Use of a secretagogue for the preparation of a medicament for the treatment of steatohepatitis associated with metabolic dysfunction

Abstract

This invention relates to the application of secretin in the preparation of drugs for treating steatohepatitis associated with metabolic dysfunction, belonging to the field of biomedical technology. This invention provides the application of secretin in the preparation of drugs for treating steatohepatitis associated with metabolic dysfunction. Cell model, gene knockout, in vivo complementation, and exogenous protein intervention experiments were conducted, revealing that secretin has significant in vitro and in vivo protective effects against steatohepatitis associated with metabolic dysfunction. In vitro, secretin can significantly improve oleic acid-induced lipid accumulation in hepatocytes, inhibit abnormal accumulation of lipid droplets in hepatocytes, reduce the expression of inflammatory factors in hepatocytes, alleviate hepatocyte inflammatory damage caused by lipotoxicity, and enter hepatocytes through endocytosis. It can alleviate high-fat diet-induced hepatic steatosis and inflammatory infiltration in MASH mice, and reduce the degree of liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of secretagogues in the preparation of medicaments for treating steatohepatitis associated with metabolic dysfunction. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a prevalent metabolic disease in clinical practice, and its onset is closely related to the increased risk of diabetes, lipid metabolism disorders, cardiovascular and cerebrovascular diseases, and various malignant tumors. However, the scientific community currently lacks a comprehensive and in-depth understanding of the pathogenesis and development mechanisms of MASH, resulting in a relative scarcity of specific biomarkers and effective methods for assessing disease improvement and guiding precise interventions in clinical practice.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide the application of secretagogue in the preparation of a drug for treating steatohepatitis related to metabolic dysfunction, in order to solve the problem of the lack of treatment methods for steatohepatitis related to metabolic dysfunction in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of secretagogues in the preparation of medicaments for treating steatohepatitis associated with metabolic dysfunction.

[0006] This invention provides the application of secretin in the preparation of drugs that inhibit abnormal accumulation of lipid droplets in hepatocytes and reduce the expression of inflammatory factors in stem cells.

[0007] This invention provides the application of secretin in the preparation of drugs that alleviate hepatic steatosis, inflammatory infiltration of liver tissue, and reduce the degree of liver fibrosis.

[0008] This invention provides the application of secretin in the preparation of drugs that alleviate insulin resistance and improve metabolic status.

[0009] Preferably, the nucleotide sequence encoding secretin is shown in SEQ ID NO.1.

[0010] Preferably, the amino acid sequence of the secretin is shown in SEQ ID NO.2.

[0011] Preferably, the secretin includes natural secretin, recombinant secretin, or gene fragments encoding secretin.

[0012] The present invention has the following technical effects and advantages: This invention conducted cell model, gene knockout, in vivo supplementation, and exogenous protein intervention experiments, revealing that secretin has significant in vitro and in vivo protective effects against metabolic dysfunction-related steatosis. In terms of the pathway of action, SCGN itself is not expressed in hepatocytes but enters hepatocytes via clathrin-mediated endocytosis, laying the structural basis for its hepatoprotective effect. Exogenously supplemented recombinant SCGN can also enter hepatocytes through this pathway and exert its biological function. In terms of effects: in vitro, secretin significantly improves oleic acid-induced lipid accumulation in hepatocytes, inhibits abnormal lipid droplet accumulation in hepatocytes, reduces the expression of inflammatory factors in hepatocytes, and alleviates hepatocyte inflammatory damage caused by lipotoxicity. In vivo, secretin can also alleviate high-fat diet-induced hepatic steatosis and inflammatory infiltration in MASH mice, reduce the degree of liver fibrosis, improve glucose and lipid metabolism disorders, alleviate insulin resistance, and improve overall metabolic status, thus exerting a protective effect at the level of disease inducing factors. Attached Figure Description

