| Objective：To observe the effect of small interfering RNA (small interfering RNA,siRNA) inhibit chloride ion channel protein3(ClC-3) gene expression on apoptosis of ratadrenal pheochromocytoma cells (rat pheochromocytoma cells, PC-12), and to explore themechanism of ClC-3on hydrogen peroxide (H2O2)-induced apoptosis of PC-12.Methods：(1) PC-12cells subculture.(2)12h after the intervention of differentconcentrations of H2O2,Cell counting kit-8(CCK-8) kit was used to detect PC-12cellsurvival rate and to determine the concentration of H2O2required to build models ofapoptosis.(3) PC-12cells in logarithmic phase were randomly divided into four groups:normal control group, model group, empty plasmid group and RNA interference groupwith six wells for each group. Normal control group was heightened with high glucoseDMEM complete medium; model group was added with H2O2intervention; empty plasmidgroup was transfected with empty vector after adding H2O2intervention; RNA interferencegroup was transfected plasmid plus interference H2O2intervention. After12hours, CCK-8was used to detect cell survival rate; Hochest33342staining to observe cell morphology;Western Blot to detect the expression of Caspase-3and ClC-3protein; real-time PCR todetect the expression of ClC-3mRNA and Caspase-3; flow cytometry (Flow cytomet, FCM)to detect apoptosis rate.Results：(1) Compared with0nmol/L model group,50,100,200,300,400,500,600,700nmol/L survival rate of PC-12cells in model group decreased (P<0.05),and negativelycorrelated with the concentration of H2O2(r=-0.961,P<0.05).In which the cell survivalrate of400nmol/L model group is (49±1.20)%,the concentration was used to establish thePC-12cell apoptosis model.(2) Hochest33342of nuclear staining showed that comparedwith normal control group, cell survival rates of model group, empty plasmid group andRNA interference group decreased and apoptosis rates increased (P<0.05). Compared withthe model group, cell survival and apoptosis rates of empty plasmid group did not changesignificantly (P>0.05), while cell survival rate of RNA interference group increased andapoptosis rate decreased (P<0.05).Compared with the empty plasmid group, the cellsurvival rate of RNA interference group increased and apoptosis rate decreased(P <0.05).(3) Western blot showed that compared with normal control group, the expression ofCaspase-3and ClC-3protein in model group and empty plasmid group increased;while theexpression of Caspase-3and ClC-3protein in RNA interference goup decreased(P<0.05).Compared with model group,the expression of Caspase-3and ClC-3protein inempty plasmid group was no significantly different (P>0.05);compared with emptyplasmid group,the expression of Caspase-3and ClC-3protein in RNA interference groupdecreased (P<0.05).(4) real-time PCR results showed that compared with normal controlgroup, the expression of Caspase-3and ClC-3mRNA in model group and empty plasmidgroup increased;RNA interference group decreased(P<0.05).Compared with model group,the expression of Caspase-3and ClC-3mRNA in empty plasmid group was no significantdifference (P>0.05); compared with empty plasmid group,the expression of Caspase-3andClC-3mRNA in RNA interference group decreased (P<0.05).(5) Flow cytometry showedthat:compared with normal control group, apoptosis rate of model group and emptyplasmid group was significantly higher(P<0.05).Compared with model group, apoptosisrate of empty plasmid group was no significant difference (P>0.05).Compared with emptyplasmid group,RNA interference group apoptosis was significantly lower (P<0.05).Conclusion：(1) H2O2can activate apoptotic signaling pathways and induce apoptosis inPC-12cells through enhancing the expression of Caspase-3.(2) Silencing ClC-3Gene,reducing the expression of ClC-3and inhibiting caspase-3-mediated apoptotic pathwaywould reduce the occur of H2O2-induced apoptosis in PC-12cells, this phenomenonindicates that ClC-3chloride channel man be involved in the apoptotic process. |
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