The Protective Role Of Silymarin Against Liver Fibrosis Via Attenuating The Infiltration Of Monocytes-derived Macrophages

BACKGROUNDLiver fibrosis is caused by imbalances between liver inflammation and repair owing to many chronic liver diseases,including viral infection,toxic damage.Metabolic disorders and alcohol abuse,characterized by excessive deposition of extracellular matrix(ECM).Fibrous deposition in liver,especially collagen-I,can protect hepatocytes against various toxic stimuli.However,dysregulated and excessive fibrous deposition can lead to liver structural damage,liver malfunction and liver cirrhosis.Up until now,there is no effective drugs to treat liver fibrosis during clinical practice.To discover effective therapeutic targets,the pathophysiological mechanism of liver fibrosis has been further studied in recent years.Previous studies have shown that innate immunity,especially macrophages,plays a key role in the development of liver fibrosis.Liver macrophages,so called Kuffer cells,can enhance liver fibrosis via multiple pathways,for example,releasing pro-inflammatory factors,such as TNF-α,IL-6,and IL-1β,to induce hepatocyte necrosis and cytokines which promote fibrosis,such as transforming growth factor(TGF)-β1,to directly activate hepatic stellate cells(HSC).There are two major sources of macrophages in the liver:one is the long-lived,self-renewing resident Kupffer cells(KFs)which are seeded from embryonic progenitors.In the steady state,KCs maintain without the contribution of circulating bone marrow-derived monocytes.The second is the migration of monocytes from bone marrow and peripheral blood under pathological conditions.Following injury,the monocytes in the peripheral blood are largely chemotactic into the liver and activated into macrophages which released proinflammatory and pro-fibrogenic cytokines to aggravate liver injury and fibrosis.These findings suggest that inhibition of monocytes infiltration may be a novel target for the treatment of liver fibrosis.Further researches revealed that monocytes are mainly divided into two major subsets according to cell surface molecules Ly6C:classical monocytes(Ly6Chi monocytes)and nonclassical monocytes(Ly6Clo monocytes).Classical monocytes express high levels of CCR2 and Ly6C but low levels of CX3CR1,with a pro-inflammatory and pro-fibrotic effect.On the other hand,nonclassical monocytes express high levels of CX3CR1 and low levels of CCR2 and Ly6C with anti-inflammatory and anti-fibrosis effect.High expression of CCR2 receptor of Ly6Chi monocytes can combine with chemoattractant protein 1(MCP-1),which is elevated in acute and chronic liver diseases,resulting in cnemotaxis of Ly6Chi monocytes into the liver.Compared to wild mice,MCP-1-/-and CCR2-/-mice exhibited less liver fibrosis in both murine models of toxic(carbon tetrachloride(CCl4))and metabolic(methioninecholine-deficient diet)liver fibrosis.Similar phenomenon were observed in mice administrated with pharmacological inhibitio1 of MCP-1.Thus,the strategy to decrease infiltration of Ly6Chi monocytes through MCP-1/CCR2 axis has become an important target for the treatment of liver fibrosis.Silybum marianum(L.Gaertn)is a medicinal plant of the genus compositae.Silymarin is a compound which contains the total medicinal components of silybum marianum,mainly containing silybin,isosilybin,silydianin and silychristin.As a traditional protecting-liver drug,silymarin has specific efficacy and low toxicity,which was widely used in the treatment of liver disease.Many clinical and animal studies have confirmed that silymarin was able to protect liver cells and relieve liver fibrosis.But the underlying mechanism remains obscure.Therefore,in present study we investigated whether silymarin can alleviate CCl4-induced liver fibrosis by inhibiting the infiltration of Ly6Chi monocytes.Further clarifification of the mechanism of silymarin against liver injury and liver fibrosis wouldprovide a theoretical basis for the application of silymarin,and provide new ideas for the treatment of liver fibrosis.With a well-established mouse model of liver damage and liver fibrosis induced by carbon tetrachloride(CCl4).To investigate the association between monocyte derived macrophages and liver injury and fibrosis.The protective role of of silymarin in anti-hepatitic and anti-fibrotic effects were investigated.We subsequently determined whether silymarin couldprevent the liver from CCl4-induced liver injury and fibrosis by suppressing Grlhi monocyte-derived macrophages migtrating into CCl4-injured liver viaattenuating MCP-1 secretion.These new findings provide robust evidence regarding the immunological anti-inflammatory effect of silymarin and antifibrotic effect and sufficient data support.The prospective for the therapeutic strategy of liver fibrosis.METHODSThis paper is divided into two main parts,including the theory of Chinese and Western medicine collation and induction and the study of animal and cellular experiment.Given ancient and modern publication,the part of theory research reveals the current prospective of traditional Chinese medicine for treating hepatic fibrosis and describe the etiology and pathogenesis of liver fibrosis.At the same time,our understanding of the pathogenesis,animal model as well as current therapeutic strategy of liver fibrosis,which provide a solid foundation for the experimental research.In this study,liver