葡萄糖调节蛋白75对棕榈酸诱导的MIN6细胞脂毒性的影响及机制研究  

Effect and mechanism of glucose regulatory protein 75 on palmitic acid-induced lipotoxicity in MIN6 cells

作　　者：宋林阳 代喆[1] 冯杰媛 薛君力[2] 王华蔚 童贝儿 张政伟 柯孟婷 何光珍 徐焱成[1] Song Linyang;Dai Zhe;Feng Jieyuan;Xue Junli;Wang Huawei;Tong Beier;Zhang Zhengwei;Ke Mengting;He Guangzhen;Xu Yancheng(Department of Endocrinology,Zhongnan Hospital,Wuhan University,Wuhan 430071,China;Department of Endocrinology,Jingzhou Central Hospital,Jingzhou 434020,China)

机构地区：[1]武汉大学中南医院内分泌科,武汉430071 [2]荆州市中心医院内分泌科,荆州434020

出　　处：《中华糖尿病杂志》2023年第6期547-556,共10页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(81970718)。

摘　　要：目的探讨葡萄糖调节蛋白75(Mortalin)对棕榈酸诱导的MIN6细胞脂毒性的影响和机制。方法无特定病原体(SPF)级雄性C57BL/6J小鼠,20只,6~8周,20~24 g。采用随机数字表法将小鼠分为标准饮食组(SD)和高脂饮食组(HFD),每组10只。使用免疫荧光染色法、免疫组织化学法检测小鼠胰腺组织中Mortalin的表达情况。通过慢病毒转染构建Mortalin敲低(Sh-Mortalin)的MIN6稳转细胞株,根据使用牛血清白蛋白(BSA)还是棕榈酸(PA)处理敲低和空载细胞将其分为Sh-Mortalin组、Mortalin敲低空载组(ShNC-Mortalin组)、Sh-Mortalin+PA组、ShNC-Mortalin+PA组,按应用细胞外调节蛋白激酶(ERK)抑制剂U0126还是二甲基亚砜(DMSO)将细胞分为Sh-Mortalin+DMSO组、Sh-Mortalin+PA+DMSO组、Sh-Mortalin+U0126组、Sh-Mortalin+PA+U0126组。使用5-乙炔基-2′-脱氧尿苷(EdU)法检测细胞增殖率,流式细胞仪检测细胞凋亡率和活性氧(ROS)水平,酶联免疫吸附测定法检测胰岛素水平,Western blotting检测ERK通路相关蛋白[磷酸化细胞外调节蛋白激酶1/2(p-ERK1/2)、磷酸化原癌基因丝苏氨酸蛋白激酶-1(p-Raf-1)]的表达。2组间比较采用t检验,多组间比较采用单因素方差分析。结果体内实验结果表明,与SD组相比,HFD组小鼠胰腺的Mortalin表达明显增加(P<0.01)。体外实验结果表明,与ShNC-Mortalin+PA组相比,Sh-Mortalin+PA组的细胞增殖水平更高,细胞凋亡率更低,细胞ROS水平更低,胰岛素水平更高(P<0.05)。Western blotting结果显示,ShNC-Mortalin和Sh-Mortalin组p-ERK1/2、p-Raf-1蛋白表达均以PA时间梯度降低,且与ShNC-Mortalin组相比,Sh-Mortalin组的p-ERK1/2蛋白水平更高(P<0.05)。与Sh-Mortalin+DMSO组相比,Sh-Mortalin+U0126组EdU阳性率和p-ERK1/2水平均更低,且细胞脂毒性损伤加重(P<0.05)。结论Mortalin参与调控了MIN6细胞的脂毒性损伤过程,且敲低Mortalin可通过激活Raf-1/ERK信号通路减轻PA诱导的MIN6细胞脂毒性损伤。Objective To investigate the effect and mechanism of glucose-regulated protein 75(Mortalin)on palmitic acid-induced lipotoxicity in MIN6 cells.Methods Twenty 6-8 weeks male C57BL/6J(20-24 g)with specific pathogen free(SPF)mice were randomly divided into standard diet(SD)and high-fat diet(HFD)groups,with 10 mice in each group.Mortalin expression levels in pancreatic tissues were detected by immunofluorescence and immunohistochemistry.Mortalin knockdown stable cell lines were constructed by lentivirus transfection using MIN6 cells,and cells were divided into Mortalin short hairpin(Sh-Mortalin)group,Mortalin knockdown and null group(ShNC-Mortalin group),Sh-Mortalin+PA group,and ShNC-Mortalin+PA group according to whether they were treated with bovine serum albumin(BSA)or palmitic acid(PA).Moreover,cells were divided into Sh-Mortalin+dimethyl sulfoxide(DMSO)group,Sh-Mortalin+PA+DMSO group,Sh-Mortalin+U0126 group,and Sh-Mortalin+PA+U0126 group according to whether the extracellular regulated protein kinase(ERK)inhibitor U0126 or DMSO was applied.Cell proliferation rate was then measured using 5-ethynyl-2′-deoxyuridine(EdU).Apoptosis rate and reactive oxygen species(ROS)levels were measured by flow cytometry,and insulin levels were measured by enzyme-linked immunosorbent assay.Expression levels of ERK pathway-related proteins[phosphorylated extracellular regulatory protein kinase 1/2(p-ERK1/2),phosphorylated v-raf-leukemia viral oncogene 1(p-Raf-1)]were detected by Western blotting.The t-test was used for comparisons between two groups,and one-way analysis of variance(ANOVA)was used for comparisons between multiple groups.Results The results of in vivo experiments showed that Mortalin expression was significantly increased in the pancreas of mice in the HFD group compared with the SD group(P<0.01).The results of in vitro experiments showed that compared with the ShNC-Mortalin+PA group,the Sh-Mortalin+PA group showed significantly higher proliferation levels,lower apoptosis rates,lower ROS levels and higher insulin le

关 键 词：葡萄糖 葡萄糖调节蛋白75 胰岛Β细胞 脂毒性 细胞外调节蛋白激酶1/2 

分 类 号：R363[医药卫生—病理学]