Effects of Senegenin against Hypoxia/Reoxygenation-Induced Injury in PC12 Cells

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 (Ca²⁺]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and 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 Ca²⁺]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 Ca²⁺]_i, thereby suppressing the mitochondrial pathway of cellular apoptosis.