Nrf2通路对日本脑炎病毒感染小鼠神经瘤细胞引起氧化应激反应的影响

doi:10.11843/j.issn.0366-6964.2023.01.029

摘要/Abstract

摘要： 旨在研究Nrf2/HO-1通路在日本脑炎病毒(Japanese encephalitis virus,JEV)感染引起神经细胞损伤中的作用机制，本试验建立Nrf2激动剂叔丁基对苯二酚(tert-butylhydroquinone，TBHQ)处理JEV感染的小鼠神经瘤母N2a细胞的体外感染模型。N2a细胞四种处理分别为空白DMEM对照组(Control)、JEV感染组(JEV)、TBHQ处理组(TBHQ)、JEV感染+TBHQ处理组(JEV+TBHQ)。不同处理后观察细胞病变，检测ROS水平及MDA、SOD、CAT含量变化，利用qPCR和WB技术检测Nrf2、HO-1的mRNA和蛋白的表达，并通过免疫荧光法检测各组细胞Nrf2蛋白表达情况，以及炎性因子及病毒含量。结果显示，JEV感染N2a细胞后随着时间延长ROS水平逐渐升高，且在36h ROS水平最高。JEV感染后36h Nrf2、HO-1的mRNA均升高，炎性因子水平升高。JEV感染N2a细胞后细胞皱缩，胞膜破裂，ROS、MDA水平升高，SOD、CAT水平降低；用40μmol·L^-1TBHQ处理6h后减轻了细胞损伤，降低了ROS、MDA水平，提高了SOD、CAT活性。JEV和TBHQ均激活了Nrf2、HO-1的mRNA和蛋白的表达，用JEV+TBHQ联合处理Nrf2/HO-1通路的相关蛋白表达水平明显升高，且免疫荧光法检测细胞内Nrf2入核现象明显，且TNF-α、IL-6的mRNA水平及病毒的含量下降。上述结果表明，TBHQ能够通过Nrf2/HO-1通路减轻JEV引起的细胞氧化应激及炎症水平。

关键词: 日本脑炎病毒, 氧化应激, TBHQ, N2a细胞, Nrf2/HO-1信号通路

Abstract: To investigate the role of the Nrf2/HO-1 pathway in neuronal damage caused by Japanese encephalitis virus (JEV) infection, an in vitro infection model was developed in which Nrf2 agonist tert-butylhydroquinone (TBHQ) was used to treat JEV-infected mouse neuroblastoma (N2a) cells. N2a cells were divided into following four groups: DMEM control group (Control), JEV-infected group (JEV), TBHQ-treated group (TBHQ), and JEV-infected +TBHQ -treated group (JEV+TBHQ). The cytopathic lesions were observed after different treatments and the changes in ROS levels and MDA, SOD and CAT contents were detected. The mRNA and protein expression of Nrf2 and HO-1 were detected by qPCR and WB techniques, and Nrf2 protein expression in each group of cells was detected by immunofluorescence, and inflammatory factors and viral content were measured. The results showed that the level of ROS gradually increased with time after JEV infection of N2a cells and was reached the highest at 36 h. The mRNA of Nrf2 and HO-1 increased at 36 h after JEV infection, and the level of inflammatory factors increased. JEV infection of N2a cells resulted in cell crumpling, cell membrane rupture, increased ROS and MDA levels and decreased SOD and CAT levels; treatment with 40 μmol·L^-1 TBHQ for 6 h reduced cell damage, decreased ROS and MDA levels and increased SOD and CAT activities. Both JEV and TBHQ activated the mRNA and protein expression of Nrf2 and HO-1, and the expression levels of proteins related to the Nrf2/HO-1 pathway were significantly increased by combined treatment with JEV+TBHQ, and the nucleation of Nrf2 in the cells by immunofluorescence was evident, and the mRNA levels of TNF-α and IL-6 and the levels of viruses decreased. The above results suggest that TBHQ is able to reduce cellular oxidative stress and inflammation levels induced by JEV through the Nrf2/HO-1 pathway.

Key words: Japanese encephalitis virus, oxidative stress, TBHQ, N2a cells, Nrf2/HO-1 signalling pathway

中图分类号:

S852.3

刘泽霖, 郭晓艳, 李佳欢, 高明星, 程国富, 胡薛英, 张万坡, 谷长勤. Nrf2通路对日本脑炎病毒感染小鼠神经瘤细胞引起氧化应激反应的影响[J]. 畜牧兽医学报, 2023, 54(1): 317-327.

LIU Zelin, GUO Xiaoyan, LI Jiahuan, GAO Mingxing, CHENG Guofu, HU Xueying, ZHANG Wanpo, GU Changqin. Effect of the Nrf2 Pathway on Oxidative Stress Induced by Japanese Encephalitis Virus Infection of Mouse Neuroblastoma Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 317-327.

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