摘要
目的:探讨脂多糖(LPS)联合z-VAD-FMK介导M1亚型巨噬细胞程序性坏死的机制。方法:使用佛波酯(PMA)和干扰素γ(IFN-γ)诱导THP-1细胞系分化得到M0和M1亚型巨噬细胞。以LPS(100μg/L)分别处理M0和M1巨噬细胞,检测不同时点的乳酸脱氢酶释放和DNA断裂情况,并观察不同抑制剂的影响。采用Western blot法检测受体相互作用蛋白(RIP)1、RIP3、细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、P38和NOD样受体蛋白3(NLRP3)的蛋白水平。结果:LPS能够诱导M1亚型巨噬细胞发生死亡,联合z-VAD-FMK可特异性介导M1亚型巨噬细胞发生程序性坏死,而对M0亚型巨噬细胞无此作用。IFN-γ能够上调RIP3表达,并增强LPS介导的JNK磷酸化,RIP3和JNK抑制剂可部分阻断LPS联合z-VAD-FMK介导的M1亚型巨噬细胞程序性坏死。结论:IFN-γ上调RIP3和增强LPS介导的JNK磷酸化,使得LPS联合z-VAD-FMK能特异性诱导经过IFN-γ预处理的巨噬细胞发生程序性坏死。
AIM:To explore the mechanism of necroptosis in M1 macrophages mediated by lipopolysaccharide(LPS)combined with z-VAD-FMK.METHODS:THP-1 cells were induced to differentiate into M0 and M1 macrophages with phorbol 12-myristate 13-acetate(PMA)and interferon-γ(IFN-γ).The release of lactate dehydrogenase(LDH)and TUNEL-positive cells at different time points after LPS(100μg/L)treatment were detected,and the effects of different inhibitors were observed.The protein levels of receptor-interacting protein(RIP)1,RIP3,extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),P38 and NOD-like receptor protein 3(NLRP3)were determined by Wes-tern blot.RESULTS:LPS mediated cell death,and its combination with z-VAD-FMK specifically mediated necroptosis in M1 macrophages rather than M0 ones.The expression of RIP3 and NLRP3 was upregulated by IFN-γ,and LPS-mediated phosphorylation of JNK was also enhanced by IFN-γ.The inhibitors against RIP3 and JNK partly blocked LDH release mediated by LPS combined with z-VAD-FMK.CONCLUSION:Combination of LPS and z-VAD-FMK mediates necroptosis in IFN-γ-pretreated macrophages possibly by upregulation of RIP3 and enhancement of LPS-mediated JNK phosphorylation.
作者
章述军
肖晴
黄文祥
ZHANG Shu-jun;XIAO Qing;HUANG Wen-xiang(Department of Infectious Diseases,The First Affiliated Hospital of Chongqing Medical University,Chongqing Key Laboratory of Infectious Diseases and Parasitic Diseases,Chongqing 400016,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第9期1653-1659,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81300317)。
