Use of an asprosin small molecule inhibitor in the preparation of a medicament for preventing and / or treating diabetic nephropathy

Abstract

The application relates to the technical field of biological medicine, and discloses an application of an Asprosin small molecule inhibitor in preparation of a medicine for preventing and / or treating diabetic nephropathy, wherein the Asprosin small molecule inhibitor comprises ganoderic acid DM. The inhibitor ganoderic acid DM with Asprosin as a target is screened from a bioactive compound library through high-throughput virtual drug screening combined with bioactivity screening, it is found that the ganoderic acid DM can be specifically combined with Asprosin, and shows strong affinity, and is expected to provide an experimental basis for clinically treating and preventing DKD and new drug research and development.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of an Asprosin small molecule inhibitor in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy. Background Technology

[0002] Diabetic kidney disease (DKD) is a microvascular complication of diabetes and a leading cause of kidney failure (KF) requiring kidney transplantation (KT). Current basic treatment for DKD includes lifestyle interventions, target management of blood pressure and glycated hemoglobin (HbA1c), and the use of organ-protective drugs, including aspirin, statins, angiotensin-converting enzyme inhibitors / angiotensin receptor blockers (ACEIs / ARBs), and sodium-glucose cotransporter 2 inhibitors (SGLT-2i). While these drugs can effectively protect the kidneys and slow disease progression, some are either ineffective in certain patients or have been shown to have significant toxic side effects, failing to reduce the incidence of end-stage renal disease. Exploring new targets for specific treatment of DKD and developing highly effective drugs for both treatment and prevention has significant application value.

[0003] Asprosin (ASP), an adipokine hormone, stimulates appetite and regulates glucose and lipid metabolism. Studies have shown that serum ASP levels in patients with disseminated intravascular coagulation (DKD) are significantly higher than normal. Furthermore, ASP plays a crucial role in various metabolic diseases, including obesity, metabolic syndrome, and atherosclerotic cardiovascular disease. Therefore, developing asprosin inhibitors not only helps overcome the limitations of traditional drugs that focus solely on lowering blood sugar or lipids, but also holds promise for achieving multi-organ protective effects, providing important experimental evidence for the development of new drugs for the treatment of DKD.

[0004] Significant progress has been made in the treatment of disseminated intravascular coagulation (DKD), yet many limitations remain. Asprosin (ASP) is an important target for the treatment and prevention of DKD, and it plays a crucial role in regulating glucose and lipid metabolism homeostasis. However, no effective asprosin-specific inhibitors have yet been discovered. Summary of the Invention

[0005] To overcome the problems existing in the background technology, the present invention provides an application of an Asprosin small molecule inhibitor in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy. By using high-throughput virtual drug screening combined with bioactivity screening, an inhibitor of ganoderic acid DM targeting Asprosin was screened from a library of bioactive compounds. It was found that ganoderic acid DM can specifically bind to Asprosin and exhibits strong affinity, which is expected to provide an experimental basis for the clinical treatment and prevention of DKD and the development of new drugs.

[0006] In this invention, previous studies have shown that asprosin-regulated excessive SUMO1 modification of Drp1 leads to mitochondrial homeostasis disturbances, exacerbating renal tubular epithelial cell damage and phenotypic transformation in DKD. Therefore, the inventors believe that inhibiting asprosin activity may be an effective means of preventing and treating DKD. Developing inhibitors for DKD targeting asprosin not only ensures compatibility with traditional treatment strategies but may also offer superior efficacy and a lower risk of adverse reactions.

[0007] To achieve the above objectives, the present invention provides, in one aspect, the use of an asprosin small molecule inhibitor in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy, wherein the asprosin small molecule inhibitor comprises ganoderic acid DM, and the structural formula of ganoderic acid DM is as follows:

[0008] .

[0009] Compared with the prior art, the present invention has at least the following beneficial effects:

[0010] (1) The small molecule inhibitor of Asprosin provided by the present invention can not only specifically target Asprosin, but also has a high affinity for Asprosin, which can significantly reduce the level of Asprosin and effectively inhibit its biological activity, reverse the glucose and lipid metabolism disorder caused by Asprosin and alleviate renal dysfunction, thereby providing a new strategy for drug development for the prevention and / or treatment of DKD.

[0011] (2) Experimental results show that the Asprosin small molecule inhibitor described in this invention can reverse the ASP-induced phenotypic transformation of HK2 cells in HK2 cell experiments; the Asprosin small molecule inhibitor described in this invention can significantly improve and reverse renal function abnormalities and glucose metabolism disorders in diabetic mice after being used in diabetic mouse models, thereby preventing and / or treating DKD in mice. Attached Figure Description

[0012] Figure 1 This is a technical roadmap of a screening method for small molecule inhibitors of Asprosin in one embodiment of the present invention;

[0013] Figure 2 This is a graph showing experimental data on the bioactivity of ganoderic acid DM (GA-DM) in inhibiting asprosin in one embodiment of the present invention;

[0014] Figure 3 This is a graph showing experimental data on the binding mode and affinity of ganoderic acid DM (GA-DM) to asprosin in one embodiment of the present invention.