[0013] Figure 1 The results show the changes in SCGN protein expression levels under different treatments; Figure 2 Oil Red O staining and Nile Red fluorescence staining results for hepatocytes in different treatment groups; Figure 3 Results of Western blot processing of hepatocyte proteins in different treatment groups; Figure 4 Gel imaging banding results for DNA amplification products from different mice; Figure 5 Immunoblot and islet immunofluorescence results for pancreas in wild-type and knockout mice; Figure 6 Results of liver physiological parameters in wild-type and knockout mice; Figure 7 The results of lipid marker detection in wild-type and knockout mice; Figure 8 The results show the mRNA expression of different genes in the livers of wild-type and knockout mice. Figure 9 The validation results are for mice in different treatment groups; Figure 10 Results of liver physiological indicators in different mice; Figure 11 Results of lipid index detection in different mice; Figure 12 Results of mRNA expression of different genes in the livers of different mice. Detailed Implementation

[0014] This invention provides the use of secretagogues in the preparation of medicaments for treating steatohepatitis associated with metabolic dysfunction.

[0015] This invention provides the application of secretin in the preparation of drugs that inhibit abnormal accumulation of lipid droplets in hepatocytes and reduce the expression of inflammatory factors in stem cells.

[0016] This invention provides the application of secretin in the preparation of drugs that alleviate hepatic steatosis, inflammatory infiltration of liver tissue, and reduce the degree of liver fibrosis.

[0017] This invention provides the application of secretin in the preparation of drugs that alleviate insulin resistance and improve metabolic status.

[0018] In this invention, the nucleotide sequence encoding secretin is shown in SEQ ID NO.1;

[0019] In this invention, the amino acid sequence of the secretin is shown in SEQ ID NO.2; The SEQ ID The amino acid sequence shown in NO.2 is: MDSSREPTLGRLDAAGFWQVWQRFDADEKGYIEEKELDAFFLHMLMKLGTDDTVMKANLHKVKQQFMTTQDASKDGRIRMKELAGMFLSEDENFLLLFRRENPLDSSVEFMQIWRKYDADSSGFISAAELR NFLRDLFLHHKKKAISEAKLEEYTGTMMKIFDRNKDGRLDLNDLARILALQENFLLQFKMDACSTEERKRDFEKIFAYYDVSKTGALEGPEVDGFVKDMMELVQPSISGVDLDKFREILLRHCDVNKDGKIQKSELALCLGLKINP.

[0020] In this invention, the secretin includes natural secretin, recombinant secretin, or gene fragments encoding secretin.

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example 1: Mechanism of SCGN entering hepatocytes

[0023] Experimental materials: HepG2 human liver cancer cells (catalog number FH0076) purchased from Shanghai Fuheng Biotechnology Co., Ltd. were used. They were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin dual antibiotics in a 37℃, 5% CO2 saturated humidity incubator to obtain cultured human liver cancer cells HepG2.

[0024] Experimental groups: The experiment was divided into a control group (CON), a treatment group with recombinant SCGN protein (rSCGN, recombinant secretin protein, purchased from Huaan Biotechnology, sterile liquid stock solution, specification 7 mg / mL, buffer system PBS, pH 7.4, purity ≥95%, endotoxin level <1 EU / μg) at concentrations of 125 nM, 250 nM, and 500 nM, and a treatment group with recombinant SCGN protein at a concentration of 500 nM combined with MβCD (methyl-β-cyclodextrin, an inhibitor of clathrin endocytosis) (combined treatment group). The combined treatment group was further divided into gradient treatment groups with MβCD at concentrations of 0, 5, and 10 mM for mechanism verification.

[0025] Experimental protocol: Cultured HepG2 human liver cancer cells were seeded into 24-well plates (with sterile coverslips for immunofluorescence) and 6-well plates (for Western blotting), respectively. After adhesion, recombinant SCGN protein was added to the treatment and combined treatment groups for 32 h. After treatment, the 24-well plates were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA. Then, they were sequentially incubated with SCGN primary antibody and fluorescent secondary antibody. After DAPI staining, the subcellular localization of SCGN was observed using a laser confocal microscope. For the 6-well plates, after removing the culture medium, total protein was extracted from each group. After SDS-PAGE electrophoresis and membrane transfer, Western blotting was performed using SCGN antibody and α-Tubulin internal control antibody to analyze the changes in SCGN protein expression levels under different treatments. The results of SCGN protein expression level changes under different treatments are shown below. Figure 1 As shown in the figure, Figure A shows the immunofluorescence map of SCGN protein in different treatment groups, and Figure B shows the immunoblot map of SCGN protein in different treatment groups.

