氯氰菊酯通过抑制Nrf2/ARE信号通路诱导原代C57BL/6小鼠大脑皮层神经元损伤

Cypermethrin induces cell injury in primary cortical neurons of C57BL/6 mice by inhibiting Nrf2/ARE signaling pathway

目的探讨Nrf2/ARE信号通路在CYP诱导的小鼠大脑皮层神经元损伤中的作用及其机制。方法原代培养并鉴定C57BL/6小鼠大脑皮层神经元细胞,建立CYP诱导细胞损伤模型。将不同剂量的CYP(0、25、50、100μmol/L)分别暴露细胞48 h。CCK-8分析CYP对细胞活性的影响、荧光探针DCFH-DA检测细胞内活性氧、流式细胞术检测细胞凋亡率,q PCR及Western blotting检测Nrf2及其下游基因HO-1、NQO1的m RNA和蛋白表达。结果与0μmol/L组相比,CYP各剂量组神经元活性均受抑制,随着CYP剂量增加,神经元细胞活性逐渐下降、细胞内ROS随之增加、细胞凋亡率逐渐升高,且CYP剂量越高,神经元损伤程度越严重。与0μmol/L组相比,CYP 50μmol/L组HO-1、NQO1,CYP 100μmol/L组HO-1、NQO1、Nrf2 m RNA及蛋白表达下调(P<0.01)。结论 CYP暴露抑制Nrf2/ARE信号通路,下调Nrf2及其下游基因HO-1、NQO1 m RNA和蛋白表达,诱导神经元细胞氧化损伤和凋亡,CYP对神经元的毒性呈现剂量效应关系。

Objective To study the role of Nrf2/ARE signaling pathway in cypermethrin-induced oxidative stress and apoptosis of cerebral cortex neurons in C57 BL/6 mice. Methods The cortical neurons of C57 BL/6 mice were cultured and identified, and a cypermethrin-induced cell injury model was established by treating the cells with 0, 25, 50 and 100 μmol/L of cypermethrin for 48 h. CCK-8 assay was used to analyze the effects of cypermethrin on the cell viability, and the fluorescence probe DCFH-DA was used for detecting intracellular reactive oxygen species(ROS);flow cytometry was performed for determining the apoptosis rate of the cells. The m RNA and protein expression levels of Nrf2 and its downstream genes HO-1 and NQO1 were detected using q PCR and Western blotting. Results Exposure to cypermethrin at different doses inhibited the viability of the cultured cortical neurons. With the increase of cypermethrin dose, the viability of the neurons decreased progressively, the intracellular ROS and the cell apoptosis rate increased, and the neuronal injury worsened. At the dose of 50 and 100 μmol/L,cypermethrin significantly down-regulated the expressions of HO-1, NQO1 and Nrf2 at both the m RNA and protein levels in the cells(P<0.01). Conclusion Cypermethrin exposure shows a dose-dependent neurotoxicity by inhibiting Nrf2/ARE signaling pathway, down-regulating the expression of Nrf2 and its downstream genes HO-1, NQO1 m RNA and protein, and inducing oxidative damage and apoptosis in primary mouse cortical neurons,.