摘要
目的:探究牛结核分枝杆菌减毒株卡介苗(bacillus Calmette-Guérin,BCG)感染人单核-巨噬细胞THP-1后,S100钙结合蛋白A4(S100 calcium-binding protein A4,S100A4)通过磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路对BCG诱导的巨噬细胞自噬的调控作用。方法:采用S100A4小干扰RNA(S100A4 small interfering RNA,si-S100A4)转染或PI3K抑制剂LY294002预处理THP-1巨噬细胞,并经BCG感染,设置不同处理组和对照组,通过Western blot方法检测PI3K/AKT/mTOR信号通路相关蛋白和自噬相关蛋白[微管相关蛋白1轻链3-II(microtubule-associated protein 1 light chain 3-II,LC3-II)、自噬相关蛋白7(autophagy-related protein 7,Atg7)和beclin-1]的表达,mRFP-GFP-LC3自噬双标腺病毒方法检测自噬流,透射电子显微镜观察细胞中自噬体和自噬溶酶体数量变化。结果:与未感染组相比,BCG感染组中p-PI3K、p-AKT和p-mTOR蛋白水平显著降低(P<0.05)。与BCG感染组相比,si-S100A4+BCG组上述磷酸化蛋白水平显著升高(P<0.05),LC3-II、Atg7和beclin-1蛋白水平显著降低(P<0.01);LY294003+BCG组上述磷酸化蛋白水平显著降低(P<0.01),LC3-II、Atg7和beclin-1蛋白水平显著升高(P<0.05),自噬体和自噬溶酶体数量显著增多(P<0.01)。与si-S100A4+BCG组相比,si-S100A4+LY294002+BCG组中p-PI3K、p-AKT和p-mTOR蛋白水平显著降低(P<0.01),LC3-II、Atg7和beclin-1蛋白水平显著升高(P<0.01),自噬体和自噬溶酶体数量显著增多(P<0.01),胞内菌载量显著减少(P<0.01)。结论:干扰S100A4经由PI3K/AKT/mTOR信号通路对BCG诱导的巨噬细胞自噬具有抑制作用。
AIM:This study investigates the regulatory effect of S100 calcium-binding protein A4(S100A4)on bacillus Calmette-Guérin(BCG)-induced autophagy in human monocyte-macrophage THP-1 cells through phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/AKT)/mammalian target of rapamycin(mTOR)signaling pathway.METHODS:The THP-1 macrophages were transfected with S100A4 small interfering RNA(si-S100A4)or pretreated with PI3K inhibitor LY294002,followed by their infection with BCG.Different treatment and control groups were established.Western blot was carried out to assess the expression of PI3K/AKT/mTOR pathway-related proteins and autophagyrelated proteins,microtubule-associated protein 1 light chain 3-II(LC3-II),autophagy-related protein 7(Atg7)and beclin-1.The mRFP-GFP-LC3 double-labeling adenovirus method was employed to evaluate the autophagic flux.A transmission electron microscope was employed to observe the changes of autophagosomes and autolysosomes in cells.RESULTS:Compared with uninfected group,the protein levels of p-PI3K,p-AKT and p-mTOR in BCG-infected group were significantly decreased after BCG infection(P<0.05).Compared with BCG-infected group,the protein levels of p-PI3K,p-AKT and p-mTOR in si-S100A4+BCG group were increased(P<0.05),while those in LY294002+BCG group were significantly reduced(P<0.01).The protein levels of LC3-II,Atg7 and beclin-1 in si-S100A4+BCG group were reduced(P<0.01),while those in LY294002+BCG group were increased(P<0.05),and autophagosome and autolysosome numbers were increased(P<0.01).Compared with si-S100A4+BCG group,the levels of p-PI3K,p-AKT and p-mTOR were significantly down-regulated in si-S100A4+LY294002+BCG group(P<0.01),while those of LC3-II,Atg7 and beclin-1 were up-regulated(P<0.01),the numbers of autophagosomes and autolysosomes increased(P<0.01),and the intracellular bacterial load was reduced(P<0.01).CONCLUSION:Knockdown of S100A4 blocked BCG-induced autophagy in macrophages through the PI3K/AKT/mTOR signaling pathway.
作者
李梦媛
刘悦阳
聂雪伊
马伯利
杨易
徐金瑞
LI Mengyuan;LIU Yueyang;NIE Xueyi;MA Boli;YANG Yi;XU Jinrui(Key Laboratory of Ningxia Ethnomedicine Modernization,Ministry of Education,Ningxia Medical University,Yinchuan 750010,China;Key Laboratory of Conservation and Utilization of Characteristic Biological Resources in Western China,Ministry of Education,Ningxia University,Yinchuan 750021,China)
出处
《中国病理生理杂志》
北大核心
2025年第12期2348-2355,共8页
Chinese Journal of Pathophysiology
基金
宁夏自然科学基金资助项目(No.2023AAC03201,No.2023AAC03100)
宁夏医科大学校级项目特殊人才启动项目(No.XT2025038)
宁夏医科大学实验室主任基金项目(No.4303250034)。
