The Effects Of SIRT1 On LPS Induced INS-1 Cells Oxidative Stress Dysfunction

Objective To investigate the effects of silent mating type information regulation 2homolog 1 in lipopolysaccharide(LPS)-induced oxidative stress injury in INS-1 cells.Methods Rat insulinoma cell lines-1(INS-1)was obtained from department of Endocrinology,Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine,and the institute of endocrinology of Ruijin Hospital.INS-1 cells were divided into 5 groups at logarithmic growth phase: CON group(normal control group),LPS group(1 mg/L LPS-treated cells for 24 h),RSV + LPS group(after 10 μmol/L RSV pretreatment for 1 h,cultured INS-1 cells with 1 mg/L LPS for 24 h),EX527+LPS group(after 20 μmol/L EX527 pretreatment for 1 h,cultured INS-1 cells with 1 mg/L LPS for 24 h),EX527+RSV+LPS group(after 20 μmol/L EX527 pretreatment for 1 h,cultured INS-1 cells with 10 μmol/L RSV and 1 mg/L LPS for24 h).We detected cell viability by Cell Counting Kit-8 and measured the content of SOD activity,MDA and ATP production,superoxide level.Detected mitochondrial membrane potential and apoptosis rate by loading with JC-1 and AnnexinⅤrespectively in each group.Mitochondrial structural damages were observed under transmission electron microscope.Then total,cytoplasmic and mitochondrial proteins were isolated from INS-1 cells.Detected the protein expression of SIRT1,TLR4,acetylated-FoxO1,cytochrome C(CytC),Mfn1(Mitofusion1),Mfn2(Mitofusion2),Fis1(Fission1)by western blot and gene level of SIRT1,FoxO1,Mfn1,Mfn2,Fis1 by real-time PCR.Results 1.Incubated with 1 mg/L LPS for 24 h,LPS decreased cell viability(P<0.01),ATP production(P <0.05),SOD activity(P <0.05),increased MDA content(P <0.01)and superoxide generation(P <0.05),but RSV pretreatment reversed LPS-induced decreased cell viability and SOD activity,alleviated MDA content and promoted ATP generation.2.Compared with LPS group,EX527 did not significantly reduce the production of ATP(P >0.05).RSV could increase ATP levels,but it did not reverse the effect of EX527(P >0.05).RSV could increase gene and protein expression of SIRT1,Mfn1 and Mfn2,decrease gene and protein expression of Fis1(P <0.01),decrease protein expression of TLR4 and CytC released in cytoplasm(P<0.01),but there was no significant changes about FoxO1(P >0.05),however,the gene level was decreased(P <0.01).3.LPS decreased mitochondrial membrane potential may take part in leading to apoptosis.Mitochondria became swollen and ridges became obscure.RSV pretreatment could alleviate LPS induced cells dysfunction.Conclusion 1 mg/L LPS could induce oxidative stress in INS-1 cells.RSV may regulate the acetylation of FoxO1,a transcription factor downstream transcription factor by activiting SIRT1,and partially participate in the regulation of LPS-induced oxidative stress and mitochondrial structural and functional damages in INS-1 cells.