Objective:To investigate the effect and the potential mechanism of Senegenin(Sen) against injury induced by hypoxia/reoxygenation(H/R) in highly differentiated PC12 cells.Methods:The cultured PC12 cells were treated with H/R in the presence or absence of Sen(60 μmol/L).Four groups were included in the experiment:control group,H/R group,H/R+Sen group and Sen group.Cell viability of each group and the level of lactate dehydrogenase(LDH) in culture medium were detected for the pharmacological effect of Sen.Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate.Moreover,mitochondrial membrane potential(△Ψm),reactive oxygen species(ROS) and intracellular free calcium([Ca~(2+)]i) were measured by fluorescent staining and flow cytometry.Cleaved caspase-3and activity of NADPH oxidase(NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay,respectively.Results:Sen significantly elevated cell viability(P<0.05),decreased the leakage of LDH(P<0.05) and apoptosis rate(P<0.05) in H/R-injured PC12 cells.Sen maintained the value of△Ψm(P<0.05) and suppressed the activity of caspase-3(P<0.05).Moreover,Sen reduced ROS accumulation(P<0.05) and[Ca~(2+)]i increment(P<0.05) by inhibiting the activity of NOX(P<0.05).Conclusion:Sen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and[Ca~(2+)]_i,thereby suppressing the mitochondrial pathway of cellular apoptosis. Objective: To investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia / reoxygenation (H / R) in highly differentiated PC12 cells. Methods: The cultured PC12 cells were treated with H / R in the presence or absence of Sen (60 μmol / L) .Four groups were included in the experiment: control group, H / R group, H / R + Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V / propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and intracellular free calcium ([Ca ~ (2 +)] i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 activity and NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively. Results: cell viability (P (P <0.05) and decreased the leakage of LDH (P <0.05) in H / R-injured PC12 cells.Sen maintained the value of △ Ψm (P <0.05) and suppressed the activity of caspase- 3 (P <0.05) .Moreover, Sen reduced ROS accumulation (P <0.05) and [Ca ~ (2 +)] i increment (P <0.05) by inhibiting the activity of NOX exert cytoprotection against H / R injury by decreasing the levels of intracellular ROS and [Ca ~ (2 +)] _i, thereby suppressing the mitochondrial pathway of cellular apoptosis.