acetylation in DKD mice promotes the transcription of inflammatory factors.

[0052] Figure 12 The PCR technology in Example 3 of the present invention verified that TFAM K76 acetylation in DKD mice increased chemokine levels.

[0053] Figure 13This is a flow chart for screening small molecule compounds according to Example 4 of the present invention.

[0054] Figure 14 This is the Western blot technique used in Result 4 of Example 4 of the present invention to screen small molecule compounds that inhibit TFAM K76 acetylation.

[0055] Figure 15 The Western blot technique was used to verify the intervention concentration of the small molecule compound that inhibited TFAM K76 acetylation in Example 4 of the present invention.

[0056] Figure 16 The protein thermal migration technology in Example 4 of the present invention verifies the binding of small molecule compounds to TFAM.

[0057] Figure 17 The BLI technology in Example 4 of the present invention verifies that the small molecule compound specifically targets TFAM.

[0058] Figure 18 The BLI technology in Example 4 of the present invention verifies that the small molecule compound specifically targets the TFAM K76 site.

[0059] Figure 19 The PCR technology in Example 5 of the present invention verifies that the small molecule compound rescues the mitochondrial DNA replication inhibited by high glucose.

[0060] Figure 20 The Western blot technique in Example 5 of the present invention verifies that the small molecule compound alleviates high glucose-induced mitochondrial damage.

[0061] Figure 21 The PCR technology in Example 5 of the present invention verifies that the small molecule compound alleviates high glucose-induced inflammation and fibrosis.

[0062] Figure 22 The PCR technology in Example 5 of the present invention verifies that the small molecule compound alleviates the increase of TGF-β induced by high glucose.

[0063] Figure 23 The Western blot technique was used to detect the effect of small molecule compounds on alleviating kidney damage in DKD mice in Example 6 of the present invention.

[0064] Figure 24 The PCR technology in Example 6 of the present invention verifies that the small molecule compound reduces the level of inflammatory factors in DKD mice.

[0065] Figure 25 The PCR technology in Result 6 of Example of the present invention verified that the small molecule compound reduced the chemokine level in DKD mice. DETAILED DESCRIPTION

[0066] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0067] Example

[0068] Materials and methods

[0069] Cell transfection

[0070] 24 hours before transfection, seed cells into culture plates / dishes to a confluency of 70%-90% at the time of transfection. Replace the culture medium with fresh complete medium (containing serum) 1 hour before transfection. Prepare the transfection complex: A. Dilute DNA: In a sterile EP tube, dilute the plasmid DNA with Opti-MEM medium.

[0071] B. Dilute Lipofectamine 3000 transfection reagent: In another EP tube, dilute Lipofectamine 3000 transfection reagent with Opti-MEM medium.

[0072] C. Mix DNA and transfection reagent: Gently mix the diluted DNA with diluted Lipofectamine 3000 transfection reagent (1:1 volume ratio). Incubate at room temperature for 10-15 minutes to allow the DNA-liposome complex to form. Add the mixture dropwise to the cell culture plate and gently shake the plate to mix thoroughly. Continue culturing in a 37°C, 5% CO2 incubator.

[0073] Immunohistochemical staining

[0074] 1) Deparaffinization and Rehydration: Immerse sections in xylene I and II for 10 minutes each to deparaffinize. Rehydrate with graded ethanol (100% → 95% → 80% → 70%) for 5 minutes each. Finally, soak in PBS (phosphate-buffered saline) for 5 minutes. 2) Antigen Retrieval: Immerse sections in antigen retrieval solution (e.g., pH 6.0 citrate buffer), microwave until boiling, hold for 10-15 minutes, and cool to room temperature. 3) Block endogenous peroxidases: Incubate with 3% H₂O₂ at room temperature for 10 minutes, then rinse three times with PBS for 5 minutes. 4) Blocking: Apply blocking solution (e.g., 5% normal goat serum) dropwise and incubate at room temperature for 30 minutes to reduce nonspecific binding. 5) Primary Antibody Incubation: Aspirate the blocking solution and apply diluted primary antibody dropwise. Incubate at 4°C overnight or at room temperature for 2 hours. Rinse three times with PBS for 5 minutes to eliminate residues. 6) Secondary Antibody Incubation: Apply HRP- or fluorescently labeled secondary antibody dropwise and incubate at room temperature for 1 hour. Rinse three times with PBS for 5 minutes. 7) DAB staining: Prepare DAB working solution according to the reagent instructions and add dropwise to the sections. Monitor the color development time under a microscope. Rinse with running water to terminate the reaction. 8) Counterstain: Stain the nuclei with hematoxylin for 1 minute, rinse with running water to return to blue. Dehydrate with graded ethanol (70% → 80% → 95% → 100%), and clear with xylene. 9) Mounting and Observation: Mount the sections with neutral gum and observe under a microscope.

