Application of Prss8m6A modification regulation in hepatic fibrosis diseases

Abstract

The invention provides application of Prss8m6A modification regulation in hepatic fibrosis diseases, a primary parenchymal hepatic cell and HSCs co-culture model is taken as a research object, Prss8m6A modification is taken as an entry point, TLR4-mediated inflammation cascade reaction induced HSCs activation is taken as a target point, various modern means are utilized, regulation of TLR4 expression, mediation of inflammation cascade reaction and promotion of HSCs over-activation by Prss8m6A modification are discussed, and the application of Prss8m6A modification regulation in hepatic fibrosis diseases is realized. The traditional Chinese medicine composition participates in the liver fibrosis occurrence and development mechanism and the intervention effect of the liver soothing and spleen tonifying prescription. Results show that Prss8m6A modification can reduce mRNA stability, inhibit Prss8 expression level, promote TLR4 expression, mediate inflammation cascade reaction, induce HSCs overactivity and participate in liver fibrosis morbidity. Therefore, inhibition of Prss8m6A modification can play a role in treating hepatic fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of treatment of liver fibrosis diseases, and specifically relates to an application of Prss8 m6A modification regulation in liver fibrosis diseases. Background Art

[0002] Liver fibrosis (LF) is a compensatory response in the process of tissue repair after chronic persistent liver inflammation or injury caused by a variety of pathogenic factors. Hepatic stellate cell (HSCs) activation is the core event in the pathogenesis of LF, and excessive accumulation of extracellular matrix (ECM) is its main pathological feature. LF is a key step in the further transformation of various chronic liver diseases into cirrhosis and liver cancer, which seriously affects the quality of life of patients and their families and has become a major challenge to global health. LF is a reversible lesion, and timely and effective anti-fibrosis treatment is a necessary measure to prevent the transformation of chronic liver disease to end-stage liver disease. However, the specific pathogenesis of LF is not yet fully understood, and there is no radical drug. Therefore, exploring the molecular mechanism of LF has important theoretical significance and clinical value.

[0003] The liver is composed of hepatocytes and non-parenchymal cells. Hepatocytes are specialized epithelial cells that account for more than 80% of the total number of liver cells. They perform important functions in the liver, including protein synthesis, lipid and carbohydrate metabolism, biotransformation of xenobiotics in the body, and toxic degradation. Non-parenchymal cells include macrophages, Kupffer cells, HSCs, sinusoidal endothelial cells, immune cells, etc. HSCs are key non-parenchymal cells in the process of liver fibrosis. After being activated by inflammatory cytokines, they produce type I collagen, resulting in excessive deposition of ECM, leading to the onset of LF.

[0004] N6-methyladenosine (m6A) modification refers to a dynamic and reversible methylation process that occurs on the sixth N atom of base A. It is the most common RNA post-transcriptional modification in eukaryotes. m6A modification can affect gene transcription, splicing, localization, translation, stability and post-transcriptional regulation at the RNA level. In the early stage, the research group screened out the key mRNA associated with the pathogenesis of LF, and there were significant differences in both m6A modification and RNA expression, namely Serine Protease 8 (Prss8), through m6A-Seq and RNA-Seq, combined with co-expression analysis, differential multiples, functional enrichment and other criteria. Prss8 is a glycosylphosphatidylinositol-anchored serine protease, derived from chromosome 16, with a genome number of 31131433-31135727 and a total length of 4.3kb.

[0005] Toll-like receptor 4 (TLR4) is located on the cell membrane and is a type I transmembrane receptor. It is composed of an extracellular region, a transmembrane region, and an intracellular region. It is an important promoter of the inflammatory cascade. The inflammatory cascade has been shown to be involved in the pathological process of LF, such as stimulating the activation and proliferation of HSCs, and promoting the occurrence and development of LF. Studies have found that activated inflammasomes can aggravate liver inflammatory reactions, overactivate HSCs, lead to a large amount of collagen fiber deposition, and accelerate the process of LF. Therefore, inhibiting the inflammatory cascade in the LF process will inhibit the overactivation of HSCs.

[0006] The research team believes that the basic pathogenesis of liver fibrosis is "liver failure, blood vessels disharmony, qi stagnation, and spleen failure to function properly", and proposed the treatment principle of "soothing the liver and relieving depression, strengthening the spleen and replenishing qi", and created a special hospital preparation "Shuganjianpi formula (SGJPF)". Previous studies have shown that SGJPF can significantly improve liver damage and collagen fiber deposition in the LF model, and reduce the expression level of inflammatory factors. However, the specific molecular mechanism and target of SGJPF in the treatment of LF are not yet fully understood. Therefore, further exploration of the molecular mechanism of SGJPF in the treatment of LF can provide new ideas and insights for the prevention and treatment of LF. Summary of the invention

[0007] This invention is based on literature survey and previous research, takes the co-culture model of primary hepatocytes and HSCs as the research object, takes Prss8m6A modification as the entry point, and TLR4-mediated inflammatory cascade reaction-induced HSCs activation as the target. By using a variety of modern methods, this invention explores the mechanism by which Prss8 m6A modification regulates TLR4 expression, mediates inflammatory cascade reaction, promotes excessive activation of HSCs, participates in the occurrence and development of liver fibrosis, and the intervention effect of Soothing Liver and Strengthening Spleen Recipe, in order to provide new ideas and potential targets for the clinical diagnosis and treatment of liver fibrosis.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention proposes an application of Prss8 m6A modification regulation in liver fibrosis diseases, specifically: Prss8m6A modification will reduce its mRNA stability, inhibit the expression level of Prss8, promote TLR4 expression, mediate the inflammatory cascade reaction, induce HSCs overactivation, and participate in the pathogenesis of liver fibrosis.

[0010] At the same time, the present invention also proposes the use of a preparation for inhibiting Prss8 m6A modification in the preparation of a liver fibrosis treatment preparation, specifically: inhibiting Prss8 m6A modification, reducing TLR4-mediated inflammatory cascade reaction, inhibiting HSCs activation, and exerting a therapeutic effect on liver fibrosis.

[0011] The present invention establishes a mouse model of liver fibrosis, separates primary hepatocytes and HSCs, takes the co-culture of the two cells as the research object, and uses real-time quantitative PCR (RT-qPCR), immunoglobulin technology (WB) and RNA methylation immunoprecipitation PCR (MeRIP-qPCR) and other technologies to detect the Prss8 mRNA and protein expression levels and Prss8m6A modification levels in hepatocytes; uses RMVar and SRAMP tools to determine the Prss8 m6A modification site, and constructs a Prss8m6A modification site mutation model through the mutation site; uses RT-qPCR, WB and MeRIP-qPCR technologies to detect the changes in Prss8 mRNA and protein expression levels and m6A modification levels in hepatocytes after the Prss8 m6A modification site mutation; uses a cell counting kit (CCK-8) and flow cytometry to detect the effects of the Prss8 m6A modification site mutation on the proliferation and cycle of HSCs; uses RT-qPCR and WB technologies to detect the Prss8 The expression levels of smooth muscle actin and type I collagen, activation markers of HSCs, were detected after the m6A modification site mutated. The expression level of TLR4, a key factor in the inflammatory cascade, was detected by WB technology. The expression levels of interleukin (IL)-1β and IL-18, two inflammatory indicators, were detected by enzyme-linked immunosorbent assay. The change of Prss8 mRNA half-life after the Prss8 m6A modification site mutated was detected by actinomycin D experiment. The recovery experiment verified that Prss8 m6A modification affected the functional phenotype of HSCs by regulating the TLR4-mediated inflammatory cascade reaction and participated in the pathogenesis of liver fibrosis.

[0012] At the same time, through the intervention of Shugan Jianpi prescription medicated serum, the effects of SGJPF medicated serum on Prss8 mRNA, protein and m6A modification levels, and mRNA half-life were observed; the effects of SGJPF medicated serum on HSCs activation markers, TLR4 expression and inflammatory indicators were observed; through a collaborative verification experiment, on the basis of Prss8 m6A modification site mutation, SGJPF medicated serum was given to intervene, and the changes in HSCs proliferation, cell cycle, activation markers and inflammatory indicators were observed.