[0026] according to Figure 1 It was found that no SCGN protein expression was detected in HepG2 cells without rSCGN treatment, indicating that HepG2 hepatocytes themselves do not express SCGN protein. However, after treatment with different concentrations (0, 125, 250, 500 nM) of recombinant SCGN protein, the SCGN protein level in HepG2 hepatocytes increased significantly in a concentration-dependent manner, indicating that hepatocytes can effectively take up exogenous SCGN. Immunoblotting and immunofluorescence staining results of HepG2 cells treated with different concentrations (0, 5, 10 nM) of MβCD combined with recombinant SCGN protein showed that the uptake of rSCGN by HepG2 cells decreased in a gradient with increasing MβCD concentration. This result confirms that SCGN mainly enters hepatocytes through the clathrin-mediated endocytosis pathway.

[0027] Example 2: Effects of recombinant SCGN protein on OA-induced lipid accumulation and inflammatory damage in hepatocytes in vitro.

[0028] Experimental materials: Human hepatocellular carcinoma HepG2 cells and mouse AML12 hepatocytes were selected and cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C and 5% CO2 saturated humidity incubator to obtain cultured human hepatocellular carcinoma HepG2 cells and mouse AML12 hepatocytes, respectively.

[0029] Experimental groups: Cells were divided into a phosphate-buffered saline (PBS) control group, a recombinant SCGN pretreatment group, an oleic acid (OA) treatment group, and an oleic acid + recombinant SCGN protein treatment group. Both cell types were treated with 2 × 10⁻⁶ cells / mL. 5 Seeds were seeded per well in 6-well plates (for Western blot), at a rate of 2 × 10⁻⁶.4 Cells / well were seeded and cultured in 24-well plates (for staining) containing sterile coverslips. The oleic acid treatment group received oleic acid to a final concentration of 500 nM; the oleic acid + recombinant SCGN protein treatment group received both oleic acid and rSCGN to a final concentration of 500 nM; the recombinant SCGN pretreatment group received rSCGN to a final concentration of 500 nM; and the control group received an equal volume of PBS. After 24 hours of treatment, Oil Red O staining, Nile Red fluorescence staining, and Western blotting were used to detect lipid deposition and the expression levels of genes regulating lipid synthesis in hepatocytes. The results of Oil Red O staining and Nile Red fluorescence staining of hepatocytes in different treatment groups are shown below. Figure 2 As shown, the results of Western blot processing are as follows: Figure 3 As shown.

[0030] according to Figure 2 It is known that oleic acid stimulation can significantly induce lipid droplet formation and lipid deposition in hepatocytes, while pretreatment with recombinant SCGN can effectively alleviate oleic acid-induced abnormal lipid accumulation and significantly reduce the number of intracellular lipid droplets. Figure 3 It is known that oleic acid stimulation can significantly upregulate the expression of key lipid synthesis proteins acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and core transcription factor sterol regulatory element binding protein 1 (SREBP1), while recombinant SCGN pretreatment can significantly downregulate the expression levels of the above lipid synthesis-related proteins, while the intracellular SCGN protein expression level is significantly increased.

[0031] In summary, recombinant SCGN can reduce oleic acid-induced lipid deposition by inhibiting the expression of key proteins in lipid synthesis in hepatocytes, thereby exerting a protective effect against lipid metabolism disorders at the cellular level.

[0032] Example 3: The effect of SCGN knockout on MASH disease

[0033] CRISPR / Cas9 gene editing technology was used to specifically knock out exons 2 and 3 of the mouse SCGN gene, constructing SCGN systemic knockout (KO) mice with a C57BL / 6J background, with wild-type (WT) mice without gene editing in the same background as negative controls.