[0075] RNA extraction and real-time polymerase chain reaction (qRT-PCR)

[0076] Total RNA was isolated from LUAD cell lines using TRIzol reagent according to the manufacturer's instructions. cDNA synthesis was then performed using the PrimeScript RT Reagent Kit (TaKaPa, Dalian, China) according to the manufacturer's instructions. qRT-PCR was performed using SYBR Green Premix EXTaq (TaKaPa, Dalian, China). The primers used are listed in Table 1 below. −ΔΔCt Methods All mRNA expression levels were calculated and normalized to GAPDH expression.

[0077] Table 1 Primer sequences used for qRT-PCR analysis.

[0078]

[0079] Western Blotting

[0080] Cells were lysed using RIPA buffer supplemented with protease inhibitors, and protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). The membranes were blocked in 5% milk and Tris-buffered saline (Tris-buffered saline) containing 0.05% Tween 20 for 2 h at room temperature. Target antigens were probed with specific antibodies overnight at 4°C. The following antibodies were used: TFAM K76 acetylation antibody (3683; QYAOBI), BAX (50599-2-Ig; Tri-Tianjin Biotechnology, Wuhan, China); Bcl-2 (12789-1-AP; Tri-Tianjin Biotechnology, Wuhan, China); N-cadherin (22018-1-AP; Tri-Tianjin Biotechnology, Wuhan, China); Vimentin (10366-1-AP), KIM-1 (30948-1-AP), and β-actin (81115-1-RR). The membrane was washed in TBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Signal detection was performed using an enhanced chemiluminescence (ECL) detection system. The density of the β-actin band was normalized to the density of the band representing the specific protein.

[0081] Immunofluorescence experiments

[0082] Cells were seeded onto culture dishes or coverslips. Experiments were performed when the cells reached a density of 60%-80% adherent. Cells were then treated with high glucose. The culture medium was aspirated and the cells were gently rinsed twice with ice-cold PBS for 5 minutes. 4% paraformaldehyde (prepared in PBS) was added and fixed at room temperature for 15-20 minutes. After fixation, the cells were rinsed three times with PBS for 5 minutes. 0.3% Triton X-100 (prepared in PBS) was added dropwise for permeabilization for 10-15 minutes at room temperature. The cells were rinsed three times with PBS for 5 minutes. 3% BSA blocking solution was added dropwise for 30 minutes at room temperature to reduce nonspecific binding. The blocking solution was aspirated and the diluted primary antibody was added dropwise. The cells were incubated overnight at 4°C. The next day, the cells were rinsed three times with PBS for 5 minutes in the dark. A fluorescently labeled secondary antibody was added dropwise and incubated at room temperature for 1 hour in the dark. The cells were rinsed three times with PBS for 5 minutes (protected from light). DAPI (a fluorescent dye, 1 μg / mL, diluted in PBS) was added dropwise and incubated at room temperature for 5-10 minutes in the dark. Rinse with PBS three times for 5 minutes (protect from light). Remove excess liquid with filter paper, add anti-fading mounting medium, and cover with a coverslip. After drying in the dark, observe under a fluorescence microscope or confocal microscope.

[0083] Mitochondrial DNA copy number detection

[0084] Total DNA was extracted from cells, and the relative mitochondrial DNA copy number was estimated by detecting the mtDNA / nDNA ratio using fluorescence quantitative PCR.

[0085] Virtual screening based on TFAM crystal structure

[0086] 1) A virtual screening model was constructed based on the crystal structure of the TFAM complex (PDB code: 3TMM). 2) Residue K76 has been shown to be a key site for TFAM function. Therefore, a molecular docking site was defined centered on K76. A corresponding grid file was generated using Maestro software. Molecular docking was then performed against this grid file using an existing compound database (5,000 small molecules with high structural diversity). Based on the docking scores, the top 150 small molecules were selected. Cluster analysis was performed on these 150 small molecules, and based on the results, 68 were ultimately selected for subsequent activity testing.

[0087] Statistical analysis

[0088] Data were analyzed and plotted using GraphPad Prism 8. All experiments were performed at least three times. Student's t-test was used for comparisons between two groups, and analysis of variance (95% CI) was used for multiple comparisons. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) were used.