[0013] The present invention is confirmed by experiments:

[0014] 1. Study on the mechanism of Prss8 m6A modification regulating TLR4-mediated inflammatory cascade and inducing HSCs overactivation

[0015] 1.1 Expression of Prss8 m6A modification, mRNA and protein in primary hepatocytes of mice with liver fibrosis

[0016] Compared with the control group, the Prss8 m6A modification level in hepatocytes in the model group was significantly increased, and the expression of Prss8 mRNA and protein in hepatocytes was significantly decreased.

[0017] 1.2 Functional phenotype of primary HSCs from mice with hepatic fibrosis

[0018] Compared with the control group, the activity of HSCs in the model group was significantly increased, and the number of HSCs in the G1 phase was significantly reduced.

[0019] 1.3 Activation indexes of primary HSCs and TLR4 expression levels of hepatocytes in mice with hepatic fibrosis

[0020] Compared with the control group, the expressions of HSCs activation indexes α-SMA and Collagen in the model group were significantly increased, and the expressions of TLR4 mRNA and protein were significantly increased.

[0021] 1.4 Changes in the expression levels of inflammatory markers IL-1β and IL-18 in the supernatant of liver fibrosis mouse cell co-cultures

[0022] Compared with the control group, the expressions of IL-1β and IL-18 in the model group were significantly increased.

[0023] 1.5 Effects of Prss8 m6A modification site mutation on Prss8 mRNA, protein and m6A modification expression

[0024] Compared with the wild type of Prss8, after the Prss8 m6A modification site mutation, the expression of Prss8 mRNA and protein in hepatic parenchymal cells was upregulated, and the Prss8 m6A modification level was downregulated in hepatic parenchymal cells.

[0025] 1.6 Effects of Prss8 m6A modification site mutations on HSCs functional phenotypes

[0026] Compared with the wild type of Prss8, the Prss8 m6A modification site mutation reduced the viability of HSCs and increased the number of cells in the G1 phase.

[0027] 1.7 Effects of Prss8 m6A modification site mutations on HSCs activation indicators and TLR4 expression levels

[0028] Compared with the wild type of Prss8, the expression of HSCs activation indicators α-SMA and CollagenⅠ was reduced after the Prss8 m6A modification site mutation, and the expression levels of TLR4 mRNA and protein also decreased significantly.

[0029] 1.8 Effect of Prss8 m6A modification site mutation on inflammatory markers IL-1β and IL-18 in cell co-culture supernatant

[0030] Compared with the wild type of Prss8, the mutation of the Prss8 m6A modification site significantly downregulated the expression levels of IL-1β and IL-18.

[0031] 1.9 Effect of Prss8 m6A modification site mutation on Prss8 mRNA stability

[0032] Compared with the wild type of Prss8, the stability of Prss8 mRNA was significantly reduced in the Prss8 m6A modification site mutation group.

[0033] 1.10 Resume Experiment

[0034] Compared with the model group, the mutation of Prss8 m6A modification site significantly inhibited the viability of HSCs, increased the number of cells in G1 phase, reduced the expression levels of α-SMA and CollagenⅠ, and reduced the expression levels of IL-1β and IL-18. Compared with the mutation of Prss8 m6A modification site, the combined intervention of Prss8 m6A modification site mutation and up-regulation of TLR4 increased the expression levels of α-SMA and CollagenⅠ, increased the viability of HSCs, reduced the number of cells in G1 phase, and up-regulated the expression levels of IL-1β and IL-18.

[0035] 2. Study on the mechanism of Sogan Jianpi prescription regulating Prss8 m6A modification, promoting Prss8 expression, alleviating TLR4-mediated inflammatory cascade reaction, and inhibiting HSCs activation

[0036] 2.1 Effects of SGJPF-containing serum on Prss8 m6A modification, mRNA and protein expression

[0037] Compared with the control group, the expression levels of Prss8 mRNA and protein in the model group were significantly decreased; compared with the model group, the expression levels of Prss8 mRNA and protein in the SGJPF-containing serum group were significantly increased. The Prss8 m6A modification level in the model group was significantly higher than that in the control group; the Prss8 m6A modification level in the SGJPF-containing serum group was significantly lower than that in the model group.

[0038] 2.2 Effects of SGJPF-containing serum on HSCs activation markers

[0039] Compared with the control group, the expression levels of α-SMA and CollagenⅠ mRNA and protein in the model group were significantly increased; compared with the model group, the expression levels of α-SMA and CollagenⅠ mRNA and protein in the SGJPF drug-containing serum group were significantly decreased.

[0040] 2.3 Effects of SGJPF-containing serum on TLR4 mRNA and protein expression

[0041] Compared with the control group, the expression levels of TLR4 mRNA and protein in the model group were significantly increased; compared with the model group, the expression levels of TLR4 mRNA and protein in the SGJPF-containing serum group were significantly decreased.

[0042] 2.4 Effect of SGJPF-containing serum on inflammatory markers IL-1β and IL-18 in cell culture supernatant

[0043] Compared with the control group, the levels of IL-1β and IL-18 in the model group were significantly increased; compared with the model group, the levels of IL-1β and IL-18 in the SGJPF-containing serum group were significantly decreased.

[0044] 2.5 Collaborative Verification Experiment

[0045] Compared with the wild type of Prss8, the mutation of Prss8 m6A modification site reduced the viability of HSCs, increased the number of cells in the G1 phase, reduced the expression levels of α-SMA and Collage I, and reduced the expression levels of IL-1β and IL-18. Compared with the mutation of Prss8 m6A modification site, the synergistic intervention of the Prss8 m6A modification site mutation and SGJPF-containing serum group significantly reduced the viability of HSCs, increased the number of cells in the G1 phase, reduced the expression levels of α-SMA and Collage I, and reduced the expression levels of IL-1β and IL-18.

[0046] 3. Experimental conclusion

[0047] (1) Prss8 m6A modification can reduce its mRNA stability, inhibit Prss8 expression level, promote TLR4 expression, mediate inflammatory cascade reaction, induce HSCs overactivation, and participate in the pathogenesis of LF.

[0048] (2) The Soganjianpi formula can reduce Prss8 m6A modification, alleviate TLR4-mediated inflammatory cascade, inhibit HSCs activation, and play a therapeutic role in LF. This confirms that preparations that inhibit Prss8 m6A modification can play a therapeutic role in liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The expression of Prss8 in primary hepatocytes of mice with liver fibrosis.

[0050] Note: A: RT-qPCR to detect the expression of Prss8 mRNA in hepatic parenchymal cells. B: WB to detect the expression of Prss8 protein in hepatic parenchymal cells. C: Semi-quantitative analysis of Prss8 protein expression in hepatic parenchymal cells. D: MeRIP-qPCR to detect the modification level of Prss8m6A in hepatic parenchymal cells. Compared with the control group, **P<0.01, indicating that the difference was statistically significant.

[0051] Figure 2 The changes in proliferation and cell cycle of primary HSCs in mice with liver fibrosis.

[0052] Note: A: CCK8 detection of HSCs vitality. B: Cell cycle quantification statistics. C: Flow cytometry detection of cell cycle. a: control group; b: model group. Compared with the control group, ** P<0.01, indicating that the difference was statistically significant.

[0053] Figure 3 Shows the changes in HSCs activation indexes and TLR4 expression levels.

[0054] Note: A: RT-qPCR to detect the expression of α-SMA mRNA; B: WB to detect the expression of α-SMA protein; C: semi-quantitative analysis of α-SMA protein expression. D: RT-qPCR to detect the expression of CollagenⅠ mRNA; E: WB to detect the expression of CollagenⅠ protein; F: semi-quantitative analysis of CollagenⅠ protein expression. G: RT-qPCR to detect the expression of TLR4 mRNA; H: WB to detect the expression of TLR4 protein; I: semi-quantitative analysis of TLR4 protein expression. Compared with the control group, ** P<0.01, indicating that the difference was statistically significant.

[0055] Figure 4 The changes in the expression levels of inflammatory indicators IL-1β and IL-18 in the supernatant of co-cultured cells from mice with liver fibrosis.

