摘要
目的探索不同M型胆碱能受体对脂多糖诱导的脓毒症小鼠模型调控炎症反应的可能机制。方法C57雄性小鼠随机分为正常对照组、LPS组、哌仑西平组和AF-DX116组。正常对照组小鼠在实验开始0h及0.5h均腹腔注射生理盐水;LPS组、哌仑西平组、AF-DX116组小鼠在实验开始0.5h均腹腔注射LPS制造小鼠脓毒症模型,分别在实验开始0h给予生理盐水、选择性M1型胆碱能受体阻滞剂哌仑西平、选择性M2型胆碱能受体阻滞剂AF-DX116预处理。分别在注射LPS后2h及12h处死小鼠,获取小鼠肝肺组织,采用流式细胞学技术检测组织巨噬细胞的极化情况;免疫荧光双染法检测巨噬细胞的极化情况;PCR法检测肝肺组织SOCS3基因的表达情况。结果流式细胞学结果提示,与LPS组比较,哌仑西平预处理后脓毒症小鼠肝肺组织M1型巨噬细胞比例降低,在肺脏12h组(3.00±0.25vs.3.98±0.38,P<0.01)、肝脏2h组(3.50±1.34vs.5.20±0.59,P<0.01)及肝脏12h组(4.50±0.42vs.5.54±0.52,P<0.05)中差异有统计学意义;而AF-DX116预处理后,可促进脓毒症小鼠模型巨噬细胞向M1型极化,其中在肺脏2h组(5.12±1.94vs.3.64±0.41,P<0.01)、肺脏12h组(4.63±1.07vs.3.98±0.38,P<0.05)及肝脏12h组(7.68±1.81vs.5.54±0.52,P<0.01)中差异有统计学意义。免疫荧光双染结果提示,哌仑西平预处理组小鼠肝脏及肺脏组织M1型巨噬细胞浸润程度较低,而AF-DX116预处理组小鼠肝肺组织M1型巨噬细胞浸润程度增加。PCR结果提示,与LPS组比较,选择性M1型胆碱能受体阻滞剂哌仑西平预处理可抑制脓毒症小鼠肝肺组织SOCS3分子基因的表达水平(肺脏2h组,4.91±0.35vs.5.69±0.64,P<0.01),而选择性M2型胆碱能受体阻滞剂AF-DX116预处理则作用相反(肺脏2h组,6.73±0.11vs.5.69±0.64,P<0.05)。结论不同的M型胆碱能受体亚型在巨噬细胞的极化过程中发挥了不同的作用。
Objective Sepsis is a life-threatening condition with high incidence and mortality of clinical emergency.We have showed previously that different antagonists of muscarinic cholinergic receptors(mAChR)have different effects on LPS-induced septic mice,but the possible mechanism is still unclear.Methods Male C57 mice,aged 6-8 weeks,were injected with LPS at a dose of 75 mg/kg to induce sepsis model,with or without pretreatment of M1 selective mAChR antagonist(pirenzepine)or M2 selective mAChR antagonist(AF-DX116).Liver and lung tissues were harvested and were made into single-cell suspension and flow cytometry(FCM)was used to detect M1 and M2 type of macrophages.Liver and lung tissues collected for double staining immunofluorescence were used to detect the different types of macrophages infiltrated in the tissues;and to.explore the possible mechanism,the mRNA level of SOCS3 was measured in the lung and liver of all those mice.ResultsLipopolysaccharides(LPS)treatment could improve both M1 and M2 type of macrophages in the lung and liver tissues.Pirenzepine pretreatment could reduce the ratio of M1 type of macrophages,the difference between the 12-hour lung treatment group(3.00±0.25 vs.3.98±0.38,P<0.01),the 2-hour liver treatment group(3.50±1.34 vs.5.20±0.59,P<0.01)and the 12-hour liver treatment group(4.50±0.42 vs.5.54±0.52,P<0.05)was statistically significant;while AF-DX116 pretreatment promoted the macrophages polarized to M1 subtype,including 2 hours in the lung treatment group(5.12±1.94 vs.3.64±0.41,P<0.01),12-hour lung treatment group(4.63±1.07vs.3.98±0.38,P<0.05)and 12-hour liver treatment group(7.68±1.81 vs.5.54±0.52,P<0.01).Those were also confirmed by the experiment of immunofluorescent double staining.To explore the mechanism that M1 and M2 mAChRs played in the polarization of macrophages,we detected the expression of SOCS3 mRNA,and we found that pirenzepine pretreatment could reduce the SOCS3 mRNA expression[2-hour lung treatment group(4.91±0.35 vs.5.69±0.64,P<0.01)],in the contrast,AF-DX116
作者
王真
刘璐
徐龙薇
Wang Zhen;Liu Lu;Xu Long-wei(Peking University Ninth School of Clinical Medicine,Beijing 100038,China)
出处
《中国急救医学》
CAS
CSCD
北大核心
2019年第5期480-485,共6页
Chinese Journal of Critical Care Medicine
关键词
脓毒症
碱能受体
巨噬细胞
极化
SOCS3
Sepsis
Muscarinic acetylcholine receptors
Macrophages
Polarization
SOCS3
