CN115040638A - Application of BMP9 in preparation of medicine for treating hepatic fibrosis/cirrhosis related to MAFLD, NASH and NASH - Google Patents

Abstract

The invention discloses the use of BMP9 in preparing medicines for MAFLD, NASH and NASH-related liver fibrosis/cirrhosis. The invention provides a feasible selection scheme for preparing medicines for treating MAFLD, NASH medicines, NASH-related liver fibrosis medicines, and NASH-related liver cirrhosis.

Description

BMP9 in the preparation of drugs for MAFLD, NASH, NASH-related liver fibrosis/cirrhosis the use of

technical field

The invention belongs to the field of medicine, and particularly relates to the use of BMP9 in the preparation of medicines for MAFLD, NASH and NASH-related liver fibrosis/cirrhosis.

Background technique

Non-alcoholic steatohepatitis (NASH) is a key process of non-alcoholic fatty liver disease (NAFLD), with no obvious symptoms and abnormal biological indicators in the early stage, and it can progress to liver occult. However, the pathogenesis of end-stage liver diseases such as fibrosis, liver cirrhosis, and liver cancer is still very vague, and there is a lack of early non-invasive diagnostic indicators and therapeutic drugs in clinical practice.

Bone morphogenetic protein 9 (BMP9) is mainly secreted by hepatic stellate cells (HSCs) and can regulate glucose and lipid metabolism. Hepatic macrophages have its high-affinity receptors. But so far, the process of how BMP9 regulates NASH has not been clearly elucidated, thus limiting the invention of related drugs.

Regarding the relationship between BMP9 and NASH, some people have used methionine choline deficiency (MCD)-induced model mice to do preliminary exploration, suggesting a correlation between the two. However, the MCD mouse model lacks the characteristics of metabolic disorders and increases in body weight. The test prolongation was significantly reduced, which was significantly different from the metabolic process of obesity-induced NASH common in humans, so the effect of BMP9 on obesity-related NASH has not been reported so far.

The Chinese patent application with publication number CN113198002A discloses the application of a hepatocyte factor BMP9 in the preparation of drugs for delaying and/or treating non-alcoholic fatty liver disease. The invention adopts the high-affinity carrier AAV8 of the liver as the drug carrier, and through gene therapy, the specific and high-efficiency overexpression of BMP9 in the liver tissue can be realized, the related symptoms of fatty liver can be alleviated, and the fatty liver can be effectively treated. It provides a new treatment idea for the treatment of non-alcoholic fatty liver; and through research, it is also found that the specific and high overexpression of BMP9 in liver tissue can also be effective for obesity symptoms caused by low expression or deletion of BMP9 cytokines. ease. However, this invention is aimed at non-alcoholic fatty liver, which is also called simple fatty liver, and only sees fatty degeneration of liver cells. Generally speaking, it can be improved by adjusting the diet structure, aerobic exercise to lose weight, correcting bad living habits, etc. , generally do not need medication, the AAV8-BMP9 used in this invention only has the effect of reducing fatty liver in a way of regulating liver lipid metabolism mediated by PPARα, but cannot be actually clarified and applied to steatohepatitis, liver fibrosis, liver cirrhosis and other diseases.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides the use of BMP9 in the preparation of medicines for MAFLD, NASH, and NASH-related liver fibrosis/cirrhosis.

The present invention proposes the following technical solutions:

Use of BMP9 in the preparation of a medicament for MAFLD (metabolism-associated fatty liver disease).

Use of BMP9 in the preparation of medicines for NASH (non-alcoholic steatohepatitis), medicines for NASH-related liver fibrosis, and medicines for NASH-related liver cirrhosis.

As a preferred option, the drug alleviates and/or treats NASH, NASH-related liver fibrosis, and NASH-related liver cirrhosis by inhibiting the TNF signaling pathway in the liver and down-regulating the expression of TRAF3 in the liver.

Preferably, the BMP9 is rhBMP9 (recombinant human bone morphogenetic protein 9).

As a preference, the medicine is an injection.