[0015] Figure 4This is a graph showing experimental data from an embodiment of the present invention showing that ganoderic acid DM (GA-DM) can reverse ASP-induced HK2 cell phenotypic transformation.

[0016] Figure 5 This is an experimental data graph showing how ganoderic acid DM (GA-DM) can improve the pathogenesis of DKD induced by Asprosin in one embodiment of the present invention. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] This invention provides, in one aspect, the use of an asprosin small molecule inhibitor in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy, wherein the asprosin small molecule inhibitor comprises ganoderic acid DM, and the structural formula of ganoderic acid DM is as follows:

[0019] .

[0020] In this invention, the asprosin small molecule inhibitor not only specifically targets asprosin but also has a high affinity for asprosin, significantly reducing asprosin levels and effectively inhibiting its biological activity. This reverses asprosin-induced glucose and lipid metabolism disorders in DKD and alleviates renal dysfunction, thus providing a new strategy for drug development to treat DKD.

[0021] According to one embodiment of the present invention, the ganoderic acid DM is obtained by the following screening method:

[0022] S1. Using Asprosin as the receptor protein, virtual screening was performed using the MCE small molecule database as the docking ligand library.

[0023] S2. Based on the virtual screening scores, the top 56 small molecule compounds were sorted and subjected to bioactivity screening to obtain ganoderic acid DM.

[0024] S3. The binding mode and affinity of ganoderic acid DM to asprosin were evaluated, and ganoderic acid DM was preliminarily identified as an inhibitor of asprosin.

[0025] In this invention, the screening method uses high-throughput virtual drug screening combined with bioactivity screening to screen for ganoderic acid DM, an inhibitor targeting asprosin, from a library of bioactive compounds. It was found that ganoderic acid DM can specifically bind to asprosin and exhibits strong affinity, which is expected to provide an experimental basis for the clinical treatment and prevention of DKD and the development of new drugs.

[0026] According to one embodiment of the present invention, the compounds are pretreated using Open Babel v3.1.1 software before virtual screening, including dehydration and hydrogenation; and the SDF format files of the compounds are output using MOE software before docking. High-throughput virtual screening is performed from a library of small natural compound ligand molecules using the virtual screening software Autodock Vina to obtain docking scoring data for small natural compound ligand molecules that inhibit Asprosin activity.

[0027] According to one embodiment of the present invention, bioactivity screening involves screening for small molecules that can inhibit the bioactivity of Asprosin, including Asprosin level inhibition screening, cAMP level inhibition screening, and CRE transcriptional activity inhibition screening.

[0028] According to one embodiment of the present invention, the methods for evaluating the binding mode and affinity of ganoderic acid DM to asprosin include molecular docking experiments, MD (molecular dynamics simulation) experiments, SPR (surface plasmon resonance) experiments, and CETSA (cell thermal displacement) experiments.

[0029] In this invention, previous studies have shown that asprosin can exacerbate renal tubular epithelial cell damage and phenotypic transformation in diabetic mice, suggesting that asprosin inhibitors may have a positive effect on improving renal function in patients with diabetic kidney disease (DKD). Therefore, ganoderic acid DM was used in animal experiments for intervention. The results showed that ganoderic acid DM significantly inhibited asprosin-induced phenotypic transformation of renal tubular epithelial cells in diabetic mice, while simultaneously improving renal function abnormalities and glucose metabolism disorders in diabetic mice, thereby preventing and / or treating DKD in mice. Furthermore, ganoderic acid DM was able to reverse ASP-induced phenotypic transformation of HK2 cells in in vitro cell experiments.

[0030] According to one embodiment of the present invention, the detection of the therapeutic effect of the asprosin small molecule inhibitor on DKD includes: detecting the effects on fasting plasma glucose (FPG), insulin tolerance (ITT), glucose tolerance (GTT), serum creatinine (Scr), blood urea nitrogen (BUN), and renal tubular epithelial cell phenotypic transformation-related proteins in mice.

[0031] According to one embodiment of the present invention, ganoderic acid DM is further determined to be an inhibitor of asprosin through in vitro and / or in vivo experiments.

[0032] The experimental results of this invention show that the Asprosin small molecule inhibitor described in this invention can reverse the ASP-induced HK2 cell phenotypic transformation in in vitro cell experiments; and that the Asprosin small molecule inhibitor described in this invention can significantly improve renal function abnormalities and glucose metabolism disorders in diabetic mouse models, thereby preventing and / or treating DKD in mice.

[0033] According to one embodiment of the present invention, the concentration range of ganoderic acid DM in in vitro cell experiments is 10~40 μM, and it has a significant inhibitory effect on asprosin within this concentration range.

