Protective Effects Of Resveratrol On The Injury Of Alveolar Epithelial Cells Induced By Hyperoxia Via Reducing Mitochondrial Dysfunction

Objective:In this study,we used Human pulmonary alveolar epithelial cells（HPAEpiC）to establish hyperoxia model,and the effects of resveratrol（Res or R）,SIRT1 agonists SRT1 720HCl（S）and SRT1 inhibitors Ex-527（E）on the changes of mitochondrial dysfunction and cell apoptosis induced by hyperoxia were observed,and to investigate whether resveratrol can exert its anti-apoptotic effect through SRT1/PGC-1αsignaling pathway.This study aimed to provide experimental basis for the application of resveratrol in the clinical treatment of hyperoxial pulmonary injury.Methods:1.Cell culture:Human pulmonary alveolar epithelial cells were maintained at 37℃in a 5%CO₂ atmosphere,and cultured in complete medium,which was Dulbecco’s modified Eagle’s medium（DMEM,High Glucose）supplemented with 10%fetal bovine serum and 100U/ml penicillin and100μg/ml streptomycin.2.Experimental grouping:According to the results of Cell Counting Kit-8（CCk-8）and some related articles,human pulmonary alveolar epithelial cells were divided into the following groups:（1）the control group（Control）;（2）the hyperoxia group（Hyperoxia）;（3）the hyperoxia+Res group（H+R20）;（4）the hyperoxia+Res+5μM EX-527 group（H+R20+E5）;（5）the hyperoxia+Res+10μM EX-527 group（H+R20+E10）;（6）the hyperoxia+SRT1720 group（H+S2）;（7）the hyperoxia+SRT1720+5μM EX-527 group（H+S2+E5）;（8）the hyperoxia+SRT1720+10μM EX-527 group（H+S2+E10）.The control group,in which cells were incubated with complete medium and maintained at 37℃in a humidified incubator containing 5%CO_2.The hyperoxia group,in which cells were incubated with the same medium,exposed to hyperoxia（inlet the mixture gas contain O₂（950ml/L）and CO₂（50ml/L）at a speed of 3L/min for 10min）,and then maintained in the same environment as the control group.The hyperoxia+Res group,in which 20μM Res was added to the complete medium,and 2h later cells were exposed to hyperoxia and then maintained in the same environment as the control group for 24h.The hyperoxia+Res+5μM EX-527 group,in which cells were pretreated 5μM EX-527 for 30min,and then 20μM Res was added to the complete medium,2h later,cells were exposed to hyperoxia,and then maintained in the same environmentfor 24h.The hyperoxia+Res+10μM EX-527 group,in which cells were pretreated 10μM EX-527 for 30min,and then 20μM Res was added to the complete medium,2h later,cells were exposed to hyperoxia,and then maintained in the same environment for 24h.The hyperoxia+SRT1720 group,in which 2μM SRT1720 was added to the complete medium,and cells were exposed to hyperoxiaand then maintained in the same environment for 24h.The hyperoxia+SRT1720+5μM EX-527 group,in which cells were pretreated 5μM EX-527 for 30min,and then 2μM SRT1720 was added to the complete medium,2h later,cells were exposed to hyperoxia,and then maintained in the same environment for 24h.The hyperoxia+SRT1720+10μM EX-527 group,in which cells were pretreated 10μM EX-527 for 30min,and then 2μM SRT1720 was added to the complete medium,2h later,cells were exposed to hyperoxia,and then maintained in the same environmentfor 24h.All cell cultures were maintained at 37℃in a humidified incubator containing 5%CO₂ for 24h and then harvested for further analysis.3.Measurement of intracellular total ROS:We used Reactive Oxygen Species Assay Kit to evaluate the toal ROS production in each group,according to the manufacturer’s instructions.4.Measurement of mitochondrial ROS:We used the Mito SOX?Kit to evaluate the mitochondrial reactive oxgen species production in each group.5.Measurement of mitochondrial membrane potential:JC-1 Kit was used to evaluate the Mitochondrial membrace potential in each group,according to the manufacturer’s instructions.6.Flow cytometry analysis of apoptosis:FITC Annexin V Apoptosis Detection Kit I was used to measure the apoptotic population of cells according to the manufacture’s protocols and apoptotic cells were quantized using