The Antagonistic Action Of 7-difluoromethylgenistein On Oxidative Stress Injury Induced By Hydrogen Peroxide In PC12 Cell Line

Objective: After the directed reconstitution of genistein in its chemical constitution, a series of genistein derivatives were obtained. we established the model of PC12 cells cultured in vitro and injured by oxidative stress, then sieved an active new chemical entity( 7-Difluoromethylylgenistein)from genistein derivatives which has more effective protection of PC12 cells injury by oxidative stress. The protective effect of active new chemical entity on PC12 cell injury by oxidative stress was studied, and its molecular mechanism of action was also investigated. The objective is to provide the experiment basis for developing a new medicine for nervous system disease therapy.Methods: PC12 cells incubated with H2O2 for 24h in vitro, the proliferation of PC12 cells was determined by 3-[4 ,5-dimethylthiazol-2-yl ]-2 ,5-diphenyltetrazolium bromide ( MTT ) assay , the apoptosis of PC12 cells was detected by flow cytometry ( FCM) with propidium iodide stain, the LDH activity of PC12 cells function by multifunction biochemistry analysator. Then we established the model of nerve cells injury by oxidative stress and sieved an active new chemical entity. Experiment groups were divided as follows: (1) blank control group; (2) H2O2 damage group; (3) lead compound group; (4) 7-Difluoromethylylgenistein group. Cell growth and multiplication activity were measured by MTT assay and cell counting method. Cell apoptosis was determined by acridine orange staining method and flow cytometry. The expression of phosphorylated ERKI/2 and P38 in PC12 cells was examined using Western Blot.Result: In the presence of 7-difluoromethylgenistein, both the inhibitory rates of PC12 cells and the LDH release of PC12 cells decreased., and the apoptosis of PC12 cells decreased as well after 40μmol/L H2O2 for 24 h. Cells were incubated with different concentration of genistein derivatives before being injured by H2O2 (40μmmo1/L ), and we found:(1) the protective effect of 7- Difluoromethylyl-5,4'-Di-hydroxyl isoflavone is higher than lead compound.(2) 7- Difluoromethylylgenistein is the most effective article among test articles.When H2O2 cells were treated with H2O2 (40μmmo1/L) for 24h, the release of LDH was increased; the cell growth and proliferation were suppressed; the apoptosis of nervous cells was increased; the expression level of phosphorylation ERK1/2 and P38 in PC12 cells was up-regulated. After nervous cells were pretreated with 7-Difluoromethylylgenistein, experiment results showed:(l) 7-Difluoromethylylgenistein can improve cell growth and proliferation in a concentration dependent manner, reduce the release of LDH,suppress the H2O2-induced apoptosis of PC12 cell.(2) 7- Difluoromethylylgenistein can up-regulate expression of phosphorylated ERK1/2 and suppress activation of P38 in PC12 cells H2O2-induced.Conclusion:1 .7- Difluoromethylylgenistein possesses the antagonistic action of PC12 cell injury by H2O2 in vitro.2. The antagonistic action of 7- Difluoromethylylgenistein on PC12 cell injury by H2O2 is possibly through up-regulatting the expression of phosphorylation ERK1/2 and suppressing the activation of P38.