Application of F13A1 as a therapeutic target for non-alcoholic fatty liver disease

Abstract

This invention discloses the application of F13A1 as a therapeutic target for non-alcoholic fatty liver disease (NAFLD), belonging to the field of biomedicine. In a mouse model of acute liver injury, this invention alleviated liver damage by injecting adeno-associated virus (AAV) with knocked-down F13A1 expression into the portal vein of the mouse liver. In a mouse model of liver fibrosis, knocking down F13A1 expression in mouse liver tissue improved liver fibrosis, significantly reduced the expression of inflammation and liver fibrosis-related factors, and significantly improved liver function. The experimental results of this invention demonstrate that specifically inhibiting F13A1 gene expression can improve the development of NAFLD fibrosis, providing a new intervention method and means for the treatment of NAFLD. This invention provides a new target and direction for the clinical treatment of NAFLD, possessing good clinical application value and prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of F13A1 as a therapeutic target for non-alcoholic fatty liver disease. Background Technology

[0002] The liver is an important organ found only in vertebrates. It has a wide range of functions, including the detoxification of various endogenous metabolites and exogenous toxins, protein synthesis, and the production of biochemical substances required for digestion. It also plays a key role in metabolism, including regulating glycogen storage and lipid homeostasis.

[0003] Damage to liver cells leads to liver inflammation and impaired liver function, causing a range of uncomfortable symptoms and abnormal liver function indicators. There are many types of liver diseases, including viral hepatitis, alcoholic hepatitis (AH), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), autoimmune hepatitis (AIH), cholestasis, cirrhosis, and hepatocellular carcinoma (HCC). Among these, NAFLD is one of the most common liver diseases worldwide and has become a significant challenge to people's liver health. The progression from NAFLD to liver fibrosis is a crucial stage in the development of NAFLD and a landmark event signifying its irreversible progression.

[0004] F13A1 is encoded by the gene for coagulation factor XIIIA1. This protein participates in the final solidification stage of the coagulation process, stabilizing fibrin clots by catalyzing the γ-glutamyl-ε-lysine cross-linking of fibrin chains through transglutaminase activity. Currently, there are no methods or reports on intervening in the development of NAFLD fibrosis by targeting F13A1. Summary of the Invention

[0005] To address the above problems, this invention provides an application of F13A1 as a therapeutic target for non-alcoholic fatty liver disease.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The first aspect of the present invention is the use of a reagent that specifically inhibits the expression of the F13A1 gene in the preparation of a medicament for improving non-alcoholic fatty liver disease.

[0008] In a mouse model of acute liver injury, this invention showed that injecting adeno-associated virus (AAV) with knockdown of F13A1 expression into the portal vein of the liver reduced liver damage. In a mouse model of liver fibrosis, the knockdown group showed significant improvement in liver fibrosis. Therefore, inhibiting the expression of the F13A1 gene could be a new direction for improving NAFLD.

[0009] In some preferred embodiments, the reagent is an interfering RNA reagent that specifically inhibits the expression of the F13A1 gene.

[0010] In some preferred embodiments, the interfering RNA reagent is an adenovirus or adeno-associated virus that knocks down the F13A1 gene.

[0011] In some preferred embodiments, the application is in the preparation of a medicament for improving fibrosis and / or liver damage in non-alcoholic fatty liver disease.

[0012] The beneficial effects of this invention are as follows:

[0013] In a mouse model of acute liver injury, this invention demonstrated that injecting adeno-associated virus (AAV) with knocked-down F13A1 expression into the portal vein of the mouse liver reduced liver damage. In a mouse model of liver fibrosis, knocking down F13A1 expression in the liver tissue improved liver fibrosis, significantly reduced the expression of inflammation and liver fibrosis-related factors, and significantly improved liver function. Therefore, this invention, by specifically inhibiting F13A1 gene expression, can improve the development of NAFLD fibrosis, providing a new target and direction for the clinical treatment of NAFLD. Attached Figure Description

[0014] The present invention will be further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present invention. For those skilled in the art, other drawings can be obtained based on the following drawings without creative effort.