fibrosis model induced by CCL4/olive oil induced liver fibrosis in mice was used to explore the inhibitory effect of silymarin on hepatic infiltration of monocyte derived macrophages and liver fibrosis.Silymarin protects against liver fibrosis in C57BL/6 mice which was abdominal cavity was given CCl4,2 times/week,up to 4 weeks at most,possibly by attenuating monocyte-derived macrophage infiltration into liver.Correspondingly,Silymarin was orally administrated for 6 weeks.The level of serum ALT and AST were monitored.collagen-I staining,Masson staining,as well as HE staining were determined in the liver tissue using the mice model.Percentages of lymphocyte subsets,including T cells,B cells,monocyte-derived macrophage,DC cells,NK cells resident in liver were detected by flow cytometry.Further,the infiltrations of CD45+leukocytes,CDllb+ monocytes,and F4/80+ macrophages in the liver tissue were also determined by the approach of imnunohistochemistry.The mRNA expressions of TGF-β1,TNF-α,IL-6 and IL-β1 in the liver tissue were quantified using real-time PCR.The expression of hepatic MCP-1 was measured by the methods immunohistochemistry real-time PCR.The activation of HSC were also determined using a-SMA immunohistochemistry.RESULTSThe mice model of significant liver fibrosis was established by repeated CCl4 injections.The model group showed high level of serum ALT and AST than normal control group.HE staining analysis further identified extensive lobular structure abnormalities,liver cell necrosis and liver tissue invasion of a large number of inflammatory cells with CCl4.High level of collagen deposition were demonstrated by Masson staining.Collagen-I immunohistochemistry can clearly observe the state of I collagen deposition in liver tissue of CC14 induced liver fibrosis in mice.Silymarin treatment is able to alleviate serum ALT and AST levels in mice and protect the liver from the cellular damage,the infiltration of inflammatory cell and the fibrosis of liver.Monocytogenic macrophages,especially Grlhi monocytes,macrophages,were elevated in the liver tissue of CCl4 treated mice,compared to the control group.The model group showed significantly elevated CD3+T cells and DC cells were than the control group,but remarkable NK and B cells.There is no substantial difference on NKT cells,NK cells,T cells,DC cells,after silymarin treatment.However,the infiltration of m Monocytogenic macrophages,especially Grlhi monocytes,macrophages was further inhibited by the silymarin.Immunohistochemical analysis showed that significantly increased CD11b+ monocytes,CD45+ leukocytes and Compared to the control group.Model group f4/80 + macrophages,and,the silymarin administration could significantly attenuate the migration of these cells.Compared to the normal group,the model group was able to express high levels of tnf-alpha mRNA,while il-1 was the level of mRNA and il-6 was also showed an similar trend as TNF-α,upon six-weeks of CCl4 injection.with Silymarin treatment,mRNA level of TNF-awithin liver tissue was also significantly reduced in model group while the mRNA levels of TGF-1iwere significantly was increased than normal control group.Interestingly,silymarin treatment was able to substantially reduce the TGF-β1 mRNA in the liver tissue.High levels of McP-1 were expressed in the liver tissue of the model group than that of control mice revealed by the approaches of real-time PCR and immunohistochemistry.The silymarin treatment also significantly reduced the expression of MCP-1 within the liver tissue in the mice model.More importantly,silymarin might protect against the liver fibrosis by inhibiting the activation of HSC,demonstrated by a-SMA immunohistochemical analysis.CONCLUSIONSIt has been well-established that silymarin has hepatoprotective and anti-fibrotic effects.But the mechanisms are poorly understood.In recent years,the role of Ly6Chi monocytes in liver fibrosis has been well demonstrated.Thus,in present study we aimed to investigate whether silymarin can relieve liver fibrosis by reducing Ly6Chi monocytes infiltration.The mouse model of liver fibrosis was established by injected with carbon tetrachloride(CCl4)via intraperitoneal repeatedly.Mice in silymarin group received silymarin treatment by gavage.Silymarin significantly reduced liver inflammation and fibrosis of the mice induced by CCl4 injection,as revealed by liver histological and pathological analysis.Mice administrated by silymarin exhibited less infiltration of Ly6Chi monocytes.But there was no difference on other tested leukocyte subsets between CCl4 group and silymarin group.Meanwhile,further study found that silymarin significantly reduced CCl4-induced increased expression of tumor necrosis factor(TNF)-α,monocyte chemoattractant protein 1(MCP-1),and transforming growth factor-β1(TGF-β1),which was in line with the decreased numbers of intrahepatic Ly6Chi monocytes.In conclusion,our study showed that the anti-inflammatory and anti-fibrotic effects of silymadin could be derived from the prevention of Ly6Chi monocyLes infiltration into the injured livers.This give us a better understanding on the cellular mechanism of hepatoprotective,anti-inflammatory,and anti-fibrotic effect for silymarin.Such beneficial role of silymarin in liver fibrosis model could be partially explained by preventing the migration of Gr1hi monocyte into the damaged livers through suppressing MCP-1 secretion.