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

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 rhodamine 123 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.