沉默信息调节因子1在脂多糖诱导胰岛素瘤细胞-1线粒体损伤中的作用  

Role of silent mating type information regulation 2 homolog 1 on lipopolysacchride-induced mitochondrial injury in insulinoma cell lines-1

作　　者：莫兴兴 王晓[1] 葛勤敏[1] 边帆[2] Mo Xingxing, Wang Xiao, Ge Qinmin, Bian Fan(Department of Emergency, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine; Department of Nephrology, Shanghai 200092, China)

机构地区：[1]上海交通大学医学院附属新华医院急诊科,上海200092 [2]上海交通大学医学院附属新华医院肾内科,上海200092

出　　处：《中华急诊医学杂志》2018年第11期1224-1231,共8页Chinese Journal of Emergency Medicine

基　　金：上海市自然科学基金（14ZR1427000）;上海市卫生和计划生育委员会科研课题（201740081）;国家临床重点专科

摘　　要：目的 探讨沉默信息调节因子（silent mating type information regulation 2 homolog1，SIRT1）在脂多糖（lipopolysaccharide，LPS）诱导胰岛素瘤细胞-1（insulinoma cell lines-1, INS-1）细胞线粒体损伤中的作用。方法 将对数生长期的INS-1 细胞分为5 组：正常对照组、1 mg/L LPS组（LPS 干预细胞24 h）、白藜芦醇（resveratrol，RSV）＋LPS 组（10 μmol/L RSV 预处理1 h 后，加入1 mg/L LPS 干预24 h）、EX527＋LPS 组（20 μmol/L EX527 预处理1 h 后，加入1 mg/L LPS干预24 h）、EX527＋RSV＋LPS 组（20 μmol/L EX527 预处理1 h 后，加入10 μmol/L 的RSV 和1mg/L LPS 干预24 h）。检测细胞活力和ATP 生成；提取INS-1 细胞总蛋白、胞质蛋白和线粒体蛋白，Western-blot 法检测SIRT1、TLR4、乙酰化FoxO1、细胞色素C（cytochrome C，CytC）、线粒体融合蛋白1（mitofusion1，Mfn1）、线粒体融合蛋白2（mitofusion2，Mfn2）、线粒体分离蛋白1（fission1，Fis1）蛋白的表达； Real-time PCR 方法检测SIRT1、FoxO1、Mfn1、Mfn2、Fis1 基因水平的变化。多组间比较采用单因素方差分析，用LSD-t 法进行组间两两比较。结果 1 mg/L LPS 作用于INS-1 细胞24 h 后，细胞活力降低（n=4，F=13.98，P〈0.01），ATP 生成减少（n=4，F=27.92，P〈0.05），RSV 预处理后促进ATP 生成（P〈0.01），但不能逆转EX527 预处理后ATP 生成的降低（P〉0.05）。RSV 可以促进SIRT1、Mfn1、Mfn2 蛋白表达和基因上调，降低TLR4 和Fis1 蛋白表达，下调其基因水平，减少CytC 在细胞质中的释放（P〈0.01），而各组间FoxO1 蛋白表达差异无统计学意义（P〉0.05）。结论 白藜芦醇可能通过改变SIRT1 的活性，调控FoxO1 的乙酰化，部分参与调节LPS 诱导的INS-1 细胞线粒体的损伤。Objective To investigate the role of silent mating type information regulation 2 homolog l（SIRT-1） in lipopolysaccharide （LPS）-induced mitochondrial injury on INS-1 （insulinoma cell lines-1 ） cells. Methods INS-1 cells were divided into 5 groups： control group, 1 mg/L LPS group, LPS group with 10 μmol/L RSV （resveratrol） pretreatment for 1 h, LPS group with 20 gmol/L EX527 pretreatment for 1 h, LPS group together with EX527 pretreatment for 1 h, then incubated these INS- 1 cells with RSV and LPS for 24 h. The cell viability and ATP generation were detected, then total, cytoplasmic and mitochondrial protein were isolated from INS-1 cells. The protein expression of SIRT1, TLR4, acetylated FoxO1, and cytochrome C （CytC）, Mfnl （mitofusionl）, Mfn2 （mitofusion2）, and Fisl （fissionl） were tested by Western-blot. Mfnl, Mfn2, and Fisl genes expression were detected by real- time PCR. The comparison of multiple groups was performed by one-way ANOVA. LSD-t method was used to compare between every two groups. Results After 1 mg/L LPS stimulation for 24 h, there was decreased cell viability （n=4, F=13.98, P〈0.01） and ATP generation （n=4, F=27.92, P〈0.05） in INS- 1 cells. RSV pretreatment could maintain ATP production, but it could not reverse the EX527-induced ATP decrease （P〉0.05）. Furthermore, RSV upregulated gene and protein expression of SIRT1, Mfnl and Mfn2, whereas decreased TLR4 and Fisl expression. LPS-induced CytC released to cytoplasm was alleviated by RSV （P〈0.01）, but there was no significant change about FoxO1 protein expression （P〉0.05）. Conclusions RSV may regulate FoxO1 aeetylation followed by its effects on SIRT1 activity, which may be partly the mechanism ofmitochondrial damage induced by LPS on INS-1 cells.

关 键 词：沉默信息调节因子 乙酰化叉头蛋白1 TOLL样受体4 氧化应激 线粒体融合与分离 胰岛素瘤细胞-1 

分 类 号：R459.7[医药卫生—急诊医学]