[0056] Note: A: ELISA to detect the expression level of IL-1β. B: ELISA to detect the expression level of IL-18. Compared with the control group, ** P<0.01, indicating that the difference was statistically significant.

[0057] Figure 5 The expression of Prss8 in primary hepatocytes of mice with liver fibrosis after mutation of m6A modification site.

[0058] Note: A: RT-qPCR detection of Prss8 mRNA expression in hepatic parenchymal cells after mutation. B: WB detection of Prss8 protein expression in hepatic parenchymal cells after mutation (MUT: Prss8 m6A-MUT, WT: Prss8 m6A-WT). C: Semi-quantitative analysis of Prss8 protein expression in hepatic parenchymal cells. D: MeRIP-qPCR detection of Prss8 m6A expression in hepatic parenchymal cells after mutation. Compared with the WT group, ## P<0.01, indicating that the difference was statistically significant.

[0059] Figure 6 The effect of m6A modification site mutation on the proliferation and cell cycle of primary HSCs in mice with liver fibrosis.

[0060] Note: A: CCK8 detection of HSCs vitality. B: Cell cycle quantification statistics. C: Flow cytometry detection of cell cycle. a: MUT group; b: WT group. Compared with the WT group, ## P<0.01, indicating that the difference was statistically significant.

[0061] Figure 7 This is the effect of m6A modification site mutation on HSCs activation indicators and TLR4 expression.

[0062] Note: A: RT-qPCR to detect the expression of α-SMA mRNA; B: WB to detect the expression of α-SMA protein; C: semi-quantitative analysis of α-SMA protein expression. D: RT-qPCR to detect the expression of CollagenⅠ mRNA; E: WB to detect the expression of CollagenⅠ protein; F: semi-quantitative analysis of CollagenⅠ protein expression. G: RT-qPCR to detect the expression of TLR4 mRNA; H: WB to detect the expression of TLR4 protein; I: semi-quantitative analysis of TLR4 protein expression. Compared with the WT group, ## P < 0.01; # P<0.05, indicating that the difference was statistically significant.

[0063] Figure 8 This is the effect of Prss8 m6A modification site mutation on IL-1β and IL-18 in the cell co-culture supernatant.

[0064] Note: A: ELISA to detect the expression level of IL-1β. B: ELISA to detect the expression level of IL-18. Compared with the WT group, # P<0.05, indicating that the difference was statistically significant.

[0065] Fig. 9 The degradation rate of Prss8 mRNA in liver parenchymal cells of mice with liver fibrosis changes after mutation of the m6A modification site.

[0066] Fig.10 This is the effect of Prss8 m6A modification site mutation on α-SMA under TLR4 upregulation.

[0067] Note: A: RT-qPCR to detect the expression of α-SMA mRNA, a marker of HSCs activation; B: WB to detect the expression of α-SMA protein, a marker of HSCs activation; semi-quantitative analysis of α-SMA protein expression. Compared with the control group, **P<0.01; compared with the model group, ##P<0.01, # P<0.05; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant.

[0068] Fig.11 This is the effect of Prss8 m6A modification site mutation on Collagen I under TLR4 upregulation.

[0069] Note: A: RT-qPCR to detect the expression of CollagenⅠmRNA; B: WB to detect the expression of CollagenⅠprotein; Semi-quantitative analysis of CollagenⅠprotein expression. Compared with the control group, **P<0.01; Compared with the model group, ## P<0.01; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant.

[0070] Fig.12 The effect of Prss8 m6A modification site mutation on HSCs proliferation and cell cycle under TLR4 upregulation.

[0071] Note: A: CCK8 detection of HSCs vitality. B: Cell cycle quantification statistics. C: Flow cytometry detection of cell cycle. a: Control group; b: Model group; c: MUT group; d: MUT+pc-TLR4 group; e: MUT+pc-NC group. Compared with the control group, ** P<0.01; compared with the model group, ## P<0.01; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant.

[0072] Fig.13 The effect of Prss 8m6A modification site mutation on IL-1β and IL-18 in the supernatant of cell co-culture in the TLR4 upregulated state.

[0073] Note: A: ELISA to detect the expression level of IL-1β. B: ELISA to detect the expression level of IL-18. Compared with the control group, ** P<0.01; compared with the model group, ## P<0.01; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant.

[0074] Fig.14 This is the effect of SGJPF-containing serum on the expression of Prss8 in primary hepatocytes of mice with liver fibrosis.

[0075] Note: A: RT-qPCR to detect the expression of Prss8 mRNA in the hepatic parenchymal cells of mice with liver fibrosis; B: WB to detect the expression of Prss8 protein in the hepatic parenchymal cells of mice with liver fibrosis; C: Semi-quantitative analysis of Prss8 protein expression. D: MeRIP-qPCR to detect the modification level of Prss8 m6A in the hepatic parenchymal cells of mice with liver fibrosis. Compared with the control group, **P<0.01; compared with the model group, ## P<0.01, indicating that the difference was statistically significant.

[0076] Fig.15 This is the effect of SGJPF-containing serum on the expression of α-SMA, an activation marker of HSCs.

[0077] Note: A: RT-qPCR to detect the expression of α-SMA mRNA, a marker of HSCs activation; B: WB to detect the expression of α-SMA protein, a marker of HSCs activation; C: semi-quantitative analysis of α-SMA protein expression. Compared with the control group, **P<0.01; compared with the model group, ## P<0.01, indicating that the difference was statistically significant.

[0078] Fig.16 This is the effect of SGJPF-containing serum on the expression of Collagen I, an activation marker of HSCs.

[0079] Note: A: RT-qPCR to detect the expression of HSCs activation marker Collagen I mRNA; B: WB to detect the expression of HSCs activation marker Collagen I protein; C: Semi-quantitative analysis of Collagen I protein expression. Compared with the control group, **P<0.01; Compared with the model group, ## P<0.01, indicating that the difference was statistically significant.

[0080] Fig.17 This is the effect of SGJPF-containing serum on the expression of TLR4 in hepatic parenchymal cells.

[0081] Note: A: RT-qPCR detection of TLR4 mRNA expression in hepatic parenchymal cells of mice with liver fibrosis; B: WB detection of TLR4 protein expression in hepatic parenchymal cells of mice with liver fibrosis; C: Semi-quantitative analysis of TLR4 protein expression. Compared with the control group, **P<0.01; Compared with the model group, ## P<0.01, indicating that the difference was statistically significant.

[0082] Fig.18 This is the effect of SGJPF-containing serum on IL-1β and IL-18 in the supernatant of cell co-culture.

[0083] Note: A: ELISA to detect the expression level of IL-1β. B: ELISA to detect the expression level of IL-18. Compared with the control group, ** P<0.01; compared with the model group, ## P<0.01, indicating that the difference was statistically significant.

[0084] Fig.19 This is the effect of SGJPF-containing serum on the degradation rate of Prss8 mRNA in hepatic parenchymal cells.

[0085] Fig. 20 This is the effect of SGJPF-containing serum on the expression of α-SMA, an activation marker of HSCs, after mutation of the Prss8 m6A modification site.

[0086] Note: A: RT-qPCR to detect the expression of α-SMA mRNA, a marker of HSCs activation; B: WB to detect the expression of α-SMA protein, a marker of HSCs activation; C: semi-quantitative analysis of α-SMA protein expression. Compared with the control group, **P<0.01; compared with the WT group, ## P<0.01, # P<0.05; compared with the MUT group, && P<0.01, & P<0.05, indicating that the difference was statistically significant.

[0087] Fig.21 This is the effect of SGJPF-containing serum on the expression of Collagen I, an activation marker of HSCs, after mutation of the Prss8 m6A modification site.

[0088] Note: A: RT-qPCR to detect the expression of HSCs activation marker Collagen I mRNA; B: WB to detect the expression of HSCs activation marker Collagen I protein; C: Semi-quantitative analysis of Collagen I protein expression. Compared with the control group, **P<0.01; Compared with the WT group, ## P<0.01; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant.

