Application of liver secreted factor Thbs2 as target spot in treatment or prevention of metabolism-related fatty liver disease and progress of metabolism-related fatty liver disease

Abstract

The invention discloses an application of a liver secreted factor Thbs2 as a target spot in treating or preventing metabolism-related fatty liver diseases and progression of the metabolism-related fatty liver diseases. According to the application disclosed by the invention, the effect of the liver secretion factor Thbs2 in the progress of the MAFLD-MASH disease is defined for the first time, and the application reveals that the liver secretion factor Thbs2 promotes the occurrence of liver injury and liver fibrosis by activating hepatic stellate cells and M1 macrophages, so that the progress of the MASH is accelerated; liver specific over-expression Thbs2 promotes development of MAFLD-MASH, liver specific knockout Thbs2 remarkably inhibits occurrence of MASH-related liver injury and liver fibrosis, liver injury and liver fibrosis are remarkably slowed down through targeted inhibition of liver Thbs2 through GalNAc-siRNA, and the MASH process is relieved, so that a new target spot is provided for preventing or treating MASH, and the application has the advantages that the application is wide in application range and high in application value. And the method has important significance for clinically developing drugs for targeted therapy of MASH.

Description

Technical Field

[0001] The present invention relates to the application of liver secretory factor Thbs2 as a target in treating or preventing metabolism-related fatty liver disease and its progression, and belongs to the technical field of biomedicine. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) affects about 30% of the world's population and is one of the most prevalent chronic liver diseases. The overall prevalence of MAFLD in my country is close to that in the world, and the proportion in some regions is as high as 42%. As a multi-system metabolic disease involving the whole body, MAFLD not only aggravates the deterioration of liver disease, but also significantly increases the risk of complications such as diabetes, cardiovascular disease and extrahepatic tumors, which poses a serious threat to national health, quality of life and socioeconomic development. MAFLD can further progress to metabolic dysfunction-associated steatohepatitis (MASH), which is characterized by fatty degeneration of the liver, inflammatory response, hepatocyte damage and varying degrees of fibrosis. The etiology of MASH is complex, involving the combined effects of genetic and epigenetic factors, lifestyle and dietary habits, and liver and extrahepatic comorbidities. Compared with other chronic liver diseases, the prognosis of MASH progressing to cirrhosis is poor. Currently, the diagnosis of MASH mainly relies on liver biopsy, lacks effective non-invasive detection methods, and currently lacks effective drug treatment options. Although thyroid hormone receptor-β selective agonists have been approved for the treatment of MASH patients with liver fibrosis, their actual efficacy still needs further study. Non-invasive diagnostic methods and new therapeutic targets for MASH patients are clinical issues that need to be urgently addressed.

[0003] Thbs2 (Thrombospondin 2, also known as TSP2) is a member of the thrombospondin TSP family. This protein family mediates cell-cell and cell-matrix interactions, can bind to fibrinogen, fibronectin, laminin, type V collagen and integrin, plays an important role in platelet aggregation and angiogenesis, and is a natural inhibitor of new blood vessel formation. Recent studies have shown that the TSP family is closely related to the development of metabolic diseases. Among them, the increase in the level of circulating Thbs2 is closely related to the progression of liver fibrosis in patients with MAFLD and type 2 diabetes. Combining Thbs2 with clinical indicators can effectively improve the predictive ability of liver fibrosis. However, the role and mechanism of Thbs2 in the progression of MAFLD disease are still unclear. Summary of the invention

[0004] The purpose of the present invention is: currently there is a lack of effective drug treatment options for the treatment of MAFLD and its further progression to MASH. The present invention finds that the liver secretory factor Thbs2 is upregulated during the progression of MAFLD-MASH disease; based on the liver-specific knockout of Thbs2, the occurrence of MASH-related liver damage and fibrosis can be alleviated. The present invention uses GalNAc-siRNA technology to specifically target hepatocyte Thbs2, which can effectively treat MASH-related liver fibrosis and damage.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides the use of the liver secretory factor Thbs2 gene or protein as a drug target in the preparation or screening of drugs for preventing, improving and / or treating metabolism-related fatty liver disease, metabolism-related fatty hepatitis, and liver fibrosis.

[0007] Preferably, the drug is a chemical drug and / or biological molecule that can inhibit the expression of liver secretory factor Thbs2 gene and / or protein in vivo, thereby reducing plasma ALT and AST levels, inhibiting liver fibrosis and liver damage, and preventing MAFLD from progressing to MASH.

[0008] In a second aspect, the present invention provides a use of a liver secretory factor Thbs2 gene or protein inhibitor for preparing a composition or preparation, wherein the composition or preparation is used for a) preventing and / or treating metabolic-related fatty liver disease or metabolic-related fatty liver hepatitis; and / or c) preventing and / or treating liver fibrosis.

