Study On The Molecular Mechanism Of Fuzheng Huayu Recipe In Preventing And Treating Liver Fibrosis In CCl₄ Mice By Regulating The Polarization Of Macrophages In The Liver

Background and objective:Liver fibrosis is a key link in the malignant progression of chronic liver disease.Macrophages?by proper hepatic macrophages of Kupffer cells and peripheral source macrophages?in the formation of liver fibrosis and reverse plays an important role.Studies have shown that Fuzheng Huayu?FZHY?can through regulating the paracrine of Kupffer cells?KCs?to inhibit the activation of hepatic stellate cells,but the regulation of hepatic macrophage phenotypic polarization during anti-fibrosis has not been systematically studied..This topic mainly discusses the molecular mechanism of FZHY to prevent liver fibrosis by regulating the phenotype polarization of liver macrophages.Methods:1.Effect of FZHY and removing stasis on phenotype changes of liver macrophages in mice with liver fibrosis.Male C57BL/6 mice were randomly divided into normal group,normal control group,model group and FZHY group.Mice in the model group and FZHY group were administrated with CCl₄-injection intraperitoneally thrice weekly for 6 weeks.Mice in the FZHY group and normal control group were administrated with CCl₄ and FZHY daily by gavage at 5.6g/kg.Mice in the normal group and the model group were given intraperitoneal injection of the same volume of normal saline,while mice in the normal group were given intraperitoneal injection of the same volume of olive oil.After 6 weeks,Sacrificed the mice and the general conditions?body weight,liver weight,spleen weight?were observed and recorded.The serum ALT and AST levels of hepatorenal function were determined by Kits.HE staining,Sirius red staining and Hydroxyproline?Hyp?content were used to evaluate the anti-fibrosis efficacy of FZHY.The liver macrophages in the normal group,the model group and the FZHY group were isolated by portal vein perfusion.The flow cytometry was used to analyze the proportion of hepatic intrinsic macrophages-KCs?CD45⁺F4/80⁺CD11B^-?and infiltrating monocytes?CD45⁺F4/80⁺CD11B⁺?in hepatic macrophages;the proportion of CD86⁺proinflammatory type macrophages?M1 type?and CD206⁺proinflammatory type macrophages?M2 type?in the innate macrophages;the proportion of Ly6C^high proinflammatory type macrophages?M1 type?and Ly6C^low proinflammatory type macrophages?M2 type?in peripheral macrophages.2.Study on the mechanism of FZHY in regulating the polarization of liver macrophages.Liver macrophage cells were isolated from the mouse of the normal control group,model group and FZHY group.Collecting cells in cultured plate and cultured for 20 min to remove other non-parenchymal liver cells.Total RNA in hepatic macrophages was extracted using TRIzol reagent.subsequently converted to c DNA and then Rt-PCR carried on to detect changes of gene expression of CCL₂?CX₃CR₁and CX₃CL₁m RNA to investigate the mechanism of FZHY in regulating the phenotype of Hepatic macrophages.3.Regulation of M1/M2 macrophages by FZHY.1ug/ml LPS was used to activate RAW264.7 cells to induce M1-type macrophages.M2-type macrophages were induced by 10ng/ml IL-4,and FZHY at different concentrations was added for intervention,Griess reagent was used to detect the expression of NO.PCR was used to detect i NOS,IL-10,Arg1,CD206 and other related indicators to investigate the effect of FZHY on the polarization of M1/M2 type macrophages.4.Effect of FZHY on promoting the activation of hepatic stellate cells by m1-type macrophages.After the intervention of FZHY in the culture of LPS-induced M1-type macrophages for24 h,the supernatant was removed and the new culture medium was added for 24 h.Then,the supernatant was added to JS1 cell lines pre-adhered to the wall,and TGF-?was used as a positive control drug.The activation degree of JS1 was evaluated by immunofluorescence detection of the expression of?-SMA in JS1.Results:1.FZHY can significantly reduce inflammation of liver tissue and liver fibrosis in mice.AST,ALT and Hyp levels were significantly higher in the model group than in the normal group.HE staining showed a large number of inflammatory cell infiltration and hepatic cell injury.Sirius red indicates the proliferation and deposition of collagen fibers in liver tissues.Compared with the model group,the serum transaminase level of mice in the FZHY administration group was significantly reduced,liver inflammation was significantly alleviated,and collagen fibers became thinner or even disappeared.2.FZHY can significantly reduce the infiltration of bone-marrow derived macrophages?CD45⁺F4/80⁺CD11B⁺?in Hepatic macrophages,significantly reduce the proportion of pro-inflammatory Ly6C^high M1-type macrophages(CD45⁺F4/80⁺CD11B⁺Ly6C^high)in peripheral macrophages,and increase the proportion of anti-inflammatory macrophages Ly6C^low M2-type macrophages(CD45⁺F4/80⁺CD11B⁺Ly6C^low).The analysis of innate macrophages in the liver?CD45⁺F4/80⁺CD11B^-?suggested that FZHY can reduce the expression of CD86⁺M1-type macrophages and increase the expression of CD206⁺restorative macrophages.3.FZHY could promote the expression of CX₃CL₁/CX₃CR₁ gene in hepatic tissue and liver macrophages?Kupffer cells and bone marrow derived macrophages?,inhibit the expression of chemokine CCL₂ gene.4.FZHY can significantly inhibit the expression of NO,an inflammatory medium,and the expression of i NOS and CCL₂ m RNA,markers of M1-type macrophages in RAW264.7 cells?M1 type?induced by LPS.IL-4 induced RAW264.7 cells?M2 type?up-regulated the expression of IL-10,CD206 and Arg1,markers of M2-type macrophage.Meanwhile,our experiment also found that FZHY can promote the expression of CX₃CR₁,IL-10 and Arg1 in M1-type macrophages.5.FZHY can significantly reduce M1-type macrophages induce the expression of?-SMA in hepatic stellate cells.Conclusion:1.FZHY can significantly reduce the degree of liver inflammation and liver fibrosis in CCl₄ infected mice.2.FZHY inhibits peripheral pro-inflammatory(CD11B⁺Ly6C^high)macrophage infiltration by reducing the expression of CCL₂ in liver intrinsic pro-inflammatory type?CD86⁺?macrophages;Meanwhile,the expression of chemokine receptor CX₃CR₁ was promoted to promote the transformation of peripheral proinflammatory macrophage Ly6C^high into Ly6C^low.3.FZHY can inhibit the inflammatory activation of RAW264.7 cells induced by LPS,and promote its function to transform into anti-inflammatory macrophages;Meanwhile,IL-4promoted the anti-inflammatory activation of RAW264.7 cells.4.FZHY can significantly reduce M1-type macrophages induce the activation of hepatic stellate cells.