[0089] Fig. 22 This is the effect of SGJPF-containing serum on the proliferation and cell cycle of HSCs after m6A modification site mutation.

[0090] Note: A: CCK8 was used to detect HSCs activity. B: Cell cycle quantification statistics. C: Flow cytometry was used to detect cell cycle. a: Control group; b: Model group; c: MUT group; d: WT group; e: MUT+SGJPF group. Compared with the control group, **P<0.01; compared with WT group, ## P<0.01; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant.

[0091] Fig.23 This is the effect of SGJPF-containing serum on IL-1β and IL-18 in the supernatant of cell co-culture after Prss8 m6A modification site mutation.

[0092] Note: ELISA was used to detect the expression of IL-1β and IL-18 levels. ** P<0.01; compared with WT group, # P<0.05; compared with the MUT group, && P<0.01, indicating that the difference was statistically significant. DETAILED DESCRIPTION

[0093] Example 1

[0094] Prss8 m6A modification regulates TLR4-mediated inflammatory cascade and induces HSCs overactivation

[0095] Prss8 is a serine protease located on the cell membrane, derived from chromosome 16, with a genome number of 31131433-31135727 and a total length of 4.3kb. It is highly conserved between humans and rodents and is widely expressed in tissues including the liver, small intestine, stomach, and lungs.

[0096] In order to further study the relationship between Prss8 and the occurrence of LF disease and its molecular mechanism, this study established an in vitro cell model of primary hepatocytes and HSCs co-cultured in mice with liver fibrosis to simulate the in vivo environment of the liver. First, by analyzing the indicators of the co-culture system, the changes in Prss8 expression, HSCs proliferation and activation, and inflammatory factor expression were determined. Secondly, by constructing a Prss8 m6A modification site mutation model, it was determined that Prss8 m6A modification can reduce its mRNA stability, inhibit Prss8 expression level, promote TLR4 expression, promote HSCs proliferation, mediate inflammatory cascade reactions, induce HSCs overactivation, and participate in the pathogenesis of LF. The response experiment confirmed the mechanism by which Prss8m6A modification regulates TLR4-mediated inflammatory cascade reactions and induces HSCs overactivation.

[0097] 1 Experimental Materials

[0098] 1.1 Experimental animals

[0099] Male SPF C57BL / 6 mice, weighing 20±2g, 6-8 weeks old, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., with the experimental animal license number: SCXK(Zhe)2019-0004. All mice were raised in the animal facility of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, with an indoor temperature of 18-22°C, humidity of 40-60%, ventilation, and alternating day and night for 12 hours each. All mice were provided with free access to food and water.

[0100] 1.2 Experimental Ethics Approval

[0101] All animal experiments involved in this study were reviewed and approved by the Animal Ethics Committee of Anhui University of Chinese Medicine, with ethics approval number: AHUCM-mouse-2023085.

[0102] 2 Experimental methods

[0103] 2.1 Establishment of liver fibrosis

[0104] SPF male C57BL / 6 mice were adaptively fed for 1 week, and then injected subcutaneously with a mixture of carbon tetrachloride (CCl4) and olive oil (ratio of 1:4) at the back at a rate of 0.1 mL / 10 g. The model was established for a total of 12 weeks, twice a week.

[0105] 2.2 Isolation of primary hepatocytes

[0106] 2.2.1 Mouse liver perfusion

[0107] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. The skin was prepared, the mice were fixed on the operating table and the abdomen was fully exposed. The abdomen was disinfected and sodium heparin injection was injected for anticoagulation. The abdominal cavity was opened by a midline incision in the lower abdomen to expose the liver and portal vein. A perfusion solution containing collagenase was prepared. 50 mg of collagenase IV was dissolved in 25 mL of DMEM culture medium and diluted 4 times to 0.05% collagenase perfusion solution. Perfusion solution 1 Hanks' balanced salt solution and collagenase IV perfusion solution were preheated in a 37°C water bath in advance. The front end of the syringe was equipped with a perfusion needle to puncture the inferior vena cava, and the Hanks' balanced salt solution was perfused at a uniform speed. When the liver turned gray, the vein was cut open and the perfusion volume was 50 mL. After that, 25 mL was perfused at a uniform speed. The 0.05% collagenase perfusion solution was replaced and 30 mL was perfused at a uniform speed within 6 minutes. The liver was cut with scissors and placed in a culture dish. The surface of the liver was washed with PBS, and the liver was torn into a paste. 0.05% collagenase perfusion solution was added and digested in a 30°C incubator for 30 minutes, during which the culture dish was shaken every 5 minutes.

[0108] 2.2.2 Isolation of hepatocytes

[0109] Add pre-cooled PBS to the digested liver tissue to terminate digestion, and filter with a 200-mesh filter. Centrifuge the filtrate at 80×g, 50×g, and 40×g for 3 min each time, and collect the cell precipitate. Resuspend the cells with serum-containing DMEM and plate them on a culture dish plated with rat tail collagen. After 4 hours, discard the culture medium and continue culturing with serum-free DMEM culture medium to obtain hepatic parenchymal cells.

[0110] 2.2.3 Isolation of HSCs

[0111] Collect the supernatant after gradient centrifugation, centrifuge at 40×g for 4 minutes, collect the supernatant again, centrifuge at 400×g for 10 minutes, and discard the supernatant. Resuspend the cells in serum-free RPMI-1640 medium, centrifuge at 400×g for 10 minutes, and discard the supernatant. Resuspend the cells in 4mL of PBS, add 2mL of cell suspension, 4mL of 35% percoll and 4mL of 50% percoll to two 15mL centrifuge tubes in turn, and centrifuge at 900×g for 30 minutes. Collect the turbid band at the junction of 35% percoll and PBS, dilute it 3 times, and centrifuge it again at 400×g for 10 minutes, discard the supernatant, culture the cells with RPMI-1640 medium containing 10% FBS, and inoculate them in a culture dish plated with rat tail collagen to obtain HSCs.

[0112] 2.3 Establishment of the co-culture model of hepatocytes and HSCs

[0113] Hepatocytes or HSCs were seeded in six-well plates and cultured overnight. After transfection with the corresponding overexpression plasmid or siRNA, the cells were cultured for 48 h, digested, centrifuged, resuspended and counted, and the cell density was adjusted to 1×10 6 1 mL of the cell suspension was added to the Transwell chamber and cultured on hepatocytes or HSCs for 48 h. After the co-culture, the hepatocytes and HSCs were collected for subsequent experimental testing.

[0114] 2.4 Synthesis of overexpression plasmid

[0115] The Prss8 overexpression plasmid vector and the control plasmid empty vector were constructed by General Biotechnology (Anhui) Co., Ltd. The overexpression vector type was pcDNA3.1, with a total length of 1698 bp.

[0116] 2.5 Real-time fluorescence quantitative PCR detection of gene expression

[0117] 2.5.1 RNA extraction

[0118] Collect the cell pellet and add 1mL TRIzol to lyse. Add 0.2mL chloroform, shake vigorously for 15s, and leave at room temperature for 5min. Centrifuge at 4℃, 12000rpm for 10min, take the supernatant (about 500μL) and transfer it to another EP tube. Add 0.5mL pre-cooled isopropanol and mix well, incubate on ice for 30min. Centrifuge at 4℃, 12000rpm for 15min, discard the supernatant. Add 1mL pre-cooled 75% ethanol, centrifuge at 4℃, 12000rpm for 5min, discard the supernatant, and repeat the above steps. Dry the RNA precipitate at room temperature, add 20 to 50μLDEPC water, and store at -80℃ for later use.

[0119] 2.5.2 RT reaction

[0120] Remove genomic DNA, add RNA (mass 1μg), 5×gDNA Eraser Buffer 2.0μL, gDNA Eraser 1.0μL, DEPC water to 10μL in a 0.2mL EP tube, mix gently, and centrifuge. Heat at 42℃ for 2min on a PCR instrument, and immediately place on ice for 1min. Add 5×RT MasterMix 4.0μL, 20×Oligo dT 1.0μL, DEPC water to 20μL in the above EP tube, mix gently, centrifuge, react at 37℃ for 30min on a PCR instrument, heat at 85℃ for 5min, and immediately place on ice for 1min. Take out the above reaction solution, which is cDNA, and store it at -20℃ for later use.