[0009] Preferably, the composition or preparation is also used for i) inhibiting the occurrence of liver fibrosis; and / or ii) inhibiting the activation of hepatic stellate cells (HSC); and / or iii) preventing the progression of MAFLD to MASH.

[0010] Preferably, the liver secretion factor Thbs2 inhibitor is selected from one or more combinations of the following a)-d):

[0011] a) Small molecule compounds;

[0012] b) siRNA, or a modified product or delivery vector thereof that specifically inhibits Thbs2 gene expression;

[0013] c) shRNA or its delivery vector that specifically inhibits Thbs2 gene expression;

[0014] d) Thbs2 protein inhibitor.

[0015] Preferably, the liver secretion factor Thbs2 inhibitor is an N-acetylgalactosamine-conjugated modified siRNA (GalNAc-siThbs2) or a delivery vector thereof, and the sequence of the siRNA is shown in SEQ ID NO: 3-4, SEQ ID NO: 5-6 or SEQ ID NO: 7-8. N-acetylgalactosamine (GalNAc) is a ligand of asialoglycoprotein receptor (ASGPR), which is highly specifically expressed on the membrane surface of hepatocytes (about 500,000 ASGPR per cell). The GalNAc-siRNA conjugate specifically binds to ASGPR and transports siRNA from the cell surface to the cell under cellular endocytosis. Subsequently, the GalNAc-siRNA conjugate separates from ASPGR, and ASPRGR returns to the cell surface while the GalNAc-siRNA conjugate further dissociates, and the released free siRNA silences the gene in the cytoplasm to exert its pharmacological effect. The 3' end of the siRNA sense strand is chemically bonded to GalNAc with a trivalent structure, and the stability of the GalNAc-siRNA conjugate is enhanced by ESC (enhanced stabilization chemistry) technology, thereby increasing liver exposure and prolonging gene sinking time.

[0016] More preferably, the sequence of the siRNA is shown in SEQ ID NO: 3-4.

[0017] In a third aspect, the present invention provides a pharmaceutical composition comprising:

[0018] a1) a first active ingredient for preventing and / or treating metabolism-related fatty liver disease, metabolism-related fatty hepatitis, and liver fibrosis, wherein the first active ingredient comprises: a liver secretory factor Thbs2 gene inhibitor, or a liver secretory factor Thbs2 protein inhibitor;

[0019] a2) a second active ingredient for preventing and / or treating metabolism-related fatty liver disease, metabolism-related steatohepatitis, and liver fibrosis, wherein the second active ingredient includes: other drugs for preventing and / or treating metabolism-related fatty liver disease, metabolism-related steatohepatitis, and liver fibrosis; and

[0020] b) a pharmaceutically acceptable carrier.

[0021] In a fourth aspect, the present invention provides a method for inhibiting the activation of hepatic stellate cells (HSC), comprising:

[0022] Hepatic stellate cells are cultured in the presence of a liver secretion factor Thbs2 gene inhibitor or a liver secretion factor Thbs2 protein inhibitor, thereby inhibiting the activation of hepatic stellate cells (HSC).

[0023] In a fifth aspect, the present invention provides a method for screening potential therapeutic agents for metabolism-related fatty liver disease, metabolism-related fatty liver hepatitis, and liver fibrosis, comprising:

[0024] A) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing the liver secretory factor Thbs2 gene for a period of time, and detecting the expression amount E1 of the liver secretory factor Thbs2 gene and / or protein in the culture system of the test group;

[0025] And in a control group without the test compound and with other conditions being the same, detecting the expression level E2 of the liver secretory factor Thbs2 gene and / or protein in the culture system of the control group; and

[0026] B) Compare E1 and E2. If E1 is significantly lower than E2, it indicates that the test compound is a potential therapeutic agent for liver fibrosis.