Beneficial effects:

The non-alcoholic fatty liver hepatitis (NASH) and related liver fibrosis and liver cirrhosis in the metabolic-related fatty liver disease of the present invention refer to liver cell necrosis, inflammatory cell infiltration, elevated transaminase, central Fibrosis around veins and around hepatocytes, formation of pseudolobules and regenerative nodules, etc., are different from the pathogenesis of simple fatty liver disclosed in the prior art.

The rhBMP9 used in the present invention achieves the effect of relieving and/or treating non-alcoholic steatohepatitis (NASH) and its related liver fibrosis and liver cirrhosis by inhibiting the TNF signaling pathway in the liver and down-regulating the expression of TRAF3 in the liver, and further application on drugs for related diseases.

It is to be understood that all combinations of the foregoing concepts, as well as additional concepts described in more detail below, are considered to be part of the inventive subject matter to the extent that such concepts are not mutually inconsistent.

Description of drawings

The drawings are not intended to be drawn to scale unless specifically stated. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by the same reference numeral. For clarity, not every component is labeled in every figure.

Figure 1 shows the comparison of BMP9 levels in NASH mice and NASH patients.

Figure 2 shows the comparison of the effect of exogenous rhBMP9 supplementation on liver steatosis and NASH progression in HFD mice.

Figure 3 is a graph showing the comparison of the effects of rhBMP9 on oleic acid (OA)-induced hepatocyte steatosis.

Figure 4 shows the comparison of the effect of rhBMP9 on the TNF signaling pathway in the liver and the expression of TRAF3 in the liver.

Figure 5 shows the comparison of the effect of rhBMP9 on the infiltration of macrophages in the liver of mice in the HFD model group.

Figure 6 shows the comparison of rhBMP9 regulating the polarization of mouse liver macrophages M1 and M2 and the cytokine secretion of macrophage cell lines in vitro.

Figure 7 shows the primers described in real-time PCR.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are some, but not all, embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention. Unless otherwise defined, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

The terms "first", "second" and similar terms used in the description and claims of the present invention do not denote any order, quantity or importance, but are only used to distinguish different components. Also, unless the context clearly dictates otherwise, the singular forms "a," "an," or "the" and the like do not denote a limitation of quantity, but rather denote the presence of at least one. Words like "including" or "comprising" mean that the elements or items appearing before "including" or "including" cover the features, integers, steps, operations, elements and/or recited after "including" or "including" or components, does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or sets thereof. "Up", "Down", "Left", "Right", etc. are only used to indicate relative positional relationship, and when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

Although the prior art discloses the application of BMP9 in the preparation of drugs for delaying or/and treating non-alcoholic fatty liver disease, the prior art is different from the present invention in terms of the site of action, pharmacological action, symptom improvement and other therapeutic mechanisms. The differences make substantial differences between NAFLD medications and metabolic-related fatty liver disease. Specifically reflected in:

Previous inventions are aimed at non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease is also known as simple fatty liver. Only fatty degeneration of liver cells is seen. Generally speaking, it can be improved by adjusting diet structure, aerobic exercise to lose weight, and correcting bad living habits. Generally, no medication is required. The AAV8-BMP9 used in this invention only has the effect of alleviating fatty liver by regulating liver lipid metabolism mediated by PPARα. Sclerosis and other diseases. The focus of the present invention is on non-alcoholic steatohepatitis (NASH) and its related liver fibrosis and liver cirrhosis in metabolic-related fatty liver disease. High, pericentral venous and perihepatocyte fibrosis, pseudolobular and regenerative nodule formation, etc. It is different from the pathogenesis of simple fatty liver. The rhBMP9 used in the present invention achieves the effect of relieving and/or treating non-alcoholic fatty liver hepatitis (NASH) and its related liver fibrosis and liver cirrhosis by inhibiting the TNF signaling pathway in the liver and down-regulating the expression of TRAF3 in the liver.

