Application of small molecule compound N106 in preparation of medicine for treating fatty liver disease related to metabolic dysfunction

Abstract

The invention discloses an application of a SUMO agonist N106 (the molecular formula is C17H14N4O3S, and the molecular weight is 354.38) in treatment or prevention of metabolic dysfunction related fatty liver disease (MASLD). The early-stage research of the invention finds that the liver SUMOylation level in MASLD clinical specimens and animal models is significantly reduced. In-vivo experiments show that in a high-fat diet induced C57BL / 6 mouse model, N106 (10mg / kg, intraperitoneal injection, once a day) can significantly improve liver lipid deposition and blood fat level of a high-fat diet mouse; in in-vitro experiments, N106 (10 mu M for 24 hours) can effectively reduce lipid droplet accumulation of HepG2 cells. The research reveals that N106 regulates lipid metabolism by activating an SUMOylation modification pathway for the first time, and a novel treatment strategy is provided for MASLD.

Description

Technical Field

[0001] The present invention relates to the field of the treatment of metabolic diseases, and particularly to the application of the SUMOylation agonist N106 in the treatment of metabolic dysfunction-related fatty liver disease, and its molecular mechanism of improving lipid metabolism by activating the SUMOylation modification pathway. Background Art

[0002] MASLD is characterized by excessive liver lipid deposition, and its global prevalence has been increasing year by year, but the existing treatment methods are limited. More and more evidence shows that SUMOylation modification can affect the stability and activity of key proteins in metabolic pathways and plays an important role in the occurrence and development of MASLD. Existing drugs mostly target single pathways and cannot globally regulate the SUMOylation network. As a novel SUMOylation small molecule agonist, N106 can directly enhance the SUMOylation modification efficiency, providing a new idea for the treatment of MASLD. Summary of the Invention

[0003] The present invention provides a new application of the SUMOylation agonist N106 in the treatment of metabolic dysfunction-related fatty liver disease (MASLD), and reveals its role in regulating the SUMOylation modification network to improve liver lipid metabolism disorder through systematic experiments. Specifically, the following core findings are included:

[0004] 1. The SUMOylation level in the liver of MASLD patients is significantly reduced

[0005] Through the analysis of clinical liver biopsy specimens, it is found that the protein levels of SUMO1 and SUMO2 / 3 in the liver of MASLD patients are significantly decreased compared with those in the healthy control group. This result indicates that the impaired SUMOylation function in the liver is an important pathological feature of MASLD.

[0006] 2. N106 significantly enhances the SUMOylation level in cells and animal models

[0007] Cell experiment: In HepG2 cells treated with palmitate (PA, 0.25 mM, 24 hours), the intervention of N106 (10 μM, 24 hours) can reverse the decreased protein levels of SUMO1 / 2 / 3 proteins due to PA treatment.

[0008] Animal experiment: In C57 mice fed a high-fat diet (HFD) for 12 weeks, starting from the 8th week, N106 (10 mg / kg) was intraperitoneally injected daily for 4 consecutive weeks. After that, the protein levels of SUMO1 / 2 / 3 in the liver were significantly increased compared with those in the model group.

[0009] 3. N106 inhibits lipid deposition through the SUMOylation-dependent pathway

[0010] Cell model: After HepG2 cells treated with PA (for 24 hours) were intervened with N106, Oil Red O staining showed that the lipid droplet area was significantly reduced.

[0011] Animal model: The liver lipid deposition area of mice in the N106 intervention group was significantly reduced compared with that in the model group, and the serum triglyceride (TG) and total cholesterol (TC) also decreased significantly.

[0012] The beneficial effects of the present invention are as follows: The present invention first reveals that the SUMOylation agonist N106 realizes the therapeutic effect of multi-dimensional regulation of lipid metabolism by targeting the SUMOylation modification network of hepatocytes, providing an innovative and clinically translatable therapeutic strategy for MASLD. N106 can be developed into an injection or oral preparation for the clinical treatment of MASLD. Brief Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0014] Figure 1 : Chemical structure formula and molecular weight of N106;

[0015] Figure 2 : WB detection of SUMO1 and SUMO2 / 3 proteins in clinical MASLD patients and healthy control groups;

[0016] Figure 3 : WB detection of SUMO1 and SUMO2 / 3 proteins in the liver of mice after intraperitoneal injection of N106 and control solvent respectively; Figure 4 : WB detection of SUMO1 and SUMO2 / 3 proteins in HepG2 cells after treatment with N106 (for 12 and 24 hours) and control solvent respectively;