[0027] Preferably, the cells include hepatocytes

[0028] The present invention detects the plasma Thbs2 level of 75 subjects (58 MAFLD patients and 17 healthy controls), and finds that the Thbs2 level of MAFLD patients is significantly higher than that of the healthy control group, and is positively correlated with the subject's BMI and clinical metabolic data (such as ALT, AST, γ-glutamyl transpeptidase). In addition, in two fatty liver mouse models and a chemical drug-induced liver fibrosis mouse model, the present invention also observed that the level of mouse plasma Thbs2 is significantly increased, and is significantly positively correlated with ALT and AST. Further research reveals that Thbs2 secreted by hepatocytes plays an important role in the progression of MAFLD disease. Liver-specific overexpression of Thbs2 promotes the development of MAFLD-MASH, while liver-specific knockout of Thbs2 significantly inhibits the occurrence of MASH-related liver damage and liver fibrosis. In vitro experiments show that liver secretory factor Thbs2 can activate hepatic stellate cells and promote M1 macrophage activation. The present invention has been shown through animal experiments that the targeted inhibition of liver Thbs2 by GalNAc-siRNA significantly slows down liver damage and liver fibrosis, and alleviates the process of MASH.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention clarifies for the first time the role of the liver secretory factor Thbs2 in the progression of MAFLD-MASH disease, and reveals that it promotes liver damage and liver fibrosis by activating hepatic stellate cells and M1 macrophages, thereby accelerating the progression of MASH; targeted inhibition of liver Thbs2 expression can significantly slow the occurrence of MASH liver fibrosis; therefore, the present invention provides a new target for the prevention or treatment of MASH, and is of great significance for the clinical development of targeted drugs for the treatment of MASH. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Results of plasma Thbs2 level detection and analysis in MAFLD patients: (A) Elisa detection of plasma Thbs2 levels in MAFLD (n=58) and healthy subjects (n=17); (B-) Correlation analysis between plasma Thbs2 and body mass index (BMI); (CE) Correlation analysis between plasma Thbs2 and liver injury indicators ALT (C), AST (D) and GTT (E).

[0032] Figure 2 Expression analysis of Thbs2 in liver tissue in MAFLD and MASH model mice: (A, B) Real-time PCR detection results (A) and Western-blot detection (B) of Thbs2 expression in liver of high-fat diet-induced obesity and diet-switch weight loss model mice; (C) Detection of plasma Thbs2 levels; (D) Correlation analysis between plasma Thbs2 and liver injury indicators ALT and AST; (EG) Detection of Thbs2 levels in liver tissue (E, F) and plasma (G) of MASH model mice induced by MCD-HFD diet; (HI) Detection of Thbs2 levels in liver tissue (H, I) and plasma (J) of liver fibrosis injury model mice induced by carbon tetrachloride.

[0033] Figure 3 Analysis of the cellular expression sources of Thbs2 in MAFLD liver: (A, B) Analysis of Thbs2 expression in hepatocytes and non-hepatocytes; (CE) Fatty acid treatment upregulated the expression and release of Thbs2 in hepatocytes; (F, G) After Thbs2 was knocked out in primary hepatocytes, the Thbs2 levels in fatty acid-treated hepatocytes (F) and culture supernatant (G) were detected.

[0034] Figure 4Liver-specific overexpression of Thbs2 promotes MASH-related liver fibrosis: (A) Mouse animal experiment model; (BC) Detection of liver-specific overexpression of Thbs2 levels; (D) Elisa detection of plasma Thbs2 levels; (E) Detection of liver injury indicators ALT and AST; (F) H&E staining, Sirius red staining and a-SMA immunohistochemistry of mouse liver tissue and quantitative analysis of related indicators; (G) Immunofluorescence staining analysis of the distribution and polarization of macrophages in liver tissue; (HI) Real-time PCR detection of gene expression related to liver tissue fibrosis (H) and inflammatory response (I).

[0035] Figure 5 Liver-specific knockout of Thbs2 alleviates MASH-related liver fibrosis injury: (A) Mouse animal experiment model; (BC) Detection of liver-specific knockout of Thbs2 levels; (D) Elisa detection of plasma Thbs2 levels; (E) Detection of liver injury indicators ALT and AST; (F) H&E staining, Sirius red staining and α-SMA immunohistochemistry of mouse liver tissue and quantitative analysis of related indicators; (G) Immunofluorescence staining analysis of the distribution and polarization of macrophages in liver tissue; (HI) Real-time PCR detection of gene expression related to liver tissue fibrosis (H) and inflammatory response (I).

[0036] Figure 6 Thbs2 promotes hepatic stellate cell activation and M1 macrophage activation: (A) Real-time PCR was used to detect the expression of related genes after LX2 was treated with Thbs2; (B) LX2α-SMA immunofluorescence staining was used for detection; (C) Western-blot was used to detect the signaling pathways related to LX2 activation; (D) Cell migration assay; (E) Real-time PCR was used to detect the expression of related genes after LX2 was pretreated with Anti-CD47; (F) Western-blot was used to detect the signaling pathways related to LX2 activation; (G) After blocking CD47, the migration ability of LX2 cells was detected; (H, I) After Thbs2 was used to treat Raw264.7, the expression of genes related to inflammatory response (H) and M1 polarization (I) was detected by Real-time PCR; (J) After blocking CD47 of Raw264.7 cells, the expression of related genes was detected by Real-time PCR.

[0037] Figure 7GalNAc-siRNA targeting liver Thbs2 alleviates MASH-related liver fibrosis injury: (A) Mouse animal experiment model diagram; (BC) Detection of liver-specific knockout Thbs2 levels; (D) Elisa detection of plasma Thbs2 levels; (E) Detection of liver injury indicators ALT and AST; (F) H&E staining, Sirius red staining and a-SMA immunohistochemistry of mouse liver tissue and quantitative analysis of related indicators; (G) Immunofluorescence staining analysis of the distribution and polarization of macrophages in liver tissue; (HI) Real-time PCR detection of gene expression related to liver tissue fibrosis (H) and inflammatory response (I).

