摘要
目的探讨健脾消渴方对高糖诱导min6细胞内质网应激(ERS)信号通路蛋白激酶R样内质网激酶(PERK)-真核启动因子(eIF)2α-C/EBP同源蛋白(CHOP)和细胞凋亡的影响。方法将min6细胞随机分为正常对照组(Con组,5.5 mmol/L葡萄糖)、模型组(Model组,25.0 mmol/L葡萄糖)、健脾消渴方组(Jpxk组,25.0 mmol/L葡萄糖+最佳干预浓度的健脾消渴方)和阳性对照药利拉鲁肽组(Li组,25.0 mmol/L葡萄糖+最佳干预浓度的利拉鲁肽),用CCK8法检测细胞活力,酶联免疫吸附试验(ELISA)检测胰岛素分泌;Hoechst33258染色和膜联蛋白Ⅴ-异硫氰酸荧光素(AnnexinⅤ-FITC)/碘化丙啶(PI)双染法流式检测细胞凋亡;Western印迹检测PERK、eIF2α、活化转运酶(ATF)4、CHOP、葡萄糖调节蛋白(GRP)78、胱天蛋白酶(Caspase)12蛋白表达水平;实时荧光定量-聚合酶链反应(RT-qPCR)检测PERK、eIF2α、ATF4、CHOP mRNA表达。结果与Con组相比,Model组min6细胞活力显著减弱,胰岛素水平明显降低,细胞凋亡率明显升高(P<0.01),且凋亡小体增多,染色质和细胞核固缩;明显上调PERK、eIF2α、ATF4、CHOP mRNA及蛋白表达、GRP78、Caspase12蛋白表达(P<0.05,P<0.01)。与Model组相比,Jpxk组min6细胞活力明显提高,胰岛素水平明显升高,凋亡细胞比例明显降低(P<0.05,P<0.01),min6细胞内凋亡小体减少;且明显下调PERK、eIF2α、CHOP蛋白及mRNA、GRP78、Caspase12蛋白及ATF4 mRNA表达(P<0.05,P<0.01)。与Li组相比,健脾消渴方能显著降低PERK及CHOP蛋白表达(P<0.05)。结论健脾消渴方可能通过抑制PERK、eIF2α、CHOP活性,改善内质网应激以保护min6细胞,PERK-eIF2α-CHOP信号通路可能是健脾消渴方发挥作用的主要机制之一。
Objective To investigate the effect of Jianpi Xiaoke decoction on endoplasmic reticulum stress(ERS)signaling pathway protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic promoter factor(eIF)2α-C/EBP homologous protein(CHOP)and apoptosis in min6 cells induced by high glucose.Methods min6 cells were randomly divided into normal control group(Con group,5.5 mmol/L glucose),Model group(25.0 mmol/L glucose),Jianpi Xiaoke decoction group(Jpxk group,25.0 mmol/L glucose+the best intervention concentration of Jianpi Xiaoke decoction)and positive control drug liraglutide group(Li Group,25.0 mmol/L glucose+optimal intervention concentration of Liraglutide),cell viability was measured by CCK8,insulin secretion was measured by enzyme linked immunosorbent assay(ELISA),and apoptosis was detected by Hoechst33258 staining and AnnexinⅤ-FITC/propyliodide(PI)double staining.The expression levels of PERK,eIF2α,activating transcription factor(ATF)4,CHOP,glucose regulated protein(GRP)78 and Caspase-12 protein were detected by Western blotting,and the mRNA expressions of PERK,eIF2α,ATF4 and CHOP were detected by real-time quantitative polymerase chain reaction(RT-qPCR).Results Compared with Con group,the activity of min6 cells was significantly decreased,the level of insulin was significantly decreased,the rate of apoptosis was significantly increased in Model group(P<0.01),and the apoptotic corpuscles increased,chromatin and nucleus shrank;up-regulated the expressions of PERK,eIF2α,ATF4,CHOP mRNA and protein,GRP78 and Caspase12 protein(P<0.05,P<0.01).Compared with Model group,the activity of min6 cells in Jpxk group was significantly increased,the level of insulin was significantly increased,and the proportion of apoptotic cells was significantly decreased(P<0.05,P<0.01),the number of apoptotic bodies in min6 cells was decreased,and significantly down-regulated the expressions of PERK,eIF2α,CHOP protein and mRNA,GRP78,Caspase-12 protein and ATF4 mRNA(P<0.05,P<0.01).Compared with Li Group,the expressions of PERK and
作者
王小芳
魏代浩
邓欢
边文飞
洪甜影
黄延芹
WANG Xiao-Fang;WEI Dai-Hao;DENG Huan(Shandong University of Traditional Chinese Medicine,Jinan 250011,Shandong,China)
出处
《中国老年学杂志》
CAS
北大核心
2024年第12期2911-2917,共7页
Chinese Journal of Gerontology
基金
国家自然科学基金项目(81974562,81603613)
山东省泰山学者工程专项(tsqn202211354)
济南市科技创新计划(202019029)
钱秋海全国名老中医药专家传承工作室(国中医药人教函[2022]75号)
