Resveratrol Protects RLE-6TN Cells From Apoptosis Induced By Endoplasmic Reticulum Stress Via Sirt1-Derlin1 Mechanism

Objective: To investigate whether Sirt1 agonist resveratrol can inhibit CSE induced apoptosis in RLE-6TN cells by up regulating the expression of derlin1.Methods: RLE-6TN cell lines were cultured in vitro and treated with 10%CSE,At the same time with resveratrol intervention.Treatment of CSE induced RLE-6TN cells with different concentrations of resveratrol,Effect of different concentrations of resveratrol on CSE induced RLE-6TN cell viability by CCK8 assay,To explore the optimal concentration of resveratrol to protect RLE-6TN cells.The RLE-6TN cells were induced by 10%CSE at different time points in group CSE(0h,3h,6h,12 h,24h,48h),At the same time,resveratrol treatment at each time point was resveratrol intervention group(0h,3h,6h,12 h,24h,48h),Flow cytometry was used to detect the apoptosis rate of each group,Western blot was used to detect the expression of Sirt1,Derlin1,CHOP,caspase12,JNK and p-JNK in each group of cells,Realtime-PCR was used to detect the expression of Sirt1 m RNA and Derlin1 m RNA in each group of cells.Select the peak point of Sirt1 expression,Treatment of CSE induced RLE-6TN cells with different concentrations of resveratrol,Detection of Sirt1 and Derlin1 protein expression by Western blot.Results: 1.cell viability assay:After RLE-6TN cells were induced by 10%CSE,With the increasing of resveratrol concentration,Cell viability also showed an upward trend,When the concentration was 40 mol/L,the cell viability reached the platform.2.detection of apoptosis rate:In the CSE group and resveratrol intervention group,The apoptosis rate increased gradually with time,There was significant difference between the each groups at each two time point(P<0.05),Except for 0h,Resveratrol intervention group with the low rate of apoptosis compared with the CSE group at the same time point,with statistically significant(P<0.05).3.activation of endoplasmic reticulum stress-induced apoptosis pathway:(1)CHOP protein expression:In the CSE group,the expression of CHOPprotein increased gradually with time,Except between 3h and 6h,the difference was statistically significant at every two time points(P<0.05);The resveratrol intervention group,the expression of CHOP protein also increased gradually with time,and 3h/6h/12h/ 24h/ 48 h compared with 0h,12h/24h/ 48 h compared with 3h,12h/24h/ 48 h compared with 6h,48 h compared with 12 h,with statistically significant(P<0.05).However,compared with the CSE group at the same time point(except 0h),The expression of CHOP protein was lower in the resveratrol intervention group,and the difference had statistically significant(P<0.05).(2)Caspase12 protein expression:In the CSE group,The expression of Caspase12 protein increased at 3h,and increased slowly after 12 h,and3h/6h/ 12h/ 24h/ 48 h compared with 0h,12h/ 24h/ 48 h compared with 3h,12h/ 24h/48 h compared with 6h,48 h compared with 12 h,with statistically significant(P<0.05);The expression of Caspase12 protein also increased in the resveratrol intervention group,Except between 12 h and 24 h,the difference had statistically significant at every two time points(P<0.05);However,compared with the CSE group at the same time point(except for 0h),the expression of Caspase12 protein was lower,and in 3h/12h/24 h had statistically significant(P<0.05).(3)total JNK and p-JNK protein expression:The expression of total JNK protein in the CSE group and resveratrol intervention group did not change significantly with time,but,p-JNK protein in the CSE group increased with time,Except between 24 h and 48 h,the difference had statistically significant at every two time points(P<0.05);The resveratrol intervention group also increased with time,Except between 12 h and 24 h,the difference also had statistically significant at every two time points(P<0.05);However,compared with the CSE group at the same time point(except for 0h),the expression of p-JNK protein was lower,and in 12 h,24h and 48 h had statistically significant(P<0.05).4.Expression of sirt1: western blot results revealed:In the CSE group,the expression of sirt1 protein decreased with time,Except between 24 h and 48 h,the difference had statistically significant at every two time points(P<0.05);The expression of sirt1 protein increased in the resveratrol group,and reach peak about 12 h,then slightly lower,but still higher than 0h,Except between 12 h and 24 h,the difference had statistically significant at every two time points(P<0.05);Except for 0h,there was significant difference between the resveratrol intervention group and CSE group at the same time points(P < 0.05).Realtime-PCR detection showed that the results of Sirt1 m RNA were consistent with the results of Western blot.5.Expression of derlin1:western blot results revealed:In the CSE group,the expression of derlin1 protein increased gradually with time,the difference was statistically significant at every two time points(P<0.05);Resveratrol intervention group also increased with time,and reach peak about 24 h,Except between 24 h and 48 h,the difference had statistically significant at every two time points(P<0.05);But resveratrolintervention group than CSE group increased significantly,except for 0h,the difference had statistically significant at the same time point compared with CSE group(P < 0.05).The expression trend of Derlin1 m RNA was consistent with the results of Western blot.6.After treatment of CSE induced RLE-6TN cells with different concentrations of resveratrol,The results of Western blot showed that the expression of Sirt1 and Derlin1 protein was positively correlated with the concentration of resveratrol.Conclusion:Sirt1 agonist resveratrol can protect against CSE induced apoptosis of RLE-6TN cells,The mechanism may be mediated by up regulation of Derlin1 expression.