[0089] Experimental results:

[0090] Results 1 The acetylation level of TFAM K76 site was significantly increased in DKD patients and mouse tissues.

[0091] In this example, high glucose was used to induce renal tubular epithelial cells. Immunoprecipitation revealed that high glucose significantly increased the acetylation of TFAM ( Figure 1 ), but there are no reports on TFAM acetylation in diabetic nephropathy. The TFAM K76 site is a key site for regulating TFAM function. Previous studies of the present invention have found that TFAM K76 acetylation inhibits mitochondrial biogenesis and reduces oxidative phosphorylation levels. In this example, using cell immunofluorescence technology, it was found that high glucose-induced TFAM K76 acetylation in renal tubular epithelial cells was significantly increased ( Figure 2 ), and immunohistochemistry revealed that TFAM K76 acetylation was significantly increased in the kidney tissues of DKD patients and DKD mice ( Figure 3 , Figure 4 Subsequently, this example detected a significant increase in TFAM K76 acetylation in the kidney tissue of DKD mice using Western Blotting technology ( Figure 5 ).

[0092] Results 2 Acetylation of TFAM K76 site increased inflammation, fibrosis and mitochondrial damage in renal tubular epithelial cells.

[0093] In this example, we constructed a TFAM K76 site-mutant plasmid to simulate the acetylation and deacetylation of TFAM K76. Plasmid transfection was performed in HK-2 cells to express wild-type TFAM, acetylated TFAM K76, and deacetylated TFAM K76. Western blotting revealed that overexpression of acetylated TFAM K76 significantly increased fibrosis in HK-2 cells, whereas overexpression of deacetylated TFAM K76 significantly reduced fibrosis in HK-2 cells ( Figure 6 PCR detection showed that overexpression of TFAM K76 acetylation significantly increased the mRNA level of TGF-β1 in HK-2 cells, while overexpression of TFAM K76 deacetylation significantly decreased the mRNA level of TGF-β1 in HK-2 cells ( Figure 7 TFAM K76 site acetylation affects mitochondrial biogenesis. In the present invention, overexpression of TFAM K76 acetylation significantly reduced the protein level of electron transport complex subunits in HK-2 cells, while overexpression of TFAM K76 deacetylation significantly increased the protein level of electron transport complex subunits in HK-2 cells ( Figure 8 At the same time, this example detected the mitochondrial DNA (mtDNA) copy number and determined that overexpression of TFAM K76 acetylation significantly reduced the mtDNA copy number in HK-2 cells, while overexpression of TFAM K76 deacetylation significantly increased the mtDNA copy number in HK-2 cells ( Figure 9 ).

[0094] Results 3 Acetylation of TFAM K76 increased inflammation and fibrosis in the kidney tissue of DKD mice.

[0095] This example determined the effect of TFAM K76 site acetylation on HK-2 cells. To further verify the role of this site in mice, this example constructed a diabetic nephropathy mouse model. The successfully constructed mice were divided into 4 groups (8 mice in each group), and adeno-associated virus was injected into the tail vein of the 4 groups of diabetic mice to overexpress TFAM wild type, TFAM K76 acetylation, and TFAM K76 deacetylation in the kidneys. Subsequently, the present invention extracted total protein and total RNA from the mouse kidney tissues, and detected by Western Blotting technology that overexpression of TFAM K76 acetylation significantly increased the expression of kidney injury factor-1 (Kim-1) and increased kidney damage in diabetic mice, while overexpression of TFAM K76 deacetylation significantly reduced the expression of kidney injury factor-1 (Kim-1) and alleviated kidney damage in diabetic mice ( Figure 10In addition, this example also used qPCR technology to detect inflammatory factors in mouse tissues ( Figure 11 ) and chemokines ( Figure 12 ) mRNA expression levels, and found that overexpression of deacetylated TFAMK76 significantly reduced inflammation in the kidneys of diabetic mice. Therefore, this example demonstrates the important role of TFAM K76 acetylation in diabetic mice and suggests it is a key target for the treatment of diabetic nephropathy.

[0096] Results 4 Screening of small molecule compounds targeting the TFAM K76 acetylation site.