[0038] Figure 8 In vitro GalNAC-siThbs2 screening and validation: (A) RT-PCR detection of siThbs2 knockout efficiency; (B) Western-blot detection of Thbs2 protein level after siThbs2 knockout. DETAILED DESCRIPTION

[0039] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0040] Example

[0041] 1. Materials and Methods

[0042] 1. Blood samples from healthy people and patients

[0043] All plasma samples of healthy subjects and NAFLD patients were obtained from Zhongshan Hospital Affiliated to Fudan University. All subjects signed informed consent during sample collection and the process was approved by the Ethics Committee.

[0044] 2. Animal Source

[0045] All mice were housed at a temperature of 20-22°C and a relative humidity of 40-60%, and had free access to water and food. All mouse strains were of C57 / BL6J background, of which Thbs2 flox / flox mice were constructed by Jicui Pharmaceuticals. Male mice aged 6-8 weeks were fed HFD (Research Diets, D12492), MCD-HFD (Research Diets, A06071302), or GAN diet (Research Diets, D09100310) to induce MAFLD or MASH for the specified time. Animal studies have been approved by the Animal Care and Use Committee of Fudan University.

[0046] (III) Detection methods

[0047] 1. Antibody Source

[0048] anti-Thbs2 (abcam, ab112543), anti-Thbs2 (abcam, ab84469), anti-CD47 (Proteintech, 66304), anti-Erk (CST, 46 95T), anti-p-Erk (CST, 4370S), anti-Yap1 ((Proteintech, 66900), anti-p-Yap1 (Ser397) ((Proteintech, 29018).

[0049] 2. Elisa detection of plasma Thbs2 levels (Ciobo BIO Kit)

[0050] (1) Addition of standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well.

[0051] (2) Sample addition: Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40 μl of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10 μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0052] (3) Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank well.

[0053] (4) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 min.

[0054] (5) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0055] (6) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard. Repeat this process 5 times and pat dry.

[0056] (7) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and color development at 37°C in the dark for 15 minutes.

[0057] (8) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0058] (9) Determination: Use the blank well as the zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be performed within 15 minutes after adding the stop solution.

[0059] 3. Extraction of Primary Hepatocytes

[0060] (1) Prepare perfusion buffer (50 ml Hanks balanced salt solution (HBSS; Ca-free) 2+ / Mg 2+ , Gibco, 14175095) containing 500 μl 7.5% NaHCO3 and 71.17 μl 0.5 M EDTA (Thermo Fisher, AM9260G)) and digestion buffer (30 ml HBSS with Ca 2+ / Mg 2+ (Gibco, 14025134), containing 300 μl 7.5% sodium bicarbonate, 60 μl 2.5M CaCl2 and 2% type II collagenase (Worthington, LS004176). The above buffer was preheated to 37°C.

[0061] (2) After anesthetizing the mouse, expose the liver, heart, and portal vein. Insert the injection needle into the right atrium to start perfusion, and then cut the portal vein. The perfusion rate is 1-2 ml / min and continues for 5 minutes. Stop the perfusion buffer and replace it with collagenase solution. Continue perfusion at a rate of 1 ml / min for 5 minutes until the liver becomes soft.

[0062] (3) Transfer the liver to a 10 cm dish and remove the gallbladder. Drop about 300 μl of collagenase solution on the liver and digest it in a 37°C incubator for 3 minutes.

[0063] (4) Resuspend the cells in 15 ml of DMEM, filter into a 50 ml centrifuge tube using a 100 μm filter, and then centrifuge at 50 g for 3 min at room temperature. Discard the supernatant, resuspend the precipitate and centrifuge again, count and detect cell viability. Inoculate in a culture dish (coated with 0.1% type I collagen solution for 15 min), and replace the supernatant after 2 h.

[0064] 4. Immunofluorescence

[0065] (1) Soak the coverslip in anhydrous ethanol for 30 min, dry it on an alcohol burner, place it in a 24-well plate, and inoculate the cells;

[0066] (2) After the cells have reached an appropriate density, remove the cell culture medium and wash the coverslip twice with PBS buffer at room temperature;

[0067] (3) Add 1 ml of 4% paraformaldehyde to fix for 10 min at room temperature, and wash the coverslips twice with PBS buffer, each time for 5 min;

[0068] (4) Add 1 ml of methanol to permeabilize the membrane for 10 min, and then wash the slides twice with room temperature PBS buffer, each time for 5 min;

[0069] (5) Add 1 ml of 5% BSA solution (prepared with PBST solution) and block at room temperature for 1 h to block nonspecific proteins;