[0034] According to one embodiment of the present invention, ganoderic acid DM can reverse ASP-induced HK2 cell phenotypic transformation.

[0035] According to one embodiment of the present invention, the half-maximal inhibitory concentration (IC50) of the small molecule inhibitor ganoderic acid DM on HK2 cells is 27.08 μM.

[0036] According to one embodiment of the present invention, the ganoderic acid DM is used as the sole active ingredient in the preparation of a drug for the prevention and treatment of diabetic nephropathy.

[0037] According to one embodiment of the present invention, ganoderic acid DM is administered orally at a daily dose of 5 mg / kg in mice.

[0038] According to one embodiment of the present invention, ganoderic acid DM prevents and treats diabetic nephropathy by targeting and inhibiting the activity of asprosin.

[0039] In this invention, the asprosin inhibitor ganoderic acid DM shows potential for combination therapy in DKD patients with multiple metabolic abnormalities.

[0040] The rationale is as follows: First, asprosin has been proven to be a key regulator of glucose and lipid metabolism. Multiple studies have shown that asprosin can exacerbate glucose and lipid metabolism disorders in diabetic patients; therefore, asprosin inhibitors hold promise for effectively improving this pathological condition. Second, clinical studies have shown that asprosin has a negative regulatory effect on renal function in DKD patients, and it can promote or exacerbate renal dysfunction in these patients.

[0041] According to one embodiment of the present invention, the drug screening process is as follows:

[0042] S1. Using Asprosin as the receptor protein, 3D homology modeling of Asprosin was performed on the Swiss Model website using the amino acid sequence of Asprosin to obtain the 3D structure of Asprosin; the small molecule database of MCE (HY-L1001) was used as the small molecule of natural compound ligand to complete the preparation of receptor and ligand.

[0043] Before screening, the compounds were pretreated using Open Babel v3.1.1 software, including dehydration and hydrogenation. Before docking, the SDF format files of the compounds were output using MOE software. The virtual screening software Autodock Vina was used to perform high-throughput virtual screening from a library of natural compound ligand small molecules to obtain docking scoring data of natural compound ligand small molecules with inhibitory Asprosin activity.

[0044] S2. Select the top 56 compounds with the lowest binding energy (the 56 compounds with the highest virtual screening scores) for further research, screening for small molecules that can inhibit the biological activity of Asprosin; the research mainly includes screening for Asprosin level inhibition, cAMP level inhibition, and CRE transcriptional activity inhibition; select compounds that can significantly inhibit the above indicators for further screening.

[0045] S3. To evaluate the binding mode and affinity between ganoderic acid DM and asprosin, molecular docking was performed using asprosin protein as the acceptor and small molecule ganoderic acid DM as the ligand. The surface structure of the binding between asprosin and ganoderic acid DM was displayed using PyMOL software, and the amino acid sites in asprosin protein that can form hydrogen bonds and van der Waals forces with ganoderic acid DM were displayed using Discovery Studio software. MD, SPR, and CETSA experiments were also performed.

[0046] In this invention, the MD experiment mainly simulates the stability and binding free energy of the Asprosin-DM complex under real solution conditions. The SPR experiment mainly detects the binding affinity between Asprosin and DM; a lower value indicates a higher affinity. The CETSA experiment detects the thermal stability of the Asprosin-DM complex; a significant increase in the thermal stability of Asprosin indicates a higher affinity.

[0047] like Figure 1As shown, the inventors screened the Asprosin inhibitor ganoderic acid DM (GA-DM) from a library of 27,015 small molecule compounds using virtual drug screening technology, asprosin level inhibition screening, cAMP level inhibition screening, CRE transcriptional activity inhibition screening, molecular docking, molecular dynamics simulation (MD), surface plasmon resonance (SPR), and cell thermal displacement (CETSA) experiments.

[0048] The present invention will be described in detail below through embodiments. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. All reagents or instruments whose manufacturers are not specified are conventional products that can be purchased through legitimate channels.

[0049] Exploration Example 1

[0050] Induction and culture of 3T3-L1 adipocytes

[0051] 3T3-L1 preadipocytes (ATCC, Catalogue No. CRL-1730, USA) were cultured in high-glucose medium (H-DMEM) containing 10% fetal bovine serum in a 37°C, 5% CO2 cell incubator. When the cells reached 95% confluence, adipogenesis induction was initiated. The induction method was as follows: Freshly prepared induction medium I (H-DMEM solution containing DEX (1 μmol / L), IBMX (0.5 mmol / L), INS (10 μg / mL), and 10% FBS) was used for 2 days, followed by induction medium II (H-DMEM solution containing INS (10 μg / mL) and 10% FBS) for 4 days, changing the medium every two days. Finally, the cells were cultured in complete H-DMEM medium containing 10% FBS for 48 hours to obtain adipogenesis-induced 3T3-L1 adipocytes. The cells were then divided into the following groups:

[0052] Normal control group (Ctrl);

[0053] Solvent control group (DMSO);

[0054] Drug treatment group (GA-DM (10 μM)), treated with 10 μM GA-DM for 48 h;

[0055] Drug treatment group (GA-DM (20 μM)), treated with 20 μM GA-DM for 48 h;

[0056] Drug treatment group (GA-DM (40 μM)), treated with 40 μM GA-DM for 48 h;

[0057] The level of asprosin in cell supernatant was detected using a commercially available ELISA kit (Nanjing, Camillo, 2M-KMLJM221034m).