[0121] 2.5.3 Fluorescence quantitative PCR reaction

[0122] The above cDNA was used as a fluorescent quantitative template to establish the reaction system in Table 1, and the fluorescent quantitative PCR reaction was performed under the reaction conditions in Table 2. β-actin was used as an internal reference to detect the expression level of each index. The primer information of each detection index is shown in Table 3. The result analysis method was the relative quantitative method, and the calculation method was 2 -△△CT The results include amplification curve, melting curve and relative expression level.

[0123] Table 1 Fluorescence quantitative PCR reaction system

[0124]

[0125] Table 2 Reaction conditions

[0126]

[0127] Table 3 Primer sequences

[0128]

[0129] 2.6 Protein expression detection by WB

[0130] After collecting cells, add 100 μL of RIPA cell lysis buffer (containing 1 mM PMSF) to each 6-well plate cell, place on ice for lysis for 30 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant to obtain total cell protein. Add 5×SDS-PAGE protein loading buffer (1:4) to the protein sample and heat it in a boiling water bath for 10 minutes to fully denature the protein. Prepare SDS-PAGE gel, load the protein sample into SDS-PAGE according to 30 μg per well, adjust the voltage to 80v, electrophoresis for 30 minutes, and then adjust the voltage to 120v, electrophoresis for 1 hour. Immerse the cut filter paper and PVDF membrane (pre-soaked in methanol for 3 minutes) in the transfer buffer for 5 minutes. The transfer device is arranged from top to bottom in the order of anode plate, filter paper, PVDF membrane, gel, filter paper, and cathode plate, and placed in the transfer tank for 300mA constant current transfer.

[0131] After the transfer, the PVDF membrane was transferred to 5% skim milk powder, shaken slowly on a shaker, and blocked at room temperature for 2 hours. The PVDF membrane was then transferred to the corresponding diluted primary antibody and incubated overnight at 4°C with slow shaking. The primary antibodies and dilution ratios used are detailed in Table 4. After the incubation, the PBST buffer was washed 3 times, 10 minutes each time. The PVDF membrane was transferred to the diluted corresponding secondary antibody, and the horseradish peroxidase (HRP) labeled secondary antibody was diluted with the secondary antibody diluent at 1:10000, and incubated at room temperature for 2 hours. The PBST buffer was washed 3 times, 10 minutes each time. ECL A solution and ECL B solution were mixed (1:1) to prepare ECL luminescent solution, and the PVDF membrane was placed in the center of the exposure plate of the automatic exposure instrument, and the ECL luminescent solution was added for 1-2 minutes to develop and take pictures. The gray value of the protein band was analyzed using Image J software, and the gray ratio of the target protein to β-actin was calculated.

[0132] Table 4 Antibody information

[0133]

[0134] 2.7 Detection of Prss8 m6A modification level in hepatocytes by MeRIP-qPCR

[0135] 2.7.1 RNA extraction

[0136] Same as experimental method 2.5.1.

[0137] 2.7.2 Immunocapture and lysis

[0138] Prepare the immunocapture solution by adding the reagents according to Table 5 to a 0.2 mL PCR tube and mix well. Vortex the tubes on a vortexer or on a rolling shaker at room temperature for 90 minutes. After 90 minutes of incubation, add 10 μL NDE and 2 μL CEM to each tube and incubate at room temperature for 4 minutes. Place the tubes on a magnetic rack until the solution is clear (approximately 2 minutes). Carefully remove and discard the supernatant. Add 1.5 μg of RNA to 20 μL ICB, then add 1.5 μL NDE, 1 μL CEM and incubate at room temperature for 2 minutes. Add 25 μL RPS to the input tube, then add 200 μL of absolute ethanol for RNA release / recovery. Keep the PCR tubes in the magnetic rack and wash each reaction tube three times with 150 μL WB (Wash Buffer) and once with 150 μL PDB.

[0139] Table 5 Preparation of immune capture solution

[0140]

[0141] 2.7.3 Release / recovery of enriched RNA

[0142] Prepare the protein digest solution by mixing Proteinase K and PDB at a ratio of 1:10. After the final wash, remove the tubes from the magnet. Add 20 μL of the protein digest solution to each sample and negative control. Mix and incubate at 55°C in a thermal cycler for 15 min. Place the tubes on the magnet until the solution is clear (approximately 2 min). Carefully transfer the solution from each sample to an unused PCR tube.

[0143] Add 20 μL of RPS to each sample and negative control tube, then add 160 μL of absolute ethanol. Add 2 μL of RNA Binding Beads to each tube. Mix thoroughly by pipetting up and down at least 10 times. Incubate at room temperature for 5 minutes to allow RNA to bind to the beads. Place the PCR tubes in a magnetic rack until the solution is clear (about 2 minutes), then carefully remove and discard the supernatant. Place the PCR tubes in a magnetic rack and fill the tubes with 150 μL of freshly prepared 90% ethanol, then carefully remove and discard the ethanol. Repeat step 3h once for a total of two washes. Resuspend the beads in 13 μL of Elution Buffer and incubate at room temperature for 5 minutes to release RNA from the beads. Catch the beads by placing the tubes in a magnetic rack until the solution is completely clear (about 1 minute). Transfer 13 μL from each sample to a new 0.2mL PCR tube and use immediately or store at -20℃.

[0144] 2.7.4 RT reaction

[0145] Add the following reactants to the EP tube in sequence: template RNA 10 μL, Random N6 1 μL, RTBuffer4μL, RNA Inhibitor 1μL, dNTPs 2μL, Reverse Transcriptase 1μL, RNase Free Water 1μL. Gently mix and centrifuge. Incubate the reaction at 42℃ for 20min and 75℃ for 5min on a PCR instrument. Take out the reaction solution, which is cDNA, and store it at -20℃ for later use.

[0146] 2.7.5 PCR reaction

[0147] The above cDNA was used as a fluorescent quantitative template to establish the reaction system in Table 6, and the fluorescent quantitative PCR reaction was performed under the reaction conditions in Table 7 to detect the expression level of each index. The information of the detection index primer Prss8 is shown in Table 3. The result analysis method was the relative quantitative method, and the calculation method was 2 -△△CT The results include amplification curve, melting curve and relative expression level.

[0148] Table 6 Fluorescence quantitative PCR reaction system

[0149]

[0150] Table 7 Reaction conditions

[0151]

[0152] 2.8 Cell transfection

[0153] 2.8.1 RNA transfection

[0154] The cells were digested normally and the cells were counted at 1×10 6 Inoculate in a 6-well plate. Transfection was performed the next morning, when the cell density in the plate was 70%. Centrifuge the tube containing dry powder siRNA at 1000 rpm for 1 min, and add 125 μL of DEPC water to the siRNA to prepare the transfection siRNA. Dissolve A: 5 μL of siRNA in 250 μL of serum-free medium, and B: 10 μL of lipo2000 in 1000 μL of serum-free medium. Mix well and let stand at room temperature for 5 min; C: Add B to tube A, mix well, and let stand at room temperature for 20 min to prepare the transfection reagent. Wash the 6-well plate 2-3 times with serum-free medium. Add the mixed medium (500 μL) to the 6-well plate, shake gently for 1-2 min, make up the volume to 2 mL, place it in the incubator for 4 h, replace it with the original complete medium, and continue culturing. Collect cells after 48 h to detect transfection efficiency by qPCR.

[0155] 2.8.2 Co-transfection

[0156] Prepare transfection reagent: Dissolve 2.5μL of overexpression plasmid and 2.5μL of siRNA in 250μL serum-free medium; dissolve 10μL of GP-transfect-Mate in 1000μL serum-free medium. Mix well and let stand at room temperature for 5 minutes; C: add B to tube A, mix well and let stand at room temperature for 20 minutes. The subsequent method is the same as 2.8.1.

