丙烯腈暴露通过NF-κB信号通路诱导大鼠脑组织氧化应激
Acrylonitrile Exposure Induced Oxidative Stress in Rat Brain Tissues through the NF-κB Signaling Pathway
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摘要: 探究丙烯腈（ACN）暴露对大鼠脑组织NF-κB信号通路相关基因和蛋白的影响。选取60只SPF级健康成年雄性SD大鼠，随机分为对照组（玉米油）、11.5 mg·kg-1 ACN组、23.0 mg·kg-1 ACN组、46.0 mg·kg-1 ACN组、N-乙酰半胱氨酸（NAC）组（46.0 mg·kg-1 ACN+300 mg·kg-1 NAC），灌胃染毒，每天1次，每周6次，连续28 d。通过分光光度法检测大鼠脑组织中谷胱甘肽（GSH）、丙二醛（MDA）含量以及谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）酶活性；ELISA法测定脑组织内炎症细胞因子浓度；RT-qPCR法检测脑组织中NF-κB信号通路相关基因IKKα、IκB、NF-κB和TLR4 mRNA相对表达水平；Western Blot法检测脑组织NF-κB信号通路相关蛋白TLR4、NF-κB、IKKα、p-IKKα/β、IκBα和p-IκBα相对表达水平。ACN染毒后，与对照组相比，各暴露组大鼠脑组织MDA含量均升高（P<0.05）；23.0 mg·kg-1和46.0 mg·kg-1的ACN暴露导致大鼠脑组织GSH含量降低（P<0.05）；46.0 mg·kg-1的ACN导致GSH-Px活性下降（P<0.05）；11.5 mg·kg-1的ACN暴露导致CAT活性下降（P<0.05）。而抗氧化剂NAC的加入后，使得大鼠脑组织SOD活性升高，MDA含量和CAT活性均下降（P<0.05）。与对照组比较，各暴露组大鼠脑组织TNF-α浓度均升高（P<0.05）；23.0 mg·kg-1和46.0 mg·kg-1的ACN暴露导致IL-1β浓度均升高（P<0.05）；46.0 mg·kg-1的ACN暴露导致IL-6浓度升高（P<0.05）；而抗氧化剂NAC的加入后，使得大鼠脑组织中IL-6、IL-1β和TNF-α浓度水平均降低（P<0.05）。RT-qPCR结果表明，23.0 mg·kg-1和46.0 mg·kg-1ACN暴露导致大鼠脑组织IKKα和NF-κB mRNA表达均上调（P<0.05），46.0 mg·kg-1的ACN暴露导致TLR4 mRNA表达上调；而抗氧化剂NAC的加入后，大鼠脑组织IKKα和NF-κB mRNA表达均下调（P<0.05）。Western Blot结果表明，各暴露组p-IKKα/β、IKKα和NF-κB蛋白相对表达量均升高（P<0.05）；23.0 mg·kg-1和46.0 mg·kg-1的ACN暴露导致p-IκBα蛋白相对表达量升高（P<0.05），46.0 mg·kg-1 ACN暴露导致TLR4 mRNA和蛋白相对表达水平升高；而抗氧化剂NAC的加入后，大鼠脑组织中p-IKKα/β和NF-κB蛋白相对表达量均升高（P<0.05）。结果表明，ACN通过诱导大鼠脑组织氧化/抗氧化失衡和炎症反应，进而激活NF-κB信号通路，NAC干预可拮抗ACN诱导脑组织发生氧化应激，抑制NF-κB的活化。Abstract: To investigate the acrylonitrile (ACN) induced alterations of genes and proteins related to NF-κB signaling pathway in rat's brain, 60 SPF SD male rats were orally administrated with 11.5, 23.0 and 46.0 mg·kg-1 ACN, and pretreated with 300 mg·kg-1 NAC+46 mg·kg-1 ACN, respectively. ACN or N-acetylcysteine (NAC) was diluted in corn oil and administrated once per day, 6 d per week for total 28 d. Vehicle control group was orally administrated with the same volume of corn oil at the same dosing frequency. At the end of the experiments, rats were euthanized and brain tissue was isolated for further examination. The contents of glutathione (GSH) and malondialdehyde (MDA), and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were measured by biochemical kit according to the kit's instruction. The levels of inflammatory cytokines IL-6, IL-1β and TNF-α were determined by ELISA. Real-time PCR was used to detect the mRNA expression levels of IKKα, IκB and NF-κB. Western Blot was used to detect the protein expression of NF-κB, IKKα, p-IKKα/β, IκBα and p-IκBα in brain tissue. Compared with the control group, the MDA content in each ACN single treated group increased, while GSH content and GSH-Px activity decreased in dose dependent manner (P<0.05). Pretreated with antioxidant NAC, the SOD content increased, while the MDA content and CAT activity decreased (P<0.05) accordingly. Compared with the control group, the level of IL-1β and TNF-α increased in the exposure group of 23.0 mg·kg-1 and 46.0 mg·kg-1 ACN (P<0.05), and the level of IL-6 increased in the 46.0 mg·kg-1 ACN group (P<0.05). Pretreated with antioxidant NAC, the level of inflammatory cytokines IL-6, IL-1β and TNF-α in brain tissue decreased (P<0.05). Compared with the control group, the relative mRNA expression of IKKα and NF-κB was upregulated in the 23.0 mg·kg-1 and 46.0 mg·kg-1 ACN group (P<0.05), and the TLR4 mRNA expression increased in the 46.0 mg·kg-1 ACN group. Pretreated with antioxidant NAC, the IKKα and NF-κB mRNA expression in brain tissue was downregulated (P<0.05). Compared with the control group, the protein expression of p-IKKα/β, IKKα and NF-κB in ACN single treated groups was increased (P<0.05), and the protein expression of p-IκBα was increased in the 23.0 mg·kg-1 and 46.0 mg·kg-1 ACN treatment group (P<0.05). In addition, the protein expression and mRNA expression of TLR4 were increased when rats were exposed to 46.0 mg·kg-1 ACN. Pretreated with antioxidant NAC, the protein expression of p-IKKα/β and NF-κB in brain tissue increased (P<0.05). ACN activates NF-κB signaling pathway by inducing oxidative/antioxidative imbalance and inflammatory response in rat brain tissue. NAC pretreatment can antagonize can-induced oxidative stress in brain tissue thus inhibit the activation of NF-κB.
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Key words:
- acrylonitrile /
- rat /
- oxidative stress /
- inflammatory response /
- NF-κB signal pathway
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