The Regulative Role Of Rkip In CCl₄-induced Liver Fibrosis

Liver fibrosis characterized by excessive accumulation of extra-cellular matrix(ECM)in liver is a consequence of many chronic liver diseases,which may result in a high risk of mortality and morbidity.Many studies have shown that liver fibrosis is the key joint during chronic liver injury to cirrhosis and even hepatoma,which could be regarded as herald stage of cirrhosis.Once cirrhosis develops,it is difficult to recover ordinary state.Therefore,prevention of liver fibrosis is a crucial step for protecting the liver against the occurrence of chronic liver disease,especially cirrhosis.The Raf-1/MEK/ERK1,2 signaling pathway is known to be essential for HSCs proliferation,differentiation and migration.The deregulation of the pathway leads to various diseases including liver fibrosis,cirrhosis and malignancies.Raf kinase inhibitory protein(RKIP),a member of the phosphatidylethanolamine binding proteins family,is an inhibitor of Raf-1/MEK/ERK1,2 pathway.It has been reported that RKIP binds to Raf-1 and disrupts downstream of the MAP kinase pathway,and overexpression of RKIP can act as a competitive inhibitor of MEK.However,it is not clear whether RKIP plays an important role in hepatic fibrosis via regulation of the Raf-1/MEK/ERK1,2 signaling pathway.Objective:In the present study,the hepatic fibrosis model was induced by CCl4,and the expression and distribution of RKIP,p-RKIP,ERK and p-ERK as well as the activation of Raf-1/MEK/ERK1,2 were investigated in different stage of liver fibrogenesis,which would illustrate the role of RKIP in liver fibrosis.Method:Hepatic fibrosis was induced by intragastric administration with CCl4 in male Sprague-Dawley(SD)rats.A total of 105 male rats were randomly divided into four groups:? group(60 animals),? group(15 animals),? group(15 animals)and ? group(15 animals).The rats in the ? and ? groups received 2 ml/kg CCl4(mixed 1:1 in peanut oil)intragastrically twice per week for 15 w,while the animals in the ? and? groups received an equivalent peanut oil.Additionally,the rats in ?and ? groups were intraperitoneally injected with 0.5mg/kg Locostatin(the inhibitor of RKIP)twice per week from weeks 7 to 15.After a period of 6 w,10 w,13 w and 15 w,some animals were randomly selected from ? group and were killed,and the serum and liver samples were then collected.In addition,all the rats in ?,? and ?groups were killed at 15 w,and the serum and liver samples were also collected.The liver samples were divided into two parts:one immediately stored at 80? for future analysis,and another excised and fixed in 10%formalin solution for H&E and Masson staining.The activities of serum ALT and AST were measured by an automatic biochemical analyzer.The expressions of hepatic IRKIP,p-RKIP,ERK,p-ERK were determined using the method of immunohistochemistry.The expressions of RKIP,ERK,Col-? and Col-? mRNA in liver were analyzed by RT-PCR.And the levels of hepatic Col-?,RKIP,p-RKIP,ERK,p-ERK were detected by western blot immunoassay.Results:1.Construction of the CCl4-induced liver fibrosis modelIn this study,the liver fibrosis was induced by 50%CCl4 gavage in SD rats.Our results revealed that the extent of weight gain was lower in the model control group than that in the normal control group.Animals exposed to CCl4 presented significant increase of ALT and AST activities.Moreover,in the model control group,severe changes were observed in liver morphology including fatty degeneration,ballooning,necrosis and infiltration of inflammatory cells.These data indicated that the liver fibrosis model in rats was successfully established.2.The expressions of RKIP,p-RKIP,ERK and p-ERK in liverOur results showed that RKIP is expressed in hepatocytes and sinusoidal endothelial cells in normal rat liver.With the development of hepatic fibrosis,the positive cells of RKIP in CCl4-treated rats were significantly decrease compared to the normal control group(p<0.05 or p<0.01).There was few expression of p-RKIP in the cytoplasm and plasma membrane in the normal rats.Whereas,the expressions of p-RKIP increased remarkably in the model group.In the central veins and portal area of the normal liver tissues,the levels of ERK and p-ERK were very low.However,a large number of ERK and p-ERK positive cells were found in the portal area of liver after CCl4 treatment.Moreover,experimental results showed that the expressions of ERK and p-ERK were increasingly enhanced with the advance of hepatic fibrosis.In addition,the results revealed that the expression of RKIP was markedly inhibited after treatment with locostatin.However,the hepatic p-RKIP,ERK,and p-ERK levels in locostatin-intervened group were significantly higher than those in the model control group.3.Western blot for RKIP,p-RKIP,ERK and p-ERK in liverWestern blot analysis showed a lower expression of RKIP in the CCl4-treated group at week 13 and week 15 when compared to the normal control.Moreover,the expressions of p-RKIP,ERK,p-ERK,Raf-1,p-Raf-1,MEK-1,p-MEK-1,?-SAM,Col-? and Col-? increased remarkably in the model group.In addition,the expression of RKIP decreased considerably after locostatin administration.On the contrary,the levels of p-RKIP,p-ERK,MEK-1,p-MEK-1??-SAM and Col-?increased significantly in the locostatin-intervened groups.4.The expressions of RKIP,ERK,Col-? and Col-? mRNACompared to the normal control group,a notable elevation of ERK,Col-? and Col-? mRNA genes levels was found in the model control group.The results of RT-PCR showed that treatment with locostatin significantly increased the expression of ERK?Col-? and Col-? mRNA,while markedly inhibited the RKIP expression.Conclusion:Raf-1/MEK/ERK1,2 signaling pathway plays an important role in liver fibrogenesis.The activation of the signaling pathway is consistent with the stage of liver fibrosis.In addition,low expression of RKIP or high level of p-RKIP can lead to the activation of ERX pathway.Thus,RKIP overexpression might alleviate liver fibrosis via inactivation of Raf-1/MEK/ERK1,2 signaling pathway,which may be regarded as a target for drug discovery against hepatic fibrosis.