Although non-alcoholic fatty liver disease and metabolic-related fatty liver disease are not literally different, they are induced by two different mechanisms and can exhibit distinguishable symptoms. Nonalcoholic fatty liver disease is only the least symptomatic part of metabolic-related fatty liver disease, and only hepatocyte steatosis is seen. In metabolic-related fatty liver disease, steatohepatitis is accompanied by degeneration and necrosis of hepatocytes and inflammatory cell infiltration on the basis of steatosis, and may be accompanied by Mallory bodies and fibrosis. Subsequent hepatic fibrosis, on the basis of steatohepatitis, appeared pericentral venous and perihepatocyte fibrosis, and even portal fibrosis and central portal fibrosis were connected. The final liver cirrhosis is the remodeling of the hepatic lobular structure secondary to fatty liver, and the formation of pseudolobules and regenerative nodules. Because the pathogenesis of nonalcoholic fatty liver disease and metabolic-related fatty liver disease are different, they can be treated with different drugs.

The action mechanism and therapeutic effect of the present invention will be further introduced in detail below through specific experimental methods and results.

The experimental method and analysis of the present embodiment are as follows:

Experimental patient grouping:

For healthy control group (n=67), NAFL (n=75) and NASH (n=26), the serum of patients was collected to detect BMP9 concentration. All patients were from Shanghai Dongfang Hospital and were approved by the Ethics Committee of Shanghai Dongfang Hospital. n represents the number of samples.

Experimental animal grouping:

6-week-old male C57BL/6 mice (16-18 g) were purchased from Shanghai Jihui Laboratory Animal Co., Ltd. (Shanghai). Animals were housed in the Experimental Animal Center of the Second Military Medical University. All animal experiments were approved by the Scientific Investigation Committee of the Second Military Medical University.

Randomly divided into three groups: High-fat diet (HFD) group (n=8), HFD+BMP9 group (n=8) and control group (n=6).

HFD group: fed with high-fat diet HFD (protein 14.1%; fat 60%; carbohydrate 25.9%), intraperitoneally injected polybutylene succinate (PBS) once a day for 4 consecutive weeks.

HFD+BMP9 group: HFD was fed, and recombinant bone morphogenetic protein 9 (rhBMP9, PEPROTECT, batch number: 553204) was intraperitoneally injected at a dose of 200 ng per animal, once a day for 4 consecutive weeks.

Control group: fed with common feed and intraperitoneally injected with PBS once a day for 4 consecutive weeks.

After 12 weeks, all mice were sacrificed and liver, serum and visceral adipose tissue (VAT) were collected. The BMP9 in this example is rhBMP9.

Histological Analysis:

Livers and VAT were fixed in 10% formalin and 4% paraformaldehyde and then embedded in paraffin or frozen following standard procedures. Liver sections were stained with hematoxylin and H&E, Sirius Red and Oil Red O. The extent of NAFLD was estimated by the NAFLD Activity Score (NAS). Image analysis software (IMAGE-PRO Plus 6.0; Media Cybernetics, Rockville, MD, United States) was used to calculate the intensity of steatosis, ie, the percentage of positive area of Oil Red O staining in the corresponding area of liver tissue.

Immunohistochemistry:

Immunohistochemical staining was performed on 4 μm thick paraffin sections of tissue fixed in buffered formalin. Sections were dewaxed in xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked with 3% H2O2, followed by antigen retrieval. Sections were incubated with primary antibodies overnight at 4°C and secondary antibodies for 60 min at room temperature. The following primary antibodies were used. BMP9 (sc514211, Santa Cruz) and F4/80 (70,076, Cell Signaling Technologies). Staining was performed using EnVision Detection Rabbit/Mouse Kit (GK500710, GeneTech, Shanghai, China).

Determination of triglycerides (TG) and cholesterol (CHOL):

The contents of TG and CHOL in mouse liver were measured using commercial kits (TG: A0-10017; CHOL: A111-2-1, Jiancheng Company, Nanjing, China).

Real-time PCR:

Total RNA from liver tissue was extracted with Trizol (Invitrogen, Carlsbad, CA, United States). Transcription levels were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) using SYBR Green PCR kit (Takara, Tokyo, Japan).

Figure 7 shows the primer sequences for qRT-PCR.