[0034] The specific gene knockout mouse identification process was as follows: Approximately 5 mm of tail tip tissue was taken from 10-day-old mice. Genomic DNA was extracted using a tissue genomic DNA extraction kit. The tissue was incubated sequentially with a mixture of 180 μL Buffer GTL and 20 μL Proteinase K at 56°C for 1 hour until completely dissolved, followed by denaturation at 90°C for 1 hour. An equal volume of Buffer GL and anhydrous ethanol were added, vortexed, and transferred to an adsorption column. The column was centrifuged at 12000 rpm for 1 minute, and the waste liquid was discarded. The tissue was then washed and purified sequentially with 500 μL Buffer GW1 and GW2. Finally, 60 μL Buffer GE was added, and the tissue was allowed to stand at room temperature for 3 minutes, followed by centrifugation at 12000 rpm for 1 minute to elute and obtain genomic DNA. Specific identification primers (Primer A: F:ATGTCCCTCTACTTCAAACCACG (SEQ ID NO.3), R:CATTGCATTGTTGGGATCAAACTC (SEQ ID NO.4); Primer B: F:ATAACCTTTCTTTCTCCCACTTGC (SEQ ID NO.4)) were used for identification. PCR amplification was performed using NO.5, R:CATTGCATTGTTGGGATCAAACTC (SEQ ID NO.6). The reaction system was 25 μL (containing 12.5 μL Green Taq Mix, 9 μL ddH2O, 1 μL each of forward and reverse primers, and 1.5 μL DNA template). The amplification program was set as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 60 s, and 35 cycles followed by a final extension at 72℃ for 5 min. The amplified products were subjected to 1% agarose gel electrophoresis (120V constant voltage electrophoresis for 45 min). The band patterns were observed using a gel imaging system. At the same time, the expression levels of SCGN protein and mRNA in mouse pancreatic and liver tissues and the serum SCGN content were detected by WB, RT-qPCR and ELISA, respectively, to verify the success of gene knockout from multiple dimensions.

[0035] Gel imaging banding results of different mouse DNA amplification products are as follows: Figure 4 As shown, with α-Tubulin Using the gene as a reference gene, DNA was extracted from pancreatic tissue samples of wild-type and knockout mice, respectively, and amplified using the extracted DNA as a template. SCGN Genes were analyzed using Western blotting; simultaneously, islet tissue from wild-type and knockout mice was analyzed using immunofluorescence; the results of Western blotting and islet immunofluorescence of pancreas from wild-type and knockout mice are as follows. Figure 5 As shown, A represents the pancreatic immunoblotting result, and B represents the islet immunofluorescence result.

[0036] according to Figure 4 and Figure 5 It can be seen that, SCGN Gene knockout mice were successfully constructed, and pancreatic tissue from gene knockout (KO) mice showed positive results. SCGN The mRNA and protein expression levels were significantly reduced in wild-type mice (WT) and normal in wild-type mice. SCGN Gene.

