Protective Mechanism Of Liuwei Dihuang Pill On Oxidative Damage Induced By H ₂ 2 In PC12 Cells

Purpose: To made the mode of oxidative damage pheochromocytoma cell lines(PC12 cells) induced by H2O2, while to observe the effects of sera contaning Liuwei Dihuang Pill on proliferation and apoptosis, total antioxidant capacity, the m RNA and the protein expression of SOD1, SOD2, by the methods of serum pharmacology and molecular ect. The aim of this experiment was to further explore the protective mechanism of Liuwei Dihuang Pill against H2O2-induced oxidative damage on the molecular level and provided experimental evidence of liuwei Dihuang Pill as the prevention of Alzheimer,s disease(AD).Material and method: PC12 cells were cultured 24 h in serm-free DMEM. After adherented, cells were randomly divided into four groups: control group, mode group, Liuwei Dihuang Pill group and VE group. Except Liuwei Dihuang Pill group, other groups were replaced by the 10% rats serum, Liuwei Dihuang Pill group was replaced by the 10% concentration of serume contaning Liuwei Dihuang Pill as well as VE group treated by 20μg/ml VE. After 24 h, except the control group, other groups were added with 200μmol/L H2O2; the cell morphology in each group was observed under inverted microscope; the cell proliferation rate was measured by MTT; the apoptotic rate was detected by flow cytometry; the expression of total antioxidant capacity was checked by ABTS; the m RNA and protein expression of SOD1, SOD2 were detected by RT-PCR and Western blot.Results: 1.The effects of Liuwei Dihuang Pill on H2O2-induced PC12 cells morphology Compared with the control group, the number of cells in the model group was significantly reduced and the cell density was decreased, the cell was rounded shrinkage or float off, the cell gap significantly increased and showed obviousdamage cell morphology; compared with the model group, damage morphology of Liu Wei Huang Pill group and VE group was returned to normal, damaged cells were reduced, cell density was significantly increased; There was no difference between the Liuwei Dihuang Pill group and the VE group in microscopic morphological. 2.The effects of Liuwei Dihuang Pill on H2O2-induced PC12 cell proliferation rate Compared with the control group, the cell proliferation rate was significantly lower(P<0.05), there was statistically significant; compared with the model group, the cell proliferation rate was significantly higher, respectively(P <0.05); There was no difference between the Liuwei Dihuang Pill group and the VE group(P>0.05). 3.The effects of Liuwei Dihuang Pill on H2O2-induced PC12 cells apoptosis rate Flow cytometry results showed that the apoptosis rate significantly decreased in model group, Compared with the control group, there was statistical significance(P<0.05); compared with the model group, Liuwei Dihuang Pill group and VE group cell apoptosis was significantly lower, respectively(P<0.05); There was no difference between the Liuwei Dihuang Pill group and the VE group(P>0.05). 4.The effects of Liuwei Dihuang Pill on H2O2-induced PC12 cells total antioxidant capacity(ABTS method) Total antioxidant capacity results showed that, the total antioxidant capacity was significantly lower in model group(P<0.05); compared with the model group, the total antioxidant capacity of Liu Wei Huang Pill group and VE group was significantly higher, respectively(P<0.05). There was no difference between the Liu Wei Huang Pill group and the VE group(P>0.05). 5.The effects of Liuwei Dihuang Pill on H2O2-induced PC12 cells m RNA expression RT-PCR results showed that the m RNA expression of SOD1, SOD2 were significantly reduced in model group(P<0.05); the m RNA expression were significantly increased in Liu Wei Huang Pill group and VE group, respectively(P<0.05); there was no significantly difference in Liu Wei Huang Pill group and VE group(P>0.05).6.The effects of Liuwei Dihuang Pill on H2O2-induced PC12 cell SOD1、 SOD2 protein expression Western blot results showed that the protein expression of SOD1, SOD2 were significantly reduced in model group(P<0.05); the protein expression were significantly increased in Liu Wei Huang Pill group and VE group, respectively(P<0.05); there was no significantly difference in Liu Wei Huang Pill group and VE group(P>0.05).Conclusion: 1.Liuwei Dihuang Pill could promote cell proliferation, reduce cell apoptosis. 2.Liuwei Dihuang Pill could enhence total antioxidant capacity, increase the expression of SOD1, SOD2, play the effect of anxidention.