The Protective Effect Of Astaxanthin On The Oxidative Damage Of COV434 Human Ovarian Granulosa Cells Induced By H₂O₂

Part Ⅰ Hydrogen peroxide stimulates human ovarian granulosa cells COV434 to establish an in vitro oxidative stress modelObjective To establish an oxidative stress model and explore its conditions by using hydrogen peroxide（H₂O₂）to stimulate human ovarian granulosa cells（COV434）in vitro.Methods COV434 cells were treated with 0.2mm/L,0.4mm/L,0.5mm/L,0.6mm/L,0.8mm/L H₂O₂ concentration gradients for 0.5h,1h,2h,4h,6h.CCK-8was used for detection Method to determine cell proliferation.Using reactive oxygen species（ROS）kit to detect the content of intracellular ROS.And at the same time,using relative kit to detect the content of lactate dehydrogenase（LDH）,estradiol and progesterone in the cell culture medium.And detecting the level of caspase-3 by using the immune group chemical method.Making sure the best conditions for modeling.Results As the concentration of H₂O₂ increased and the action time increased,the cell proliferation rate showed a downward trend.After treatment with 0.4-0.6mmol/L H₂O₂ for 2h,the cell proliferation rate decreased significantly,the intracellular reactive oxygen species（ROS）content increased most significantly,and caspase-3 increased most significantly.The content of estradiol and progesterone in the cell culture medium showed a downward trend as the concentration of H₂O₂ increased and the action time increased,and the content of LDH showed an upward trend.Conclusion H₂O₂ can induce oxidative damage of human ovarian granulosa cells to establish an oxidative stress model.The most suitable concentration is 0.4-0.6 mmol/L,and the action time is 2 hours.Part 2 The protective effect of astaxanthin on H₂O₂ induced oxidative damage of COV434 human ovarian granulosa cellsObjective To evaluate the protective effects and mechanisms of Astaxanthin（AST）on oxidative injury of human ovarian granulosa cells（COV434）induced by H₂O₂.Methods The model of oxidative injury was established by supplement with H₂O₂（0.5mmol/L）.COV434 cells were divided into control group,model,group（H₂O₂ 0.5mmol/L）,the low,medium,and high dose group of AST（AST2、4、8umol/L+H₂O₂ 0.5mmol/L）.Cell proliferation activity was evaluated by CCK8 assay.Intracellular reactive oxygen species（ROS）production in the cell was measured by DCFH-DA.The activity of lactic dehydrogenase（LDH）in cell culture medium were determined by microenzyme labeling.The apoptosis and cell cycle of COV434 were detected using Annexin V-FITC/7-DDA.The expression of m TOR,PI3 K and AKT related genes in the PI3K/AKT signaling pathway were detected using q RT-PCR analysis.Results Compared with the control group,the cell proliferation activity of the model group decreased,the level of intracellular ROS and the release of LDH in the cell supernatant increased,and the percentage of S phase,early apoptosis,late apoptosis and total apoptosis were significantly increased（P<0.01）.The m RNA expression of PI3 K,AKT,and m TOR related genes decreased（P<0.05）.Compared with the model group,the levels of intracellular ROS,LDH release in cell supernatant and the percentage of S phase in the AST low,medium and high dose groups decreased,while the percentage of G2/M phase increased（P<0.05）,The expression of PI3 K,AKT,m TOR related gene m RNA was significantly increased.And as the concentration of AST increases,the effect increases.Conclusion Astaxanthin has a significant protective effect on oxidative injury of human ovarian granulosa cells（COV434）induced by H₂O₂ within a certain dose range.The protective effect may be related to the elimination of intracellular ROS,reducing DNA oxidative damage,and regulating PI3K-AKT signaling pathway and mitochondrial pathway mediated apoptosis.