PKCα-NLRP3信号通路在小鼠急性肺损伤过度炎症反应中的作用机制  

Mechanism of PKCα-NLRP3 signaling pathway in excessive inflammato⁃ry response of acute lung injury in mice

作　　者：陈杨 施强强 金光勇 陈蕾妃 李玉苹[3] 顾南媛[1] CHEN Yang;SHI Qiangqiang;JIN Guangyong;CHEN Leifei;LI Yuping;GU Nanyuan(Department of Critical Care Medicine,Chengbei Branch of Hangzhou First People's Hospital(Hangzhou Geriatric Hospi-tal),Hangzhou 310000,China;Department of Respiratory Medicine,Affiliated Dongyang Hospital of Wenzhou Medical University,Dongyang 322100,China;Department of Respiratory and Critical Care Medicine,The First Affiliated Hospi-tal,Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]杭州市第一人民医院城北院区(杭州市老年病医院)重症医学科,浙江杭州310000 [2]温州医科大学附属东阳市人民医院呼吸内科,浙江东阳322100 [3]温州医科大学附属第一医院呼吸与危重症医学科,浙江温州325000

出　　处：《中国病理生理杂志》2023年第6期996-1004,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81970066);浙江省医药卫生科技计划项目(No.2020KY773)。

摘　　要：目的:研究细胞焦亡在脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)过度炎症反应中的作用,并探讨蛋白激酶Cα(PKCα)-核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路在细胞焦亡发生发展中的调节机制。方法:(1)雄性C57BL/6J小鼠经腹腔注射0.15 mg/kg钙磷蛋白C(calphostin C)干预PKCα表达,经气道内滴注LPS(10 mg/kg)构建ALI模型,设置对照组、calphostin C组、LPS组和LPS+calphostin C组,每组12只。检测各组小鼠支气管肺泡灌洗液(BALF)中炎症因子分泌和氧化应激活性,以及肺内细胞焦亡水平。(2)RAW264.7细胞分别用calphostin C(100 nmol/L)及活性氧(ROS)清除剂N-乙酰半胱氨酸(NAC;2.5 mmol/L)干预,再经1 mg/L LPS刺激建立细胞模型,设置对照组、LPS组、LPS+calphostin C组和LPS+NAC组。高内涵成像分析系统检测各组细胞内ROS水平,免疫荧光检测细胞硫氧还蛋白互作蛋白(TXNIP)表达量,Western blot检测PKCα-NLRP3信号通路相关蛋白的表达。结果:(1)气道内滴入LPS诱导小鼠BALF中白细胞介素1β(IL-1β)、IL-18和肿瘤坏死因子α(TNF-α)分泌水平显著升高,丙二醛(MDA)含量显著增加,超氧化物歧化酶(SOD)和谷胱甘肽(GSH)活性显著降低(P<0.01),肺组织中NLRP3炎症小体相关蛋白表达水平显著升高(P<0.01)。与LPS组相比,LPS+calphostin C组小鼠BALF中炎症因子水平显著降低(P<0.05),MDA含量显著减少,SOD和GSH活性显著增强(P<0.01),肺组织中NLRP3炎症小体相关蛋白表达水平显著降低(P<0.05)。(2)细胞模型中,与对照组相比,LPS组巨噬细胞ROS生成显著增加(P<0.01)。与LPS组相比,LPS+calphostin C组和LPS+NAC组巨噬细胞ROS水平显著降低(P<0.01),TXNIP表达下调(P<0.01),NLRP3、cleaved caspase-1和含caspase募集结构域的凋亡相关斑点样蛋白(ASC)蛋白水平降低,IL-1β和IL-18分泌减少(P<0.01)。结论:PKCα通过介导TXNIP-NLRP3信号通路而调控细胞焦亡过程,从而参与小鼠ALI中的过度炎症反应。AIM:To investigate the role of pyroptosis in excessive inflammatory response of lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice,and to explore the regulatory mechanism of protein kinase Cα(PKCα)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway in pyroptosis.METHODS:(1)Male C57BL/6J mice were intraperitoneally injected with calphostin C(0.15 mg/kg)to interfere the expression of PKCα,and the ALI model was established by intratracheal instillation of LPS(10 mg/kg).The mice were ran‐domly divided into 4 groups:control group,calphostin C group,LPS group and LPS+calphostin C group(n=12 per group).The secretion of inflammatory cytokines and oxidative stress activity in bronchoalveolar lavage fluid(BALF)as well as pyroptosis-related protein expression levels in lung tissue were detected.(2)In vitro,RAW264.7 cells were pre‐treated with 100 nmol/L calphostin C or 2.5 mmol/L N-acetylcysteine[NAC;a reactive oxygen species(ROS)scaven‐ger],and then were stimulated by 1 mg/L LPS for 6 h.Control group,LPS group,LPS+calphostin C group and LPS+NAC group were set.The intracellular ROS levels were assessed using high-content imaging analysis system,the thioredoxin-in‐teracting protein(TXNIP)expression was detected by immunofluorescence staining,and the PKCα-NLRP3 signaling path‐way-related protein expression was measured by Western blot.RESULTS:(1)Intratracheal instillation of LPS resulted in increased secretion of interleukin-1β(IL-1β),IL-18 and tumor necrosis factor-α(TNF-α),increased content of malondi‐aldehyde(MDA),and decreased activity of superoxide dismutase(SOD)and glutathione(GSH)in BALF(P<0.01).Besides,increased expression of NLRP3 inflammasome-related proteins in lung tissues was found in LPS group(P<0.01).Compared with LPS group,the levels of inflammatory cytokines,MDA content and NLPR3 inflammasome-related proteins expression were down-regulated in LPS+calphostin C group(P<0.05).Furthermore,calphostin C significantly enhanced activity of antioxid

关 键 词：PKCα-NLRP3信号通路 肺损伤 炎症 

分 类 号：R363.21[医药卫生—病理学] R563.1[医药卫生—基础医学]