摘要
目的研究丙戊酸钠(VPA)诱发HAPI小胶质细胞炎症反应的可能机制。方法体外培养大鼠HAPI小胶质细胞,分别给予(0、0.25、0.5、1、2.5、5)mmol/L VPA处理24 h。显微镜观察各组细胞生长及形态变化,CCK-8法检测细胞存活率,ELISA检测各组细胞培养上清液中肿瘤坏死因子α(TNF-α)的含量。以2.5 mmol/L VPA作为最适浓度,Western blot法检测核因子κB抑制物(IκB)激酶α/β(IKKα/β)、磷酸化的IκB激酶(pIKKα/β)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)的蛋白表达,激光共聚焦显微镜技术观察NF-κB p65蛋白核转位情况。结果随着VPA浓度增高,HAPI细胞逐渐伸出丝状伪足,并最终激活形成阿米巴样形态,且HAPI细胞的存活率不断降低;当VPA的浓度增高至1 mmol/L后,小胶质HAPI细胞分泌的TNF-α明显高于正常对照组。2.5 mmol/L VPA处理的HAPI细胞NF-κB通路相关蛋白IKKα/β、pIKKα/β、TNF-α、IL-1β表达明显增高,同时伴随HAPI细胞中NF-κB p65蛋白表达由胞质中高表达转为主要在细胞核内表达。结论高浓度VPA通过激活HAPI小胶质细胞NF-κB通路,引起炎症反应。
Objective To investigate the possible mechanism of sodium valproate(VPA)inducing HAPI microglia inflammatory response.Methods HAPI microglia were cultured in vitro and treated with VPA of 0,0.25,0.5,1,2.5,5 mmol/L for 24 hours.The cell growth and morphological changes in each group were observed under inverted microscope,the cell viability determined by the CCK-8 assay,and the level of tumor necrosis factorα(TNF-α)in the cell culture supernatant of each group measured by ELISA.The protein expressions of IκB kinaseα/β(IKKα/β),phosphorylated IκB kinase(pIKKα/β),TNF-α,and interleukin-1β(IL-1β)were detected by Western blot with 2.5 mmol/L of VPA as the optimal concentration.The nuclear translocation of NF-κB p65 was observed by confocal laser scanning microscopy.Results With the increase of VPA concentration,HAPI cells gradually extended filopodia and finally became activated to form amoeboid microglia,and the viability of HAPI cells decreased continuously;when the concentration of VPA increased to 1 mmol/L,the TNF-αsecreted by HAPI microglia was significantly higher than that by cells in the normal control group.The expression levels of NF-κB pathway related proteins IKKα/β,pIKKα/β,TNF-α,and IL-1βin HAPI cells treated with 2.5 mmol/L of VPA were significantly increased,and the NF-κB p65 in those HAPI cells was highly expressed in the nucleus rather than in the cytoplasm.Conclusion High concentration of VPA induces HAPI microglia inflammatory response by activating NF-κB pathway.
作者
廖爱玲
王岩
兰俊英
杨戎
王莎莉
LIAO Ailing;WANG Yan;LAN Junying;YANG Rong;WANG Shali(Department of Physiology,School of Basic Medical Sciences,Chongqing Medical University,Chongqing 400016,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2021年第5期421-426,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81801506)。