Effects And Apoptotic Mechanisms Of Glycodeoxycholate On The Human Normal Hepatocyte HL-7702 Line

Objective: To investigate the effects of glycodeoxycholate(GCDC) on theapoptosis of human normal hepatocyte HL-7702 line and to elucidate itsmolecular mechanisms.Methods: (1) HL-7702 cells were incubated with various concentrations ofGCDC(final concentration of 50μmol/L,100μmol/L,150μmol/L,200μmol/L,250μmol/L,respectively) or with 250μmol/L GCDC for 4h,8h,12h,24h.The apoptotic effects of GCDC on HL-7702 cells were investigatedby light microscope and agarose gel electrophoresis.The apoptotic ratio ofHL-7702 was determined by AnnexinV-FITC/PI double staining. Thechanges of HL-7702 cells intracelluar [Ca²⁺]_i were determined withFluo-3/AM load technique.(2) The activities of caspase-3/-9 in HL-7702cells were performed by spectrophotometer method and the mRNAexpression levels of caspase-3/-9,Bcl-2,Bax in HL-7702 cells were analyzedby RT-PCR after HL-7702 cells were treated by GCDC for 24 hours whichfinal concentration was 100μmol/L,150μmol/L,200μmol/L,250μmol/L,respectively.Results: (1)A typical apoptotic morphological changes were observed afterHL-7702 cells had been treated by 150μmol/L GCDC for 24h.(2) When HL-7702 cells were treated by 50μmol/L,100μmol/L,150μmol/L GCDCrespectively or 150μmol/L GCDC for 8h,12h,24h, Typical DNA Ladder ofttL-7702 cells were detected by agarose gel electrophoresis.(3)The resultsdetected by Annexin V-FITC/PI double staining showed that HL-7702 cellscould be induced to undergo apoptosis in a concentration dependent mannerafter 50μmol/L,100μmol/L,150μmol/L,200μmol/L,250μmol/L GCDCtreatment for 24 hours. The apoptotic ratio was 9.26±2.6%(t=4.58),13.16±2.9%(t=6.41), 20.3±3.0%(t=10.22), 25.02±2.1%(t=18.11), 45.02±3.5%(t=28.89), respectively,which were markedly higher (P＜0.05)than controlgroup. The apoptotic ratio of HL-7702 cells was dependent on time.Theapoptotic ratio was 10.35±3.1%(t=4.48), 19.37±2.9%(t=10.04), 25.07±3.1%(t=12.57), 45.02±2.5% (t=28.89) when HL-7702 cells were incubated withGCDC for 4h,8h,12h,24h, respectively, which were significantly higher(P＜0.05) than the control gtoup.(4) The intracellular [Ca²⁺]_i increased with atime-and concentration-dependent manner when HL-7702 were incubatedwith GCDC.(5)GCDC could markedly activate caspase-3/-9 in HL-7702cells.The activities of caspase-3/-9 were higher than the control group(P＜0.05).(6)The mRNA expression of caspase-3/-9 in HL-7702 cells wereactivated by GCDC.(7)GCDC could down-regulate the expression of Bcl-2mRNA and up-regulate the expression of Bax mRNA.Conclusions:(1)GCDC could induce apoptosis of HL-7702 cells in a time-and concentration-dependent manner.(2) GCDC can induce HL-7702 cellsapoptosis through increasing intracellular [Ca²⁺]_i.(3)GCDC can obviouslyactivate the activities of caspase-3/-9 and upregluate its mRNA expression inHL-7702.Apoptosis of HL-7702 cells induced by GCDC can be initiated bymitochondrial pathway. (4) GCDC can inhibit the expression of Bcl-2 mRNAand up-regulate the expression of Bax mRNA. Bcl-2 family members are very important in apoptosis of HL-7702 cells induced by GCDC.(5) Ourresearch explore the apoptotic mechanisms of HL-7702 cells induced byGCDC,which might be useful for clinical therapy and treatment of cholestaticliver disease.