[0157] 2.9 Actinomycin D detection of changes in mRNA half-life

[0158] After the cells were treated normally, 4 μM actinomycin D was used to treat the cells for 0 h, 2 h, 4 h and 8 h. The cells were collected at different time points, and RNA of each group was extracted. The cDNA in method 2.5.2 was used as a fluorescent quantitative template to establish the reaction system in Table 1. Fluorescent quantitative PCR reaction was performed under the reaction conditions in Table 2, and GAPDH was used as an internal reference to detect the expression level of each indicator. The primer information of each detection indicator is shown in Table 8. The result analysis method was the relative quantitative method, and the calculation method was 2 -△△CT The results include amplification curve, melting curve and relative expression level.

[0159] Table 8 Primer sequences

[0160]

[0161] 2.10CCK8 detection of cell viability

[0162] After the normal and model hepatocytes were extracted, complete medium was added to resuspend the cells and the cell density was adjusted to 1×10 5 / mL. The cell suspension was gently mixed, normal cells were added to 1 column, model cells were added to 5 columns, 100 μL of cell suspension was added to each well, the edge wells were filled with sterile PBS, and blank wells containing only culture medium were set up. The inoculated cell culture plate was placed in a 37°C, 5% CO2 incubator for culture. The model cells in the transfection group were transfected, and the complete culture medium was replaced after 6 hours. The remaining cells were also replaced. The cell supernatant of each well was collected. After normal and model stellate cells were digested normally, they were cultured as above. The HSCs culture medium was removed, and the supernatant of normal hepatic parenchymal cells was added to normal HSCs, and the supernatant of hepatic parenchymal cells in the model group and the treatment group was added to the model HSCs. After 48 hours of co-culture, 10 μL of CCK8 was added to each well and cultured for another 1 hour. The absorbance of each well was measured at OD 450nm in an enzyme-linked immunosorbent assay, and blank wells (culture medium, CCK8) were set up at the same time.

[0163] 2.11 Flow cytometry detection of cell cycle

[0164] Wash the cells with pre-cooled PBS, digest the cells normally with trypsin, centrifuge at 2000rpm for 5min, resuspend the cells with PBS, remove the supernatant and keep about 50μL remaining. Then add 0.3mL PBS to fully resuspend, separate the cell clumps into single cells, gently vortex the cells and slowly add 2.7mL pre-cooled anhydrous ethanol to adjust the final concentration to 90%, and fix at -20℃ for 1h. The fixed cells were washed twice with pre-cooled PBS, centrifuged at 2000rpm for 5min, discarded PBS and added 500μL PBS again, and transferred to a 1.5mL EP tube, flicking the bottom of the tube to properly disperse the cells to avoid cell clumping. Add 100μL RNase to resuspend the cells, water bath at 37℃ for 30min, then add 400μL PI dye to resuspend the cells, stain at 4℃ in the dark for 30min to 1h, mix the cells thoroughly, filter with a 200-mesh filter, and detect the cell cycle by flow cytometry.

[0165] 2.12ELISA detection of IL-1β and IL-18 expression

[0166] Set up standard wells, 0 value wells, blank wells and sample wells; add 50 μL of standard of different concentrations to each standard well, add 50 μL of sample diluent to 0 value wells, add nothing to blank wells, and add 50 μL of sample to be tested to sample wells; add 100 μL of HRP-labeled detection antibody to standard wells, 0 wells and sample wells (excluding empty wells); cover the reaction plate with a sealing film and incubate in a constant temperature oven in the dark for 60 min; discard the liquid, fill each well with washing solution, leave for 20 s, remove the washing solution, and repeat 5 times; mix substrates A and B in a 1:1 ratio and add 100 μL to all wells; cover the reaction plate with a sealing film and incubate at a constant temperature for 15 min; add 50 μL of stop solution to all wells and read the absorbance of each well on an ELISA reader.

[0167] 2.13 Statistical analysis

[0168] The experiments involved in this study included at least 3 independent repeated experiments, and the experimental data were statistically analyzed using Spss 23.0 software. The results are expressed as mean ± standard deviation The differences between the two groups were compared using the t-test, and when p < 0.05, it was considered statistically significant. The differences between more than two groups were compared using one-way analysis of variance, and when p < 0.05, it was considered statistically significant.

[0169] 3 Experimental results

[0170] 3.1 Expression of Prss8 mRNA, protein and m6A modification in primary hepatocytes of mice with liver fibrosis

[0171] In order to investigate the expression of Prss8 in LF, RT-qPCR, WB and MeRIP-qPCR were used to detect the changes in the expression levels of Prss8 mRNA, protein and m6A modification in hepatocytes. Figure 1 As shown in Figures AC, compared with the control group, the expression levels of Prss8 mRNA and protein in the liver parenchymal cells of the model group were significantly decreased. Figure 1 As shown in D, compared with the control group, the Prss8 m6A modification level in the hepatic parenchymal cells of the model group was significantly increased.

[0172] 3.2 Changes in proliferation and cell cycle of primary HSCs in mice with liver fibrosis

[0173] like Figure 2 As shown in A, compared with the control group, the activity of HSCs in the model group was significantly increased. Figure 2 As shown in B and 2C, the number of G1 HSCs in the model group was significantly reduced compared with the control group.

[0174] 3.3 Activation indexes of primary HSCs and expression level of TLR4 in primary hepatocytes of mice with liver fibrosis

[0175] RT-qPCR and WB were used to detect the expression levels of HSCs activation markers α-SMA, CollagenⅠ and TLR4 in mice with liver fibrosis. Figure 3 As shown in A, 3D and 3G, the expression levels of α-SMA, CollagenⅠ and TLR4 mRNA in the model group were significantly increased compared with the control group. Figure 3 As shown in BC, 3E-F, and 3H-I, the expressions of α-SMA, CollagenⅠ, and TLR4 proteins were increased in the model group compared with the control group.

[0176] 3.4 Expression levels of inflammatory markers IL-1β and IL-18 in the supernatant of liver fibrosis mouse cell co-cultures

[0177] ELISA results showed that the IL-1β level in the model group was significantly increased compared with the control group ( Figure 4 A). Compared with the control group, the IL-18 level in the model group was significantly increased ( Figure 4 B).

[0178] 3.5 Expression of Prss8 in hepatocytes after mutation of Prss8 m6A modification site

[0179] In order to study the expression of Prss8 in hepatocytes after the mutation of Prss8 modification sites in LF, RT-qPCR, WB and MeRIP-qPCR were used to detect the changes in the expression levels of Prss8 mRNA, protein and m6A modification in hepatocytes under the mutation of Prss8 m6A modification sites. Figure 5 As shown in AC, compared with the WT group, the expression levels of Prss8 mRNA and protein in the Prss8 m6A modification site mutation group were significantly increased. Figure 5 As shown in D, the Prss8 m6A modification level in the Prss8 m6A modification site mutation group was significantly reduced compared with the WT group.

[0180] 3.6 Effects of Prss8 m6A modification site mutation on HSCs proliferation and cell cycle

[0181] like Figure 6 As shown in A, compared with the WT group, the activity of HSCs in the MUT group was significantly reduced. Figure 6 As shown in B and 6C, the number of HSCs in the G1 phase increased significantly after the Prss8 m6A modification site mutation compared with the WT group.

[0182] 3.7 Effects of Prss8 m6A modification site mutation on HSCs activation indicators and TLR4 expression

[0183] RT-qPCR and WB were used to detect the effects of Prss8 m6A modification site mutation on HSCs activation markers α-SMA, CollagenⅠ and TLR4. Figure 7 As shown in A, 7D, and 7G, the mRNA expression levels of α-SMA, CollagenⅠ, and TLR4 in the Prss8 m6A modification site mutation group were significantly reduced compared with the WT group. Figure 7 As shown in BC, 7E-F, and 7H-I, the expression levels of α-SMA, CollagenⅠ, and TLR4 proteins in the Prss8 m6A modification site mutation group were significantly decreased compared with the WT group.

[0184] 3.8 Effects of Prss8 m6A modification site mutation on inflammatory markers IL-1β and IL-18

[0185] ELISA results showed that the expression level of IL-1β in the Prss8 m6A modification site mutation group was significantly decreased compared with the WT group ( Figure 8 A). Compared with the WT group, the IL-18 expression level in the Prss8 m6A modification site mutation group was significantly decreased ( Figure 8 B).