[0058] Exploration Example 2

[0059] Culture of HepG2 liver cancer cells

[0060] HepG2 cells (ATCC, Catalogue No. CRL-1730, USA) were cultured in high-glucose medium (H-DMEM) containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 cell incubator. When the cells reached 70%-80% confluence, they were divided into the following groups:

[0061] Control group (Ctrl);

[0062] The Asprosin group (ASP) was treated with 50 nM recombinant Asprosin protein (Qiangyao Biotechnology, Suzhou, China) for 48 h.

[0063] The ASP+GA-DM (10 μM) group was treated with 50 nM Asprosin recombinant protein for 48 h, and then treated with 10 μM GA-DM for 48 h.

[0064] The ASP+GA-DM (20μM) group was treated with 50nM Asprosin recombinant protein for 48h and then with 20μM GA-DM for 48h.

[0065] The ASP+GA-DM (40μM) group was treated with 50nM Asprosin recombinant protein for 48h and then with 40μM GA-DM for 48h.

[0066] After receiving appropriate treatment, HepG2 cells in each group were lysed and the lysate supernatant was collected. The cAMP level in HepG2 cells was detected using a commercially available ELISA kit (Nanjing, Camillo, 2H-KMLJh310709).

[0067] Exploration Example 3

[0068] Dual-luciferase reporter gene plasmid transfection

[0069] HepG2 cells were seeded in 24-well plates (6 × 10⁻⁶ cells per well). 4Cells / well were treated according to the method described in Example 2. Then, using lip-3000 liposomes, 250 ng of a CRE overexpression plasmid containing firefly luciferase (vector element sequence: pGL4.29-luc2P-3*CRE-Hygro) and 25 ng of a Renilla luciferase internal control plasmid (vector element sequence: TK promoter-Renilla_Luciferase) were added to the cells of each experimental group at a ratio of 10:1 for co-transfection. The specific transfection steps are as follows:

[0070] (1) Use a pipette to take 250 μL of Opti-MEM Medium into an EP tube, then add 250 ng of CRE overexpression plasmid and 25 ng of Renilla luciferase internal control plasmid, and gently shake to mix thoroughly. This is solution A.

[0071] (2) Then take 9 μL of lipofectamine 3000 liposomes into another new EP tube, add 250 μL of Opti-MEM medium, and gently shake to mix thoroughly, which is solution B;

[0072] (3) Use a pipette to aspirate solution A into solution B, gently shake to mix, centrifuge, and let stand at room temperature for more than 10 minutes but no more than 30 minutes;

[0073] (4) The liposome-DNA complex was obtained and then added dropwise to the cell culture dish. After shaking slowly and evenly, it was placed on the clean bench for several minutes and then transferred to the incubator for 24 hours of further culture.

[0074] Dual-luciferase reporter gene assay

[0075] The dual-luciferase reporter gene assay was performed using a commercially available kit (Yisheng Biotechnology, Shanghai, 11402ES60). The specific steps are as follows:

[0076] (1) Use a pipette to remove the cell culture medium, add PBS to wash, and then aspirate the residual solution;

[0077] (2) Add 200 μL of cell lysis buffer to the culture wells, shake gently to ensure that the lysis buffer fully covers the cells, place the cell culture plate on an ice box, and shake gently on a shaker at room temperature and low speed for 5 minutes to fully lyse the cells;

[0078] (3) Take 20 μL of lysis buffer and add it to another black microplate. Then add 100 μL of Firefly luciferase reaction solution, vortex gently to mix, and immediately detect the activity of firefly luciferase.

[0079] (4) Add 100 μL of Renilla luciferase reaction solution, immediately detect the activity of Renilla luciferase, and finally perform statistical analysis.