[0015] Figure 1-6 This study analyzed samples from patients with liver fibrosis and healthy individuals; among them, Figure 1 This is a UMAP diagram of myeloid cell subsets. Healthy represents healthy individuals, NAFLD represents non-alcoholic fatty liver disease, NASH represents non-alcoholic steatohepatitis, Cirrhosis represents individuals with cirrhosis, and End stage represents the end stage of cirrhosis. Figure 2 A graph showing GO enrichment analysis of complement-related pathways in myeloid cell subsets; Figure 3 Cell differentiation progression constructed for pseudo-temporal analysis, using Healthy / MKL1+ COL4A3BP + MCs are the starting point of differentiation towards Cirrhosis / F13A1 + PLTP + MCs and NAFLD / NASH / End stage / PIK3R3 + DSCAM + MC differentiation (left: grouped by sample, right: grouped by macrophage subset); Figure 4 To identify branch-dependent genes (including C3, C5, F13A1, and PLTP). Figure 5 H&E (hematoxylin-eosin staining) and C3d immunohistochemical staining (IHC of C3d) of liver tissues from healthy individuals and patients with NASH-related liver fibrosis. Control represents the healthy control group, and NASH & Fibrosis represents patients with non-alcoholic steatohepatitis-related fibrosis. Scale bar: 250 μm. Figure 6 Multiplex immunofluorescence staining of CSF1R, F13A1, and PLTP in liver tissues of healthy individuals and patients with NASH-associated liver fibrosis to show F13A1 + PLTP + Localization of MCs subsets in the liver, DAPI for nuclear staining, PLTP for phospholipid transport protein, CSF1R for colony-stimulating factor 1 receptor, F13A1 for coagulation factor XIII A1, Merge for merged plot, scale bar: 100 μm.

[0016] Figure 7-10 This study investigated the complement C3-dependent macrophage subsets in a mouse model of liver fibrosis; among which... Figure 7 Liver H&E staining, Oil-Red O staining, and Sirius red staining of WT (wild-type) mice after 12 weeks of induction with ND+Oil (normal diet + oil), HFD+Oil (high-fat diet + oil), and HFD+CCl4 (high-fat diet + carbon tetrachloride). Scale bar: 250 μm. Figure 8 The levels of serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) in mice; Figure 9 Immunohistochemical staining of mouse liver for F4 / 80 and C3d, scale bar: 250 μm; Figure 10 Multiple immunofluorescence staining of CSF1R, F13A1 and PLTP in the control group (ND+Oil) and the NASH-related liver fibrosis model group (HFD+CCl4) shows the localization of complement C3-dependent macrophage subsets in the liver. Scale bar: 100 μm.

[0017] Figure 11-15A study aimed at alleviating CCl4-induced acute liver injury in mice by knocking down F13A1 in the liver; among which, Figure 11 The mRNA expression levels of F13A1 in the liver tissues of control mice (shCon) and AAV-F13A1 mice (shF13A1) are shown. Figure 12 The protein expression levels of F13A1 in the liver tissues of control mice (shCon) and AAV-F13A1 mice (shF13A1) were determined, with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as an internal reference protein. Figure 13 H&E staining of livers in control mice (shCon) and AAV-F13A1 mice (shF13A1) acute liver injury models. Scale bar: 250 μm. Figure 14 The levels of serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) in mice; Figure 15 The values ​​represent the mRNA expression levels of inflammation-related factors in liver tissue of an acute liver injury model. F4 / 80 is a specific surface marker of mature macrophages. TNF-α is α-tumor necrosis factor, IL-6 is interleukin-6, and IL-1β is interleukin-1β.

[0018] Figures 16-22 This study aimed to investigate the effect of knocking down F13A1 expression in mouse liver tissue on liver fibrosis; among which, Figure 16 The mRNA expression level of F13A1 in liver tissue of a mouse model of liver fibrosis is shown in the figure. shCon is the control group and shF13A1 is the experimental group. Figure 17 The localization of F13A1 immunofluorescence staining in liver tissues of the control and experimental groups is shown. DEPI is used for cell membrane staining. Scale bar: 100 μm. Figure 18 H&E staining, Picro-Sirius Red staining, and Masson staining were performed on the liver of a mouse model of liver fibrosis. Figure 19 The serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels in mice with liver fibrosis model; Figure 20 The values ​​represent the mRNA expression levels of liver fibrosis-related factors in the liver of a mouse model of liver fibrosis. α-sma is α-smooth muscle actin, Col1α1 is type I collagen α1, TIMP-1 is tissue inhibitor of metalloproteinases-1, and TGF-1β is transforming growth factor-β. Figure 21 The values ​​represent the mRNA expression levels of inflammation-related factors in the liver of a mouse model of liver fibrosis, with MCP-1 being monocyte chemoattractant protein-1. Figure 22 The image shows the localization of α-SMA immunofluorescence staining in liver tissue for the control group (shCon) and the F13A1 adeno-associated virus group (shF13A1). DEPI is used for cell membrane staining. Scale bar: 100 μm. Detailed Implementation

[0019] The present invention will be further described in conjunction with the following embodiments.

