Application of C3 complement inhibitors in the preparation of drugs to improve non-alcoholic steatohepatitis

Abstract

This invention discloses the application of a C3 complement inhibitor in the preparation of drugs to improve non-alcoholic steatohepatitis (NASH), belonging to the field of biomedicine. This invention utilizes sn-RNA sequencing and analysis technology to discover that NASH-related liver fibrosis is associated with elevated C3d expression and F13A1... + PLTP + Increased levels of MC subsets were associated with this condition. Cellular and mouse models confirmed that targeting complement inhibitors or C3 gene deletion can reduce F13A1 levels. + PLTP + The differentiation and formation of MCs subsets are dependent on complement C3 and can significantly reduce lipid stasis and liver fibrosis. The therapeutic effect of the C3 complement inhibitor CR2-Crry was studied in a mouse liver fibrosis model. The results showed that blocking the C3-activated complement pathway by CR2-Crry can improve lipid stasis and liver fibrosis caused by non-alcoholic steatohepatitis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of C3 complement inhibitors in the preparation of drugs for improving non-alcoholic steatohepatitis. 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 non-alcoholic hepatitis to liver fibrosis is a crucial stage in the development of NAFLD and a landmark event signifying its irreversible progression. Liver fibrosis is a dynamic process of liver damage and repair. The production of extracellular matrix (ECM), mainly composed of collagen, exceeds the liver's degradation capacity, leading to excessive deposition of ECM in the liver and the formation of so-called fibrous "scars." Currently, there are no drugs or methods specifically for liver fibrosis. Summary of the Invention

[0004] To address the above problems, this invention provides the application of a C3 complement inhibitor in the preparation of a drug for improving non-alcoholic steatohepatitis.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The first aspect of the present invention is to provide the use of a C3 complement inhibitor in the preparation of a medicament for improving non-alcoholic steatohepatitis.

[0007] In some preferred embodiments, the C3 complement inhibitor is CR2-Crry.

[0008] In some preferred embodiments, the application is in the preparation of a medicament for improving fibrosis and / or lipid stasis in non-alcoholic steatohepatitis.

[0009] In some preferred embodiments, the application is in the preparation of F13A1 inhibitors + PLTP + Applications of drugs in the differentiation of MCs subpopulations.

[0010] Activated hepatic stellate cells (HSCs) transforming into fibroblasts are the primary source of the extracellular matrix (ECM), but the mechanism of HSC activation remains incompletely understood. With ongoing research and the development of single-cell techniques, the roles of different immune cells in liver fibrosis are receiving increasing attention, particularly macrophages. Macrophages are considered central coordinators of tissue responses to injury, and different macrophage subsets and activation states play crucial roles in fibrosis progression and regression. Targeting liver fibrosis-related specific macrophage subsets may lead to new therapeutic approaches for liver fibrosis.

[0011] The inventors discovered in their research that elevated expression of C3d (a marker of complement system cleavage products) and F13A1 was observed in tissue samples from human NASH-associated liver fibrosis. + PLTP + The elevated levels of MCs subsets were specifically observed by the inventors through a rigorous quality control process. This involved integrating multiple Single-Nucleus RNA Sequencing (sn-RNA) sequencing data, including liver samples from healthy individuals and various stages of NAFLD progression, and identifying F13A1. + PLTP + MCs (myeloid cells, a type of innate immune cell derived from pluripotent stem cells in the bone marrow) increase significantly during the liver fibrosis stage (see appendix). Figure 1 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 2 , 3 ), prompt F13A1 + PLTP + MCs subsets are specifically expressed in patients with NASH-related liver fibrosis.

[0012] The inventors confirmed through cell and mouse models that targeting complement inhibitors or C3 gene deletion can reduce F13A1. + PLTP + The differentiation and formation of MCs subsets are dependent on complement C3 and can significantly reduce lipid accumulation and liver fibrosis. The ameliorative effect of the C3 complement inhibitor CR2-Crry on liver fibrosis was verified in a mouse liver fibrosis model.

[0013] CR2-Crry is a C3 complement inhibitor that blocks all complement pathways in the C3 activation process. It has two key structures: CR2 and Crry. These two proteins form a fusion protein. CR2 targets the target region (complement activation region), and Crry exerts its inhibitory effect to inhibit complement activation in the target region. It is prepared by linking complement receptor 2 (CR2) to a membrane-binding regulator (Crry). For details on the preparation, please refer to the preparation of CR2-Crry fusion protein in the article "Atkinson C, Song H, Lu B, et al. Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection. J ClinInvest. 2005; 115(9):2444-2453".

[0014] While disclosing the application of the above-mentioned C3 complement inhibitor in the preparation of a drug for improving non-alcoholic steatohepatitis, this invention also provides a drug for improving non-alcoholic steatohepatitis, the drug comprising a C3 complement inhibitor.

[0015] In some preferred embodiments, the C3 complement inhibitor is CR2-Crry.

