摘要
[目的]探讨灯盏花乙素(Scu)调控蛋白激酶R样内质网激酶(PERK)-核因子E2相关因子2(Nrf2)/激活转录因子4(ATF4)-C/EBP同源蛋白(CHOP)通路拮抗2,2-偶氮二(2-甲基丙基咪)二盐酸盐(AAPH)诱导的人主动脉内皮细胞(HAEC)损伤的具体机制。[方法]用Scu对HAEC进行预保护,再用AAPH诱导HAEC损伤,探究Scu对HAEC损伤的具体分子机制。将细胞分为对照组、AAPH组、AAPH+Scu低、中、高剂量组,测定细胞中超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽-硫转移酶(GSH-ST)的含量,荧光探针检测细胞内活性氧(ROS)含量,Annexin V-FITC/PI法检测细胞凋亡率,RT-qPCR检测细胞中PERK、真核起始因子2α(eIF2α)mRNA的表达,Western blot测定细胞中葡萄糖调节蛋白78(GRP78)、p-PERK、PERK、eIF2α、p-eIF2α、ATF4、CHOP、Nrf2、Bcl-2、Caspase-3及Caspase-12蛋白的表达。为了进一步探究Scu拮抗HAEC损伤的分子机制,采用基因沉默技术抑制HAEC中PERK的表达。将细胞分为五组,分别为对照组、AAPH+si-con组、AAPH+Scu+si-con组、AAPH+si-PERK组、AAPH+si-PERK+Scu组,采用RT-qPCR检测si-PERK干扰后细胞中PERK、eIF2αmRNA的表达,Western blot测定si-PERK干扰后细胞中PERK、p-eIF2α、eIF2α、ATF4、CHOP、Nrf2、Bcl-2、p53正向调节因子(PUMA)、Caspase-3及Caspase-12蛋白的表达。[结果]Scu干预后细胞内ROS含量及细胞凋亡率显著降低(P<0.01)。Scu可下调PERK、eIF2αmRNA的表达,并下调GRP78、p-PERK、p-eIF2α、ATF4、CHOP、PUMA、Caspase-3、Caspase-12以及上调Nrf2、Bcl-2蛋白的表达(P<0.01)。用si-PERK干扰后,细胞中PERK、p-eIF2α、ATF4、CHOP、Nrf2、Bcl-2、PUMA、Caspase-3、Caspase-12蛋白的表达以及PERK和eIF2αmRNA的表达与未干扰前有显著差异(P<0.01),证明Scu发挥抗内质网应激及细胞凋亡与其调控PERK-Nrf2/ATF4-CHOP通路密切相关。[结论]Scu通过调控PERK-Nrf2/ATF4-CHOP通路抗内质网应激及细胞凋亡,从而有效减轻AAPH诱导的HAEC损伤。
Aim To explore the specific mechanism by which scutellarin(Scu)antagonizes the injury of human aortic endothelial cells(HAEC)induced by 2,2-azobis(2-methylpropylimidate)dihydrochloride(AAPH)by regulating the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-nuclear factor erythroid 2-related factor 2(NRF2)/activating transcription factor 4(ATF4)-C/EBP homology protein(CHOP)pathway.Methods HAEC were pre-protected by Scu and then injured by AAPH to explore the molecular mechanism of Scu on HAEC injury.The cells were divided into control group,AAPH group,AAPH+Scu low,medium and high groups.The contents of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-Px)and glutathione S-transferase(GSH-ST)in the cells were measured.The content of reactive oxygen species(ROS)in cells was detected by fluorescent probe,and the apoptosis rate was detected by Annexin V-FITC/PI method.The mRNA expression of PERK and eIF2αin cells was detected by RT-qPCR.The protein expression of glucose regulated protein 78(GRP78),PERK,p-PERK,eukaryotic initiation factor 2α(eIF2α),p-eIF2α,ATF4,CHOP,Nrf2,Bcl-2,p53 up-regulated modulator of apoptosis(PUMA),Caspase-3 and Caspase-12 in cells was detected by Western blot.In order to further study the molecular mechanism of Scu against HAEC injury,gene silencing technology was used to inhibit the expression of PERK in HAEC.The cells were divided into five groups:control group,AAPH+si-con group,AAPH+Scu+si-con group,AAPH+si-PERK group,AAPH+si-PERK+Scu group.The mRNA expression of PERK and eIF2αin cells after si-PERK interference was detected by RT-qPCR.The protein expression of PERK,p-eIF2α,eIF2α,ATF4,CHOP,Nrf2,Bcl-2,PUMA,Caspase-3 and Caspase-12 in cells after si-PERK interference was detected by Western blot.Results The content of ROS and the rate of apoptosis were significantly reduced after Scu intervention(P<0.01).Scu could down-regulate the mRNA expression of PERK and eIF2α,and down-regulate the protein expression of GRP78,p-PERK,p-eIF2α,ATF4,CHOP,PUMA,Caspase-3,Caspase-12 and up-regulate the protein expression of Nrf2 and Bcl-2(P<0.01).After interference with si-PERK,there were significant differences in the protein expression of PERK,p-eIF2α,ATF4,CHOP,Nrf2,Bcl-2,PUMA,Caspase-3,Caspase-12,as well as the mRNA expression of PERK and eIF2αin cells compared to before interference(P<0.01).It is proved that Scu could anti-endoplasmic role in reticulum stress and apoptosis,which is closely associated with the regulation of the PERK-Nrf2/ATF4-CHOP pathway.Conclusion Scu can effectively alleviate AAPH-induced injury to HAEC by regulating PERK-Nrf2/ATF4-CHOP pathways to inhibit endoplasmic reticulum stress and cell apoptosis.
作者
赵芮琪
鲍柳池
单诗淇
金越
ZHAO Ruiqi;BAO Liuchi;SHAN Shiqi;JIN Yue(College of Pharmacy,Dalian Medical University,Dalian,Liaoning 116044,China)
出处
《中国动脉硬化杂志》
2025年第3期227-234,共8页
Chinese Journal of Arteriosclerosis
基金
辽宁省教育厅面上项目(LJKMZ20221260)。
