Trx-1过表达通过NF-κB信号通路减轻MPP^+所致PC12细胞的氧化应激损伤

Trx-1 over-expression attenuates oxidative stress injury in MPP+-induced PC12 cells by regulating NF-κB signaling pathway

目的:探讨硫氧还蛋白1(Trx-1)过表达通过NF-κB信号通路减轻1-甲基-4-苯基吡啶离子(MPP^+)诱导的大鼠嗜铬细胞瘤PC12细胞氧化应激损伤的作用,探讨帕金森病的发病机制。方法:采用1、3和5 mmol/L MPP^+损伤PC12细胞,MTT法检测细胞活力,商用试剂盒检测细胞上清液中氧化应激指标乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,Western blot检测细胞中Trx-1蛋白的表达。以3 mmol/L MPP^+损伤PC12细胞,以含Ad-Trx-1-GFP序列的慢病毒感染建立Trx-1过表达的帕金森病细胞模型,采用MTT法、商用试剂盒和Western blot分别检测Trx-1过表达对PC12细胞活力、氧化应激反应和NF-κB信号通路的影响。给予NF-κB信号通路激活剂佛波酯(PMA)作用于经MMP^+处理的P12细胞,观察激活NF-κB信号通路对PC12细胞活力和氧化应激反应的影响;给予NF-κB信号通路抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)作用于Trx-1过表达的MPP^+损伤PC12细胞,观察Trx-1过表达通过NF-κB信号通路对PC12细胞活力和氧化应激反应的影响。结果:1、3和5 mmol/L MPP^+能够明显降低PC12细胞活力、细胞上清液中SOD活性和细胞内Trx-1蛋白的表达,升高细胞上清液中LDH活性和MDA含量,且3 mmol/L MPP^+和5 mmol/L MPP^+的作用明显大于1 mmol/L MPP^+(P<0.05),而3 mmol/L MPP^+和5 mmol/L MPP^+间的差异无统计学显著性。Trx-1过表达能够明显减弱MPP^+对PC12细胞活力的抑制作用及其诱导的氧化应激损伤和NF-κB信号通路活化。NF-κB信号通路的激活促进了MPP^+对PC12细胞活力的抑制作用及其诱导的氧化应激损伤,而抑制NF-κB信号通路则增强了Trx-1过表达对MPP^+损伤的PC12细胞的保护作用。结论:Trx-1过表达可通过NF-κB信号通路减轻MPP^+作用下PC12细胞的氧化应激损伤。

AIM: To investigate the inhibitory effect of thioredoxin 1(Trx-1) over-expression on oxidative stress injury in 1-methyl-4-phenylpyridinium(MPP^+)-induced rat pheochromocytoma PC12 cells by regulating NF-κB signaling pathway.METHODS: The PC12 cells were damaged by treatment with MPP^+ at 1, 3 and 5 mmol/L, and the optimal concentration of 3 mmol/L was selected. The cell viability was measured by MTT assay. The oxidative stress indexes lactate dehydrogenase(LDH) activity, superoxide dismutase(SOD) activity and malondialdehyde(MDA) content in the cell culture supernatant were detected, and the protein expression of Trx-1 was determined by Western blot. Lentiviral infection with Ad-Trx-1-GFP sequence was used to establish a model of MPP^+-treated PC12 cells with Trx-1 over-expression. The effects of Trx-1 over-expression on the cell viability, oxidative stress responses and NF-κB signaling pathway were determined by MTT assay, commercial kits and Western blot. The effects of phorbol 12-myristate 13-acetate(PMA), an activator of NF-κB signaling pathway, on the viability and oxidative stress of PC12 cells were observed. The NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate(PDTC) was used in MPP^+-treated PC12 cells with Trx-1 over-expression, and the cell viability and oxidative stress responses were measured. RESULTS: The viability of PC12 cells, SOD activity in the supernatant and protein expression of Trx-1 were decreased, while LDH activity and MDA content in the supernatant were increased significantly by treatment with MPP^+ at 1, 3 and 5 mmol/L. The effect of MPP^+ at 3 mmol/L and 5 mmol/L was significantly greater than that at 1 mmol/L(P<0.05), and no significant difference between 3 mmol/L and 5 mmol/L was observed(P>0.05). The inhibitory effect of MPP^+ on the viability of PC12 cells, and the oxidative stress injury and activation of NF-κB signaling pathway induced by MPP^+ were significantly attenuated by over-expression of Trx-1. The inhibitory effect of MPP^+ on the viability of PC12 cells and the oxidative stress injury induced by MPP^+ were promoted by the activation of NF-κB signaling pathway, while the protective effects of Trx-1 over-expression on the MPP^+-treated PC12 cells were enhanced by the inhibition of NF-κB signaling pathway. CONCLUSION: Over-expression of Trx-1 protects MPP^+-treated PC12 cells from oxidative stress injury by regulating NF-κB signaling pathway.