HBSP Inhibits Fas-mediated Hepatocyte Apoptosis Through Enhancing Phosphorylation Of AKT

The main pathways of hepatocyte apoptosis include FasL/Fas,TRAIL/DR5,and TNF-/ TNF-R,among which FasL/Fas pathway plays a vital role in death and regeneration of hepatocytes.The mFas combines with FasL to form a polymer that recruits FADD and procaspase8 to form the death inducing signal complex(DISC)to induce the apoptosis of hepatocytes by the cascade reaction of the caspase family.It has been reported that HBSP is closely related to the persistent infection and the pathogenicity of hepatitis B virus.HBSP induce the phosphorylation of AKT,while FasL,Fas polymer and FLIP all have been regulated with AKT.But now,it is not clear weather HBSP affects Fas mediated apoptosis of hepatocytes through enhancing the phosphorylation of Akt.Thus,this study aims to investigate the effect of HBSP on Fas mediated apoptosis of hepatocytes through enhancing the phosphorylation of Akt.The first part of this study investigates the effect of HBSP on Fas mediated apoptosis of hepatocytes though the phosphorylation of AKT.To address these issues,we screen stable HepG2(wildtype p53)cell lines,HepG2-Flag and HepG2-HBSP cells,which harbo the empty vector pcDNA3.1/Hygro(+)-Flag and pcDNA3.1/Hygro(+)-HBSP-Flag respectively.The expression of HBSP in thses two cell lines was detected by western blot.Cell viability and apoptosis were confirmed by CCK8 and AnnexinV/PI.The results demonstrate that HBSP inhibit the decrease of HepG2 cell activity and the apoptosis of cells induced by anti-Fas CH11.The second part of this study investigates the mechanismon the pathway of Fas effected by HBSP.The control group makes no difference with HepG2-HBSP on the expression of total Fas,mFas,sFas and P53 at mRNA or protein levels,which were quantified or qualitied by Real-time PCR or Western blot analysis.In order to detect the expression of HBSP on Fas level,we investigate the expression of FasL,FLIP and Fas ploymers.The results show that HBSP down-regulate the expression of FasL and inhibit the formation of Fas polymers and resist the down regulation of FLIP at mRNA or protein levels though AKT.This study is to elucidate that HBSP reduce FasL and Fas polymer but enhance FLIP duing to the change of DISC.Then co-immunoprecipitation(Co-IP)was used to confirm the interaction between FADD,Fas and caspase8.The results demonstrate that HBSP down-regulate the expression of FasL,inhibit the formation of Fas polymers and resist the down regulation of FLIP which because that HBSP play a negative role in DISC levels.The third part of this study is to elucidate the mechanism of HBSP inducing the phosphorylation of AKT.HBSP enhance phosphorylation of AKT at Ser473 and Thr308 sites,and promote phosphorylation of mTOR and PDK1 but have no effect on the quantity of mTOR and PDK1.Then co-immunoprecipitation(Co-IP)was not discoveried the interaction between HBSP and AKT,PDK1,mTOR.