[0070] (6) Take a clean 24-well plate, add 20-30 μl of 1:100 diluted primary antibody (prepared with 5% BSA solution), place the cell surface of the slide on the primary antibody, add 1 ml of PBS buffer to the surrounding blank wells, wrap with plastic wrap, and incubate at 4°C overnight;

[0071] (7) Place the slide with the cells facing up in another clean 24-well plate, add 1 ml of PBS buffer, wash on a shaker for 5 min, and repeat 3 times;

[0072] (8) Remove the PBS buffer and add 200 μl of fluorescent secondary antibody diluted 1:200 (prepared with 5% BSA solution) to the 24-well plate. Incubate at room temperature in the dark for 1 h. Add 1 ml of PBS buffer to the 24-well plate, wrap it with tin foil, and wash it on a light-proof shaker for 5 min. Repeat 3 times.

[0073] (9) Add 5-10 μl of mounting medium containing DAPI to the slide, place the coverslip cell side upside down on the slide, incubate at 37°C for 15 min, and then observe and photograph under a confocal microscope.

[0074] 5. Cell Migration Assay

[0075] (1) Preparation of cell suspension: Digest the cells with trypsin, discard the culture medium after serum digestion is terminated, wash twice with sterile PBS solution, and resuspend in serum-free culture medium.

[0076] (2) Inoculation of cells: Count the number of cells using a cell counter and dilute the cell suspension to an appropriate density. Take 100 μl of cell suspension (e.g. 5×10 LX2 cells). 4 ) were added to the upper Transwell chamber of the 24-well plate. Then 600 μl of cell culture medium containing 10% FBS was added to the lower chamber of the 24-well plate (be careful not to allow bubbles to form in the culture medium in the lower chamber), and cultured for 24 h and 48 h, respectively.

[0077] (3) Cell fixation and staining: Remove the Transwell chamber, discard the culture medium in the well, rinse twice with sterile PBS solution, add 1 mL of methanol to the chamber, fix at room temperature for 15 minutes, discard the methanol in the chamber, and stain with 0.1% crystal violet stain for 30 minutes.

[0078] (4) Cleaning nonspecific staining in the chamber: Wash the chamber with PBS solution, then discard the PBS solution, repeat 5 times. Then wipe the PBS solution in the chamber with dust-free paper and blow dry in a fume hood.

[0079] (5) Result statistics: Place the blow-dried chamber under a microscope and take a 4x wide field image. Then, randomly select five 10x fields of view under the microscope and count the cells captured.

[0080] 6.Thbs2-siRNA design and in vitro screening

[0081] The following Thbs2 siRNA fragments were transfected into the Hepa1-6 mouse liver cell line, and the cell RNA and protein were collected 48 hours after transfection to detect the knockdown efficiency.

[0082] Thbs2-Mus-1410

[0083] sense:5'-GCACCUGCAAGAAAUUUAATT-3'(SEQ ID NO: 1);

[0084] antisense:5'-UUAAAUUUCUUGCAGGUGCTT-3' (SEQ ID NO: 2);

[0085] Thbs2-Mus-1358

[0086] sense:5'-GGCCGAAUCUUUGCAGAAATT-3'(SEQ ID NO: 3);

[0087] antisense:5'-UUUCUGCAAAGAUUCGGCCTT-3' (SEQ ID NO: 4);

[0088] Thbs2-Mus-2500

[0089] sense:5'-CCUGGUGUGUGCUACUAAUTT-3' (SEQ ID NO: 5);

[0090] antisense:5'-AUUAGUAGCACACACCAGGTT-3' (SEQ ID NO: 6);

[0091] Thbs2-Mus-2821

[0092] sense:5'-GCGAGACAAUUGUCCAAUTT-3' (SEQ ID NO: 7);

[0093] antisense:5'-AUAUGGACAAUUGUCUCGCTT-3' (SEQ ID NO: 8);

[0094] 7. Subcutaneous injection of Galnac-siRNA

[0095] (1) Dissolve ESC+ (Enhanced Stabilization Chemistry Plus) Galnac-modified siNC and siThbs2 in saline and prepare for use immediately.

[0096] Thbs2-Mus-1358

[0097] sense:5'-GGCCGAAUCUUUGCAGAAATT-3'(SEQ ID NO: 3);

[0098] antisense:5'-UUUCUGCAAAGAUUCGGCCTT-3' (SEQ ID NO: 4);

[0099] (2) Starting from day 0 after feeding 8-week-old C57 wild-type male mice with MCD-HFD, 10 mg / kg GalNAc-siThbs2 was subcutaneously injected into the back. The injection was repeated 5 times on days 0, 3, 7, 14, and 21.

[0100] (3) Mice were killed on day 28 after feeding MCD-HFD, and samples were collected for subsequent experiments.

