Resveratrol Inhibits Hypeixia-induced Cell Apoptosis Through Up-regulating SIRT1 Expression In HPAEpiC Cells

Objective: To explore the role of SIRT1 oxidative stress signal pathway in mediating apoptosis of hyperoxia-exposed HPAEpi C cells. To explore the role of SIRT1 agonist Res in protecting HPAEpi C cells apoptosis induced by hyperoxia.Method: This study is based on alveolar epithelial cell line HPAEpi C cells cultured in vitro as research object. First, the optimal concentration of Res was detected by MTT assay. And the HPAEpi C lines were randomly divided into 3groups: control group, hyperoxia group,Res group. Cells in control group were conventional cultured in DMEM medium with 10% serum and 1% streptomycin.The hyperoxia group was exposed to a mixture of O2(900ml/L) and CO2(50ml/L)for 10 ~ 15 minutes, then cultured in a closed environment with 5%CO2. Res group was treated with specific SIRT1 agonist Res of 100umol/L and was exposed to a mixture of O2(900ml/L) and CO2(50ml/L) for 10~15minutes, then cultured in a closed environment with 5%CO2. Cells were collected, and then the change of morphology were observed under invered phasecontrast microscope;the expression of caspase 9/XIAP/SIRT1 proteins was measured by sp IHC; the changes of reactive oxygen species and membrane potential in mitochondrion were detected by Laser scanning confocal microscope, the expression of SIRT1 protein was measured by western blot, the change of cell apoptosis was analyzed by flow cytometer.Result:(1) The optimal concentration of Res was 100umol/L.(2) In control group, cells had a well-growth state, a high-adherence rate, well-vigor, was flat irregular polygon. Circle nucleus was in the center of cell, was about 1/3~1/2.Cells had a large number, were arranged tightly, were connected with pieces and a low suspension cells. In hyperoxic group, cell had a bad-growth state, a low-adherence rate, bad-vigor. There were many ovai and round cells. The gap of cells increased and connection of cells loosed. The number of survival cells and suspension cells was decreased and increased obviously. In Res group, the state、adherence rate and the vigor of cells were not very well. The number of survival cells and suspension cells was increased and decreased, compared with hyperoxia group,s, but it was less than control group,s.(3) According to the result of IHC, compared with control group, there was statistical significance in the expression differences of caspase-9/XIAP/SIRT1 of HPAEpi C cells after cultured for 24 hs in hyperoxia group(P<0.05). And there was statistical significance in the expression differences of caspase-9/XIAP/SIRT1 of HPAEpi C cells after cultured for 24 hs in Res group(P<0.05), compared with hyperoxia group.(4)According to the result of Laser scanning confocal microscope, the expression of reactive oxygen in control group was lowest and highest in hyperoxic group, and in Res group the expression of reactive oxygen was between them. There was statistical significance in their expression differences(P<0.05).(5)The result of Laser scanning confocal microscopecleared that membrane potential of mitochondrion in hyperoxic group was decreased obviously, compared with control group. There was statistical significance in their expression differences(P<0.05). Membrane potential of mitochondrion in Res group was increased a bit, compared with hyperoxia group.There was statistical significance in their expression differences(P<0.05). But it was less than that in control group(P<0.05).(6)The result of western blot showed that the expression of SIRT1 in hyperoxia group was decreased obviously, compared with control group. There was statistical significance in their expression differences(P<0.05). The expression of SIRT1 in Res group was increased obviously, compared with control group. There was statistical significance in their expression differences(P<0.05).(7)The result of FCM indicated that the cell apoptosis rate in hyperoxia group was increased clearly compared with control group. There was statistical significance in their expression differences(P<0.05).And the cell apoptosis rate was reduced significantly in Res group compared with hyperoxia group. There was statistical significance in their expression differences(P<0.05). But it was less than that in control group(P<0.05).Conclusion: By up-regulating the expression of SIRT1 in alveolar epithelial cells and depressing the generating of ROS, Res inhibits the apoptosis of alveolar epithelial cells and maintains the cell membrane potential, thus effectively alleviating hyperxia-induced lung injury.