[0020] The embodiments of the present invention relate to the use of a reagent that specifically inhibits F13A1 gene expression in the preparation of a medicament for improving non-alcoholic fatty liver disease.

[0021] Example 1

[0022] Through a rigorous quality control process, the inventors integrated multiple Single-Nucleus RNA Sequencing (sn-RNA) sequencing data, including liver samples from healthy individuals and multiple stages of the NAFLD process, and discovered F13A1. + PLTP + MCs are significantly increased in the liver fibrosis stage (see appendix) Figure 1 , 2 By sequencing four macrophage groups using pseudo-temporal analysis, the inventors discovered F13A1. + PLTP + Complement-related genes are significantly activated in MCs (especially C3, C5, F13A1, and PLTP) (see appendix) Figure 3 , 4 The inventors also examined complement expression and F13A1 in human liver tissue samples. + PLTP + The results showed that C3d expression was elevated and F13A1 expression was increased in tissue samples from human NASH-associated liver fibrosis. + PLTP + Increased levels of MCs subgroups (with appendix) Figure 5 , 6 ), prompt F13A1 + PLTP + MCs subsets are specifically expressed in patients with NASH-related liver fibrosis.

[0023] To better understand the role of the aforementioned macrophage subsets in NASH-related liver fibrosis, the inventors constructed an HFD+CCl4-induced model using wild-type (WT) mice. (See appendix) Figure 7-10 In the induced mouse model, immunohistochemistry of liver tissue sections showed that a large number of C3d cells were deposited in lipid accumulation and fibrosis areas in the liver fibrosis group. Furthermore, immunolabeling of this cell subset in pathological sections of the mouse model revealed that the content of complement C3-dependent macrophages was significantly higher in the NASH-related fibrosis group than in the control group, indicating the presence of F13A1 in the liver fibrosis mouse model. + PLTP + MCs subgroup.

[0024] To elucidate the role of F13A1, the inventors constructed an adenovirus shRNA (sequence: 5'-AGCTAATTGTGACCAAGCAAA-3', SEQ ID NO: 1) for knocking down F13A1 expression and a negative control adenovirus shRNA (sequence: 5'-TTCTCCGAACGTGTCACGT-3', SEQ ID NO: 2). F13A1 adeno-associated virus (provided by Shanghai Jikai Gene Technology Co., Ltd., using the broad-spectrum promoter CMV, serotype 9, 100 μL, titer 2e12 vg / mL) was injected via portal vein into the liver of mice to knock down wild-type mice (AAV-F13A1). After stable expression, an acute injury model was constructed, and 25% CCl4 was injected intraperitoneally. Samples were collected 24 hours later, and the liver tissue of this model was analyzed for H&E, F13A1 expression levels, and inflammatory markers, including F4 / 80, TNF-α, IL-6, and IL-1β. See Appendix. Figure 11-15 The results showed that, compared with the control group (shCon), the AAV-F13A1 group (shF13A1) had a significantly smaller area of ​​liver necrosis, significantly improved liver function, and significantly reduced expression of inflammatory factors.

[0025] To further investigate the role of F13A1 in liver fibrosis, a mouse model of liver fibrosis was established using F13A1 adeno-associated virus knockdown mice via HFD+CCl4. The mice were injected with CCl4 twice weekly for 4 weeks. Compared with the control group (shCon), the results are shown in the appendix. Figure 16-22 The AAV-F13A1 group (shF13A1) showed significant improvement in liver fibrosis, a significant decrease in the expression of inflammation and liver fibrosis-related factors, and a significant improvement in liver function. This indicates that inhibiting F13A1 expression in mouse liver tissue can improve HFD+CCl4-induced liver fibrosis. The above results suggest that specific inhibition of F13A1 gene expression can improve the development of NAFLD fibrosis.

[0026] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of a reagent that specifically inhibits F13A1 gene expression in the preparation of drugs to improve non-alcoholic fatty liver disease, characterized in that, The reagent is an interfering RNA reagent that specifically inhibits the expression of the F13A1 gene. The interfering RNA reagent is an adenovirus or adeno-associated virus that knocks down the F13A1 gene. The expression shRNA of the adenovirus or adeno-associated virus targeting the F13A1 gene sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, Application in the preparation of drugs for improving fibrosis and / or liver damage in non-alcoholic fatty liver disease.

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