The Role And Potential Mechanisms Of Discoidin Domain Receptor 1 In The Development Of Hepatic Fibrosis

Background and Objectives:Fibrosis is a common pathological feature of most liver diseases,which is usually due to a repair imbalance caused by chronic liver injury of various causes.Although mild fibrosis is largely asymptomatic,it can progress to cirrhosis and eventually lead to liver failure,which is a major cause of poor prognosis in patients with liver disease.So far,there is no direct anti-fibrosis therapy,and further exploration of the related molecular pathological mechanism is of great significance for the development of efficient and specific anti-fibrosis drugs.Discoidin domain receptor 1(DDR1)is a collagen receptor function by binding to collagen.Several studies have shown that DDR1 is closely related to the occurrence and development of fibrosis diseases,but the underlying mechanism of DDR1 in hepatic fibrosis remains unclear.In this study,we investigated the role and the potential mechanisms of DDR1 during liver fibrogenesis in vivo and in vitro.Methods:1.We performed in vivo studies with wild type(WT)mice and DDR1knockout(DDR1-KO)mice of the same background to identify the role of DDR1 in liver fibrosis induced by carbon tetrachloride(CCL4)(0.5mL/kg).Blood and livers were collected from mice for analyses.The correlation between DDR1 and liver fibrosis was analyzed by the determination of serum biochemical indexes,histopathological observation and the detection of the expression of fibrotic indicators.2.Indirect co-culture system of hepatocytes and hepatic stellate cells(HSC)was established to study the effect of DDR1 on HSC function in vitro.Lo2 and hepG2 cells with DDR1 gene overexpression(DDR1-OE)or knockdown(DDR1-KD)were constructed by transfection of the corresponding lentivirus.LX2 cells were incubated with conditioned medium(CM)of the above stable transfected cells treated with collagen.Cells and supernatants were collected for molecular and biochemical analyses.Further,LX2 cells were incubated with the above CM which added with neutralizing antibody,and then the cells were collected for molecular detection.3.To further elucidate the molecular mechanism of DDR1 regulating the expression of pro-fibrosis mediators in hepatocytes,we detected the activation of DDR1 and related pathway proteins in stable transfected cells when stimulated by collagen,and after the cells pretreated with pathway protein inhibitors,we further detected the expression level of pro-fibrosis cytokines in CM from stable transfected cells under the stimulation of collagen.Through the above experiment to explore relevant signaling pathways involved in the regulation of the secretion of pro-fibrosis cytokines in hepatocytes when DDR1 is activated.Results:1.DDR1 expression was increased in hepatocytes from CCL4-induced fibrotic livers compared to normal livers in WT mice.Liver fibrosis was relieved,the number of αSMA positive staining HSCs decreased,and the protein and mRNA expression levels of αSMA and COL1 were reduced in CCL4-treated DDR1-KO mice compared with CCL4-treated WT mice.2.In indirect co-culture studies,it was found that LX2 cells cultured in CM of LO2 DDR1-OE cells showed the increased expression of αSMA and COL1,and increased cells proliferation.Meanwhile,the expression levels of αSMA and COL1 in LX2 cells cultured in CM of hepG2 DDR1-KD cells decreased,and cell proliferation decreased.Moreover,IL6,TNFα and TGFβ1 in CM of DDR1-OE cells appeared to promote LX2 cells activation and proliferation.3.In order to clarify the relationship between signaling pathway activation possibly induced by activated DDR1 and the secretion of IL6,TNF-α,TGFβ1 in hepatocytes,pathway inhibitors have been used to pretreat DDR1-OE cells,and then stimulation the cells with collagen,the results showed that BAY 11-7082 decreased the content of IL6 and TNFα in the above cell supernatant,while MK2206 decreased the content of TGFβ1 in the above cell supernatant.Further detection of collagen-stimulated DDR1 phosphorylation and the activation of NF-κB and Akt pathway protein showed that IL6,TNFα and TGFβ1 in CM of DDR1-OE cells was regulated by NF-κB and Akt pathways.Conclusion:The above results indicated that DDR1 is closely related to the progression of hepatic fibrosis.DDR1 in hepatocytes promoted HSC activation and proliferation,and paracrine factors IL6,TNFα and TGFβ1 induced by DDR1 through activating NFκB and Akt pathways may be the underlying mechanisms.Our study suggests that collagen-receptor DDR1 may be a potential therapeutic target for hepatic fibrosis.