Role Of Senegenin In Hypoxia/Reoxygenation-induced Injury In PC12 Cells

Objective:To study the effect of Senegenin(Sen) on the hypoxia/reoxygenation(H/R) model, and to investigate the protective mechanism of Sen in H/R injury and provide the experimental basis for the treatment of ischemia/ reperfusion injury.Methods:Our experiments include three parts:Part 1:Observe the effect of Sen on viability in highly differentiated PC12 cells by MTT assay.Part 2:Observe the effect of Sen on the H/R model:There were several experimental groups:control group; H/R group; H/R+Sen(15,30,60μmol/L) group; Sen(60μmol/L) group. The viability of PC 12 cells was observed by MTT assay. The level of lactate dehydrogenase (LDH) in the culture solution was determined. The apoptosis of PC 12 cells was analysed by Hoechst 33258 staining and AnnexinV/PI double staining.Part 3:Study the protective mechanism of Sen in H/R model:analyzed the activity of NADPH oxidase(NOX) by ELISA; detected the fluorescence intensity of reactive oxygen species(ROS) and Ca2+ by flow cytometry; the mitochondrial membrane potential(ATm) of PC 12 cells was measured by JC-1 as a fluorescent molecular probe; observed the activity of Caspase 3.Results:1. MTT assay showed that Sen at concentrations of 15,30,60μmol/L did not affect the cell viability after 6 h incubation(P>0.05), while Sen at concentration of 80μmol/L reduced cell viability remarkably(P<0.05).2. Compared with control group, viability of PC 12 cells were reduced remarkably (P<0.05), while the release of LDH and apoptosis rate were increased significantly (P<0.05) in H/R group. Following treatments of different concentrations of Sen, compared with H/R group, viability of PC12 cells in H/R+Sen(30μmol/L) group and H/R+Sen(60μmol/L) group were increased remarkably(P<0.05), the release of LDH and apoptosis rate in H/R+Sen(15μmol/L) group,H/R+Sen(30μmol/L) group and H/R+Sen(60μmol/L) group were reduced significantly(P<0.05), and Sen takes effect in a concentration-dependent manner. Hoechst 33258 staining showed that the nuclei of normal cells appeared the same size and regular conformation, while in H/R group some cells showed pyknosis and karyorrhexis and this phenomenon changed after treatments of different concentrations of Sen.3. Compared with control group, the level of ROS,the concentration of Ca2+,activity of Caspase 3 and NOX were increased significantly (P<0.05),ΔΨm of PC12 cells were reduced remarkably(P<0.05) in H/R group. Compared with H/R group, the level of ROS,the concentration of Ca2+ and activity of Caspase 3 were reduced significantly(P<0.05) after treatments of different concentrations of Sen, and Sen takes effect in a concentration-dependent manner, the activity of NOX in H/R+Sen(60μmol/L) group was reduced significantly(P<0.05), theΔΨm in H/R+Sen(30μmol/L) group and H/R+Sen(60μmol/L) group were increased significantly(P<0.05).Conclusion:1. Sen can protect PC12 cells against H/R injury, promote the cell viability, reduce the release of LDH and apoptosis rate.2. Sen has protective role against H/R-induced cell apoptosis. The mechanism may be related to inhibiting the activation of NOX, reducing the level of ROS, reducing the concentration of intracellular Ca2+, relieving the degree of Ca2+ overloading, and maintainingΔΨm, and then inhibiting the activation of Caspase 3.