[0016] In some preferred embodiments, pharmaceutically acceptable excipients are included.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention utilizes a targeted complement inhibitor to block the C3-activated complement pathway, thereby reducing the dependence of F13A1 on complement C3. + PLTP +The differentiation and formation of MC subsets, which in turn inhibits lipid stasis and liver fibrosis caused by non-alcoholic steatohepatitis, suggests that complement inhibition via the C3 activation step may be a potential and precise treatment for NASH. Attached Figure Description

[0019] 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.

[0020] Figure 1-3 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 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 3 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.

[0021] Figure 4-7 To investigate the complement C3-dependent macrophage subsets in a mouse model of liver fibrosis; Figure 4 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 5 The levels of serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) in mice; Figure 6 Immunohistochemical staining of mouse liver for F4 / 80 and C3d, scale bar: 250 μm; Figure 7Multiple 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.

[0022] Figure 8-10 The effect of complement C3-dependent macrophage subset inhibition on liver fibrosis in a mouse model; Figure 8 Wild-type mice (WT) and C3 knockout mice (C3) - / - Liver H&E staining, Oil-Red O staining, Sirius red staining, C3d and F4 / 80 immunohistochemical staining after 12 weeks of HFD+CCl4 induction, scale bar: 250μm; Figure 9 Multiple immunofluorescence staining of CSF1R, F13A1 and PLTP in the WT group and C3 knockout group shows the localization of complement C3-dependent macrophage subsets in the liver. Scale bar: 100 μm. Figure 10 F13A1 in myeloid cell subsets of single-cell sequencing WT group (left) and C3 knockout group (middle: sample 1, right: sample 2) + PLTP + MCs subgroup UMAP diagram;

[0023] Figure 11-13 To study the therapeutic effect of the complement inhibitor CR2-Crry in a mouse model of liver fibrosis; Figure 11 Liver H&E staining, Oil-Red O staining, and Sirius Red staining were performed on the saline control group (NS) and the CR2-Crry treatment group (CR2-Crry). Figure 12 Immunohistochemical staining for F4 / 80 and C3d, scale bar: 250μm; Figure 13 Multiplex immunofluorescence staining of CSF1R, F13A1, and PLTP in the NS and CR2-Crry groups showed F13A1. + PLTP + Localization of MCs subsets in liver tissue, scale bar: 100 μm. Detailed Implementation

[0024] The present invention will be further described in conjunction with the following embodiments.

[0025] Embodiments of the present invention relate to the use of a C3 complement inhibitor in the preparation of a medicament for improving non-alcoholic steatohepatitis.

[0026] Example 1

[0027] To better understand the role of macrophage subsets in NASH-related liver fibrosis, the inventors constructed an HFD+CCl4-induced model using wild-type (WT) mice. (See appendix) Figure 4-7 In the induced mouse model, immunohistochemistry of liver tissue sections showed that a large number of C3d cells were deposited in the lipid accumulation and fibrosis areas of the liver fibrosis group. At the same time, immunolabeling of this subpopulation of cells in the pathological sections of the mouse model showed that the content of complement C3-dependent macrophage subpopulation was significantly higher in the NASH-related fibrosis group than in the control group.

[0028] Example 2

[0029] To reveal the F13A1 + PLTP + The role of MCs subset differentiation in liver fibrosis: The inventors constructed an HFD+CCl4 model with WT and C3 knockout backgrounds, and analyzed F13A1 in liver pathological sections of this model. + PLTP + Immunofluorescence colocalization and single-cell sequencing of MCs subsets. Results are shown in the appendix. Figure 8-10 Compared to the WT group, the C3 knockout group showed a lack of complement C3d deposition, significantly reduced lipid accumulation and liver fibrosis; single-cell sequencing results showed that F13A1 + PLTP + The content of MC subgroups was significantly reduced. These results suggest that C3... - / - Reduce F13A1 + PLTP + The differentiation and formation of MCs subsets is dependent on complement C3 and can alleviate liver fibrosis.

[0030] Example 3

[0031] The therapeutic effect of the complement inhibitor CR2-Crry was investigated in a mouse model of liver fibrosis. A mouse liver fibrosis model was established using HFD+CCl4. CR2-Crry (25 mg / kg) was injected every other day for 4 weeks concurrently with CCl4 injections (twice a week). The control group received normal saline (NS). Results are shown in the appendix. Figure 11-13 After CR2-Crry treatment, C3d expression decreased, complement activation was inhibited, lipid stasis and liver fibrosis were significantly improved, and the expression of macrophage marker F4 / 80 was significantly reduced, as was F13A1. + PLTP + The number of MCs subsets was significantly reduced. These results suggest that blocking the C3-activated complement pathway via CR2-Crry can improve lipid accumulation and liver fibrosis caused by non-alcoholic steatohepatitis.

[0032] 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. Use of a C3 complement inhibitor, which is CR2-Crry, in the manufacture of a medicament for ameliorating fibrosis in nonalcoholic steatohepatitis.

2. Use according to claim 1, characterized in that, In the manufacture of a medicament for inhibiting F13A1 + PLTP + MCs subpopulation differentiation forming a medicament for use in the treatment of a disease.

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