Effect Of Senegenin On H₂O₂ Induced Damage In Hippocampal Neurons Of SD Rat

Objective:The research is to observe the protective effects of senegenin on oxidative stress induced by H2O2 in vivo, and disclose the related mechanisms for further developing senegenin in the treatment of dementia diseases such as AD.Methods:Part11. Hippocampal neurons were isolated from neonatal SD rats and cultured in the serum-free culture media. The purity of neurons was identified by immunofluorescence method, MTT assay for the determination of cell viability by H2O2. The cell viability of senegenin was determined by MTT analysis. The primarily cultured neurons were divided into control group, H2O2 group, Sen+H2O2 group and Sen group.2. The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were determined by using commercially available kits to evaluate the influence of antioxidative abilities of senegenin on hippocampal neurons.Part 21. The morphological changes of nucleus of the neurons were observed by Hoechst 33258 staining.2. In order to make clear the relative mechanisms of antiapoptosic effects of Sen, the mRNA expression of bcl-2 and bax was quantified by real time PCR.3. The protein level of bcl-2 and bax was measured by Western blot.4. The activation of caspase-3 in the hippocampal neurons was determined by caspase-Glo3 Assay kit. Results:Part11. The purity of hippocampal neurons reached 90%. We can use it for the following study. Preincubation with H2O2 can decrease the neurons survival rate to about 30% after 12 h.2. Compared with the control group, the activity of SOD was decreased and the contents of MDA were increased treated with H2O2. But,20μmol/L senegenin pretreatment attenuated the changes of SOD activities and reduced the contents of MDA(P<0.05).Part 21. After Hoechst 33258 staining, apoptotic cells increased which contained heterogeneous intensity, chromatin condensation in the H2O2 group, however, treated with senegenin, the morphology of apoptosic nuclei were improvement.2. Real time PCR showed that mRNA expression of bcl-2 increased and that of bax decreased (P<0.05) in Sen+H2O2 group.3. Compared with the control group, western blot results showed that the expression bcl-2 protein of H2O2 group was decreased, protein of bax was increased (P<0.05). Compared with the H2O2 group, the expression of bcl-2 protein was up-regulated and bax protein was down-regulated in 20μmol/L Sen+H2O2 group (P<0.05).4. Compared with the H2O2 group, the activity of caspase-3 reduced after Sen cultivating (P<0.05).Conclusions:The protective effect of senegenin on hippocampal neurons under the condition of H2O2 injury may be involed in the mechanisms of neuronal antioxidative response, the regulation of bcl-2 family and suppression of caspase-3 activity by this Chinese medicine. The results provide the experimental basis for the treatment of AD.