[0037] Experimental protocol: Subsequently, 8-week-old KO mice and WT mice with no abnormalities were selected, with 12 mice in each group. All mice were acclimatized for 1 week in an SPF-grade animal room (temperature 20-26℃, relative humidity 40%-70%, 12h light and dark alternation). After that, they were uniformly fed a GAN-Diet high-fat, high-fructose, and high-cholesterol diet (40kcal% fat, 20kcal% fructose, 2% cholesterol) that had been sterilized by cobalt-60 irradiation. The mice had free access to food and water and were fed continuously for 28 weeks to establish the MASH model. During the modeling period, the fasting weight of the mice was measured at fixed times every week and the feed consumption was recorded. After modeling, mice were fasted for 5 hours and anesthetized intraperitoneally with 1% sodium pentobarbital (50 mg / kg). Blood was collected via the orbital fossa, and after standing at room temperature for 4 hours, the blood samples were centrifuged at 3000 rpm for 20 minutes. The serum was separated and stored at -80℃. Serum total triglyceride (TG), liver total triglyceride (TG), serum total cholesterol (TC), serum high-density lipoprotein (HDL-C), serum low-density lipoprotein (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured. After dissection, the gross morphology of the liver was observed and photographed, the liver weight was measured and the liver weight ratio was calculated, and liver tissue samples were processed (fixed in 4% paraformaldehyde, embedded in OCT, cryopreserved, and stored at -80℃ for molecular detection). Liver fat was assessed by H&E staining and Oil Red O staining. The degree of degeneration, F4 / 80 and α-SMA immunohistochemistry were used to detect inflammatory infiltration and hepatic stellate cell activation; total RNA and total protein were extracted from liver tissue, and RT-qPCR was used to detect the mRNA expression levels of fatty acid synthesis-related genes (Srebp-1c, Fasn, Pparα and Acc1), fatty acid β oxidation-related genes (Pparα, Cpt1a, Acox1 and Mcad), lipid transport-related genes (Cd36, Fapp1 and Mttp), inflammation-related genes (Tnf-α, IL-6 and IL-1β) and fibrosis-related genes (α-SMA, Col1a1) (with GAPDH as an internal reference gene, the relative expression level was calculated using the 2^(-ΔΔCt) method), and Western blotting was used to verify the differences in expression of related proteins.

[0038] The results of liver physiological parameters detection in wild-type mice and knockout mice are as follows: Figure 6As shown in the figure, A represents the liver morphology of different mice, B represents the bar chart of food intake, liver weight, and liver-to-body weight ratio of different mice, C represents the H&E staining and Oil Red O staining of liver tissue of different mice, and D represents the immunohistochemical staining of F4 / 80 and α-SMA of different mice; the lipid index detection results of wild-type mice and knockout mice are as follows. Figure 7 As shown, the mRNA expression results of different genes in the livers of wild-type mice and knockout mice are as follows: Figure 8 As shown.

[0039] according to Figures 6-8 Physiological tests revealed that, compared to wild-type mice, gene knockout mice exhibited significantly whiter, larger, and oily-looking livers with a granular texture. Liver weight and liver-to-body weight ratio were significantly increased. HE and Oil Red O staining showed a significant increase in the number, size, and number of vacuolar steatosis cells in the liver. F4 / 80 and α-SMA immunohistochemical results showed significantly increased macrophage infiltration and hepatic stellate cell activation. Biochemical tests showed significantly elevated levels of lipid markers such as TG, TC, and LDL in the liver and serum of gene knockout mice, and abnormal HDL levels. The levels of ALT and AST, liver injury markers, were significantly increased, and the mRNA expression levels of fatty acid synthesis-related genes (Srebp-1c, Fasn, Pparα, and Acc1), inflammation-related genes (Tnf-α, IL-6, and IL-1β), and fibrosis-related genes (α-SMA and Col1a1) in liver tissue were all upregulated to varying degrees. This fully demonstrates that SCGN gene knockout significantly aggravates GAN-Diet-induced glucose and lipid metabolism disorders in MASH mice, and further exacerbates hepatic steatosis, inflammatory response, and liver fibrosis.

[0040] Example 4: Effects of exogenous SCGN on gene knockout MASH mice

[0041] Virus Construction: Using AAV9-CMV-MCS-EGFP as the backbone plasmid, the full-length coding sequence of mouse SCGN (GenBank accession number NM_021455.2, nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) was amplified by PCR. After double digestion (EcoRI and BamHI), the sequence was inserted downstream of the CMV promoter in the backbone plasmid to construct the recombinant plasmid AAV9-CMV-SCGN-EGFP, which served as the treatment group (AAV9-SCGN). Simultaneously, an empty vector plasmid AAV9-CMV-EGFP without the SCGN gene was constructed as a control (AAV-CTRL). After the plasmids were verified to be correct by sequencing, they were purified using an endotoxin-free plasmid extraction kit and extracted via NanoDrop. Plasmid concentration was measured at 2000 μL to ensure a final concentration ≥1 mg / mL and an OD260 / OD280 ratio of 1.9. Subsequently, the recombinant plasmid was mixed with pAAV-RC (serotype 9) and pHelper helper plasmid at a 1:1:1 ratio and co-transfected into HEK293T cells using liposome transfection. Cells and supernatant were collected 72 h post-transfection. After repeated freeze-thaw cycles, the virus was purified by density gradient centrifugation with iodixanol. After ultrafiltration concentration, the viral titer was determined to ensure that the titers of both AAV9-CTRL and AAV9-SCGN reached 1 × 10⁻⁶. 12 AAV9-CTRL virus and AAV9-SCGN virus were obtained by vg / mL.

