丙咪嗪经酸性鞘磷脂酶/神经酰胺通路抑制LPS诱导的RAW264.7细胞NLRP3炎症小体的激活  

Imipramine inhibits LPS-induced NLRP3 inflammasome activation through Cer/ASM pathway in RAW264.7 macrophages

作　　者：蒋建军 杨进[1] 金永梅 操基玉[2] 陆友金[1] Jiang Jianjun;Yang Jin;Jin Yongmei(Dept of Respiratory Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601)

机构地区：[1]安徽医科大学第二附属医院呼吸内科,合肥230601 [2]安徽医科大学公共卫生学院劳动卫生与环境卫生学系,合肥230032

出　　处：《安徽医科大学学报》2019年第7期1042-1046,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81400058);安徽省科技攻关计划项目(编号:1401045016)

摘　　要：目的研究丙咪嗪对脂多糖(LPS)诱导的小鼠单核巨噬细胞(RAW264.7)NLRP3炎症小体激活的影响并探讨其可能的作用机制。方法 CCK-8检测丙咪嗪对RAW264.7细胞活性的影响;用不同浓度丙咪嗪预先处理RAW264.7细胞2 h后再用LPS处理细胞8 h,Western blot法检测酸性鞘磷脂酶(ASM)、NLRP3、caspase-1、白细胞介素(IL)-1β和IL-18的蛋白表达水平;细胞免疫荧光法检测神经酰胺(Cer)的含量变化;ELISA法检测炎性因子IL-1β和IL-18的释放水平。结果与对照组相比,LPS(1 mg/L)能增加RAW264.7细胞ASM的蛋白表达和Cer的含量(P<0.01);上调细胞中NLRP3、caspase-1、IL-1β和IL-18的蛋白表达水平以及细胞上清液IL-1β和IL-18的释放水平(P<0.01)。而经丙咪嗪预先处理能够显著抑制LPS所致ASM和Cer的改变(P<0.01);可明显抑制细胞中NLRP3、caspase-1、IL-1β和IL-18的蛋白表达以及细胞上清液中IL-1β和IL-18的释放(P<0.05,P<0.01)。结论丙咪嗪可通过调控ASM/Cer通路,抑制NLRP3炎症小体的激活从而发挥抗炎活性。Objective To investigate the protective effect and possible mechanism of imipramine on LPS-induced NLRP3 inflammasome activation in RAW264.7 macrophages. Methods The effect of imipramine on RAW264.7 cells viability were detected by CCK-8 method. RAW264.7 cells were pretreated with different concentrations of imipramine for 2 hours and then treated with LPS for 8 hours. The protein expression of acid sphingomyelinase(ASM), NLRP3, caspase-1, interleukin-1β(IL-1β) and IL-18 were determined by Western blot;the content of ceramide(Cer) was detected by the method of cell immunofluorescence;the release levels of inflammatory cytokine IL-1β and IL-18 were measured by ELISA. Results Compared with the control group, LPS(1 mg/L) significantly increased ASM protein expression and the content of Cer in RAW264.7 cells(P<0.01);LPS(1 mg/L) significantly up-regulated the protein expression levels of NLRP3, caspase-1, IL-1β and IL-18 as well as the release levels of IL-1β and IL-18 in the supernatants(P<0.01). However, pretreatment with imipramine significantly inhibited ASM and Cer changes induced by LPS(P<0.01);It significantly inhibited the protein expression levels of NLRP3, caspase-1, IL-1β and IL-18 as well as the release levels of IL-1β and IL-18 in the supernatants(P<0.05, P<0.01). Conclusion Imipramine exerts anti-inflammatory activity by inhibiting the activation of NLRP3 inflammasome through the regulation of ASM/Cer pathway.

关 键 词：丙咪嗪 酸性鞘磷脂酶 神经酰胺 NLRP3炎症小体 炎症 巨噬细胞 

分 类 号：R3[医药卫生—基础医学]