Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress

摘要

BACKGROUND:Previous studies have shown that rifampicin exhibits neuroprotective effects,but the precise mechanisms remain unclear.Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger.OBJECTIVE:To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN,TIME AND SETTING:A repeated measure,cell-based study was performed at the Department of Neurology,Second Affiliated Hospital,Sun Yat-sen University,China between December 2007 and November 2008.MATERIALS:PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School,Sun Yat-sen University,China.Rotenone and rifampicin were purchased from Sigma,USA.METHODS:PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco's modified Eagle's medium/Nutrient Mix F12(DMEM/F12) supplemented with 10% fetal bovine serum.The cells were assigned to six groups according to various treatment conditions:control,cultured with normal media;rifampicin group,treated with 300 μmol/L rotenone for 26 hours;rotenone group,treated with 2.5 μmol/L rotenone for 24 hours;rifampicin pretreatment groups,pretreated with 100,200,and 300 μmol/L rifampicin for 2 hours,respectively,followed by 2.5 μmol/L rotenone for 24 hours.MAIN OUTCOME MEASURES:Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry,respectively,using rhodamine123 staining.Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2',7'-dichlorofluorescin-diacetate staining,and intracellular reduced glutathione was measured with a microplate reader.Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry.RESULTS:Increased apoptosis in rotenone-induced,differentiated,PC12 cells was accompanied by the loss of mitochondrial transmembrane potential,the formation of reactive oxygen species,and reduced glutathione depletion(P<0.01).Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin(P<0.05 or P<0.01).CONCLUSION:Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.