The Hepatic Stimulator Substance Alleviates Liver Fibrosis Via Inhibition Of Hepatic Stellate Cell Activation

Backgrounds and ObjectivesHepatic stellate cells(HSCs)are sources of excessive extracellular matrix(ECM)in the process of liver fibrosis.After injuried by chemical toxins,hepatocytes often exhibites necrosis and apoptosis,resulting in inflammatory response and producing varieties of inflammatory cytokines.HSCs are thereafter be activated by these cytokines and progressively trans-differentiate into myofibroblast(MFB)-like cells which synthesize abundant extracellular matrix(ECM)including collagens.In the light of cardinal role of HSCs during pathogensis of liver fibrosis,the attempt to inactivation on HSCs and promotion of HSC apoptosis has been considered as the essential step to breakdown liver fibrosis and reverse the fibrosis.Hepatic stimulator substance(HSS)is a bio-active substance with characteristic of protecting hepatocytes from various injuries and promoting their growth.Protection of HSS on hepatic functionality is probably related with maintenance of intracellular calcium homeostasis and suppression on cell apoptosis.The overexpression of HSS is seen in the process of liver fibrosis and cirrhosis,indicating that HSS may be closely associated liver scar.In our previous study,over-expression of HSS can evidently attenuate liver fibrosis in mice.Meanwhile,the knowdown of HSS can deteriorate liver fibrosis in mouse with CCl4-induced liver fibrosis.However,mechanisms how HSS ccan attenuate the liver fibrosis is still unclarified.In this experiment we are aiming to explore the role of HSS in the development of liver fibrosis along with HSC activation.Furthermore,we investigate relavant molecular and cellular mechanisms in the effect of HSS on the process of HSC activation.Methods1.Construction of Cell model: LX-2 cells with phenotypes of activated HSC were cultivated and stable transfected by a HSS-containing plasmid or HSS-sh RNA.2.Measurement of fibrosis indicates: determinate whether expressions of indicates of liver fibrosis are changed by HSS in LX-2 cell model or not3.Measurement of characteristics of HSC activation: test capabilities of proliferation in cell model by flow cytometry of cell cycle and immunorescence of co-localization of Ki67 and DAPI;analyze abilities of cell migration by Transwell measurement and Xcelligence test4.Research on mechanisms: probe into certain proteins of influences on proliferation and migration via cytokine arrays5.Research on mechanisms of cell motility: analyze distribution of cell microfilaments by high content analysis;analyze cell lamellipodia and stress fibers by confocal immunorescence6.Exploration on mitochondrial dynamics: analyze effects of HSS on mitochondrial dynamics by confocal immunorescence and concern software.Results1.LX-2 cells with stable over-expression of HSS(HSS-Tx)and knockdown of HSS(HSS-sh RNA)were successfully established.2.?-SMA and type ? pro-collagen expression in HSS-sh RNA LX-2 cells and HSSTx LX-2 cells by Western blot were detected.The HSS-sh RNA cells exhihited profound ?-SMA(P <0.05)and type ? pro-collagen(P <0.01)expression comparing with HSS-Tx group.3.We compare abilities of proliferation and migration in LX-2 cell model.Obviously,MTS measurement indicates that HSS-sh RNA group proliferated more faster than HSS-Tx group at 36 h(P<0.05),48 h(P<0.01),60 h(P<0.01)and 72 h(P<0.05).Flow cytometry also shows that G0/G1 phase decreased in HSS-sh RNA group compared with HSS-Tx group(P<0.05).To further confirm this hypothesis,immunofluorescence staining shows co-localiztion of Ki67 and DAPI in cells,which indicates that the proportion of Ki67 positive in HSS-sh RNA cells is higher than HSS-Tx cells(P<0.05).Moreover,Transwell assay evidently shows enhanced migration ability in HSSsh RNA group compared with HSS-Tx group(P<0.01).4.F-actins and nuclei are marked by immunofluorescence stain in LX-2 cells.Remarkably,immunofluorescence stain analyzed by high content analysis technology suggests that more quantity of F-actin in HSS-sh RNA LX-2 cells than HSS-Tx cells(P<0.01).Confocal immunorescence progressively confirms that HSS can decrease quantities of microfilaments in LX-2 cells.5.We analyze basal cytosolic Ca2+ levels and mitochondrial Ca2+ levels in LX-2 cells by using specific Ca2+ fluorescent dye-Fluo-3 and Rhod-2.Notably,HSS-sh RNA cells have lower cytosolic Ca2+ levels than HSS-Tx cells.In mitochondria,HSSsh RNA group have lower mitochondrial Ca2+ levels than HSS-Tx group.6.Mitochondrial tracker staining shows shorter(P<0.0001)and rounder(P<0.0001)Shapes of Mitochondria in HSS-Tx cells than HSS-sh RNA cells.Western blot results indicate lower MFN2 expression(P<0.05)in HSS-Tx cells than HSS-sh RNA cells.ATP measurements show significant lower ATP level in HSS-Tx group compared with HSS-sh RNA group(P<0.0001).We also measure OCR and ECAR level in the HSS-Tx cells and its control cells(vector-Tx).The result indicates lower aerobic respiration ability in HSS-Tx group than vector-Tx group at 14.62 min basal level(P<0.01)and significant lower ability in HSS-Tx group after stimulation by Oligomycin and FCCP at 21.25 min(P<0.001).7.Rescue experiments indicates that HSS-Tx cells were transfected by HSS-si RNA and its negative control(scramble si RNA).We analyze shapes of these cells after transfection,which suggesting longer(P<0.0001)and more rod-like(P<0.0001)shapes in HSS-si RNA group than scramble si RNA group.ConclusionsOur data demonstrate for the first time that HSS may down-regulate fibrogenic markers in LX-2 cells;through inducing less cytosolic Ca2+ and reducing F-actin assembly HSS can progressively attenuate cell migration,which alleviates activation of HSCs and improves liver fibrosis.