Western blot analysis:

Tissues and cells were lysed in lysis buffer (125 mM Tris-HCl pH 6.8, 25% glycerol, 5% SDS) supplemented with protease inhibitors (Roche, Switzerland). Lysates were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to methanol-activated nitrocellulose membranes (HAHY00010; Millipore, Merck KGaA, Darmstadt, Germany). Membranes were blocked for 1 h in PBS containing 1% Tween-20 and 5% milk and incubated with primary antibodies overnight at 4°C. After 1 hour incubation with donkey anti-mouse or donkey anti-rabbit secondary antibodies (IRDye 800; LI-COR Biosciences, Lincoln, NE, United States), images were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences) at 700/800 nm wavelengths. signal for imaging. The primary antibody used is. SREBP1 (ab96777, Abcam; sc-557036, Santacruz), phosphorylated Akt (4060, Cell Signaling Technologies), Akt (40D4, Cell Signaling Technologies) and GAPDH (YM3029, Immunoway).

Biochemical Analysis:

Serum biochemical indexes were measured with an automatic analyzer in the Clinical Immunology Department of the Eastern Institute of Hepatobiliary Surgery.

RNA sequencing (RNA-seq):

RNA样品制备试剂盒(Illumina，美国)按照

RNA样品制备指南合成配对端文库。文库构建和测序工作在上海生物技术公司进行。根据每个样品中的FPKM来估计倍数变化。显著差异表达的基因采用以下筛选标准：P值≤0.05，∣Log2FC∣>1。这些数据可以在NCBI-Bio项目中获得，登录号为PRJNA644055。Total RNA was isolated from liver samples using the RNeasy mini kit (Qiagen, Germany). use

RNA sample preparation kit (Illumina, USA) according to

RNA sample preparation guidelines for synthesizing paired-end libraries. Library construction and sequencing were performed at Shanghai Biotechnology Company. The fold change was estimated from the FPKM in each sample. Significantly differentially expressed genes were selected using the following screening criteria: P value ≤ 0.05, ∣Log2FC∣>1. These data are available in the NCBI-Bio project under accession number PRJNA644055.

ATAC-seq:

ATAC-seq was performed on fresh liver tissue as previously described (Buenrostro et al., 2015). MACS2 (2.1.1) was used to call the peak, and the initial threshold was defined as p<1* ^10-5 . Pairwise Spearman correlations between any pair of ATAC-seq samples were calculated from reads/signals on the ATAC-seq peaks pooled for all samples. ATAC-seq peaks were annotated using Homer's annotate Peaks.pl. Protein binding patterns were identified using HOMER software (http://homer.ucsd.Edu/homer/). Three biological replicates were used. These data are available in the NCBI-Bio project under accession number PRJNA644146.

Statistical Analysis:

Data analysis was performed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA) to assess the statistical significance of differences. Unpaired Student's t-test was performed to compare differences between groups. All data are presented as mean and/or SEM. Statistical significance was set at *p≤0.05, **p≤0.01, and ***p≤0.001. Differences with a P value of <0.05 were considered statistically significant.

The result is as follows:

Example 1

Changes of BMP9 levels in NASH mice and NASH patients.

The HFD-induced mouse NAFLD model has preliminarily shown the early pathological characteristics of NASH (hepatocyte ballooning and inflammatory cell infiltration), and the expression of BMP9 at the mRNA and protein levels in the mouse liver was analyzed.

A high-fat diet (HFD)-induced mouse model of NAFLD (with early pathological changes in NASH) was established, and liver samples were collected from mice in the control and treatment groups, and the expression of BMP9 was analyzed; research cohorts of healthy controls, NAFL and NASH patients were established. Serum BMP9 concentration was detected by ELISA.

RT-PCR showed that the expression of BMP9 mRNA in HFD mice was significantly down-regulated compared with the control group (Fig. 1A).

Western blot and immunohistochemistry showed that BMP9 protein levels were also significantly reduced in fatty livers of HFD mice (Figure 1B-E).

In addition, immunohistochemical staining further showed that BMP9 staining in the non-parenchymal cells of the liver of HFD mice was lighter than that of the control group (Fig. 1D-E).

The concentration of serum BMP9 in NAFLD patients and the control group was further detected, and it was found that the serum BMP9 level in NAFL and NASH patients was significantly lower than that in the control group, and the serum BMP9 level in the NASH group and NAFL group was also significantly decreased (Figure 1F).

