南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (8): 1158-1164.doi: 10.12122/j.issn.1673-4254.2021.08.05

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原花青素B2通过调控P13K/Akt/Nrf2信号通路减轻氯氰菊酯所致神经元损伤

周礼华，常见荣，高扬丽，王朝凯   

  1. 蚌埠医学院公共卫生学院，科研中心，安徽 蚌埠 233030
  • 出版日期:2021-08-20 发布日期:2021-09-07

Procyanidin B2 protects neurons from cypermethrin-induced oxidative stress through the P13K/Akt/Nrf2 signaling pathway

ZHOU Lihua, CHANG Jianrong, GAO Yangli, WANG Chaokai   

  1. School of Public Health, Scientific Research Center, Bengbu Medical College, Bengbu 233030, China
  • Online:2021-08-20 Published:2021-09-07

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摘要： 目的 探讨原花青素B2（PCB2）是否通过调控P13K/Akt/Nrf2信号通路保护氯氰菊酯（CYP）导致的神经元氧化损伤。方 法 原代培养C57BL/6小鼠大脑皮层神经元，将细胞随机分成5组：正常对照组；PCB2处理组（将5 μg/mL的PCB2与细胞共培养24 h）；CYP暴露组（将50 μmol/L的CYP与细胞共培养24 h）；PCB2预处理组（将5 μg/mL的PCB2 与细胞预处理30 min后加入CYP 50 μmol/L共培养24 h）；LY294002处理组（先将20 μmol/L的 LY294002与细胞预处理30 min，然后加入PCB2预处理30 min，后与CYP共培养24 h）。采用CCK-8分析各组细胞活性，采用荧光探针DCFH-DA及流式细胞术检测细胞内ROS水平，通过Hoechst 33342观察细胞核形态变化，采用JC-1检测线粒体膜电位异常，利用Western blot检测Nrf2、HO-1、p-Akt、Akt蛋白表达。结果 CYP暴露降低细胞活性（P<0.001），引起线粒体膜电位下降、促使细胞核出现固缩、浓染等凋亡特征，导致ROS产生异常。PCB2预处理提高细胞存活率（P=0.006），改善细胞核形态异常，逆转CYP导致的线粒体膜电位下降，减弱细胞内ROS产生。CYP暴露引起Nrf2核易位，Nrf2、HO-1、p-Akt蛋白表达上调（P<0.001）。PCB2预处理调节Nrf2、HO-1、p-Akt蛋白过度表达。抑制P13K/Akt信号通路消除了PCB2对CYP诱导损伤的保护作用（P<0.05）。结论 PCB2通过激活P13K/Akt信号通路，调控Nrf2/ARE通路，减轻CYP所致小鼠大脑皮层神经元氧化损伤。

关键词: 氯氰菊酯；原花青素B2；神经元；P13K/Akt/Nrf2信号通路

Abstract: Objective To explore whether procyanidin B2 (PCB2) regulates the P13K/Akt/Nrf2 signaling pathway to protect neurons from oxidative stress induced by cypermethrin (CYP). Methods Primary cultures of cerebral cortex neurons from C57BL/6 mice were randomly divided into 5 groups: normal control group (cultured in serum-free neurobasal-B27 medium), PCB2 treatment group (treated with 5 μg/mL PCB2 for 24 h), CYP exposure group (treated with 50 μmol/L CYP for 24 h), PCB2 pretreatment group (pretreated with 5 μg/mL PCB2 for 30 min followed by exposure to 50 μmol/L CYP for 24 h), and LY294002 treatment group (pretreated with 20 μmol/L LY294002 for 30 min before treatment with PCB2 for 30 min and then CYP for 24 h). CCK-8 assay was used to analyze the neuronal viability after the treatments. Reactive oxygen species (ROS) production in the cells was detected using the fluorescent probe DCFH-DA and flow cytometry. The changes in nuclear morphology and mitochondrial membrane potential of the cells were examined with Hoechst 33342 and JC-1 staining, respectively. Western blotting was performed to detect the protein expressions of Nrf2, HO-1, p-Akt and Akt in the cells. Results In CYP exposure group, the cells showed significantly decreased viability and mitochondrial membrane potential with obvious apoptotic morphological changes and abnormal ROS production. By comparison, the cells in PCB2 preconditioning group showed improved cell survival rate, reduced abnormalities in nuclear morphology, increased mitochondrial membrane potential, and lowered intracellular ROS production. CYP exposure caused Nrf2 nuclear translocation and up-regulated Nrf2, HO-1, p-Akt protein expressions in the cells, which were inhibited by PCB2 pretreatment. Inhibition of the P13K/Akt signaling pathway obviously neutralized the protective effect of PCB2 against CYP- induced neuronal injury. Conclusions PCB2 regulates the Nrf2/ARE signaling pathway by activating the P13K/Akt signaling pathway to protect mouse cerebral cortical neurons against oxidative injury induced by cypermethrin.

Key words: cypermethrin; procyanidin B2; neurons; P13K/Akt/Nrf2 signaling pathway