Effect Of Resveratrol On Oxidative Stress Damage Of Follicular Granulosa Cells

Objective:To investigate the antagonistic effect of resveratrol on oxidative stress damage of human follicular granulosa cells induced by hydrogen peroxide.Methods:1. The granulosa cells were collected by density gradient centrifugation from the follicular fluid of IVF/ICSI patients in the fourth Hospital of Hebei Medical University and the cell primary culture was carried out.2.The survival rate of granulosa cells those treated with different concentrations of hydrogen peroxide（H₂O₂）was measured by Cell Counting Kit-8（CCK-8）method,and the survival curve was drawn.The oxidative damage model of granulosa cells was established by IC50 concentration.3.The cultured granulosa cells were divided into four groups:⑴Control group:granulosa cells without anything;⑵Injury model group:granulosa cells with IC50 concentration of H₂O₂;⑶10μmol/L resveratrol group:granulosa cells with 10μmol/L resveratrol and IC50 concentration of H₂O₂;⑷50μmol/L resveratrol group:granulosa cells with 50μmol/L resveratrol and IC50 concentration of H₂O₂.4.The cell activity of each group was determined by CCK-8 method.The content of malondialdehyde was determined by thiobarbituric acid method and the level of superoxide dismutase was determined by WST-1method.The secretion of progesterone was measured by enzyme-linked immunosorbent assay.The cell cycle was determined by flow cytometry and propidium iodide single staining,and the apoptosis rate was determined by Annexin V-FITC/PI double staining.The protein expressions of Bcl-2,Caspase-3 and SIRT1 were determined by Western Blotting method.Results:1.According to the cell survival curve,the IC50 value was 65.72μmol/L,and this concentration was used to establish the oxidative damage model of granulosa cells.2. Compared with the control group,the cell survival rate,SOD level and progesterone secretion in the injury model group were significantly decreased,and the cell cycle showed an increase in sub G₁ peaks,the number of the cells in the G₁/G₀ and G₂/M phases were significantly decreased,and the content of MDA,the rate of apoptosis,the expression of Caspase-3 were all significantly increased,while the expression of Bcl-2 was significantly decreased（P<0.05）.3. There were no significant differences in the cell survival rate,the level of SOD,MDA,and Pg,the apoptosis rate,the cell cycle,the expression of caspase-3,bcl-2,and SIRT1 protein between the injury model group and the10μmol/L resveratrol group（P>0.05）.4. Compared with the injury model group,the cell survival rate,The levels of SOD and progesterone,the sub G1 peak of the cell cycle,the number of the cells in G1/G0 and G2/M phase were significantly increased,and the MDA content,apoptosis rate and expression of protein Caspase-3 were significantly decreased,while the expression of protein SIRT1 and protein Bcl-2 were significantly increased（P<0.05）in the 50μmol/L resveratrol group.Compared with the control group,the cell survival rate,The levels of SOD and progesterone,the expression of protein Bcl-2 and the number of the cells in G₁/G₀ phase were significantly decreased,and MDA content,apoptotic rate,and expression of protein Caspase-3 was significantly increased（P<0.05）.But there were no significant differences in the sub G₁,the number of the cells in G₂/M phase between the two groups（P>0.05）.Conclusions:1.The culture conditions of primary luteinized-follicular granulosa cells are harsh,which need to increase the inoculation density and need to culture for a long time to meet the experimental needs.2.The oxidative stress model of follicular granulosa cells induced by H₂O₂ is stable and successful,which can be used in oxidative and/or antioxidant studies.3.As an agonist of SIRT1,resveratrol can improve the function of granulosa cells and enhance the antioxidant capacity and anti-apoptotic capacity in granulosa cells,which provides an experimental basis for further in vivo antioxidant research.