[0186] 3.9 Effect of Prss8 m6A modification site mutation on Prss8 mRNA stability in hepatocytes

[0187] The stability of Prss8 mRNA in hepatic parenchymal cells was detected by actinomycin D assay. Fig. 9 As shown in the figure, compared with the control group, the stability of Prss8 mRNA in the model group was significantly reduced; compared with the WT group, the stability of Prss8 mRNA in the Prss8 m6A modification site mutation group was significantly increased.

[0188] 3.10 Reply Experiment

[0189] 3.10.1 Effect of Prss8 m6A modification site mutation on the expression of α-SMA, an activation marker of HSCs, under TLR4 upregulation

[0190] In order to verify the effect of Prss8 m6A modification on TLR4 on the expression of α-SMA, a marker of HSCs activation, this study conducted a recovery experiment. RT-qPCR and WB were used to detect the effect of Prss8 m6A modification site mutation on α-SMA expression under TLR4 upregulation. The RT-qPCR results are shown in Fig.10 As shown in A, compared with the Prss8 m6A modification site mutation group, the expression level of α-SMA in the Prss8 m6A modification site mutation and TLR4 overexpression co-intervention group was significantly increased. Fig.10 As shown in B, compared with the Prss8 m6A modification site mutation group, the expression level of α-SMA in the Prss8 m6A modification site mutation and TLR4 overexpression co-intervention group was significantly increased.

[0191] 3.10.2 Effect of Prss8 m6A modification site mutation on the expression of HSCs activation marker Collagen I under TLR4 upregulation

[0192] In order to verify the effect of Prss8 m6A modification on TLR4 expression of HSCs activation marker Collagen I, a response experiment was performed in this study. Fig.11 As shown in A, compared with the Prss8 m6A modification site mutation group, the expression level of Collagen I in the Prss8 m6A modification site mutation and TLR4 upregulation co-intervention group was significantly increased. Fig.11 As shown in B, compared with the Prss8 m6A modification site mutation group, the expression level of Collagen I in the Prss8 m6A modification site mutation and TLR4 overexpression co-intervention group was significantly increased.

[0193] 3.10.3 Effects of Prss8 m6A modification site mutations on HSC proliferation and cell cycle in the TLR4 up-regulated state

[0194] In order to verify the effect of Prss8 m6A modification on TLR4 expression of HSCs activation marker Collagen I, this study conducted a response experiment. Fig.12 A) and flow cytometry ( Fig.12 B) Detect HSCs proliferation and cell cycle. Fig.12 As shown in A, compared with the Prss8 m6A modification site mutation group, the HSCs activity in the Prss8 m6A modification site mutation and TLR4 overexpression co-intervention group was significantly increased, and the number of HSCs in the G1 phase was significantly reduced. Fig.12 As shown in B and 12C, compared with the Prss8 m6A modification site mutation group, the HSCs activity in the Prss8 m6A modification site mutation and TLR4 overexpression co-intervention group was significantly increased, and the number of HSCs in the G1 phase was also significantly reduced.

[0195] 3.10.4 Effects of Prss8 m6A modification site mutations on inflammatory markers IL-1β and IL-18 in the upregulated state of TLR4

[0196] In order to verify the effect of Prss8 m6A modification on TLR4 regulation on inflammatory indicators IL-1β and IL-18, this study conducted a response experiment. ELISA results showed that compared with the Prss8 m6A modification site mutation group, the expression levels of IL-1β and IL-18 in the Prss8 m6A modification site mutation and TLR4 overexpression co-intervention group were significantly increased ( Fig.13 AB).

[0197] 4 Summary

[0198] Prss8 m6A modification reduces its mRNA stability, inhibits Prss8 expression level, promotes TLR4 expression, mediates inflammatory cascade reaction, induces HSCs overactivation, and participates in the pathogenesis of liver fibrosis, providing a research basis and basis for further exploring the intervention mechanism of the serum containing the Shugan Jianpi recipe.

[0199] Example 2

[0200] Study on the mechanism of Shugan Jianpi prescription regulating Prss8 m6A modification, promoting Prss8 expression, alleviating TLR4-mediated inflammatory cascade reaction, and inhibiting HSCs activation

[0201] SGJPF can improve liver function, reduce the degree of liver tissue damage, reduce proinflammatory factors and inhibit HSCs activation, but its specific molecular mechanism is still unclear. Example 1 reveals the mechanism by which Prss8 m6A modification regulates TLR4-mediated inflammatory cascade reactions and induces overactivation of HSCs. Therefore, it is speculated that regulating Prss8 m6A modification can inhibit the production of proinflammatory factors and overactivation of HSCs, which may be the potential mechanism of action of SGJPF in the treatment of LF.

[0202] In this experiment, SGJPF-containing serum was prepared to intervene in primary hepatocytes and HSCs, aiming to observe the effects of SGJPF-containing serum on Prss8 m6A modification, HSCs proliferation, HSCs activation indicators, TLR4 and downstream inflammatory cascade reactions, in order to clarify the mechanism by which SGJPF regulates Prss8 m6A modification, promotes Prss8 expression, alleviates TLR4-mediated inflammatory cascade reactions, and inhibits HSCs activation, providing new insights and pharmacological experimental basis for the treatment of LF with SGJPF.

[0203] 1 Experimental Materials

[0204] 1.1 Experimental animals

[0205] SPF male SD rats, weighing 200±20g, 6-8 weeks old, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with the experimental animal license number: SCXK(Liao)2020-0001. All rats were raised in the animal room of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, with an indoor temperature of 18-22℃, humidity of 40-60%, ventilation, and alternating day and night for 12h each. All rats were provided with free access to food and water.

[0206] 1.2 Experimental Ethics Approval

[0207] All animal experiments involved in this study were reviewed and approved by the Animal Ethics Committee of Anhui University of Chinese Medicine, with the ethics approval number: AHUCM-rats-2024011.

[0208] 1.3 Experimental herbs

[0209] SGJPF is composed of bupleurum, atractylodes, white peony root, astragalus, tuckahoe, capillaris, coix seed, umbellate, zedoaria, fructus aurantii and isatis root, all of which were purchased from the Chinese medicine pharmacy of the First Affiliated Hospital of Anhui University of Chinese Medicine. The medicinal materials were taken in according to the prescription ratio, the drugs were checked and the materials were added. All the medicinal pieces were decocted and extracted three times with water. For the first time, 10 times the amount of water was added and decocted for 1.5h. For the second and third times, 8 times the amount of water was added and decocted for 1.0h. The combined extracts were filtered and allowed to settle for 48h. The supernatant was aspirated and concentrated under reduced pressure to obtain the SGJPF extract.

[0210] 2 Experimental methods

[0211] 2.1 Preparation of serum containing Shugan Jianpi prescription

[0212] SD rats were randomly divided into a blank group (10 rats) and a SGJPF-containing serum group (20 rats). The SGJPF-containing serum group was intragastrically administered at 20 times the clinical equivalent dose, with a dosage of 2 mL / 100 g, once a day, for 7 consecutive days. One hour after administration on the last day, the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg), and blood was collected from the abdominal aorta. The blood was centrifuged at 3000 rpm for 10 min, and the serum was collected and inactivated in a 56°C water bath for 30 min. After filtration and sterilization with a sterile filter membrane (0.22 μm), it was aliquoted and stored at -80°C for later use.

[0213] 2.2 Isolation and co-culture of primary hepatocytes

[0214] Hepatocytes and HSCs were separated by gradient centrifugation, and the specific experimental method was the same as the operation step 2.2 of Example 1. Hepatocytes and HSCs were placed in the same Transwel chamber, and the hepatocytes and HSCs were collected after co-culture, and the specific experimental method was the same as the operation step 2.3 of Example 1.

[0215] 2.3 RT-qPCR detection of related gene expression

[0216] RT-qPCR was used to detect α-SMA, Collagen I, Prss8 and TLR4 in cells after SGJPF intervention. The specific operation steps of the experiment were the same as those in 2.5 of Example 1.