[0097] The present invention confirms that K76 is a key site of TFAM, and then screens small molecule compounds targeting the K76 site. Figure 13 As shown, first, this embodiment builds a virtual screening model based on the TFAM crystal structure (PDB code: 3TMM), defines the molecular docking site with K76 as the center, and then performs molecular docking on the existing compound database (200,000 small molecule compounds with high structural diversity) and the grid file. According to the docking scores, the top 150 small molecules are selected. Next, a cluster analysis is performed on the 150 small molecules, and based on the results of the cluster analysis, 68 small molecules are finally selected. Among these 68 small molecule compounds, some compounds were eliminated due to poor drugability, and then the remaining compounds were tested for activity and function. In this embodiment, the affinity between the compounds and TFAM was detected by BLI technology, and it was found that compounds 10, 14, 25, 34, and 35 have a high affinity with the TFAM protein. Next, this embodiment further screened the functions of the compounds by Western Blotting technology, and found that in a high sugar environment, only compound 14 could significantly reduce the acetylation level of TFAMK76 ( Figure 14 ), and the screened compound 14 was named S14. Moreover, S14 had the most significant downregulation of TFAM K76 acetylation at a concentration of 50 μM ( Figure 15 In HK-2 cells, compound 14 was found to significantly increase the thermal stability of TFAM protein after S14 intervention ( Figure 16 In addition, this example further demonstrates that the affinity of S14 for binding to TFAM protein increases in a concentration-dependent manner using BLI technology ( Figure 17 ), but cannot bind to the TFAM K76R mutant protein ( Figure 18 ), which further illustrates the targeting effect of S14 on the TFAM K76 site.

[0098] Among them, the chemical structure of S14 is: .

[0099] Results 5 Small molecule compounds alleviated high glucose-induced inflammation, fibrosis and mitochondrial damage in renal tubular epithelial cells.

[0100] To further verify the effect of the screened S14, this example treated HK-2 cells induced by high glucose with S14. qPCR and Western Blotting revealed that S14 treatment significantly increased the number of mitochondrial DNA (mtDNA) copies under high glucose induction. Figure 19 ) and electron transport complex subunit protein levels ( Figure 20 ), indicating that S14 treatment increased high glucose-induced mitochondrial biogenesis in HK-2 cells. In addition, this example detected inflammation and fibrosis changes by qPCR and Western Blotting, confirming that S14 treatment reduced high glucose-induced fibrosis and apoptosis in HK-2 cells ( Figure 21 ) and reduced the mRNA level of TGF-β1 in HK-2 cells induced by high glucose ( Figure 22 ). This example screened out a new small molecule compound for treating diabetic nephropathy based on protein acetylation regulation.

[0101] Results 6 Small molecule compounds alleviated inflammation and fibrosis in the renal tissue of DKD mice.

[0102] Subsequently, the present invention used S14 to treat diabetic mice. During the treatment process, this example also set up a dapagliflozin treatment group as a control. After the treatment, the total protein and total RNA of the mouse kidney tissue were extracted respectively. Western Blotting technology detection showed that S14 treatment significantly reduced the expression of kidney injury factor-1 (Kim-1) and alleviated the kidney damage of diabetic mice ( Figure 23 At the same time, inflammatory factors in mouse tissues were detected by qPCR technology ( Figure 24 ) and chemokines ( Figure 25 ) mRNA expression levels, and found that S14 treatment significantly reduced inflammation in the renal tissue of diabetic mice. Therefore, this example further confirms the therapeutic effect of S14 in delaying the progression of renal damage in diabetic mice, representing a newly discovered effective strategy for the treatment of diabetic nephropathy.

[0103] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a compound that reduces or inhibits the expression level of TFAM K76 acetylation modification in at least one of the following b1) to b4): b1) Preparation of products for reducing inflammation and / or fibrosis of renal tubular epithelial cells; b2) preparing products for reducing mitochondrial damage in renal tubular epithelial cells; b3) preparing products for screening to slow the progression of diabetic nephropathy; b4) Preparation of medicaments for treating diabetic nephropathy; The chemical structure of the compound is shown below: 。 2. The use according to claim 1, characterized in that: The diabetic nephropathy is a disease caused by diabetes and characterized by lesions of kidney tissue or renal tubular epithelial cells.

3. The use according to claim 1, characterized in that: The product is a medicine or an experimental reagent.

4. The use according to claim 1, characterized in that: The medicine is a pharmaceutical composition and also includes pharmaceutical excipients.

5. The use according to claim 4, characterized in that: The pharmaceutical excipients are carriers and / or excipients.

6. The use according to claim 5, characterized in that: The carrier and excipient are one or more of alumina, serum protein, lecithin, maltitol, starch, gum arabic, sucrose, lactose, alginate, glucose, sorbitol, mannitol, magnesium stearate, mineral oil, xylitol, erythritol, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, phosphate, and water.

7. The use according to claim 1, characterized in that: The subjects of administration of the drug are humans and non-human mammals.

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