摘要
目的探究组织蛋白酶B(CTSB)介导NLRP3小体在砷致小胶质细胞(BV-2)炎症激活中的作用。方法取处于对数生长期的BV-2细胞,分别暴露于终浓度为0、2、4、8μmol/L亚砷酸钠(NaAsO_(2))溶液培养24 h,检测细胞活性,测定各组细胞内CTSB和细胞焦亡相关蛋白NLRP3、Caspase-1、IL-18、IL-1β的表达水平。流式细胞仪检测胞内溶酶体膜稳定性。基于实验结果,增设CTSB抑制剂组(5μmol/L CA074-Me+8μmol/L NaAsO_(2)、10μmol/L CA074-Me+8μmol/L NaAsO_(2)),检测两组细胞内炎症相关蛋白NLRP3、Caspase-1和IL-1β、IL-18的表达水平。结果与对照组比较,各染砷组细胞抑制率增高,呈现剂量效应关系,溶酶体膜稳定性下降,差异有统计学意义(P<0.01),胞内CTSB、NLRP3、IL-1β、IL-18、Caspase-1表达增高,差异有统计学意义(P<0.01);与对照组(8μmol/L NaAsO_(2))比较,抑制剂组BV-2细胞胞内CTSB、NLRP3、IL-1β、IL-18、Caspase-1水平均降低,差异有统计学差异(P<0.01)。结论NaAsO_(2)通过诱导小胶质细胞内CTSB水平的上升,介导NLRP3炎症小体激活小胶质细胞,促其释放炎性因子,致神经系统损伤。
Objective To explore the role of CTSB(cathepsin B)-mediated NLRP3 bodies in arsenic-induced inflammatory activation of mouse microglial BV-2 cell.Methods BV-2 cells in logarithmic growth phase were respectively exposed to NaAsO_(2) solution at the final concentration of 0,2,4 and 8μmol/L for 24 h.Cell viability was detected by CCK-8 assay.The levels of intracellular CTSB,NLRP3,Caspase-1,IL-18,and IL-1βwere determined by Western blot.The intracellular lysosome level was detected by flow cytometry analysis.According to the experimental results,CTSB inhibitor group(5μmol/L CA074-Me+8μmol/L NaAsO_(2),10μmol/LCA074-Me+8μmol/L NaAsO_(2))was added to detect the expression of NLRP3,Caspase-1,IL-1βand IL-18 in the two groups.Results Compared with the control group,the cell inhibition rate of all exposed groups increased in a dose-dependent manner,the level of lysosome integrity decreased,the difference was statistically significant(P<0.01).The expression of CTSB,NLRP3,IL-1βand IL-18,Caspase-1 increased significantly,the difference was statistically significant(P<0.01).Compared with the inhibitor group,the intracellular CTSB,NLRP3,IL-1βand IL-18,Caspase-1 levels of BV-2 cells exposed to 8μmol/L NaAsO_(2) without inhibitor were significantly higher(P<0.01).Conclusion NaAsO_(2) can induce the increase of CTSB level in microglia,mediate the activation of microglia by NLRP3 inflammatory bodies,promote the release of inflammatory factors and damage the central nervous system.
作者
张哲昱
皮若铮
刘吉
方惠民
杨丹
孙宝飞
ZHANG Zhe-Yu;PI Ruo-Zheng;LIU Ji;FANG Hui-Min;YANG Dan;SUN Bao-Fei(Department of Human Anatomy,College of Basic Medicine,Guizhou Medical University,Guiyang 550000,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2022年第11期2215-2223,共9页
Progress In Biochemistry and Biophysics
基金
贵州省卫健委(gzwkj2021-432)
贵州医科大学国基培育项目(20NSP017)
贵州省大学生创新创业训练计划项目(202110660002,S202010660072)资助。