[0042] Experimental protocol: Eight-week-old male SCGN knockout (SCGN-KO) mice with a C57BL / 6J background were randomly divided into an AAV-CTRL group (control group) and an AAV-SCGN group (exogenous SCGN supplementation group), with 12 mice in each group. All mice were housed in an SPF-grade animal facility (temperature 20–26°C, relative humidity 40%–70%, 12-hour light-dark cycle) and fed GAN-Diet (40 kcal% fat, 20 kcal% fructose, 2% cholesterol) for 36 weeks to establish a MASH model. At 10 weeks of age, AAV9-CTRL virus and AAV9-SCGN virus were injected via tail vein, respectively. Virus (100 μL / mouse) was administered to three randomly selected mice at 13 weeks of age. Virus distribution in organs was detected using in vivo imaging and confocal fluorescence microscopy. The mRNA expression levels of SCGN protein in liver and pancreatic tissues were detected by Western blotting and RT-qPCR, respectively. Serum SCGN levels were measured by ELISA to verify AAV transfection efficiency and SCGN expression recovery. The remaining mice continued GAN-Diet feeding until 36 weeks of age, with fasting body weight and food intake recorded weekly. At the experimental endpoint, mice were anesthetized intraperitoneally with 1% sodium pentobarbital (50 mg / kg), and serum was collected from the orbital sinus for total triglyceride (TG) measurement. The levels of total triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the liver were measured. After dissection, the gross morphology of the liver was observed and photographed. Liver weight was measured and the liver-to-body weight ratio was calculated. Liver tissue samples were collected and processed (fixed in 4% paraformaldehyde, embedded in OCT, cryopreserved at -80℃ for molecular detection). The degree of hepatic steatosis was assessed by H&E staining and Oil Red O staining. F4 / 80 and α-SMA immunohistochemistry were used to detect inflammatory infiltration and hepatic stellate cell activation. Total RNA and total protein were extracted from liver tissue and analyzed by RT-qPCR. The mRNA expression levels of fatty acid synthesis-related genes (Srebp-1c, Fasn, Pparα, and Acc1), fatty acid β-oxidation-related genes (Pparα, Cpt1a, Acox1, and Mcad), lipid transport-related genes (Cd36, Fapp1, and Mttp), inflammation-related genes (Tnf-α, IL-6, and IL-1β), and fibrosis-related genes (α-SMA and Col1a1) were detected. Western blotting was used to verify the differences in expression of related proteins. Based on the above indicators, the ameliorative effect of exogenous SCGN supplementation on glucose and lipid metabolism disorders, hepatic steatosis, inflammation, and fibrosis in SCGN knockout MASH mice was determined.

[0043] The validation results of mice in different treatment groups are as follows: Figure 9 As shown, A represents in vivo imaging images of mice in different treatment groups, and B represents confocal fluorescence images of mice in different treatment groups; the results of liver physiological index detection in different mice are as follows. Figure 10 As shown in the figure, A represents the morphology of different mouse livers, B represents the bar charts of food intake, liver weight, and liver-to-body weight ratio in different mice, C represents the H&E staining and Oil Red O staining images of different mouse liver tissues, and D represents the immunohistochemical staining images of F4 / 80 and α-SMA in different mice; the results of lipid index detection in different mice are as follows. Figure 11 As shown, the mRNA expression results of different genes in the livers of different mice are as follows: Figure 12 As shown.

