Abstract：
This study investigated the protective effect of ginsenoside Rg_1(GRg_1) 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+GRg_1(0.1, 1, 10 μmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+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 detected 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 proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently 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, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 μmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+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, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.
摘要：
本研究探讨人参皂苷Rg_1(GRg_1)对大鼠肾上腺嗜铬细胞瘤(PC12)细胞氧糖剥夺/复氧(OGD/R)损伤的保护作用及其潜在机制是否与肌醇需求酶1(IRE1)-c-JunN末端激酶(JNK)-C/EBP同源蛋白(CHOP)信号通路的调控有关。在PC12细胞中建立了OGD/R模型,将PC12细胞随机分为对照组，模型，OGD/R+GRg_1(0.1,1,10μmol·L~(-1)),OGD/R+GRg_1+雷帕霉素(自噬激动剂),OGD/R+GRg_1+3-甲基腺嘌呤(3-MA，自噬抑制剂），OGD/R+GRg_1+衣霉素(内质网应激激动剂),OGD/R+GRg_1+4-苯基丁酸(4-PBA,内质网应激抑制剂），和OGD/R+GRg_1+3,5-二溴水杨醛(DBSA，IRE1抑制剂)组。除了对照组,其他组接受OGD/R治疗，即，氧气和葡萄糖剥夺6小时，然后复氧6小时。通过3-（4,5-二甲基-2-噻唑基）-2,5-二苯基四唑溴化物（MTT）测定法检测细胞活力。Hoechst33342染色检测细胞凋亡，和通过单糖尸胺（MDC）测定自噬体的荧光强度。Westernblot检测自噬相关蛋白(Beclin1、LC3-Ⅱ、和p62）和通路相关蛋白[IRE1，p-IRE1，JNK，p-JNK,葡萄糖调节蛋白78（GRP78），和CHOP]。结果显示GRg_1剂量依赖性地增加PC12细胞的活力,下调Beclin1、LC3-Ⅱ的表达,p-IRE1,p-JNK,GRP78和CHOP,与模型组相比。此外，GRg_1降低细胞凋亡率和MDC荧光强度,上调p62蛋白表达。与OGD/R+GRg_1(10μmol·L~(-1))组比较,OGD/R+GRg_1+雷帕霉素和OGD/R+GRg_1+衣霉素组细胞凋亡率和MDC荧光强度增加,Beclin1,LC3-Ⅱ上调蛋白水平,p-IRE1,p-JNK,GRP78和CHOP,相对细胞存活率降低，和下调p62蛋白水平。3-MA,4-PBA,和DBSA组发挥相反的作用。一起来看,GRg_1可能通过IRE1-JNK-CHOP通路抑制自噬改善OGD/R诱导的PC12细胞损伤。