[0101] 2. Implementation Plan and Results

[0102] 1. Analysis of plasma Thbs2 levels in the progression of MAFLD in humans and mice

[0103] To clarify the relationship between thrombospondin 2 (Thbs2) and the progression of metabolic-associated fatty liver disease (MAFLD), we first compared the expression levels of Thbs2 in the plasma of MAFLD patients and healthy controls. The results showed that the plasma Thbs2 level of MAFLD patients was significantly higher than that of the healthy control group (see Figure 1 A). Further correlation analysis showed that plasma Thbs2 levels were significantly positively correlated with the subjects' body mass index (BMI) and liver injury indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamyl transferase (GGT) (see Figure 1 BE). Subsequently, we detected the expression levels of liver and plasma Thbs2 in diet-induced obese mice. The experimental results showed that in high-fat diet-induced obese mice, the gene and protein expression of liver Thbs2 was significantly upregulated; and after switching their diet to ordinary feed for one month, the expression of liver Thbs2 was significantly reduced (see Figure 2 A, B). At the same time, the level of plasma Thbs2 also decreased accordingly (see Figure 2C), and significantly positively correlated with liver injury indicators ALT and AST (see Figure 2 D).

[0104] To further verify the relationship between plasma Thbs2 levels and MAFLD disease progression, we constructed a metabolic-associated fatty liver disease (MASH) model induced by a methionine-choline-deficient high-fat diet (MCD-HFD) and detected the expression of Thbs2 in liver tissue and plasma of MASH model mice. In addition, the test was also performed in a carbon tetrachloride-induced liver fibrosis injury mouse model. The results showed that in both disease models, the expression of Thbs2 in liver tissue was significantly upregulated, accompanied by a significant increase in plasma Thbs2 levels (see Figure 2 EI). In summary, plasma Thbs2 levels are not only consistent with Thbs2 expression in the liver, but also closely related to the disease progression of MAFLD.

[0105] 2. Hepatocytes are the main source of upregulated Thbs2 expression in MAFLD

[0106] About 80% of the composition of liver tissue comes from hepatic parenchymal cells, while the remaining 20% ​​is composed of non-parenchymal liver cells. Non-parenchymal liver cells include various types such as hepatic stellate cells, endothelial cells, bile duct cells, Kupffer cells and immune cells. In order to clarify the specific source of upregulated Thbs2 expression in MAFLD liver, we isolated and analyzed the Thbs2 expression levels in hepatic parenchymal cells and non-parenchymal liver cells of normal and obese mice. The experimental results showed that Thbs2 expression was significantly upregulated in hepatic parenchymal cells of obese mice, while there was no significant difference in Thbs2 expression in non-parenchymal liver cells between the two groups of mice ( Figure 3 A, B). Further studies have found that free fatty acid stimulation can increase the expression and secretion of Thbs2 in hepatic parenchymal cells ( Figure 3 CE). In addition, Thbs2 flox / flox genotype mice were used to isolate and culture primary liver cells, and the Thbs2 gene was knocked out by Cre recombinant adenovirus infection to confirm that the secretion of Thbs2 mainly comes from hepatocytes. The results showed that after knocking out the Thbs2 gene, the level of Thbs2 in the cell supernatant was significantly reduced and was not regulated by free fatty acid stimulation (Figure F, G). In summary, the present invention confirms that hepatocytes are the main source of upregulated Thbs2 expression in MAFLD livers.

[0107] 3. Liver-specific overexpression of Thbs2 promotes MAFLD-MASH disease transformation

[0108] Since the previous record showed that the level of Thbs2 in plasma is closely related to the occurrence of MAFLD, and confirmed that the upregulation of Thbs2 in MAFLD liver tissue is mainly derived from hepatocytes. In order to further explore the role of liver secretory factor Thbs2 in the progression of MAFLD, the present invention injected adeno-associated virus (AAV-TBG-Thbs2) through the tail vein to achieve liver-specific overexpression of Thbs2, and fed GAN feed to establish a MASH mouse model ( Figure 4 A). The results showed that AAV could significantly increase the expression of Thbs2 in liver tissue and the level of Thbs2 in plasma ( Figure 4 BD). In addition, Thbs2 overexpression led to increased levels of liver injury indicators ALT and AST, and aggravated the degree of liver fibrosis ( Figure 4 E, F). Immunofluorescence staining analysis showed that Thbs2 overexpression significantly increased the infiltration of liver tissue macrophages and promoted the polarization of M1 macrophages ( Figure 4 G). Real-time PCR test results further proved that Thbs2 overexpression significantly upregulated the expression of fibrosis-related genes and inflammatory response-related genes ( Figure 4 H, I). In summary, these experimental results reveal that liver-specific overexpression of Thbs2 exacerbates MASH-related liver fibrosis and promotes the pathological transformation of MAFLD to MASH.

