Li Yu-Qing, Wei Liang-Li, Yuan Yu-Qi, Yang Zi-Teng, Wang Ning, Cai Biao, Wang Guang-Yun
College of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine Hefei 230012, China.
College of Pharmacy, Anhui University of Chinese Medicine Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2024 May;49(10):2745-2753. doi: 10.19540/j.cnki.cjcmm.20231128.405.
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.
本研究探讨人参皂苷Rg_1（GRg_1）对氧糖剥夺/复氧（OGD/R）损伤的大鼠肾上腺嗜铬细胞瘤（PC12）细胞的保护作用，以及其潜在机制是否与肌醇需求酶1（IRE1）-c-Jun氨基末端激酶（JNK）-C/EBP同源蛋白（CHOP）信号通路的调控有关。在PC12细胞中建立OGD/R模型，并将PC12细胞随机分为对照组、模型组、OGD/R+GRg_1（0.1、1、10 μmol·L(-1)）组、OGD/R+GRg_1+雷帕霉素（自噬激动剂）组、OGD/R+GRg_1+3-甲基腺嘌呤（3-MA，自噬抑制剂）组、OGD/R+GRg_1+衣霉素（内质网应激激动剂）组、OGD/R+GRg_1+4-苯丁酸（4-PBA，内质网应激抑制剂）组和OGD/R+GRg_1+3,5-二溴水杨醛（DBSA，IRE1抑制剂）组。除对照组外，其他组均进行OGD/R处理，即氧糖剥夺6小时后再复氧6小时。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基溴化四氮唑（MTT）法检测细胞活力。采用Hoechst 33342染色检测细胞凋亡，采用单丹磺酰尸胺（MDC）法检测自噬体的荧光强度。采用蛋白质免疫印迹法检测自噬相关蛋白（Beclin1、LC3-Ⅱ和p62）以及通路相关蛋白[IRE1、p-IRE1、JNK、p-JNK葡萄糖调节蛋白78（GRP78）和CHOP]的表达。结果显示，与模型组相比，GRg_1剂量依赖性地增加PC12细胞活力，并下调Beclin1、LC3-Ⅱ、p-IRE1、p-JNK、GRP78和CHOP的表达。此外，GRg_1降低细胞凋亡率和MDC荧光强度，并上调p62蛋白的表达。与OGD/R+GRg_1（10 μmol·L(-1)）组相比，OGD/R+GRg_1+雷帕霉素组和OGD/R+GRg_1+衣霉素组细胞凋亡率和MDC荧光强度增加，Beclin1、LC3-Ⅱ、p-IRE1、p-JNK、GRP78和CHOP蛋白水平上调，相对细胞存活率降低，p62蛋白水平下调。3-MA、4-PBA和DBSA组则产生相反的作用。综上所述，GRg_1可能通过IRE1-JNK-CHOP通路抑制自噬，从而改善OGD/R诱导的PC12细胞损伤。
