丙烯腈诱导大鼠脾的氧化应激和免疫系统响应

摘要

为探讨丙烯腈(acrylonitrile,ACN)诱导的氧化应激对大鼠脾核转录因子NF-κB(nuclear transcription factor-κB,NF-κB)信号通路的影响,将60只无特定病原体(specific pathogen free,SPF)级健康成年雄性大鼠随机分为5组:对照组(玉米油),低、中、高剂量染毒组(11.5、23、46 mg·kg-1ACN)和N-乙酰-L-半胱氨酸(N-acetylcysteine,NAC)干预组(46 mg·kg-1ACN+300mg·kg-1NAC),1次/天,6天/周,连续灌胃4周.染毒结束后,次日处死大鼠,分离大鼠脾并称重.以分光光度法检测脾组织超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)和总抗氧化能力(total antioxidant capacity,T-AOC)含量或活力;以Elisa法检测大鼠脾白介素6(IL-6)、白介素1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平;以Western Blot法检测脾NF-κB、IκB、p-IκB蛋白表达水平;以RT-PCR法检测NF-κB、IκB mRNA表达水平.结果显示,与对照组比较,ACN低、中剂量组大鼠脾湿重明显降低(P＜0.05);ACN中剂量组脾脏器系数降低(P＜0.05).中、高剂量组大鼠脾组织MDA含量显著增加(P＜0.05);高剂量组大鼠SOD、GSH-Px活力显著增加(P＜0.05);低剂量组大鼠CAT活力降低,高剂量组大鼠CAT活力增加(P＜0.05);低剂量组大鼠GSH含量、T-AOC活力均降低(P＜0.05).与高剂量组相比较,NAC组大鼠MDA、SOD、CAT、GSH、GSH-Px、T-AOC均降低(P＜0.05).与对照组比较,ACN中、高剂量组大鼠脾组织内IL-1β含量降低(P＜0.05),低、中、高剂量组大鼠TNF-α含量增加(P＜0.05).与高剂量组比较,NAC组TNF-α含量降低(P＜0.05).Western Blot结果显示,中、高剂量染毒组大鼠脾NF-κB、IκB、p-IκB蛋白表达水平与对照组比较均升高(P＜0.05).NAC组大鼠脾IκB、p-IκB蛋白表达水平与高ACN组比较降低(P＜0.05).RT-PCR结果显示,ACN各剂量染毒组大鼠脾NF-κB mRNA表达水平与对照组比较均升高(P＜0.05);中、高剂量染毒组大鼠脾IκB mRNA表达水平与对照组比较均升高(P＜0.05).NAC组大鼠脾NF-κB mRNA表达水平低于高剂量组(P＜0.05).结果表明ACN亚急性染毒可对大鼠脾产生氧化损伤和免疫系统响应,NAC可通过拮抗作用减轻损伤的程度.%To investigate the effect of acrylonitrile (ACN) -induced oxidative stress on nuclear transcription factor-κB (NF-κB) signaling pathway in rats spleen, 60 specific pathogen free (SPF) grade healthy adult male rats were randomly divided into 5 groups: control group (corn oil), low-, medium-, and high-dose groups (11.5, 23, 46 mg·kg-1 ACN) and NAC groups (46 mg·kg-1 ACN + 300 mg·kg-1 NAC), and were treated once/day, 6 days/week, by intragastric gavage for 4 weeks. The spleen was isolated and weighed. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), the contents of glutathione (GSH), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in spleen were detected by spectrophotometry. The interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α) levels were determined by Elisa.The expressions of NF-κB, IκB and p-IκB were measured using western blotting and the expressions of NF-κB and IκB mRNA were determined by RT-qPCR. The results showed that the splenic wet weight of the rats were significantly decreased in the low-and middle-dose groups compared with the control group (P＜0.05). The splenic coefficient was also significantly decreased in the middle-dose group (P＜0.05). The contents of MDA in the spleen of rats were significantly increased in the medium-and high-dose groups (P ＜ 0. 05) and the activities of SOD and GSH-Px were significantly increased in the high-dose group (P＜0.05). The activity of CAT and the contents of TAOC and GSH were all decreased in the low-dose group, whereas, the CAT activity was increased in the high-dose group (P＜0.05).Meanwhile, the activities of SOD, CAT and GSH-Px and the contents of MDA, GSH and T-AOC were significantly decreased in the NAC group compared with the high-dose group (P＜0.05). Compared with the control group, the contents of IL-1β were decreased in the middle-and high-dose groups, and the levels of TNF-αwere increased significantly in all dose groups (P＜0.05). Compared with the high-dose group, the content of TNF-α was significantly decreased in the NAC group (P＜0.05). Western Blot results showed that the expressions of NF-κB, IκB, and p-IκB were significantly increased in the medium-and high-dose groups compared with the control group (P＜0.05). The expressions of IκB and p-IκB in NAC group was decreased in the high ACN group (P ＜0.05). RT-PCR results showed that the expression of NF-κB mRNA were increased in the low-, medium-and highdose ACN groups compared with the control group (P＜0.05). And the expressions of IκB mRNA were increased in the medium-and high-dose ACN groups compared with the control group (P＜0.05). The expression of NF-κB mRNA was decreased in the NAC group compared with the high-dose group (P＜0.05). Therefore, the present study suggests that subacute exposure to ACN could produce oxidative damage and immune system response in rats 'spleen, and NAC might reduce the degree of injury through antagonism.