[0017] Figure 5 : Comparison of H&E and Oil Red O staining in the liver of mice (control group, high-fat group, high-fat + N106 group);

[0018] Figure 6 : Serum triglyceride and total cholesterol levels in mice (control group, high-fat group, high-fat + N106 group);

[0019] Figure 7 : Comparison of immunohistochemical staining of SUMO1 and SUMO2 / 3 proteins in the liver of mice (control group, high-fat group, high-fat + N106 group);

[0020] Figure 8: Oil Red O staining of HepG2 cells (PA, PA+N106). Specific implementation methods

[0021] Clinical specimens: Specimens from patients undergoing hepatic hemangioma resection at the First Affiliated Hospital of Xi'an Jiaotong University were collected. Among them, 6 cases with metabolic associated fatty liver disease (MASLD) were used as the case group, and 6 cases without MAFLD were used as the control group. Exclusion criteria included severe liver disease, digestive tract diseases, tumors, alcoholism, etc. Liver tissue sections were evaluated by blind SAF scoring. This study was approved by the ethics committee of this hospital.

[0022] Experimental animals and model construction: In this study, healthy male C57BL / 6J wild-type mice aged 6-8 weeks (18-24 g) were used. All mice were housed in the SPF environment (22 °C, 12 h day-night cycle) of the Experimental Animal Center of Xi'an Jiaotong University. The experiments followed the ethical standards of Chinese experimental animals and were approved by the medical ethics committee of this university. All mice were euthanized by isoflurane anesthesia. Male C57BL / 6J mice aged 6-8 weeks were randomly divided into a control group (normal diet), a model group (high-fat diet, HFD), and an N106 intervention group (HFD+10 mg / kg N106). From the 8th week of modeling, the N106 group was intraperitoneally injected with N106 (dissolved in DMSO) daily for 4 consecutive weeks; the control group was injected with an equal volume of solvent.

[0023] HepG2 cell culture and treatment: Cells were routinely cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin-streptomycin), placed in an incubator at 37 °C and 5% CO2, and passaged by digestion with 0.25% trypsin every 2-3 days (1:3-1:5) to keep the cells in the logarithmic growth phase. When the cell density reached 60%, 0.25 mM palmitate and 10 μM N106 were added for 24 hours, and the control group was added with 0.25 mM palmitate and the control solvent DMSO.

[0024] HE staining of tissues: Mouse liver tissues were fixed with 4% paraformaldehyde for 24 hours (4 °C), dehydrated with gradient alcohol (70%→100%) for 1 hour each, cleared with xylene and then embedded in paraffin. Sections of 4-5 μm were cut and pasted and dried. After dewaxing and rehydration, first stained with hematoxylin for 3-5 minutes, differentiated with hydrochloric acid alcohol and blued with running water, then counterstained with eosin for 1-2 minutes, dehydrated with gradient alcohol, cleared with xylene, and sealed with neutral gum for microscopic examination. The cell nuclei were blue and the cytoplasm was pink.

[0025] Oil Red O staining of tissues: Fresh mouse liver tissues were quickly frozen in liquid nitrogen and then prepared into 5-8 μm frozen sections. After being fixed with 4% paraformaldehyde for 10 minutes, they were rinsed with running water and then rinsed with 60% isopropanol. Under light-proof conditions, they were stained with freshly prepared Oil Red O working solution (3:2) at 37°C for 15-20 minutes, differentiated with 60% isopropanol for 10 seconds, counterstained with hematoxylin for 1 minute and blued with running water, and then sealed with glycerin gelatin and examined under a microscope. Lipid droplets were bright red and cell nuclei were blue.

[0026] Immunohistochemical staining: Paraffin sections were dewaxed with xylene and rehydrated with gradient ethanol, then antigen heat repair was performed (citrate buffer, pH 6.0), endogenous peroxidase was blocked with 3% H2O2, blocked with 5% BSA for 30 minutes (at room temperature), incubated with rabbit anti-SUMO1 and SUMO2 / 3 (1:50) primary antibodies (overnight at 4°C) respectively, incubated with HRP-labeled secondary antibody (1:200) at room temperature for 1 hour, developed with DAB (controlling the developing time under the microscope), counterstained with hematoxylin for cell nuclei, sealed with neutral gum, and observed under an optical microscope (positive signals were brown and cell nuclei were blue).

