7-酮基胆固醇通过激活内质网应激通路诱导胰岛β细胞凋亡的研究  

Study of 7-ketocholesterol induced pancreaticβ-cell apoptosis via endoplasmic reticulum stress pathway in vitro

作　　者：李歌 张文静 吴加华[1] 孙水雅 尹雪瑶[1] 周嘉强[1] Li Ge;Zhang Wenjing;Wu Jiahua;Sun Shuiya;Yin Xueyao;Zhou Jiaqiang(Department of Endocrinology and Metabolism,Sir Run Run Shaw Hospital,Zhejiang University School of Medicine,Hangzhou 310016,China)

机构地区：[1]浙江大学医学院附属邵逸夫医院内分泌代谢科,杭州310016

出　　处：《中华糖尿病杂志》2023年第4期329-335,共7页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(81870562,82270857);浙江省自然科学基金(LZ22H070002)。

摘　　要：目的探讨7-酮基胆固醇(7-KC)对胰岛β细胞凋亡的影响及其可能分子机制。方法用0、0.25、2.5和25μmol/L浓度的7-KC孵育INS-1细胞24 h,应用Western blotting法筛选出引起INS-1细胞凋亡的浓度。再将INS-1细胞分为对照组、25μmol/L 7-KC组、25μmol/L 7-KC+1 mmol/L 4-苯基丁酸(4-PBA)组以及1 mmol/L 4-PBA组。应用Western blotting、流式细胞仪、免疫荧光、电镜和免疫共沉淀技术,检测细胞内质网应激标志蛋白[肌醇需求酶1α(IRE-1α)、蛋白激酶R样内质网激酶(PERK)、转录激活因子4(ATF4)、磷酸化α亚基的真核起始因子2(p-eIF-2α)、α亚基的真核起始因子2(eIF-2α)、转录因子C/EBP同源蛋白(CHOP)、磷酸化IRE-1α(p-IRE-1α)、磷酸化PERK(p-PERK)和转录激活因子6(ATF6)]、凋亡蛋白[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和切割型半胱氨酸天冬氨酸蛋白酶3(cleaved-Caspase 3)]、内质网形态和内质网应激相互作用蛋白[葡萄糖调节蛋白78(GRP78)]表达水平,验证内质网应激在7-KC损害β细胞功能中的作用。两组间比较采用t检验,多组间比较采用方差分析,组内两两比较采用Bonferroni多重比较法。结果随着7-KC浓度的增加,cleaved-Caspase 3相对表达量呈剂量依赖性增加,并且在25μmol/L 7-KC时能显著增加cleaved-Caspase 3的水平(P<0.01)。与对照组相比,25μmol/L 7-KC组可引起内质网扩张和囊泡化,诱导内质网应激感受蛋白p-PERK、p-IRE-1α、ATF6及下游效应蛋白p-eIF-1α、ATF4、CHOP的表达。与25μmol/L 7-KC组比较,25μmol/L 7-KC+1 mmol/L 4-PBA组的内质网应激标志蛋白表达下降,同时凋亡蛋白表达减少(P<0.05)。免疫共沉淀结果显示,25μmol/L 7-KC组可抑制分子伴侣GRP78和PERK的结合,促进PERK自体磷酸化激活内质网应激。结论7-KC通过抑制细胞中GRP78与PERK结合来激活内质网应激,促进胰岛β细胞凋亡的发生。Objective To investigate the effect of 7-ketocholesterol(7-KC)on apoptosis and to elucidate its underlying mechanism in pancreaticβ-cells.Methods INS-1 cells were treated with 0,0.25,2.5 and 25μmol/L 7-KC for 24 h,and the concentration inducing apoptosis of INS-1 cells was detected by western blotting.Furthermore,the cells were divided into four groups:blank group,25μmol/L 7-KC treatment group,7-KC treatment and pretreated with 1 mmol/L endoplasmic reticulum(ER)stress inhibitor 4-phenylbutyric acid(4-PBA)group,and 1 mmol/L 4-PBA group.The expression of ER stress marker proteins[inosital-requiring enzyme-1α(IRE-1α),protein kinase R-like endoplasmic reticulum kinase(PERK),activating transcription factor 4(ATF4),phosphorylatedαsubunit of eukaryotic initiation factor 2(p-eIF-2α),αsubunit of eukaryotic initiation factor 2(eIF-2α),C/Ebp-homologous protein(CHOP),phosphorylated inosital-requiring enzyme-1(p-IRE-1α),phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK),activating transcription factor 6α(ATF-6α)],apoptosis proteins[B-cell lymphoma-2(Bcl-2),Bcl2-associated X(Bax),cleaved cysteinyl aspartate-specific proteinase-3(cleaved-Caspase 3)],ER morphology and interaction proteins of ER[glucose regulated protein 78(GRP78)]were elucidated via western blotting,flow cytometry and immunofluorescence,transmission electron microscopy and co-immunoprecipitation,in order to verify the role of ER stress inβ-cell function impairment by 7-KC.Two-tailed Student′s t tests were performed to compare means between two groups.ANOVA followed by Bonferroni′s multiple comparisons was used to compare means from three or more groups.Results The cleaved-Caspase3 was increased dose-dependently and upregulated obviously at 25μmol/L 7-KC treatment group(P<0.01).Meanwhile,the dilation and vesiculation of ER,and ER stress-sensing proteins p-PERK,p-IRE-1α,ATF6,p-eIF-1,ATF4,and CHOP were dramatically upregulated at 25μmol/L 7-KC treatment group.Compared with 25μmol/L 7-KC treatment group,the further study s

关 键 词：胰岛 细胞凋亡 胰岛Β细胞 7-酮基胆固醇 内质网应激 脂毒性 

分 类 号：R587.1[医药卫生—内分泌]