[0109] 4. Liver-specific knockout of Thbs2 alleviates MASH-induced liver fibrosis

[0110] To further clarify the function of Thbs2 in the transition of MAFLD to MASH disease, we used Thbs2 flox / flox mice to achieve liver-specific knockout of Thbs2 by tail vein injection of adeno-associated virus (AAV-TBG-Cre) and established a MASH mouse model by feeding them with MCD-HFD diet ( Figure 5 A). The experimental results showed that knocking out Thbs2 in the liver could significantly reduce the level of plasma Thbs2 ( Figure 5 BD). Further studies have found that liver-specific knockout of Thbs2 effectively inhibited liver injury indicators, namely plasma ALT and AST levels, and reduced the degree of liver tissue fibrosis ( Figure 5 E, F). At the same time, immunofluorescence staining analysis showed that knocking out Thbs2 significantly reduced the infiltration of liver tissue macrophages and inhibited the polarization of M1 macrophages ( Figure 5 G). In addition, the knockout of Thbs2 significantly inhibited the expression of fibrosis-related genes and inflammatory response-related genes ( Figure 5H, I). These results indicate that liver tissue-specific knockout of Thbs2 can alleviate MASH-related liver fibrosis injury.

[0111] 5.Thbs2 promotes activation of hepatic stellate cells and M1 macrophages

[0112] The activation of hepatic stellate cells (HSCs) plays a key role in MASH-induced liver fibrosis. To explore whether the liver-secreted factor Thbs2 can directly activate HSCs, we used Thbs2 recombinant protein to treat HSCs and systematically analyzed its activation-related biological functions. The experimental results showed that Thbs2 recombinant protein treatment can significantly increase the expression of type I collagen, α-SMA and TGFβ genes in HSCs, and promote the gene expression of endogenous Thbs2 in HSCs ( Figure 6 A). Immunofluorescence analysis showed that Thbs2 treatment promoted the growth of HSC synapses and up-regulated α-SMA protein levels ( Figure 6 B). In addition, Thbs2 treatment significantly reduced the phosphorylation of YAP1 (ser397) and promoted the phosphorylation of p-ERK1 / 2. Cell migration experiments showed that Thbs2 enhanced the cell migration ability of HSC ( Figure 6 D). The above research results show that Thbs2 treatment can directly activate HSC. Existing studies have found that Thbs1 in the Thbs family of proteins can bind to the CD47 receptor and participate in the progression of various diseases, including cardiovascular disease, chronic kidney disease and tumors. Thbs2 and Thbs1 have 62% homology in protein sequence, which suggests that CD47 may be a potential receptor for Thbs2 to activate HSC. The experimental results found that pre-treatment of HSC cells with anti-CD47 antibodies can block the expression of HSC activation-related genes caused by Thbs2 stimulation, as well as the activation of the ERK1 / 2 signaling pathway ( Figure 6 E, F).

[0113] Animal experiments have shown that liver-specific overexpression of Thbs2 can increase the infiltration of liver tissue macrophages and lead to the polarization of M1 macrophages. In order to further clarify the direct effect of Thbs2 on macrophages, we used Thbs2 recombinant protein to treat mouse Raw264.7 mononuclear macrophages in vitro to study its effect on the biological function of macrophages. The experimental results showed that Thbs2 treatment significantly enhanced the expression of inflammatory response-related genes and M1 polarization-related genes induced by lipopolysaccharide (LPS) ( Figure 6 H, I). However, the use of CD47 blockers can significantly inhibit the expression of related genes induced by Thbs2 ( Figure 6J). The above research results indicate that Thbs2 activates the ERK1 / 2 signaling pathway through interaction with the CD47 receptor, promoting the transformation of HSC into myofibroblasts and the activation of M1 macrophages.

[0114] 6. GalNAc-siRNA targeting liver Thbs2 alleviates liver fibrosis and damage caused by MASH

