| Background and objective:Liver fibrosis is caused by many factors(virus,drugs,alcohol,metabolic syndrome,autoimmune diseases and heredity)that lead to chronic liver injury.The widespread incidence,existence and development of liver fibrosis lead to the progression of chronic liver disease to cirrhosis and even liver cancer,which has become a global problem endangering human health.In China,regardless of the relative incidence or absolute number of cases,patients with liver fibrosis rank first in the world,which is a refractory disease that seriously endangers people's health and consumes social resources.Therefore,the treatment of liver fibrosis has become the focus of the current research on chronic liver diseases.The extensive experimental and clinical studies of anti-fibrosis of traditional Chinese medicine in recent years have provided many valuable experiences for the treatment of liver fibrosis.Therefore,further elucidation of the target mechanism of anti-fibrosis of traditional Chinese medicine,and development of safe and effective anti-fibrosis drugs will have great prospects and challenges.Method:1.The liver fibrosis models of CCl4 mice and DMN rats were established.Serum and liver tissues were collected after treatment with C02.The anti-fibrosis mechanism of C02,an active component of Cordyceps militaris mycelium,was analyzed by HE and Sirius red collagen staining,hydroxyproline(Hyp)content,serum liver function index,immunohistochemistry of fibrosis-related indicators and changes of protein and gene levels.2.Activated hepatic stellate cells(HSCs)were treated with different concentrations of C02 in vitro,and the changes of activation,apoptosis,proliferation-related genes and proteins of HSCs were observed.Different concentrations of C02 were used to interfere with LPS-activated RAW264.7 macrophages.Genes and protein levels of macrophages secretion-related factors were detected.Changes of genes and proteins related to macrophage activation and proliferation were observed.The main mechanism of C02 regulating macrophages was explored.3.LPS-induced macrophage activation model was co-cultured with normal HSC in vitro to observe the effect of activated macrophages on HSC activation and the intervention of C02 on HSC activation.The key paths between C02 affecting macrophages and regulating HSC activation were further explored in order to clarify the mechanism of C02 anti-hepatic fibrosis.Result:1.In vivo experiment: C02 can significantly improve the liver function of fibrotic mice induced by CCl4.The middle and high dose groups of C02 can effectively reduce the serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),and the high dose group is better than the low dose group(P < 0.05).The results of Sirius red staining,collagen semi-quantitative staining and HE staining of liver tissue in mice showed that C02 high dose group could effectively reduce collagen deposition and hepatic inflammatory cell infiltration.The results of hydroxyproline in liver tissue were basically consistent with those of collagen semi-quantitative staining(P <0.05).Immunohistochemical results showed that the expression of alpha-SMA and COL-I in C02 group was significantly lower than that in model group,and the efficacy of C02 high-dose group was better than that of low-dose group.The expression of fluorescence quantitative RT-PCR and Western-blot was basically consistent with that of immunohistochemistry(P <0.01 or P < 0.05).Western-blot results showed that the levels of P-ERK/ERK,PDGF and TNF-alpha protein in C02 high dose group were significantly lower than those in model group(P < 0.05).Compared with model group,the expression of phenotype-related proteins STAT1 and IRF5 in M1 macrophages in C02 high dose group was down-regulated(P <0.05).Immunohistochemical results showed that the positive expression of M1 macrophage marker CD11 b in C02 high dose group was significantly lower than that in model group,and the positive expression of M2 macrophage marker CD206 was higher than that in model group.2.In vivo experiment: C02 can improve the liver function of fibrotic rats induced by DMN.The middle and high dose groups of C02 can effectively reduce the contents of serum aspartate aminotransferase(AST),alanine aminotransferase(ALT)and alkaline phosphatase(ALP),and the high dose group is better than the low dose group(P < 0.05).The results of Sirius red staining,collagen semi-quantitative staining and HE staining of liver tissue in rats showed that C02 high dose group could effectively reduce collagen deposition and hepatic inflammatory cell infiltration.The results of hydroxyproline in liver tissue were basically consistent with those of collagen semi-quantitative staining(P < 0.05).Immunohistochemical results showed that the expression of alpha-SMA and COL-I in C02 group was significantly lower than that in model group,and the efficacy of C02 high-dose group was better than that of low-dose group(P< 0.05).The results of fluorescence quantitative RT-PCR and Western-blot were basically consistent with the results of immunohistochemistry.Western-blot results showed that the levels of P-ERK/ERK,PDGF and TNF-alpha protein in C02 high-dose group were significantly lower than those in model group(P < 0.05),and the expressions of phenotype-related protein STAT1 and IRF5 in M1 macrophages in C02 high-dose group were down-regulated compared with model group(P < 0.05).Immunohistochemical results showed that the positive expression of M1 macrophage marker CD68 in C02 high dose group was significantly lower than that in model group,and the positive expression of M2 macrophage marker CD163 was higher than that in model group.3.In vitro experiments: C02 could inhibit the proliferation of JS-1 cells and primary hepatic stellate cells in a dose-dependent manner(P < 0.05);JS-1 cells were activated by5ng/m L TGF-beta treatment and treated with different concentrations of C02.Compared with the model group,40?M C02 could significantly reduce the level of alpha-SMA/COL-I gene and protein(P < 0.05);The results of Factin staining showed that C02 could decrease the expression of Factin in hepatic stellate cells at the concentration of 20?M,40?M and 80?M,and the oil red staining of primary stellate cells showed that C02 could significantly increase the content of lipid droplets in the cytoplasm of primary hepatic stellate cells.4.In vivo experiments: RAW264.7 macrophages were activated by 100ng/m L LPS and treated with different concentrations of C02.The results of fluorescence quantitative PCR showed that the expression of TGF-beta,IL-1 and PDGF in C02 at the concentration of5?M/2.5?M was lower than that in model group(P < 0.01 or P < 0.05).Elisa results showed that C02 could continuously decrease PDGF-BB secretion by macrophages within 2h-36h(P < 0.01 or P < 0.05),and i NOS secretion by macrophages within 8h-36h(P < 0.01 or P < 0.05).5.RAW264.7 macrophages co-cultured with JS-1 cells: After 24 hours of co-culture of activated RAW264.7 macrophages and JS-1 cells,the expression of alpha-SMA in JS-1 cells increased significantly(P < 0.05).After C02 intervention,the expression of alpha-SMA in high dose group decreased(P < 0.05).The same results were obtained in cellular immunofluorescence alpha-SMA detection.STAT1 inhibitor can inhibit the expression of alpha-SMA in JS-1 cells by interfering with activated macrophages.The effect of STAT1 inhibitor combined with C02 high dose group is better than that of STAT1 or C02 high dose group alone(P < 0.01 or P < 0.05).Western-blot immunoblotting results showed that compared with model group,high dose group of C02 can inhibit the expression of p-ERK/ERK protein(P< 0.01 or P < 0.05).Conclusion:1.C02 can effectively improve the degree of fibrosis in CCl4 mice and DMN rats.Its mechanism may be related to the inhibition of M1 macrophages and the reduction of PDGF/TNF-alpha production.2.C02 can inhibit the activation of hepatic stellate cells by regulating STAT1 protein,which may be related to ERK signaling pathway. |
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