[0080] The results of Exploration Examples 1-3 are as follows Figure 2 As shown: A: Asprosin level in the culture supernatant of induced mature 3T3-L1 adipocytes; B: cAMP level in HepG2 cells; C: Effect of GA-DM on CRE transcriptional activity detected by a dual-luciferase reporter gene system. Compared with the Ctrl group or DMSO group, *** P<0.001, compared with the ASP group, ## P<0.05, ### P < 0.001, ns: no statistical significance;

[0081] Results Analysis: To evaluate whether GA-DM can act as a bioactive inhibitor of ASP, the inventors verified its efficacy from two aspects: its physiological origin and its main pathway of action. First, given that ASP is mainly synthesized and secreted by white adipocytes, the inventors tested the effect of GA-DM in a induced mature 3T3-L1 adipocyte model. The results showed that GA-DM treatment significantly reduced the ASP content in the cell culture supernatant in a concentration-dependent manner. Figure 2 (A) This suggests that GA-DM can inhibit the synthesis or secretion of ASP. Secondly, based on the fact that ASP promotes glycogen release by activating the cAMP-PKA signaling pathway, the inventors further investigated the effect of GA-DM on this pathway. In HepG2 cells, ASP treatment significantly increased intracellular cAMP levels, while GA-DM co-treatment effectively reversed the ASP-induced increase in cAMP, and the reversal effect was stronger with increasing GA-DM concentration. Figure 2 (B) To confirm whether this upstream signal inhibition is transmitted to downstream gene transcription, the inventors used a cAMP response element (CRE) dual-luciferase reporter gene system to detect the effect of GA-DM on CRE transcriptional activity. The results showed that GA-DM can, in a concentration-dependent manner, inhibit CRE-mediated transcriptional activity induced by ASP stimulation (…). Figure 2(C). In summary, this study demonstrates that GA-DM not only inhibits ASP levels at the source of secretion but also antagonizes ASP-mediated activation of the cAMP-PKA-CREB signaling pathway in target cells. These findings collectively confirm that GA-DM is an effective small-molecule inhibitor of ASP.

[0082] Exploration Example 4

[0083] Molecular docking

[0084] Based on the known amino acid sequence of Asprosin, the 3D structure of Asprosin was constructed using the SWISS-MODEL website (https: / / swissmodel.expasy.org / ). Subsequently, the 3D structure of GA-DM (CID: 11784642) was obtained from the PubChem compound database. GA-DM was then preprocessed using Open Babel v3.1.1 software. After preparing the ligand and acceptor, molecular docking was performed using Autodock Vina 1.2.5 software. Finally, data analysis and processing were performed using Pymol 1.7.6 and Discovery Studio 2019 software.

[0085] Exploration Example 5

[0086] Molecular dynamics simulation (MD)

[0087] MD simulations of Asprosin and GA-DM were performed using the GROMACS 2025.2 software package. The system was placed in a periodic boundary cubic box, solvated using the CHARMM36 force field and TIP3P water model, and ions were added to a physiological concentration (150 mM NaCl). After stepwise NVT / NPT equilibration (100 ps each) under energy minimization and position constraints, a 100 ns production simulation was performed (integration step size 2 fs). The trajectory was processed to remove periodic artifacts and the system was centered. After the simulation, key parameters of the complex, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg), were calculated using Gromacs' built-in tools.

[0088] Exploration Example 6

[0089] Surface plasmon resonance (SPR) experiment

[0090] SPR analysis was performed using the Biacore 8K system to assess the binding affinity of GA-DM to Asprosin protein. The CM5 sensor chip was activated with N-hydroxysulfosuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyliodide (EDC) and then coupled with purified Asprosin protein (MedChemExpress, HY-P7612). Asprosin was diluted to 10 μg / ml with acetate buffer (pH=5.0), and the surface was then blocked with ethanolamine to prevent nonspecific binding. A gradient concentration of GA-DM (1.5625–50.0 μM) was flowed through the chip surface at a flow rate of 30 μl / min (the flow buffer consisted of PBS buffer containing 0.05% Tween 20 and 5% DMSO) for 60 seconds. Finally, the data were processed and analyzed using Biacore evaluation software (T200 v.1.0).

[0091] Exploration Example 7

[0092] Cellular thermal displacement assay (CETSA)

[0093] 3T3-L1 adipocytes were induced to mature using the method described in Example 1, and then collected by trypsin digestion and centrifugation. The cells were resuspended in PBS buffer containing protease inhibitors, then flash-frozen in liquid nitrogen for 3 minutes, thawed at room temperature, and subjected to three freeze-thaw cycles to extract cellular proteins. After freeze-thaw cycles, the samples were centrifuged (4℃, 12,000g, 20min), and the supernatant was collected. The protein supernatant was then divided into two groups: a GA-DM group and a DMSO group. The GA-DM group was incubated with GA-DM (270μM) and the cell suspension at room temperature for 2 hours, while the control group was incubated with an equal volume of DMSO for 2 hours. The supernatant was then aliquoted into EP tubes and heated at gradient temperatures (37, 40, 43, 46, 49, 52, 55, and 56℃) for 3 minutes each, followed by centrifugation (4℃, 15,000g, 40min) to collect the supernatant. Finally, protein detection and quantification were performed using Western blot.