Figure 1 shows the level of BMP9 in NASH mice and NASH patients. In the figure, (A) qPCR detected mRNA transcription level; (B-C) Western blot detected protein translation level; (D-E) Liver immunohistochemical analysis of BMP9 in protein translation level and positioning. (F) Serum BMP9 concentrations in healthy controls, NAFL and NASH groups. Data are expressed as mean ± SEM (n=6 mice per group; healthy controls n=67; NAFL n=75; NASH n=26), vs. Control group, *p<0.05, ***p< 0.001; ##p<0.01 vs. NAFL group. All these data confirm that BMP9 levels are significantly reduced in NASH mice as well as NASH patients, that is, BMP9 expression is reduced in NASH.

Example 2

Effects of exogenous rhBMP9 supplementation on hepatic steatosis and NASH progression in HFD mice.

Figure 2 shows that exogenous supplementation of rhBMP9 significantly improved liver steatosis and NASH progression in HFD mice. In the figure, (AB) liver appearance and liver wet weight; (CI) Oil red O staining (CD), HE staining (E) analysis of intralobular inflammation (F), steatosis (G), hepatocyte ballooning (H) ) and liver NAFLD activity score (I), scale bar 100 μm; (J) intrahepatic triglyceride (TG) content. Data are expressed as mean±SEM (n=8), vs. unmodeled Control group, *p<0.05, **p<0.01, ***p<0.001; vs. HFD group, ^# p<0.05, ^## p< 0.01.

The results showed that the liver color of HFD mice was significantly paler than that of the control group, and the liver wet weight was significantly greater than that of the control group. However, the liver color of the HFD mice treated with rhBMP9 was close to that of the control group, and the liver wet weight was also significantly reduced (Figure 2A-B). Histologically, rhBMP9 treatment significantly attenuated hepatic steatosis (Fig. 2C, D, G), hepatocyte ballooning (Fig. 2E, F, H) and nonalcoholic fatty liver disease activity score (NAS) in HFD mice ) ( FIG. 2I ); in addition, rhBMP9 also significantly decreased intrahepatic triglyceride (TG) levels in HFD mice ( FIG. 2J ).

Furthermore, whether rhBMP9 inhibits hepatic steatosis was verified by hepatic cell line, and the steatosis model of HepG2 cells was induced by oleic acid (OA) to observe the effect of rhBMP9 on steatosis.

Figure 3 shows that rhBMP9 treatment significantly inhibited oleic acid (OA)-induced hepatocyte steatosis. HepG2 cells were induced to undergo steatosis by OA (0.5mM), and the treatment group was treated with rhBMP9 (10ng/ml) at the same time. After 48 hours, they were stained with Oil Red O and photographed, and the steatosis and intracellular triglyceride (TG) conditions were counted. . (AB) Statistics of oil red O staining positive area of cells in each treatment group (A) and representative graph (B), the bar is 100 μm; (C) Determination of intracellular triglyceride (TG) content in each group. Data are expressed as mean±SEM (n=8), **p<0.01, ***p<0.001 vs. Control group; ^# p<0.05, ^## p<0.01 vs. OA group.

The results showed that oleic acid could significantly induce steatosis in HepG2 cells, and the steatosis of HepG2 cells was significantly inhibited by adding rhBMP9 (Fig. This further indicated that rhBMP9 could inhibit hepatic steatosis.

Example 3

Effects of rhBMP9 on TNF signaling pathway in liver.

Experiment: In order to clarify the mechanism by which rhBMP9 treatment improves fatty liver/NASH in HFD mice, the present invention uses RNA-seq to detect changes in liver transcriptional profiles of HFD mice after rhBMP9 treatment.

Figure 4 shows that rhBMP9 significantly inhibited liver TNF signaling pathway and down-regulated TRAF3 expression in liver. The normal Control group, HFD model group, and BMP9+HFD group mouse livers were used for RNA-seq analysis of transcriptional profiles, and edgeR was used for data analysis to analyze differential genes between samples. FDR (False Discovery Rate) determines the threshold of p-value, and the corrected p-value is q-value. At the same time, we calculated the fold of differential expression according to the FPKM value, that is, Fold-change. Differential gene screening conditions were q<0.05 and Fold-change>2. (A) KEGG signal pathway enrichment analysis; (B) differential gene analysis between the rhBMP9 treatment group and the HFD group; (C) the close connection between the TNF and Toll signaling pathways screened in A and TRAF3.