[0217] 2.4WB detection of related gene protein expression

[0218] The α-SMA, Collagen I, Prss8 and TLR4 proteins in the cells after SGJPF intervention were detected by WB. The specific operation steps of the experiment were the same as those in Item 2.6 of Example 1.

[0219] 2.5 Actinomycin D detection of changes in mRNA half-life

[0220] Actinomycin D was used to detect the changes in the half-life of cell Prss8 mRNA at different time points after SGJPF intervention. The specific operation steps of the experiment were the same as those in Item 2.9 of Example 1.

[0221] 2.6CCK8 detection of cell viability

[0222] The CCK8 method was used to detect the activity of HSCs after SGJPF intervention. The specific operation steps of the experiment were the same as those in 2.10 of Example 1.

[0223] 2.7 Flow cytometry detection of cell cycle

[0224] Flow cytometry was used to detect the HSCs cycle after SGJPF intervention. The specific experimental steps were the same as those in Item 2.11 of Example 1.

[0225] 2.8ELISA detection of IL-1β and IL-18 expression

[0226] ELISA was used to detect IL-1β and IL-18 in the supernatant of HSCs after SGJPF intervention. The specific operation steps of the experiment were the same as those in Item 2.12 of Example 1.

[0227] 2.9 Statistical analysis

[0228] The experiments involved in this study included at least 3 independent repeated experiments, and the experimental data were statistically analyzed using Spss 23.0 software. The results are expressed as mean ± standard deviation The differences between the two groups were compared using the t-test, and when p < 0.05, it was considered statistically significant. The differences between more than two groups were compared using one-way analysis of variance, and when p < 0.05, it was considered statistically significant.

[0229] 3 Experimental results

[0230] 3.1 Effects of SGJPF-containing serum on the expression of Prss8 mRNA, protein and m6A modification

[0231] The results of RT-PCR and WB experiments showed that compared with the control group, the mRNA and protein expression levels of Prss8 in the model group were significantly decreased; compared with the model group, the mRNA and protein expression levels of Prss8 in the SGJPF-containing serum group were significantly increased. Fig.14 AC. Fig.14 As shown in D, compared with the control group, the Prss8 m6A modification level in the hepatic parenchymal cells of the model group was significantly increased; compared with the model group, the Prss8 m6A modification level in the SGJPF-containing serum group was significantly decreased.

[0232] 3.2 Effects of SGJPF-containing serum on α-SMA mRNA and protein expression

[0233] The results of RT-qPCR and WB experiments showed that compared with the control group, the mRNA and protein expression levels of α-SMA in the model group were significantly increased; compared with the model group, the mRNA and protein expression levels of α-SMA in the SGJPF-containing serum group were significantly decreased. Fig.15 As shown in AC.

[0234] 3.3 Effect of SGJPF-containing serum on the expression of Collagen I mRNA and protein

[0235] The results of RT-qPCR and WB experiments showed that compared with the control group, the mRNA and protein expression levels of Collagen I in the model group were significantly increased; compared with the model group, the mRNA and protein expression levels of Collagen I in the SGJPF drug-containing serum group were significantly decreased. Fig.16 As shown in AC.

[0236] 3.4 Effect of SGJPF-containing serum on TLR4 mRNA and protein expression

[0237] The results of RT-qPCR and WB experiments showed that compared with the control group, the mRNA and protein expression levels of TLR4 in the model group were significantly increased; compared with the model group, the mRNA and protein expression levels of TLR4 in the SGJPF-containing serum group were significantly decreased. Fig.17 As shown in AC.

[0238] Effect of 3.5SGJPF-containing serum on IL-1β and IL-18 in supernatant

[0239] The results of ELISA showed that compared with the control group, the levels of IL-1β and IL-18 in the model group were significantly increased; compared with the model group, the levels of IL-1β and IL-18 in the SGJPF-containing serum group were significantly decreased. Fig.18 As shown in AB.

[0240] Effect of SGJPF-containing serum on the stability of Prss8 mRNA in hepatocytes

[0241] The stability of Prss8 mRNA in hepatic parenchymal cells treated with SGJPF-containing serum was detected by actinomycin D assay. Fig.19 As shown, compared with the control group, the stability of Prss8 mRNA in the model group was significantly reduced; compared with the model group, the stability of Prss8 mRNA in the SGJPF-containing serum group was increased.

[0242] 3.7 Collaborative Verification Experiment

[0243] 3.7.1 Effect of SGJPF-containing serum on the expression of α-SMA, an activation marker in HSCs after mutation of the Prss8 m6A modification site

[0244] The results of RT-qPCR and WB experiments showed that compared with the control group, the mRNA and protein expression levels of α-SMA in the model group increased significantly; compared with the WT group, the mRNA expression level of α-SMA in the MUT group decreased significantly; compared with the MUT group, the mRNA and protein expression levels of α-SMA in the MUT+SGJPF drug-containing serum group decreased significantly. Fig. 20As shown in AC.

[0245] 3.7.2 Effect of SGJPF-containing serum on the expression of Collagen I, an activation marker in HSCs after Prss8 m6A modification site mutation

[0246] The results of RT-qPCR and WB experiments showed that compared with the control group, the mRNA and protein expression levels of Collagen I in the model group increased significantly; compared with the WT group, the mRNA expression level of Collagen I in the MUT group decreased significantly; compared with the MUT group, the mRNA and protein expression levels of Collagen I in the MUT+SGJPF drug-containing serum group decreased significantly. Fig.21 As shown in AC.

[0247] 3.7.3 Effects of SGJPF-containing serum on the proliferation and cell cycle of HSCs with Prss8 m6A modification site mutation

[0248] like Fig. 22 As shown in A, compared with the control group, the HSCs activity in the model group was significantly increased; compared with the WT group, the HSCs activity in the MUT group was significantly decreased; compared with the MUT group, the HSCs activity in the MUT+SGJPF drug-containing serum group was significantly decreased. Fig. 22 As shown in B and 22C, compared with the control group, the number of G1 HSCs in the model group was significantly decreased; compared with the WT group, the number of G1 HSCs in the MUT group was significantly increased; compared with the MUT group, the number of G1 HSCs in the MUT+SGJPF drug-containing serum group was significantly increased.

[0249] 3.7.4 Effect of SGJPF-containing serum on IL-1β and IL-18 in the supernatant of co-cultured cells after Prss8 m6A modification site mutation

[0250] ELISA results showed that compared with the control group, the levels of IL-1β and IL-18 in the model group were significantly increased; compared with the WT group, the levels of IL-1β and IL-18 in the MUT group were significantly decreased; compared with the MUT group, the levels of IL-1β and IL-18 in the MUT+SGJPF drug-containing serum group were significantly decreased. Fig.23 As shown in A and B.

[0251] 4 Summary

[0252] After SGJPF intervention, the Prss8 m6A modification level decreased, the Prss8 expression level increased, and the expression of TLR4, HSCs activation markers (α-SMA, CollagenⅠ) and inflammatory indicators (IL-1β and IL-18) decreased. The synergistic effect of Prss8 m6A modification site mutation and SGJPF intervention can further reduce the expression of α-SMA, CollagenⅠ, IL-1β and IL-18, and inhibit HSCs cell viability.

[0253] The experiment in Example 2 showed that SGJPF exerted a therapeutic effect on LF by regulating Prss8 m6A modification, alleviating TLR4-mediated inflammatory cascade reaction, inhibiting HSCs activation.

[0254] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. Application of Prss8 m6A modification regulation in liver fibrosis, characterized in that: Prss8 m6A modification reduces its mRNA stability, inhibits Prss8 expression level, promotes TLR4 expression, mediates inflammatory cascade reaction, induces HSCs overactivation, and participates in the pathogenesis of liver fibrosis.

2. Use of a preparation for inhibiting Prss8 m6A modification in the preparation of a preparation for treating liver fibrosis, characterized in that: Inhibiting Prss8 m6A modification alleviates TLR4-mediated inflammatory cascade reaction, inhibits HSCs activation, and exerts a therapeutic effect on liver fibrosis.

Images