[0044] according to Figures 9-12 It was found that after tail vein injection of SCGN knockout mice, in vivo imaging, immunoblotting, and RT-qPCR results confirmed that the virus was efficiently distributed in organs such as the liver and pancreas. The mRNA and protein expression levels of SCGN in the liver and pancreas tissues of mice were significantly restored, and the serum SCGN content returned to near physiological levels, confirming the successful AAV transfection and recovery of SCGN expression. Based on this, after feeding mice with GAN-Diet to 36 weeks of age to induce the MASH model, physiological tests showed that compared with the AAV-CTRL control group mice, the liver appearance of the AAV-SCGN group mice changed from greasy and whitish with increased volume to smooth surface and soft texture. The liver weight and liver weight ratio were significantly reduced. HE staining and Oil Red O staining showed a significant reduction in the number, size, and number of vacuolar steatosis cells in the liver. The F4 / 80 ratio and α-S... Immunohistochemical results showed that the infiltration of hepatic macrophages and the activation level of hepatic stellate cells were significantly reduced. In terms of biochemical detection, lipid indicators such as TG, TC, and LDL in the liver and serum of mice in the AAV-SCGN group were significantly reduced, HDL level returned to normal, and the contents of liver injury markers such as ALT and AST were significantly reduced. Moreover, the mRNA expression levels of fatty acid synthesis-related genes (Srebp-1c, Fasn, Pparα and Acc1), inflammation-related genes (Tnf-α, IL-6 and IL-1β), and fibrosis-related genes (α-SMA, Col1a1, etc.) in liver tissue were downregulated to varying degrees. This fully demonstrates that exogenous SCGN supplementation can effectively improve the glucose and lipid metabolism disorder in SCGN knockout MASH mice, and significantly reduce the progression of hepatic steatosis, inflammatory response and liver fibrosis in mice.

[0045] As can be seen from the above embodiments, this invention provides the application of secretin in the preparation of drugs for treating steatohepatitis related to metabolic dysfunction. This invention has conducted cell model, gene knockout mouse, in vivo complementation, and exogenous protein intervention experiments, which showed that secretin has significant in vitro and in vivo protective effects against steatohepatitis related to metabolic dysfunction. In terms of the pathway of action, SCGN itself is not expressed in hepatocytes, but it can enter hepatocytes through clathrin-mediated endocytosis, laying the structural basis for its hepatoprotective effect. Exogenously supplemented recombinant SCGN can also enter hepatocytes through this pathway and exert its biological function. In terms of effects: in vitro, secretin can significantly improve oleic acid-induced lipid accumulation in hepatocytes, inhibit abnormal accumulation of lipid droplets in hepatocytes, and reduce the expression of inflammatory factors in hepatocytes, alleviating hepatocyte inflammatory damage caused by lipotoxicity. In vivo, secretin can also alleviate hepatic steatosis and inflammatory infiltration in MASH mice induced by a high-fat diet, reduce the degree of liver fibrosis, improve glucose and lipid metabolism disorders, alleviate insulin resistance, and improve overall metabolic status, thus exerting a protective effect at the level of disease inducing factors.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of secretin in the preparation of drugs for treating steatohepatitis associated with metabolic dysfunction.

2. Application of secretin in the preparation of drugs that inhibit abnormal accumulation of lipid droplets in hepatocytes and reduce the expression of inflammatory factors in stem cells.

3. Application of secretagogues in the preparation of drugs that alleviate hepatic steatosis, inflammatory infiltration of liver tissue, and reduce the degree of liver fibrosis.

4. The application of secretagogues in the preparation of drugs that alleviate insulin resistance and improve metabolic status.

5. The application according to any one of claims 1 to 4, characterized in that, The nucleotide sequence encoding secretin is shown in SEQ ID NO.

1.

6. The application according to any one of claims 1 to 4, characterized in that, The amino acid sequence of the secretin is shown in SEQ ID NO.

2.

7. The application according to any one of claims 1 to 4, characterized in that, The secretin includes natural secretin, recombinant secretin, or gene fragments encoding secretin.

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