The results showed that compared with HFD mice, the liver of rhBMP9-treated mice showed down-regulated expression of 189 genes and up-regulated expression of 130 genes. Further analysis showed that BMP9 reversed the expression of 118 genes, including genes related to glucose and lipid metabolism and Inflammation, immune response genes.

The KEGG signaling pathway enrichment analysis found that compared with the normal Control group, the TNF and Toll signaling pathways were significantly up-regulated in the HFD model group mice, and the two pathways were significantly down-regulated in the rhBMP9-treated mice (Figure 4A), suggesting that The related pathways are involved in the possible mechanism of the therapeutic effect of rhBMP9.

After analysis, TRAF3 has the highest fold reduction among all down-regulated genes (Fig. 4B). This is because TRAF3 just mediates the TNF and Toll signaling pathways. Therefore, rhBMP9 is most likely to achieve its treatment of HFD by affecting TRAF3 and related signaling pathways. Induced fatty liver and early NASH (Fig. 4C).

Example 4

BMP9 regulates the polarization of M1/M2 and the expression of related inflammatory factors in liver macrophages of NASH mice.

Experiment: Immunohistochemistry (FIG. 5A-B) and qPCR analysis (FIG. 5C) were performed on the liver macrophage marker F4/80.

Figure 5 shows that rhBMP9 treatment can reduce the infiltration of macrophages in the liver of mice in the HFD model group. (AB) F4/80 immunohistochemical staining in each group. (C) Expression of F4/80 at the mRNA level in the livers of mice in each group. Data are expressed as mean±SEM, *p<0.05 vs. Control group; ^# p<0.05, ^### p<0.001 vs. HFD group.

The results showed that compared with normal control mice, the infiltration of macrophages in the liver of the HFD group was significantly increased, while the infiltration of macrophages was significantly inhibited after rhBMP9 treatment.

In order to further analyze the polarization of liver macrophages M1 and M2, the present invention uses RT-PCR to analyze the transcription levels of M1 and M2 polarization marker genes in mouse liver.

Figure 6 shows that rhBMP9 regulates the polarization of mouse liver macrophages M1 and M2 and the cytokine secretion of macrophage cell lines in vitro. (AD) M1 macrophage polarization genes TNF-α (A), IL-6 (B) and M2 macrophage polarization genes IL-4 (C), Arg1 (D) in the livers of mice in each group The mRNA transcription level of ; (EF) the concentration of cytokines TNF-α (E) and IL-6 (F) in the supernatant of the RAW 264.7 macrophage cell line cultured in vitro under different treatment conditions. Data are expressed as mean±SEM, vs. unmodeled or solvent Control group, *p<0.05, **p<0.01, ***p<0.001; vs. HFD or LPS group, ^# p<0.05, ^## p<0.01 , ^### p<0.001.

The results showed that the mRNA expressions of M1 macrophage-related polarization genes TNF-α (Fig. 6A) and IL-6 (Fig. 6B) in the HFD model group were significantly higher than those in the normal group, and they basically returned to normal levels after rhBMP9 treatment (Fig. Figure 6A, B); while the mRNA expressions of M2 macrophage-related genes IL-4 and Arg1 were significantly reduced in the HFD model group, and the above changes were reversed after treatment with rhBMP9 (Figure 6C, D).

In order to study the effect of rhBMP9 treatment on the polarization balance of M1/M2, the present invention further uses RAW 264.7 macrophage cell line for research.

The results showed that LPS could significantly promote the secretion of M1 macrophages' signature cytokines TNF-α and IL-6, showing typical M1 macrophage characteristics, and rhBMP9 treatment could significantly inhibit the secretion of these two cytokines ( Figure 6E,F). These preliminary results suggest that M1 macrophage polarization is enhanced during NASH, and the secreted TNF-α, IL-6 and reactive oxygen species synergistically promote the activation of HSCs, thereby aggravating NASH, while rhBMP9 intervention promotes NASH through "BMP9-macrophage polarization". -Activation and differentiation of hepatic stellate cells -BMP9 secretion "loop regulation and restoration of endogenous BMP9 production, rebuilding the immune balance in the body, and alleviating the occurrence of NASH and liver fibrosis.

