重组人突触融合蛋白4对脂多糖诱导的胰岛β细胞INS-1损伤影响和机制研究  

Effects of recombinant human syntaxin-4(STX4)on lipopolysaccharide-induced isletβ-cell INS-1 injury and underlying mechanism

作　　者：赵建林 陈玉凤 吴苏豫 李江雁 熊承云 张莹 耿秀琴 Zhao Jianlin;Chen Yufeng;Wu Suyu;Li Jiangyan;Xiong Chengyun;Zhang Ying;Geng Xiuqin(Department of Endocrinology,Xinxiang Central Hospital,The Fourth Clinical College of Xinxiang Medical College,Xinxiang 453000,China)

机构地区：[1]新乡市中心医院新乡医学院第四临床学院内分泌科,新乡453000

出　　处：《中华内分泌代谢杂志》2021年第8期738-744,共7页Chinese Journal of Endocrinology and Metabolism

摘　　要：目的:探讨重组人突触融合蛋白4(STX4)对脂多糖(LPS)诱导的胰岛β细胞INS-1损伤影响。方法:胰岛β细胞INS-1分成Control组(空白对照)、LPS组(LPS处理)、LPS+NC组(转染阴性对照载体,LPS处理)、LPS+STX4组(转染pcDNA-STX4,LPS处理),RT-qPCR和Western blot检测STX4 mRNA和蛋白质表达,流式细胞术检测凋亡,DCFHDA法检测活性氧簇(ROS)水平、黄嘌呤氧化法检测超氧化物歧化酶(SOD)水平、比色法检测谷胱甘肽过氧化物酶(GSH-Px)水平、钼酸铵比色法检测过氧化氢酶(CAT)水平、硫代巴比妥酸法检测丙二醛(MDA)水平,ELISA法检测细胞分泌的白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平和葡萄糖条件下胰岛素分泌水平,Western blot法检测Cleaved Caspase-3、Bax、p65蛋白质表达。用NF-κB信号通路激活剂处理上调STX4后的胰岛β细胞INS-1,给予LPS刺激,同样利用上述方法测定细胞凋亡、ROS、SOD、GSH-Px、CAT、MDA水平和分泌的IL-1β、TNF-α、胰岛素水平以及Cleaved Caspase-3、Bcl-2相关X蛋白(Bax)、p65蛋白质水平。结果:与Control组比,LPS组胰岛β细胞中STX4 mRNA和蛋白质表达均减少,细胞凋亡率、ROS水平、MDA水平均升高,SOD、GSH-Px、CAT水平均降低,细胞分泌的IL-1β、TNF-α水平均升高,细胞分泌的胰岛素水平降低,Cleaved Caspase-3、Bax、p65蛋白质表达水平也升高。NF-κB信号通路激活剂处理逆转上调STX4对LPS条件下胰岛β细胞凋亡、ROS、SOD、GSH-Px、CAT、MDA水平和分泌的IL-1β、TNF-α、胰岛素水平以及Cleaved Caspase-3、Bax、p65蛋白质水平影响。结论:上调STX4抑制LPS诱导的胰岛β细胞氧化损伤、细胞凋亡和炎症因子释放,机制与降低NF-κB信号通路激活水平有关。Objective To explore the effect of recombinant human syntaxin-4(STX4)on lipopolysaccharide(LPS)-induced injury in isletβ-cells(INS-1).Methods Pancreatic isletβ-cells(INS-1)were divided into Control(blank control),LPS(LPS treatment),LPS+NC(transfection of negative control vector,LPS treatment),and LPS+STX4(transfection of pcDNA-STX4,LPS treatment)groups.RT-qPCR and Western blot were used to detect STX4 mRNA and protein expression,flow cytometry to detect apoptosis,DCFHDA method to detect reactive oxygen species(ROS)level,xanthine oxidation method to detect superoxide orgotein dismutase(SOD)level,colorimetric method to detect glutathione peroxidase(GSH-Px)level,ammonium molybdate colorimetric method to detect catalase(CAT)level,thiobarbituric acid method to detect malonaldehyde(MDA)level,ELISA method to detect the level of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and insulin secretion levels under glucose conditions secreted by cells,Western blot method to detect Cleared Caspase-3,Bcl-2 Associated X Protein(Bax),p65 protein expression.After treatment with NF-κB signaling pathway activator,STX4 up-regulated isletβ-cell INS-1 was given LPS stimulation,and the same method was used to measure apoptosis,ROS,SOD,GSH-Px,CAT,MDA levels and secreted IL-1β,TNF-α,insulin levels and Cleaved Caspase-3,Bax,p65 protein levels.Results Compared with the Control group,the expression of STX4 mRNA and protein in isletβcells of the LPS group decreased,the apoptosis rate,ROS level,and MDA levels increased,and the levels of SOD,GSH-Px,and CAT decreased,the levels of IL-1β,TNF-αincreased,the level of insulin secreted by the cells decreased,and the expression levels of Cleaved Caspase-3,Bax,and p65 also increased.NF-κB pathway activator treatment reversed the effect of up-regulated STX4 on isletβ-cell apoptosis,ROS,SOD,GSH-Px,CAT,MDA levels and secreted IL-1β,TNF-αlevels,and Cleaved Caspase-3,Bax and p65 protein levels.Conclusion Up-regulation of STX4 alleviated LPS-induced isletβcell oxidative damage,apoptosi

关 键 词：突触融合蛋白4 炎症 胰岛Β细胞 脂多糖 细胞凋亡 

分 类 号：R587.1[医药卫生—内分泌]