[0094] The results of Exploration Examples 4-7 are as follows Figure 3As shown: A: Molecular docking experiments to detect the main interaction sites between GA-DM and Asprosin; BD: Molecular dynamics simulation (MD) experiments to detect the root mean square deviation (RMSD), root mean square fluctuation (RMSF) of protein residues, and radius of gyration (Rg) of the GA-DM–Asprosin complex system; EF: Surface plasmon resonance (SPR) technology to evaluate the binding ability of GA-DM and Asprosin (E), and the binding dissociation constant Kd value (F) was calculated by curve fitting; GH: Cell thermal translocation assay (CETSA) to analyze the effect of GA-DM on the thermal stability of Asprosin protein in HK2 cells.

[0095] Results Analysis: To investigate the interaction between GA-DM and ASP, the inventors first used molecular docking to evaluate their binding mode and binding free energy. The results showed that the binding free energy (ΔG) was –8.763 kcal / mol, indicating a good binding potential. Further analysis of the binding mode revealed that GA-DM can interact with multiple amino acid residues of ASP, including forming hydrogen bonds with ASP129, SER102, LEU131, TRP25, and ASN60, and generating van der Waals forces with residues such as ASN130 and SER24, indicating a strong intermolecular interaction between GA-DM and ASP. Figure 3 (A). To evaluate the dynamic stability of the GA-DM-ASP complex in solution, the inventors conducted MD simulations. The root mean square deviation (RMSD) of the system reached equilibrium after approximately 60 ns and remained stable for the remainder of the simulation time with a small fluctuation range, indicating that the complex structure had reached sufficient equilibrium and a stable state. Figure 3 (B). Root mean square fluctuation (RMSF) analysis of protein residues showed low overall conformational fluctuation of the complex, with most residues having RMSF values ​​between 0.1 and 0.5 nm, suggesting that GA-DM binding helps stabilize the structure of ASP. Figure 3 (C). Furthermore, the radius of gyration (Rg) remained stable and low throughout the simulation, further indicating that the combination of GA-DM resulted in a more compact overall conformation of the ASP, reduced local flexibility, and thus enhanced the structural stability of the complex. Figure 3 To further verify the affinity between the two at the experimental level, the inventors conducted SPR analysis. The SPR results showed that the binding dissociation constant (Kd) of GA-DM and ASP was 4.366 μM, indicating a strong affinity between the two. Figure 3 E, Figure 3(F). Meanwhile, CETSA results showed that, compared with the control group, GA-DM treatment significantly improved the thermal stability of ASP at 43℃, further confirming the direct binding of GA-DM and ASP in the cellular environment. Figure 3 China G, Figure 3 (H). In summary, GA-DM can directly bind to ASP with high affinity and effectively stabilize its protein structure, making it a promising ASP inhibitor.

[0096] Example 1

[0097] Culture of human renal proximal tubular epithelial cells (HK2)

[0098] HK2 cells (ATCC, Catalogue No. CRL-1730, USA) were cultured in F12 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 cell incubator. The effect of GA-DM on HK2 cell viability was first assessed using a CCK8 assay, as follows:

[0099] (1) After resuspending, HK2 cells were evenly seeded into 96-well plates (4×10⁻⁶ cells per well). 4 (1 cell / well) After the cells have grown to 70-80% confluence, they are treated with GA-DM at different concentration gradients (0, 5, 10, 20, 40, 80, 160, 320 μM). Each group has 3 replicates and a blank control group. The cells are then incubated at 37°C in a 5% CO2 cell incubator for 48 h.

[0100] (2) Prepare CCK-8 solution at a volume of 100 μL / well, mix 90 μL of complete culture medium with 10 μL of CCK-8 reagent thoroughly, and continue to incubate at 37°C in a 5% CO2 cell culture incubator for about 1 hour.

[0101] (3) The OD value of each well was detected at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The effect of different concentrations of GA-DM treatment on the viability of HK2 cells was statistically analyzed using Graphpad Prism software.

[0102] HK2 cells (ATCC, Catalogue No. CRL-1730, USA) were cultured in F12 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 cell incubator. When the cells reached 60% confluence, the effect of GA-DM on HK2 cell phenotypic transformation was investigated. The procedure was as follows: 40 mM high-concentration glucose was added and the cells were treated for 48 h. Then, the cells were divided into four groups: DMSO group, GA-DM (10 μM) group, DMSO+ASP (20 nM) group, and GA-DM+ASP group. After 48 h of treatment with ASP and GA-DM, corresponding indicators were measured.