[0027] Oil Red O staining of cells: HepG2 cell slides were fixed with 4% paraformaldehyde for 10 minutes, rinsed with 60% isopropanol for 1 minute, stained with freshly prepared Oil Red O working solution (Oil Red O saturated solution: water = 3:2) at 37°C for 15 minutes under light-proof conditions, differentiated with 60% isopropanol for 10 seconds, counterstained with hematoxylin for 1 minute and blued with running water, and then sealed with glycerin gelatin and examined under a microscope. Lipid droplets were bright red and cell nuclei were blue.

[0028] Protein Western Blot (WB) detection: Liver tissue or cell samples were used to extract proteins with RIPA lysis buffer and quantified by BCA method. Equal amounts of proteins were electrophoresed by SDS-PAGE (concentrating gel at 80V / separating gel at 120V) and then wet-transferred to PVDF membranes, blocked with 5% skim milk for 1 hour, incubated with primary antibody (overnight at 4°C) and HRP-labeled secondary antibody (1 hour at room temperature) successively, developed with ECL, and the expression level of the target protein was calibrated by β-actin / GAPDH internal reference. All key steps needed to be operated at low temperature throughout the process and the antibody concentration and exposure time were controlled.

[0029] Mouse blood lipid detection: Mice fasted for 6 hours were anesthetized and blood was collected from the orbital venous plexus. The blood samples were allowed to stand for 30 minutes and then centrifuged (centrifuged at 3000×g for 15 minutes at 4°C) to separate the supernatant, which was stored at 4°C in the short term or aliquoted and stored frozen at -80°C in the long term (avoiding repeated freezing and thawing). When detecting, an animal-specific kit was used, and the quantification was performed by colorimetry (measuring TG at 500nm and TC at 450nm) in comparison with the standard product.

[0030] Experimental results

[0031] 1. Global SUMOylation in cells was significantly decreased in clinical fatty liver diseases. As Figure 2Shown: The protein levels of SUMO1 and SUMO2 / 3 in liver specimens of clinical MASLD patients were significantly lower than those in the healthy control group.

[0032] 2. The small molecule compound N106 can increase the overall SUMOylation level in mouse liver and human HepG2 cells in in vivo and in vitro experiments. As Figure 3 shown, after intraperitoneal injection of N106 in mice, the protein levels of SUMO1 and SUMO2 / 3 in liver specimens were significantly higher than those in the control group; as Figure 4 shown, after treatment of HepG2 cells with N106, the protein levels of SUMO1 and SUMO2 / 3 were significantly higher than those in the control group.

[0033] 3. The small molecule compound N106 can reduce blood lipid levels and liver lipid deposition in mice in vivo. As Figure 5 shown, after 12 weeks of high-fat feeding in mice, H&E and oil red staining of the mouse liver showed that the liver lipid deposition in the N106 treatment group was significantly lower than that in the control group; as Figure 6 shown, after 12 weeks of high-fat feeding in mice, the blood lipid (triglyceride and total cholesterol) levels in the N106 treatment group were significantly lower than those in the control group; as Figure 7 shown, after 12 weeks of high-fat feeding in mice, immunohistochemical staining of the mouse liver showed that the protein levels of SUMO1 and SUMO2 / 3 in the N106 treatment group were significantly increased.

[0034] 4. The small molecule compound N106 can reduce lipid deposition in human HepG2 cells in a high-fat environment in in vitro experiments. As Figure 8 shown, under the background of PA treatment of HepG2 cells for 24 hours, cell oil red staining showed that the cell lipid deposition in the N106 treatment group was significantly lower than that in the control group.

Claims

1. Use of compound N106 in the preparation of a drug for treating or preventing metabolic dysfunction-related fatty liver disease, wherein the molecular formula of N106 is C17H14N4O3S and the molecular weight is 354.

38.

2. The application according to claim 1, characterized in that: The drug inhibits liver lipid deposition and blood lipid levels by activating the SUMOylation modification pathway.

3. The application according to claim 1, wherein: The drug exerts its effect by activating the overall SUMOylated form of hepatocytes.

4. The application according to claim 1, characterized in that, The dosage of the drug in the mouse model is 10 mg / kg, administered intraperitoneally once a day for 4 consecutive weeks.

5. The application according to claim 1, wherein The concentration of the drug used in the cell experiment is 10 μM, and the treatment time is 24 hours.

6. The application according to claim 1, characterized in that, The drug contains N106 as an active ingredient and is formulated into an injection, sustained-release preparation or oral preparation in combination with pharmaceutically acceptable excipients.

7. The application according to claim 1, characterized in that, The drug is used to reduce hepatocyte lipid deposition or reverse hepatocyte steatosis.

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