The Injury Effect Of Cholic Acid And Its Gut Microbial Metabolite On Hepatocyte And The Related Mechanism

Background & Objective : Non-alcoholic fatty liver disease(NAFLD)is a metabolic stress liver disease characterized by excessive lipid deposition in hepatocytes.Nowadays,NAFLD has been the major type of chronic liver disease that impairs Chinese health.Recently,it has reported that NAFLD patients present enlargement of bile acid pool and abnormal change of cholic acid(CA)and deoxycholic acid(DCA)both in serum and in liver.These two kinds of bile acids are suggested to play a role in the progenesis and progress of NAFLD,though less mechanism has been uncovered until now.Therefore,our study aims to treat Hep G2 cells,with or without free fatty acid(FFA)-induced steatosis,by CA and its metabolite(DCA),respectively,so as to explore the effect of CA and DCA on hepatocytes and underlying mechanism.Methods:Hep G2 cells were randomly allocated into CA group,FFA+ CA group,DCA group and FFA + DCA group,respectively.Both FFA+CA and FFA+DCA groups were exposed to 500?M FFA(oleic acid(OA): palmitic acid(PA)= 2:1)for 12 hours,and then CA/DCA of different concentrations for another 12 hours.Cells in the CA group and DCA group were cultured by normal medium for 12 hours,and then treated by CA/DCA of different concentrations for another 12 hours.The hepatocellular steatosis was detected using oil red O staining and triglyceride(TG)assay kit.Mitochondrial membrane potential(MMP)was detected by JC-1 probe in respect to mitochondrial depolorization.Reactive oxygen species(ROS)level,malondialdehyde(MDA)level and superoxide dismutase(SOD)activity were examed by DCFH-DA probe,TBA method and NBT method,respectively,to highlight the oxidative stress.Cell proliferation activity was detected by CCK-8 method.The changes of Nrf2,HO-1 and NQO1 at m RNA and protein level were detected by quantitative real time polymerase chain reaction(q RT-PCR)and western blot(WB).Results: In FFA + CA group and FFA + DCA group,numerous lipid droplets positive to oil red O staining was observed in the cells,with significant increase of intracellular TG level compared with those cells in CA group or DCA group without FFA treatment(p<0.001~p<0.05).With the increase of DCA concentration,the MMP decreased in a concentration-dependent manner,while the level of ROS and MDA increased and proliferation activity was inhibited in DCA group and FFA+DCA group.Being exposed to the same concentration of DCA,the FFA+DCA group demonstrated significantly decreased MMP(p<0.01~ p<0.05),deteriorated markers of oxidative stress(p<0.001~ p<0.01),and impaired viability(p<0.01~ p<0.05)in comparison to those of the DCA group.In CA group,CA enhanced the cellular viability siginificantly at the CA concentrations of 250 M and 500 M(p<0.001~p<0.05).Meanwhile,the cellular viability in 500 M FFA+CA group decreased siginificantly compared with 0 M FFA+CAgroup(p<0.05).CA induced hepatocellular oxidative stress in both CA and FFA+CA groups at the concentration of 500 M.Nevertheless,there was no significant difference in oxidative stress between these two groups.After the treatment of DCA,the m RAN levels,but not protein levels,of Nrf2,HO-1,and NQO1 experienced great upregulation in DCA group and FFA+DCA group(p<0.0001~p<0.05).Conclusion: DCA enhances oxidative stress and its associated hepatocellular damage by inducing mitochondrial depolarization.Steatosis increase the sensitivity of hepatocytes to DCA injury.In contrast,CA displays weaker effect on hepatocyte injury.OriginalityThis study explored the effect of primary bile acid CA and its metabolite,DCA on steatosis hepatocytes,aiming to disclose the underlying mechanism of NAFLD progression.In our study,we find that DCA displays stronger injury effect on hepatocytes in terms of mitochondrial depolorization,oxidative stress and cellular viability,compared with CA.Furthermore,steatosis tends to elevate the sensitivity of hepatocytes to DCA,which indicates that DCA plays an important role in the genesis and progress of NAFLD.Through this study,we hope to provide potential new targets in clinical diagnosis and therapies of NAFLD.PotentialitySteatosis increased the sensitivity of hepatocytes to DCA injury,suggesting that DCA may play an important role in the genesis and development of NAFLD.Therefore,it may provide a theoretical basis for NAFLD prevention and treatment via targeting the metabolites of gut microbiota.