In the present invention, in the fatty liver and early NASH models of HFD obese mice, exogenous supplementation of BMP9 improves NASH, and can reduce the infiltration of hepatic macrophages; in addition, supplementation of BMP9 can significantly down-regulate the expression of TRAF3 in mouse liver; and BMP9 Capable of suppressing M1 polarization. Therefore, exogenous supplementation of rhBMP9 is likely to have the effect of preventing and treating NASH and fibrosis, and its effect is partly by regulating the phenotype and activation of liver macrophages, affecting the interaction between macrophages and HSCs, thereby down-regulating TRAF3 and affecting HSCs. proliferation, activation, and apoptosis.

In the HFD mouse fatty liver model, exogenous supplementation of rhBMP9 can not only lose weight, improve glucose and lipid metabolism, reduce hepatic steatosis, and reduce the infiltration of macrophages in the liver. Hepatocyte steatosis regulates oleic acid-induced macrophage phenotype changes, resulting in a decrease in the secretion of TNF-α and TGF-β. Therefore, exogenous supplementation of rhBMP9 may have the effect of preventing and treating NASH and fibrosis.

Further research showed that TRAF3 can mediate TNF and Toll signaling pathways. RNA-seq experiments showed that in the liver of early NASH model mice treated with rhBMP9, the TNF signaling pathway in the liver was significantly inhibited and the expression of TRAF3 in the liver was down-regulated, indicating that rhBMP9 It is very likely to regulate the phenotype and activation of liver macrophages, affect the interaction between macrophages and HSCs, thereby downregulating TRAF3, affecting TRAF3 and its related signaling pathways, and affecting the proliferation, activation and apoptosis of HSCs to achieve its treatment of early NASH. and NASH-related liver fibrosis and cirrhosis.

Therefore, BMP9 may inhibit the secretion of the inflammatory factor TNF-α in M1 macrophages by acting on the specific receptors on macrophages, while TRAF3 mediates the function of BMP9, thereby preventing the activation and differentiation of HSCs, and finally Relief of NASH, which provides a new idea for the preparation of medicines for relieving and/or treating hepatitis/hepatic fibrosis/cirrhosis.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Those skilled in the art to which the present invention pertains can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined according to the claims.

Claims (8)

1. Use of BMP9 in the manufacture of a medicament for MAFLD.
2. 2. Use of BMP9 in the manufacture of a medicament of a MAFLD according to claim 1, wherein the medicament alleviates and/or treats the MAFLD by inhibiting TNF signaling pathways in the liver and downregulating TRAF3 expression in the liver.
3. 3. Use of BMP9 in the manufacture of a medicament for a MAFLD according to claim 1, wherein the BMP9 is rhBMP 9.
4. 4. Use of BMP9 in the manufacture of a medicament of a MAFLD according to claim 1, wherein the medicament is an injection.
5. Use of BMP9 in preparing NASH, NASH-related hepatic fibrosis and NASH-related cirrhosis medicines.
6. 6. Use of BMP9 in the manufacture of a medicament for NASH, a medicament for NASH-related liver fibrosis, a medicament for NASH-related cirrhosis of the liver in accordance with claim 5, wherein said medicament alleviates and/or treats NASH, NASH-related liver fibrosis, NASH-related cirrhosis of the liver by inhibiting TNF signaling pathways in the liver and down-regulating TRAF3 expression in the liver.
7. 7. The use of BMP9 in the manufacture of a medicament for NASH, a medicament for NASH-related liver fibrosis, a medicament for NASH-related liver cirrhosis according to claim 5, wherein BMP9 is rhBMP 9.
8. 8. Use of BMP9 in the manufacture of a medicament for NASH, a medicament for NASH-related liver fibrosis, a medicament for NASH-related cirrhosis according to claim 5, wherein the medicament is an injection.

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