flow cytometer.7.Western blot:The total cell protein of each group was extracted and the relative expression of SIRT1,Ac-p53,PGC-1α,NRF1 and TFAM were detected.Results:1.Res inhibited the growth of HPAEpiC cells in a dose-dependent manner.When the concentration of Res was greater than 20μM,it significantly inhibited the growth of HPAEpiC cells（P<0.001）.SRT1720inhibited the proliferation of HPAEpiC cells in a dose-dependent manner,and when the concentration of SRT1720 was greater than 1μM,it significantly inhibited the growth of HPAEpiC cells（P<0.05）.EX-527 inhibited the growth of HPAEpiC cells in a dose-dependent manner.When the concentration of EX-527 was greater than 2.5μM,the growth of HPAEpiC cells was significantly inhibited（P<0.05）.2.The results of total reactive oxygen species showed that the total reactive oxygen species significantly increased in the hyperoxia group comparing with the control group（P<0.001）.And the total reactive oxygen species in the hyperoxia+Res group decreased significantly（P<0.001）,when compared with the hyperoxia group.The total reactive oxygen species in the hyperoxia+Res+5μM EX-527 group and the hyperoxia+Res+10μM EX-527 group,which were pretreatment with EX-527,significantly increased comparing with the hyperoxia+Res group（P<0.001）.And the results of SRT1720 were similar to Res.3.The results of mitochondrial reactive oxygen species showed that the mitochondrial reactive oxygen species in the hyperoxia group increased significantly compared with that in the control group（P<0.001）.The mitochondrial reactive oxygen species in the hyperoxia+Res group decreased significantly when compared with that in the hyperoxia group（P<0.001）,while the mitochondrial reactive oxygen species in the hyperoxia+Res+5μM EX-527 group and the hyperoxia+Res+10μM EX-527 group increased significantly（P<0.001）,comparing with the hyperoxia+Res group.And the results of SRT1720 and Res were similar.4.The results of mitochondrial membrane potential showed that compared with the control group,the mitochondrial membrane potential of the hyperoxia group was significantly decreased（P<0.001）.And when compared with the hyperoxia group,the mitochondrial membrane potential of the hyperoxia+Res group was increased（P<0.001）.The mitochondrial membrane potential of the hyperoxia+Res+5μM EX-527 group and the hyperoxia+Res+10μM EX-527group were significantly reduced comparing with the hyperoxia+Res group（P<0.001）.And the results of SRT1720 were similar to Res.5.Flow cytometry analysis of apoptosis showed that the apoptosis rate of the hyperoxia group was significantly increased when compared with that of the control group（P<0.001）.The apoptosis rate of the hyperoxia+Res group was significantly decreased comparing with the hyperoxia group（P<0.001）.And when compared with the hyperoxia+Res group,the apoptosis rate of the hyperoxia+Res+5μM EX-527 group and the hyperoxia+Res+10μM EX-527group were significantly increased（P<0.001）.SRT1720 and Res group had similar results.6.The results of Western Blot showed that the expression of SIRT1,PGC-1α,NRF1,TFAM protein in the hyperoxia group significantly decreased when compared with the control group（P<0.05）,and the expression of Ac-p53 increased（P<0.05）.The expression of SIRT1,PGC-1α,NRF1 and TFAM protein in the hyperoxia+Res group increased（P<0.05）,and the expression of Ac-p53 decreased（P<0.05）,compared with that in the hyperoxia group.The expression of SIRT1,PGC-1α,NRF1 and TFAM in the hyperoxia+Res+5μM EX-527 group and the hyperoxia+Res+10μM EX-527group decreased（P<0.05）,and the expression of Ac-p53 increased（P<0.05）,comparing with the hyperoxia+Res group.The results of SRT1720 were similar to those of Res groups.Conclusion:This study showed that Res alleviate the injury of human pulmonary alveolar epithelial cells induced by hyperoxia via reducing apoptosis and mitochondrial dysfunction.Res may play a protective role in hyperoxia-induced injury of human pulmonary alveolar epithelial cells through SIRT1/PGC1αsignaling pathway.