| Objective: To investigate the neuroprotective effects of methyl 3,4-dihydroxybenzoate(MDHB) against t-butylhydroperoxide(TBHP)-induced oxidative damage in SH-SY5 Y cells and its underlying mechanisms.Methods: SH-SY5 Y human neuroblastoma cells were cultured in DMEM+10%FBS for 24 hours, which were then pretreated with different concentrations of MDHB or N-acetyl-L-cysteine(NAC) for 4 hours prior to addition of 40μM TBHP for 24 hours. Cell viability was analyzed by methyl thiazolyl tetrazolium(MTT) assay and lacate dehydrogenase(LDH) assay. The divided experimental groups were as follows: control group, vehicle + THBP(40μM, the same behind) group, 2μM MDHB + TBHP group, 4μM MDHB + TBHP group, 8μM MDHB + TBHP group, 100μM NAC + TBHP group. The morphological changes of cells were observed under the microscope. Annexin V-FITC assay was used to detect cell apoptosis rate. 2’,7’-Dichlorofluorescin diacetate(DCFH-DA) assay was used to determine the intracellular ROS level. The activities of antioxidative enzymes(GSH-Px and SOD) were measured by kits. The oxidative DNA damage marker 8-OHd G was detected by ELISA. Western blot was used to determine the expression of Bcl-2, Bax, caspase 3, Nrf2, p-Akt and Akt proteins in treated SH-SY5 Y cells.Results: The cell viability of SH-SY5 Y cells significantly decreased after treated with 40μM TBHP for 24 hours. Pretreatment of SH-SY5 Y cells with MDHB(2, 4, 8μM) prior to TBHP exposure could significantly increase cell viability(P<0.01), decrease the rate of cell apoptosis(P<0.01), scavenge ROS accumulation(P<0.01), increase GSH-Px activity(P<0.01), SOD activity(P<0.05), reduce 8-OHd G in cell culture medium(P<0.01). Furthermore, MDHB could increase the Bcl-2/Bax level(P<0.01), inhibite the activation of caspase-3(P<0.01), increase the expression of Nrf2 protein(P<0.01) and increase the phosphorylation of Akt protein(P<0.05).Conclusion: MDHB could protect SH-SY5 Y cells from TBHP-induced oxidative damage possibly by inhibiting ROS accumulation, reducing DNA oxidative damage, activating PI3K/Akt-Nrf2 signaling pathway and modulating the expression of apoptosis-related proteins. |
