摘要
探讨人参皂苷Rg1(ginsenoside Rg1,GRg1)对氧糖剥夺/复氧(oxygen and glucose deprivation/reoxygenation,OGD/R)损伤大鼠嗜铬细胞瘤(rat adrenal pheochromocytoma,PC12)细胞的保护作用及其作用机制是否与调控肌醇需求酶 1(inositol-re-quiring enzyme 1,IRE1)-c-Jun 氨基末端激酶(c-Jun N-terminal kinase,JNK)-C/EBP 同源蛋白(C/EBP homologous protein,CHOP)信号通路有关.在PC12 细胞中建立OGD/R模型,将PC12 细胞随机分组为对照组、模型组、OGD/R+GRg1(0.1、1、10 μmol·L-1)组,OGD/R+GRg1+雷帕霉素(rapamycin,自噬激动剂)组、OGD/R+GRg1+3-甲基腺嘌呤(3-methyladenine,3-MA,自噬抑制剂)组、OGD/R+GRg1+衣霉素(tunicamycin,内质网应激激动剂)组、OGD/R+GRg1+4-苯基丁酸(4-phenylbutyric acid,4-PBA,内质网应激抑制剂)组、OGD/R+GRg1+3,5-二溴水杨醛(3,5-dibromosalicylaldehyde,DBSA,IRE1 抑制剂)组.除对照组外,其他组都进行OGD/R处理,即氧糖剥夺 6 h再复氧 6 h.MTT法检测细胞活力,Hoechst 33342 染色检测细胞凋亡情况,MDC法检测细胞自噬体的荧光强度.Western blot检测自噬相关蛋白 Beclin1、LC3-Ⅱ、p62 和通路相关蛋白 IRE1、p-IRE1、JNK、p-JNK、葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、CHOP 的表达情况.结果显示,与模型组相比,0.1、1、10 μmol·L-1 GRg1 剂量依赖性地提高了 PC12 细胞的活力,降低了自噬蛋白 Beclin1、LC3-Ⅱ和通路相关蛋白 p-IRE1、p-JNK、GRP78、CHOP表达,降低了细胞凋亡率和MDC荧光强度,同时也增加了p62 蛋白表达.而分别干预自噬和内质网应激后,与OGD/R+GRg1(10 μmol·L-1)组相比,OGD/R+GRg1+rapamycin组和OGD/R+GRg1+tunicamycin组细胞凋亡率和MDC荧光强度增加,自噬蛋白Beclin1、LC3-Ⅱ和通路相关蛋白p-IRE1、p-JNK、GRP78、CHOP 表达增加,细胞相对存活率和p62 蛋白表达降低.3-MA、4-PBA和DBSA则发挥了相反作用.综上所述,GRg1 可能通过IRE1-JNK-CHOP通路抑制自噬而改善OGD/R诱导PC12 细胞的损伤.
Abstract
This study investigated the protective effect of ginsenoside Rg1(GRg1)on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12)cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP)signaling pathway.An OGD/R model was established in PC12 cells,and PC12 cells were randomly classified into control,model,OGD/R+GRg1(0.1,1,10 μmol·L-1),OGD/R+GRg1+rapamycin(autophagy agonist),OGD/R+GRg1+3-methyladenine(3-MA,autophagy inhibitor),OGD/R+GRg1+tunicamycin(endoplasmic reticulum stress agonist),OGD/R+GRg1+4-phenylbutyric acid(4-PBA,endoplasmic re-ticulum stress inhibitor),and OGD/R+GRg1+3,5-dibromosalicylaldehyde(DBSA,IRE1 inhibitor)groups.Except the control group,the other groups were subjected to OGD/R treatment,i.e.,oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h.Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT)assay.Apoptosis was detec-ted by Hoechst 33342 staining,and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC)assay.Western blot was employed to determine the expression of autophagy-related proteins(Beclin1,LC3-Ⅱ,and p62)and the pathway-related pro-teins[IRE1,p-IRE1,JNK,p-JNK,glucose-regulated protein 78(GRP78),and CHOP].The results showed that GRg1 dose-de-pendently increased the viability of PC12 cells and down-regulated the expression of Beclin1,LC3-Ⅱ,p-IRE1,p-JNK,GRP78,and CHOP,compared with the model group.Furthermore,GRg1 decreased the apoptosis rate and MDC fluorescence intensity and up-regu-lated the expression of p62 protein.Compared with the OGD/R+GRg1(10 μmol·L-1)group,OGD/R+GRg1+rapamycin and OGD/R+GRg1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity,up-regulated protein levels of Beclin1,LC3-Ⅱ,p-IRE1,p-JNK,GRP78,and CHOP,decreased relative cell survival rate,and down-regulated protein level of p62.The 3-MA,4-PBA,and DBSA groups exerted the opposite effects.Taken together,GRg1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.
基金项目
国家自然科学基金(82204667)
安徽省自然科学基金(2208085QH272)
安徽省高等学校自然科学研究项目(KJ2021A0587)
安徽中医药大学人才项目(2019rcyb004)
新时代育人质量工程项目(2022xscx106)
NETL
NSTL
万方数据