氯菊酯对人小胶质细胞HMC3的毒性效应及潜在机制  

Toxic effects of permethrin on HMC3 microglia and its associated mechanism

作　　者：张婉莉 单文琪 陈超 董昊炜 袁浩 周秋明 陶峰 彭恒 马雅军 ZHANG Wanli;SHAN Wenqi;CHEN Chao;DONG Haowei;YUAN Hao;ZHOU Qiuming;TAO Feng;PENG Heng;MA Yajun(Faculty of Naval Medicine,Naval Medical University,Shanghai 200433,China;College of Basic Medicine,Naval Medical University,Shanghai 200433,China)

机构地区：[1]海军军医大学海军医学系,上海200433 [2]海军军医大学基础医学院,上海200433

出　　处：《环境与职业医学》2024年第3期267-275,共9页Journal of Environmental and Occupational Medicine

基　　金：国家重点研发计划项目(2022-3.3);上海市2022年度“科技创新行动计划”启明星培育项目(22YF1458700)。

摘　　要：[背景]氯菊酯是一种常用的拟除虫菊酯类杀虫剂,研究发现其具有潜在神经系统毒性。小胶质细胞是中枢神经系统中的天然免疫细胞,参与一系列神经退行性疾病的发生。[目的]本研究观察氯菊酯在体外对人小胶质细胞HMC3的毒性效应,并探讨其机制。[方法]使用0、10、25、55μmol·L^(-1)的氯菊酯染毒HMC372 h后,使用流式细胞仪检测细胞周期和细胞凋亡,实时荧光定量PCR(qPCR)检测细胞周期蛋白依赖性激酶1基因(CDK1)、细胞周期蛋白依赖性激酶抑制因子1A基因(CDKN1A)、细胞周期蛋白B2基因(CCNB2)、肿瘤蛋白p53基因(p53)、凋亡相关因子基因(FAS)、胱天蛋白酶3基因(CASP3)和H2A变体组蛋白基因(H2AX)的表达。转录组测序(RNA-seq)检测0、25μmol·L^(-1)氯菊酯染毒后HMC3的差异基因和富集通路。再次使用0、10、25、55μmol·L^(-1)的氯菊酯染毒HMC372 h后,使用格里斯试剂法检测上清中一氧化氮(NO)含量,酶联免疫吸附法检测白细胞介素(IL)-6的分泌水平,qPCR检测丝裂原活化蛋白激酶(MAPK)通路(包括MAPK1、MAPK8、MAPK14)、IL-1β、IL-6和基质金属蛋白酶(MMP)家族(包括MMP1、MMP2、MMP3和MMP9)的m RNA表达情况,蛋白质印记法(Western blot)检测磷酸化p38(p-p38)、磷酸化细胞外信号调节激酶(p-ERK)、IL-1β、IL-6和MMP1蛋白表达情况。[结果]0、10、25、55μmol·L^(-1)氯菊酯染毒细胞后,HMC3在G2/M期阻滞,其中55μmol·L^(-1)氯菊酯染毒组与对照组差异有统计学意义(P<0.01),qPCR结果显示CDKN1A mRNA表达较对照组上调(P<0.05)。各组细胞凋亡比例差异无统计学意义(P>0.05)。RNA-seq结果提示差异基因富集于MAPK通路。q PCR结果提示55μmol·L^(-1)染毒组MAPK1、MAPK8和MAPK14的m RNA表达较对照组上调(P<0.05)。Western blot发现,与对照组相比,10μmol·L^(-1)氯菊酯染毒组的p-p38和p-ERK水平均升高(P<0.05),25μmol·L^(-1)氯菊酯染毒组的p-ERK水平升高(P<0.05),55μmol·L^(-1)氯菊酯染�[Background]Permethrin is a commonly used pyrethroid insecticide and has been found to be potentially neurotoxic.Microglia are innate immune cells in the central nervous system and are involved in the development of a range of neurodegenerative diseases.[Objective]To observe possible toxic effects of permethrin on human microglia clone 3(HMC3)in vitro and explore associated mechanism.[Methods]HMC3 were treated with 0,10,25,and 55μmol·L^(-1)permethrin for 72 h.Cell cycle and apoptosis were measured using flow cytometry.Cyclin-dependent kinase 1(CDK1),cyclin-dependent kinase inhibitor 1A(CDKN1A),cyclin B2(CCNB2),cellular tumor antigen p53(p53),factor-related apoptosis(FAS),caspase 3(CASP3),and H2A histone family member X(H2AX)were detected by quantitative real-time PCR(qPCR).The differential genes and enrichment pathways of HMC3 after 0 and 25μmol·L^(-1)permethrin treatment was analyzed by RNA sequencing.HMC3 was treated by 0,10,25,and 55μmol·L^(-1)permethrin for 72 h.The content of nitric oxide(NO)in the supernatant was detected using Griess reagent.The secretion level of interleukin-6(IL-6)was detected by enzyme linked immunosorbent assay(ELISA).The mRNA expression levels of mitogen-activated protein kinase(MAPK)pathway(including MAPK1,MAPK8,and MAPK14),interleukin-1β(IL-1β),IL-6,and matrix metalloproteinase(MMP)families(including MMP1,MMP2,MMP3,and MMP9)were detected by qPCR.The protein expressions of phosphorylated p38 mitogen-activated protein kinase(p-p38),phosphorylated extracellular signal-regulated kinase(p-ERK),IL-1β,IL-6,and MMP1 were detected by Western blot.[Results]HMC3 was arrested in G2/M phase after 0,10,25,and 55μmol·L^(-1)permethrin treatment for 72 h,of which there was a statistically significant difference between the 55μmol·L^(-1)permethrin treatment group and the control group(P<0.01),and the mRNA expression of CDKN1A was up-regulated according to the qPCR(P<0.05).There was no statistically significant difference in the proportions of apoptosis between the groups(P>0.05).The RNA

关 键 词：氯菊酯 小胶质细胞 白细胞介素-1Β 基质金属蛋白酶 丝裂原活化蛋白激酶 炎症反应 

分 类 号：R135.1[医药卫生—劳动卫生]