[0103] The results of Example 1 are as follows Figure 4 As shown: A: Effects of gradient concentrations of GA-DM on HK2 cell viability; B: Detection of IC50 of GA-DM on HK2 cells; C: Effects of GA-DM on the expression levels of ASP-induced epithelial markers E-Cadherin (C, D) and mesenchymal markers Col III (C, E), Vimentin (C, F), and α-SMA (C, G) proteins in HK2 cells. Compared with the Ctrl+DMSO group, ** P<0.01, *** P<0.001, compared with the ASP group, ## P<0.01, ns: no statistical significance;

[0104] Results Analysis: In the in vitro experiment, the inventors selected HK2 cells. First, they treated HK2 cells with gradient concentrations of GA-DM (0, 5, 10, 20, 40, 80, 160, and 320 μM) for 48 hours. Then, the cell viability was detected using the CCK8 assay, revealing that cell viability gradually decreased with increasing drug concentration. Figure 4 (A), and then the inventors fitted the IC50 curve and found that IC50 = 27.08 μM ( Figure 4 (B). The inventors then selected a cell-free concentration, 10 μM, for subsequent experiments. Phenotypic transformation is a non-adaptive alteration process that occurs after HK2 cells are damaged. Western blotting results showed that GA-DM intervention significantly reversed the ASP-induced decrease in the expression of the epithelial marker E-Cadherin and the increase in the expression of the mesenchymal markers α-SMA, ColIII, and Vimentin in HK2 cells. Figure 4 C- Figure 4 (G). This indicates that GA-DM can improve ASP-induced phenotypic transformation of HK2 in vitro.

[0105] Example 2

[0106] Animal experiments and grouping

[0107] The mice used in this study were purchased from Southern Model Biotechnology Co., Ltd. (Shanghai, China). The experimental protocol was approved by the Laboratory Animal Ethics and Welfare Committee of Nanchang University (Approval No.: NCULAE-20241213001) and strictly followed the "Laboratory Animal Care and Use Guidelines" (8th Edition) published by the National Academy of Sciences Press in 2011. The experimental animals were housed in standard cages with an ambient temperature controlled at 23±1℃, humidity maintained at 50%-60%, a 12-hour light / dark cycle, and ample drinking water provided. First, a diabetic (DM) mouse model was established using a high-fat diet (HFD, 60% high-fat diet) combined with streptozotocin (STZ, 45 mg / kg, IV). The mice were then fed HFD for 12 weeks to induce a diabetic nephropathy (DKD) mouse model. After successful induction of the DM mouse model, the mice were divided into the following four groups:

[0108] NS (normal saline) + DMSO group;

[0109] NS+GA-DM group;

[0110] ASP (0.5 μg / kg / d, IV) + DMSO group;

[0111] ASP+GA-DM (5 mg / kg / d, ig) group;

[0112] In the ASP+DMSO and ASP+GA-DM groups, recombinant asprosin protein (0.5 μg / kg / d, iv) was administered intraperitoneally for eight consecutive weeks during the subsequent feeding process. After eight weeks, mice in the NS+GA-DM and ASP+GA-DM groups were administered GA-DM by gavage (5 mg / kg / d, ig) for four weeks during the subsequent feeding process. Finally, the mice were euthanized with 3% (v / v) isoflurane. Samples were collected to investigate the therapeutic effect of GA-DM on diabetic kidney disease (DKD), including its effects on fasting plasma glucose (FPG), insulin tolerance (ITT), glucose tolerance (GTT), serum creatinine (Scr), blood urea nitrogen (BUN), and renal tubular epithelial cell phenotypic transformation-related proteins in DM mice, to determine the therapeutic effect of GA-DM on DKD.

[0113] Kidney function index testing

[0114] Serum creatinine (Scr) was measured using a creatinine assay kit (Nanjing Jiancheng, C011-2-1). Blood urea nitrogen (BUN) was measured using a urea assay kit (Nanjing Jiancheng, C013-2-1). Urinary albumin was measured using an albumin assay kit (Nanjing Jiancheng, A028-2-1). The experimental procedures strictly followed the operating specifications provided by the manufacturer.

[0115] Western Blot

[0116] Kidney tissue or HK2 cell samples were placed on ice and lysed using RIPA lysis buffer (KGI Biotech, Jiangsu, China, KGB5203-100). After lysis, the samples were centrifuged at 12000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit (Beijing Seven, SW101). Next, 30 μg of protein sample was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was then blocked in 7% skim milk for 2 hours, followed by overnight incubation with primary antibody at 4°C. Afterward, HRP-labeled secondary antibody (1:2000 dilution, Boster Biological, Wuhan, BA1050, BA1054) was added for incubation. Finally, the gel was developed using an enhanced chemiluminescence (ECL) substrate on a gel imaging system (Bio-Rad Laboratories, USA), and the results were analyzed using ImageJ software. The specific information of the antibodies used in this study is as follows: anti-E-Cadherin (1:1000, ZhengNeng Biotechnology, Chengdu, China, #340341), anti-Col III (1:1000, ZhengNeng Biotechnology, Chengdu, China, #R23957), anti-Vimentin (1:1000, HuaAn Biotechnology, Zhejiang, China, #ET1610-39), anti-α-SMA (1:1000, AiFei Biotechnology, Jiangsu, China, #AF1032), anti-β-actin (1:1000, Boster Biologics, Hubei, China, #BA2305), anti-β-Tubulin (1:2000, SanYing Biotechnology, Hubei, China, #10094-1-AP), anti-ASP (1:1000, AdipoGen, Switzerland, #Q61554);

[0117] Glucose tolerance test (GTT) and insulin tolerance test (ITT) in mice

[0118] Mice were fasted for 4 hours before the ITT experiment. They were then anesthetized with 3% isoflurane, and plasma glucose (FPG) was recorded at 0 minutes via tail vein sampling. Subsequently, mice were intraperitoneally injected with glucose solution (2 g / kg) or insulin (1 U / kg). Plasma glucose levels were measured at 15, 21, 30, 60, 90, 120, 180, and 150 minutes after injection. Time-concentration curves were plotted, and the area under the curve (AUC) was calculated. For the GTT experiment, mice were fasted for 12 hours before the experiment, and subsequent procedures were the same.