[0115] Since hepatocyte-derived Thbs2 promotes the transformation of MAFLD-MASH, and hepatocyte-specific knockout of Thbs2 can inhibit the occurrence of MASH-related liver fibrosis. Therefore, targeted inhibition of hepatocyte Thbs2 expression may alleviate liver fibrosis and damage caused by MASH. To this end, we used si-Thbs2 (SEQ ID NO: 3-4) and control si-RNA combined with N-acetylgalactosamine (GalNAc) chemical modification to achieve liver-specific knockdown of Thbs2 by subcutaneous injection, and established a MASH mouse model by giving an MCD-HFD diet ( Figure 7 A); wherein, the present invention, through Thbs2-siRNA design and in vitro screening, selected four targets (Thbs2-Mus-1410, Thbs2-Mus-1358, Thbs2-Mus-2500, Thbs2-Mus-2821) to design Thbs2-siRNA and subjected it to N-acetylgalactosamine (GalNAc) chemical modification, the results showed that siThbs2-Mus-1358 had the highest knockdown efficiency, followed by siThbs2-Mus-1358 and siThbs2-Mus-2500, while siThbs2-Mus-1410 could not achieve knockdown (such as Figure 8 As shown in Figure 2, therefore, si-Thbs2 (SEQ ID NO: 3-4) with the highest knockdown efficiency was selected in the animal experiment. The experimental results showed that subcutaneous injection of GaNAc-siRNAs could effectively inhibit the expression of Thbs2 in the liver and significantly reduce the level of Thbs2 in plasma ( Figure 7 BD). Further studies revealed that liver-specific knockdown of Thbs2 significantly reduced plasma ALT and AST levels and alleviated the degree of liver tissue fibrosis ( Figure 7 E, F). Immunofluorescence staining analysis showed that knockdown of Thbs2 significantly reduced macrophage infiltration in liver tissue and inhibited the polarization of M1 macrophages ( Figure 7 G). In addition, GalNAc-siRNA targeting liver Thbs2 significantly inhibited the expression of genes related to fibrosis and inflammatory response ( Figure 7H, I). These research results generally indicate that GalNAc-siRNA targeting liver Thbs2 can alleviate MASH-related liver fibrosis and liver damage, suggesting that the liver secretory factor Thbs2 may be a new target for the treatment of MASH.

[0116] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of the liver secretory factor Thbs2 gene or protein as a drug target in the preparation or screening of drugs for preventing, improving and / or treating metabolic-related fatty liver disease, metabolic-related fatty hepatitis, and liver fibrosis.

2. The use according to claim 1, characterized in that The drug is a chemical drug and / or biological molecule that can inhibit the expression of the liver secretory factor Thbs2 gene and / or protein in vivo, thereby reducing plasma ALT and AST levels, inhibiting liver fibrosis and liver damage, and preventing MAFLD from progressing to MASH.

3. A use of a liver secretion factor Thbs2 gene or protein inhibitor, characterized in that: Used for preparing a composition or preparation, wherein the composition or preparation is used for a) preventing and / or treating metabolism-related fatty liver disease; and / or b) preventing and / or treating liver fibrosis.

4. The use according to claim 3, characterized in that The composition or preparation is also used for i) inhibiting the occurrence of liver fibrosis; and / or ii) inhibiting the activation of hepatic stellate cells (HSC); and / or iii) preventing the progression of MAFLD to MASH.

5. The use according to claim 3, characterized in that The liver secretion factor Thbs2 inhibitor is selected from one or more combinations of the following a)-d): a) Small molecule compounds; b) siRNA, or a modified product or delivery vector thereof that specifically inhibits Thbs2 gene expression; c) shRNA or its delivery vector that specifically inhibits Thbs2 gene expression; d) Thbs2 protein inhibitor.

6. The use according to claim 5, characterized in that The liver secretion factor Thbs2 inhibitor is N-acetylgalactosamine coupled modified siRNA (GalNAc-siThbs2) or its delivery vector, and the sequence of the siRNA is shown in SEQ ID NO: 3-4, SEQ ID NO: 5-6 or SEQ ID NO: 7-8.

7. A pharmaceutical composition, characterized in that include: a1) a first active ingredient for preventing and / or treating metabolism-related fatty liver disease, metabolism-related fatty hepatitis, and liver fibrosis, wherein the first active ingredient comprises: a liver secretory factor Thbs2 gene inhibitor, or a liver secretory factor Thbs2 protein inhibitor; a2) a second active ingredient for preventing and / or treating metabolism-related fatty liver disease, metabolism-related steatohepatitis, and liver fibrosis, wherein the second active ingredient includes: other drugs for preventing and / or treating metabolism-related fatty liver disease, metabolism-related steatohepatitis, and liver fibrosis; and b) a pharmaceutically acceptable carrier.

8. A method for inhibiting the activation of hepatic stellate cells (HSC), characterized in that: include: Hepatic stellate cells are cultured in the presence of a liver secretion factor Thbs2 gene inhibitor or a liver secretion factor Thbs2 protein inhibitor, thereby inhibiting the activation of hepatic stellate cells (HSC).

9. A method for screening potential therapeutic agents for metabolic-related fatty liver disease, metabolic-related fatty liver hepatitis, and liver fibrosis, characterized in that: include: A) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing the liver secretory factor Thbs2 gene for a period of time, and detecting the expression amount E1 of the liver secretory factor Thbs2 gene and / or protein in the culture system of the test group; And in a control group without the test compound and with other conditions being the same, detecting the expression level E2 of the liver secretory factor Thbs2 gene and / or protein in the culture system of the control group; and B) Compare E1 and E2. If E1 is significantly lower than E2, it indicates that the test compound is a potential therapeutic agent for liver fibrosis.

10. The method according to claim 9, characterized in that The cells include hepatocytes.

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