Role Of Endoplasmic Reticulum Stress-induced Autophagy In Hepatocyte Apoptosis

Objective: To observe the changes of autophagy-related index during the process of endoplasmic reticulum stress(ERS)induced by dithiothreitol(DTT)and its effect on apoptosis in human normal hepatocytes.Methods: LO2 cells were treated with DTT at 2.0 mmol/L for 0,6,12 and 24 h to induce ERS.The proliferation of LO2 cells was detected by real time cellular analysis(RTCA).The expression of glucose regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),and activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP)and autophagy related gene 12(Atg12),autophagy related gene 5(Atg5)and microtubule-associated protein 1 light chain 3(LC3)at mRNA and protein levels was determined by real time PCR and Western blot.The apoptosis was analyzed by flow cytometry.The formation of autophagosomes was observed under transmission electron microscope.After the LO2 cells were pretreated with rapamycin(RAP)and 3-methyladenine(3-MA)for 1 h and treated with DTT for 24 h,the effects of RAP and 3-MA pretreatment on endoplasmic reticulum stress and apoptosis were observed.In order to elucidate the mechanism of ERS regulating autophagy,CHOP siRNA was used to transfect LO2 cells and to observe the effect of CHOP gene silencing on autophagy and apoptosis.Results: After treatment with DTT at 2.0 mmol/L for 6,12 and 24 h,the mRNA and protein levels of GRP78,PERK,ATF4,CHOP,Atg12,Atg5 and LC3 in the LO2 cells were significantly higher than those in 0 h group.At the same time,the ration of LC3II/LC3 I was also increased after DTT treatment.Observation under transmission electron microscope showed that autophagosomes were found in the LO2 cells treated with DTT for 6,12 and 24 h.After DTT treatment for 6,12 and 24 h,the apoptotic rate of LO2 cells was significantly higher than that in DTT 0 h group.Pretreatment with RAP could significantly decrease the apoptosis in LO2 cells,whereas pretreatment with 3-MA could significantly elevate the apoptosis.After knocking down the expression of CHOP gene in LO2 cells by small interference RNA technique,it was found that the down-regulation of CHOP expression significantly increased the expression level of autophagy-associated molecules and decreased the apoptosis induced by DTT.Conclusion: ERS could induce autophagy in hepatocytes,and the increase of autophagy has protective effect on hepatocytes under ERS.The upregulation of ERS-specific transcription factor CHOP could inhibit autophagy and promote hepatocyte apoptosis,while knock-down CHOP gene could attenuate hepatocyte apoptosis induced by DTT.