[0119] The results of Example 2 are as follows Figure 5As shown: A: Effect of GA-DM on fasting plasma glucose (FPG) in Asprosin-induced diabetic mice; BC: Effect of GA-DM on glucose tolerance (GTT) in Asprosin-induced diabetic mice; DE: Effect of GA-DM on insulin sensitivity (ITT) in Asprosin-induced diabetic mice; F: Effect of GA-DM on serum creatinine (Scr) levels in Asprosin-induced diabetic mice; G: Effect of GA-DM on blood urea nitrogen (BUN) levels in Asprosin-induced diabetic mice; H: Effect of GA-DM on urinary albumin to creatinine ratios (UACRs) in Asprosin-induced diabetic mice; IM: Effect of GA-DM on the expression levels of epithelial markers E-Cadherin (I, J) and interstitial markers Col III (I, K), Vimentin (I, L), and α-SMA (I, M) proteins in Asprosin-induced diabetic mice. Compared with the NS+DMSO group, ** P<0.01, *** P<0.001, compared with the ASP+DMSO group, ## P<0.01, ns: no statistical significance;

[0120] Results analysis: GA-DM can improve the pathogenesis of ASP-induced DKD.

[0121] The inventors used GA-DM on diabetic mice and found that GA-DM not only significantly reduced fasting plasma glucose (FPG) in diabetic mice, but also improved the further increase in blood glucose induced by ASP intervention in diabetic mice. Figure 5 (A). Detection of GTT and ITT in mice revealed that GA-DM intervention significantly improved glucose tolerance in DM mice or ASP-interventional DM mice. Figure 5 B, Figure 5 (C) and improve insulin sensitivity ( Figure 5 D, Figure 5 (E). This indicates that GA-DM plays a positive regulatory role in glucose metabolism in DM mice. Next, the inventors found that GA-DM can significantly improve ASP-induced renal function abnormalities in DM mice, mainly manifested in a lower circulating Scr (Scr) compared to the ASP+DMSO group. Figure 5 Medium F), BUN ( Figure 5 The levels of urinary globulin (G) and the urinary protein to creatinine ratio (UACRs) were significantly reduced. Figure 5The results (H) indicate that GA-DM reversed the progression of DKD. The inventors extracted proteins from the renal cortex of mice and performed Western blotting experiments to observe the effect of GA-DM on the phenotypic transformation of renal tubular epithelial cells in ASP-induced diabetic mice. They found that after GA-DM intervention, the phenotypic transformation in ASP-induced diabetic mice was weakened, mainly manifested by increased levels of the epithelial marker E-Cadherin, while the levels of interstitial markers Col III, Vimentin, and α-SMA decreased. These results indicate that GA-DM can improve the progression of ASP-induced DKD.

[0122] In one embodiment of the present invention, the data analyzed are expressed as mean ± standard error. Statistical analysis between two groups uses an unpaired t-test, and comparisons among multiple groups use one-way or two-way ANOVA combined with Tukey's multiple comparison test. P < 0.05 is considered statistically significant. Statistical analysis is performed using GraphPad Prism 8.0 (GraphPad Software).

[0123] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The use of an asprosin small molecule inhibitor in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy, characterized in that, The Asprosin small molecule inhibitor is ganoderic acid DM, and the structural formula of ganoderic acid DM is as follows: 。 2. The application according to claim 1, characterized in that, Ganoderic acid DM was further confirmed as an inhibitor of asprosin through in vitro and / or in vivo experiments.

3. The application according to claim 2, characterized in that, In in vitro cell experiments, the concentration range of ganoderic acid DM was 10~40 μM.

4. The application according to claim 2, characterized in that, Ganoderic acid DM can reverse the phenotypic transformation of HK2 cells induced by Asprosin.

5. The application according to claim 4, characterized in that, The half-maximal inhibitory concentration (IC50) of ganoderic acid DM on HK2 cells was 27.08 μM.

6. The application according to claim 2, characterized in that, Ganoderma lucidum DM is administered orally at a daily dose of 5 mg / kg in mice.

7. The application according to claim 2, characterized in that, Ganoderic acid DM prevents and treats diabetic nephropathy by targeting and inhibiting the activity of asprosin.

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