Taurocholic Acid Promotes Hepatic Stellate Cell Activation Via S1PR2/p38 MAPK/YAP Signaling Under Cholestatic Conditions

Cholestasis is an important factor in liver fibrosis.It is generally believed that the degree of disease progression is related to the accumulation of bile acids,but the mechanism has not been fully elucidated.Sphingosine 1-phosphate receptor 2(S1PR2)is a recently discovered bile acid G-protein-coupled receptor,which plays an important role in the regulation of cell proliferation,migration and differentiation.In this study,we use taurocholic Acid(TCA),which changes the most during cholestasis,to explore the role and mechanism of S1PR2 in the activation of Hepatic stellate cells(HSC)and the progression of liver fibrosis.In order to provide a new potential target for the intervention of cholestatic liver fibrosis.The main research contents and conclusions are as follows:(1)TCA stimulation was given on HSC lines LX-2 and JS-1.The effects of TCA on HSC proliferation,migration,cell contraction and ECM secretion,such as Collagen I and α-SMA,were investigated by cell viability assay,wound healing assay,collagen gel contraction assay,RT-PCR and Western blotting analysis.The results showed that TCA promoted HSC cell proliferation,migration and contraction,and significantly increased the expression of Collagen I and α-SMA,confirming that TCA directly promoted HSC activation.(2)The role of S1PR2 in promoting HSC activation by TCA.Firstly,the expression of S1PR2 in HSC and liver fibrosis was significantly increased by RT-PCR,DNA gel electrophoresis and immunofluorescence staining.Secondly,S1PR2 expression was silenced or inhibited by S1PR2 sh RNA and S1PR2 specific inhibitor JTE-013 in hepatic stellate cell lines LX-2 and JS-1.It was confirmed that blocking S1PR2 inhibited TCA and promoted the proliferation,migration,contraction and secretion of ECM(Collagen I and α-SMA)of HSC.Finally,in 0.1%DDC induced cholestatic liver fibrosis model,mouse liver S1PR2 was specifically knocked out by caudal injection of adeno-associated virus(AAV sh S1PR2),or S1PR2 specific inhibitor JTE-013 was given intraperitoneal injection.Determination of hepatic enzyme levels(ALT/AST),H&E staining,Masson’s Trichrome staining,hydroxyproline levels and α-SMA fluorescence staining were used to confirm that blocking or reducing S1PR2 can significantly reduce DDC-induced liver tissue damage,reduce collagen deposition and inhibit the activation of HSC,and effectively alleviate the progression of cholestatic liver fibrosis.(3)YAP signaling pathway involved in TCA-S1PR2 mediated HSC activation.First,S1PR2 expression was silenced or inhibited by S1PR2 sh RNA and S1PR2 specific inhibitor JTE-013 in HSC lines LX-2 and JS-1.The intracellular distribution of YAP and the expression of YAP target genes(CTGF and Cyclin D1)were investigated by Western blotting,immunofluorescence staining and RT-PCR.The results showed that: Blocking or silencing S1PR2 can inhibit the promotion of YAP nuclear translocation by TCA and the expression of CTGF and Cyclin D1.Subsequently,ROCK inhibitor Y27632,ERK inhibitor U0126,and p38 MAPK inhibitor SB203580 were used to confirm that p38 MAPK mediated TCA-S1PR2 promoted HSC activation.Finally,YAP expression was knocked down by YAP si RNA and other methods,confirming that TCA-mediated HSC activation was inhibited after YAP signaling pathway was blocked.In conclusion,TCA can directly promote HSC activation through S1PR2/p38MAPK/YAP signaling pathway during cholestasis and participate in the progression of cholestatic liver fibrosis.