JNK/AP-1信号通路在百草枯诱导小胶质细胞NLRP3炎症小体活化中的作用
Role of JNK/AP-1 signaling pathway in paraquat-induced activation of NLRP3 inflammasome in microglia
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摘要:
背景 环境化学物百草枯（PQ）是一种能够引起散发性帕金森病（PD）的环境因素之一，其中小胶质细胞介导的神经炎症反应在PD发生发展中发挥重要作用。前期研究发现低剂量PQ可将BV-2小胶质细胞激活为M1表型并发挥促炎作用，但这种激活的小胶质细胞介导的神经炎症反应机制尚不清楚。
目的 探讨c-Jun氨基酸末端激酶（JNK）/激活蛋白-1（AP-1）信号通路在PQ诱导小胶质细胞NOD样受体热蛋白结构域相关蛋白（NLRP3）炎症小体活化中的作用。
方法 建立体外BV-2小胶质细胞模型，采用0、0.03、0.06、0.12 μmol·L−1 PQ处理细胞24 h后提取细胞全蛋白，通过免疫印迹法测定JNK、AP-1组成蛋白（c-Jun、c-Fos）、NLRP3、含胱天蛋白酶募集结构域凋亡相关斑点蛋白（ASC）、胱天蛋白酶-1前体（pro caspase-1）、白介素-18（IL-18）、白介素-1β（IL-1β）的相对表达水平，以观察PQ暴露对JNK/AP-1信号通路和NLRP3炎症小体的影响。通过20 μmol·L−1 JNK抑制剂SP600125干预后，再次检测上述蛋白以探究JNK/AP-1信号通路对NLRP3炎症小体活化的驱动作用。
结果 PQ暴露后，0.06 μmol·L−1 PQ组和0.12 μmol·L−1 PQ组JNK、c-Jun、c-Fos、NLRP3、ASC、pro caspase-1的表达水平高于0 μmol·L−1 PQ组（
P ＜0.05），炎症因子（IL-18、IL-1β）的相对表达水呈逐渐增加（P ＜0.05）。经JNK抑制剂SP600125干预后，对照组、20 μmol·L−1 SP600125组、20 μmol·L−1 SP600125+0.06 μmol·L−1 PQ组JNK/AP-1信号通路关键蛋白、NLRP3炎症小体关键蛋白及IL-18、IL-1β的相对表达水平低于0.06 μmol·L−1 PQ组（P ＜0.05）。结论 PQ暴露能够激活JNK/AP-1信号通路驱动BV-2小胶质细胞NLRP3炎症小体激活介导神经炎症反应。
Abstract:Background Paraquat (PQ) is one of the environmental factors that can cause sporadic Parkinson's disease (PD). Microglia-mediated neuroinflammation plays an important role in the occurrence and development of PD. Our previous studies have found that low doses of PQ can activate BV-2 microglia to the M1 phenotype and exert pro-inflammatory effects, but the associated mechanism is not clear yet.
Objective To explore the role of c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) signaling pathway in PQ-induced activation of the NOD-like receptor thermal protein domain associated protoin 3 (NLRP3) inflammasome in microglia.
Methods An
in vitro microglia model was established. The cells were treated with 0, 0.03, 0.06,and 0.12 μmol·L−1 PQ for 24 h, the whole cell protein was extracted. The relative expression levels of JNK, AP-1 constituent proteins (c-Jun, c-Fos), NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspasse-1 precursor (pro caspase-1), interleukin-18 (IL-18), and interleukin-1β (IL-1β) were evaluated by Western blotting, to observe the effects of PQ exposure on JNK/AP-1 signaling pathway and NLRP3 inflammasome. After the treatment of 20 μmol·L−1 JNK inhibitor SP600125, the above proteins were detected again, to explore the driving effect of JNK/AP-1 signaling pathway on NLRP3 inflammasome activation.Results After PQ exposure, the relative expression levels of key proteins of JNK, c-Jun, and c-Fos, NLRP3, ASC, and pro caspase-1 in the 0.06 μmol·L−1 PQ group and the 0.12 μmol·L−1 PQ group were higher than those in the 0 μmol·L−1 PQ group (
P ＜0.05), and the relative expression levels of IL-18 and IL-1β increased with higher exposure (P ＜0.05). After the treatment of JNK inhibitor SP600125, the relative expression levels of key proteins of JNK/AP-1 signaling pathway (JNK, c-Jun, and c-Fos), NLRP3 inflammasome (NLRP3, ASC, and Pro caspase-1), and inflammatory factors (IL-18 and IL-1β) in the control group, the 20 μmol·L−1 SP600125 group, and the 20 μmol·L−1 SP600125+0.06 μmol·L−1 PQ group were lower than those in the 0.06 μmol·L−1 PQ group (P ＜0.05).Conclusion PQ exposure can activate the JNK/AP-1 signaling pathway and subsequently drive the activation of NLRP3 inflammasome in BV-2 microglia to mediate neuroinflammatory responses..
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