Study On The Protective Mechanism Of Bicyclol On Liver Injury Via Autophagy And Anti-oxidative Stress

ObjectsAcute liver injury is caused by many factors,with higher aminotransferase and abnormal liver function.Bicyclol has been widely used in clinical practice and could effectively decrease aminotransferase and protect liver function.The liver protectant mechanism of bicyclol is strongly linked to the protection of membrane and mitochondrial functions,suppression of endoplasmic reticulum stress,elimination of free radicals,induction of heat shock protein,and inhibition of inflammatory cytokines.Autophagy is a self-protection mechanism when eukaryotic cells faced with starvation,hypoxia,damage.Autophagy was observed in acute liver injury in past studies.There is few study about whether bicyclol protect liver from injury via promoting autophagy.In this study,CCl₄ was delivered by intraperitoneal injection to induce acute liver injury in C57BL/6 mice,with or without bicyclol.The expression of genes related autophagy and anti-oxidative stress in liver tissue were detected.The study was aimed to evaluate the role of autophagy and anti-oxidative stress in liver protection by bicyclol.Methods1.Animal experiment:40ul 50%CCl₄ was administered by intraperitoneal injection to induce acute liver injury.Overall 30 six-eight-week old C57BL/6 male mice were randomly assigned into five groups,the control group,the CCl₄ 24h group,the CCl₄48h group,the CCl₄+bicyclol 24h,the CCl₄+bicyclol 48h.Mice were sacrificed after CCl₄ given 24h or 48h.Blood samples were collected to detect ALT level.Pathological changes of liver tissue were assessed after hematoxylin-eosin staining.Western Blot was used to detect the expression of Atg5,Atg7,Atg12,p62,LC3,Beclin-1,and mTOR in liver tissue of mice.The variation of autophagosomes and autolysosomes was assessed by transmission electron microscopy.The mRNA expression levels of HO-1,NQO1,GSTA-1 in liver tissue were analyzed by qRT-PCR.2.Cell experiment:Pre-treated with bicyclol or 3-MA,HepG2 was stimulated by CCl₄.The cells were gathered after 2 hours for protein extraction.Western Blot was used to test the protein level of p62,LC3,and mTOR.The HepG2 cells were transiently transfected with the GFP-LC3 expression vector,and then treated with bicyclol or 3-MA.The GFP-LC3 fluorescence was watched using a fluorescence microscope,and the autophagosomes in each cell were calculated.3.The experimental data was analyzed by SPSS 20.0 and p<0.05 was considered to be statistically significantResults1.In animal experiment,serologic test showed that the serum ALT level of C57BL/6mice was significantly increase after injected CCl₄ compared with the control group（P<0.001）,the ALT level of mice was apparently decrease after bicyclol was given by gavage（P<0.001）compare with CCl₄ group.Pathological section showed obviously degeneration and necrosis in CCl₄ group,while damage of hepatic cell was repaired in bicyclol group.2.In animal experiment,the CCl₄+bicyclol group were compared with the control group and CCl₄ 48h group,the levels of Atg7,LC3 II,Beclin-1,p62 increased,P-mTOR decreased significantly（P<0.05）.The levels of p62 and LC3 II also increased in CCl₄ 24h than the control group.No statistical difference in Atg5 or Atg12 was found among the five groups（P>0.05）.3.In animal experiment,compared with the control group and CCl₄ group,the mRNA expression levels of HO-1,NQO1,GSTA-1,Keap-1,p62 was increased obviously in CCl4+bicyclol group（P<0.01）.4.Compared with the control group,the levels of p62,P-mTOR decreased,LC3 II increased in CCl₄+bicyclol group,3-MA as an inhibitor of autophagy could ameliorate these change（P<0.05）.The GFP-LC3 plasmid was transfected into HepG2cells.Compared with control group,the amount of green dots（indicating autophagosomes）were increased significantly after treatment with bicyclol,pre-treated with 3-MA the green dots in cells decreased（P<0.01）.ConclusionsBicyclol could obviously decrease ALT level and repair hepatic cell in acute liver injury.Autophagy associated protein such as Atg7,LC3 II,Beclin-1,p62,P-mTOR play an important role in this variation.p62 was induced by bicyclol and then affected Keap-1/Nfr2 anti-oxidative stress signal pathway,leading to up-regulation of HO-1,NQO1,GSTA1.Bicyclol could alleviate acute liver injury induced by CCl₄ through promotion of autophagy and induction of anti-oxidative stress.