加味四妙勇安汤通过ERS通路对高糖诱导的人视网膜内皮细胞的保护作用

Protective effect of Jiawei Simiao Yong'an Decoction on human retinal endothelial cells induced by high glucose through ERS pathway

目的:分析加味四妙勇安汤(Jiawei Simiao Yong’an Decoction,JSY)通过内质网应激(endoplasmic reticulum stress,ERS)信号通路对人视网膜内皮细胞(human retinal vascular endothelial cell,HRCEC)的保护作用机制。方法:将细胞分为阴性对照组(LG组)、高糖诱导组(HG组)、阳性对照组(二甲双胍,MET组)以及JSY组,CCK-8法筛选JSY对HRCEC作用的最适宜浓度;AnnexinⅤ-FITC/PI法检测各组细胞凋亡;RT-qPCR法测定细胞中eIF2α、CHOP、VEGF、IL-1β、IL-6及TNF-α的mRNA表达水平;ELISA法检测细胞上清中IL-6、IL-1β、TNF-α和AGE的水平;蛋白免疫印迹法检测细胞中eIF2α、CHOP、JNK、caspase12及NF-κB的蛋白水平。结果:与LG组相比,HG组细胞凋亡比率显著增加(P<0.05),eIF2α、CHOP、VEGF、IL-1β、IL-6及TNF-α的mRNA表达水平均增加(P<0.05),IL-6、IL-1β、TNF-α和AGE的含量均增加(P<0.05),eIF2α、CHOP、JNK、caspase12及NF-κB的蛋白相对表达量均显著增加(P<0.05);与HG组相比,MET组及JSY组细胞凋亡比率明显减少(P<0.05),eIF2α、CHOP、VEGF、IL-1β、IL-6及TNF-α的mRNA表达均明显减少(P<0.05),IL-6、IL-1β、TNF-α和AGE的含量均明显下降(P<0.05),eIF2α、CHOP、JNK、caspase12及NF-κB的蛋白相对表达量均显著减少(P<0.05)。结论:JSY可能通过抑制ERS信号通路和下游NF-κB通路的激活,降低相关mRNA和蛋白的表达,从而降低高糖诱导的HRCEC炎症因子水平,抑制细胞凋亡,发挥保护HRCEC的作用。

OBJECTIVE To explore the protective mechanism of Jiawei Simiao Yong'an Decoction(JSY) on human retinal vascular endothelial cells(HRCEC) through endoplasmic reticulum stress(ERS) signaling pathway.METHODS The cells were assigned into four groups of negative control(LG),high glucose induction(HG),positive control(metformin,MET) and JSY.CCK-8 method was employed for screening the optimal concentration of JSY on HRCEC cells.Annexin Ⅴ-FITC/PI method was utilized for detecting cell apoptosis in each group.The mRNA expression levels of eIF2α,CHOP,VEGF,IL-1β,IL-6 and TNF-α were determined by reverse transcription-polymerase chain reaction(RT-qPCR).And the levels of IL-6,IL-1β,TNF-α and AGE in supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Protein levels of eIF2α,CHOP,JNK,caspase12 and NF-κB were detected by Western blot.RESULTS As compared with LG group,apoptotic rate rose markedly in HG group(P<0.05).The mRNA expression levels of eIF2α,CHOP,VEGF,IL-1β,IL-6 and TNF-α were significantly up-regulated(P<0.05).The contents of IL-6,IL-1β,TNF-α and AGE spiked markedly(P<0.05).The relative expression levels of eIF2α,CHOP,JNK,caspase12 and NF-κB increased markedly(P<0.05).As compared with HG group,apoptotic rate declined markedly in MET/JSY group(P<0.05).The mRNA expressions of eIF2α,CHOP,VEGF,IL-1β,IL-6and TNF-α dropped sharply(P<0.05).The contents of IL-6,IL-1β,TNF-α and AGE were markedly elevated(P<0.05).The relative expression levels of eIF2α,CHOP,JNK,caspase12 and NF-κB dropped markedly(P<0.05).CONCLUSION JSY may arrest ERS signaling pathway and downstream NF-κB pathway,down-regulate the expressions of related mRNA and protein,lower the levels of HG-induced HRCEC inflammatory factors and stunt cell apoptosis.It play some roles in protecting HRCEC.