摘要
目的观察LPS耐受巨噬细胞自噬水平以及抑制内质网Ca 2+释放是否具有上调自噬水平和逆转LPS耐受的作用。方法分离Balb/c小鼠腹腔巨噬细胞,LPS 5 ng/ml诱导耐受4 h,随后给予LPS 100ng/ml刺激,ELISA检测TNF-α和IL-6水平,Western blot和免疫荧光检测自噬蛋白(Beclin-1和LC3-Ⅱ/Ⅰ)表达水平,Fluo-4 AM探针检测胞内Ca 2+浓度;在LPS刺激的同时给予胞内Ca 2+螯合剂(BAPTA)或内质网IP3通道阻断剂(2-APB)或CaMKⅡ抑制剂(KN-93),检测对自噬蛋白LC3-Ⅱ/Ⅰ表达以及TNF-α释放的影响。结果LPS耐受细胞经LPS再次刺激后,TNF-α、IL-6释放明显减少,自噬水平也显著下降,同时胞内Ca 2+水平异常增高;给予BAPTA或2-APB或KN-93能够降低胞内Ca 2+水平,上调LC3-Ⅱ/Ⅰ表达,增加TNF-α释放。结论LPS耐受的巨噬细胞自噬水平显著降低,抑制内质网Ca 2+通路是上调自噬水平并逆转LPS耐受的有效途径。
This study was designed to detect the autophagic level of LPS tolerant macrophage and explore whether the inhibition of endoplasmic reticulum(ER)Ca 2+release could enhance autophagy and reverse LPS tolerance.Peritoneal macrophages(PMs)were isolated from Balb/c mice and tolerated with 5 ng/ml LPS for 4 h and then stimulated with LPS 100 ng/ml.The release of TNF-αand IL-6 were detected by ELISA;the levels of LC3B and Beclin-1 were analyzed by immunofluorescence and Western blotting.Intracellular Ca 2+was detected by the Fluo-4 AM probe,while intracellular Ca 2+release was inhibited by chelator BAPTA,inhibitors 2-APB or KN-93.Data showed that the release of TNF-αand IL-6 were declined and autophagy was inhibited in LPS tolerance PMs.However,the intracellular Ca 2+was dramatically elevated.Treatment with BAPTA,2-APB or KN-93 reduced intracellular Ca 2+,up-regulated the level of LC3-Ⅱ/Ⅰand enhanced the release of TNF-αin LPS tolerance PMs.In summary,autophagy is impaired in LPS tolerant macrophages,while suppression of the ER Ca 2+dependent pathway may be an effective strategy to up-regulate autophagy and reverse LPS tolerance.
作者
吴嘉麒
刘鑫
王宁
郑江
WU Jiaqi;LIU Xin;WANG Ning;ZHENG Jiang(Medical Research Center,First Affiliated Hospital of Army Medical University,Chongqing 400038,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2020年第1期11-16,共6页
Immunological Journal
基金
国家自然科学基金（81772137,81601716）
关键词
LPS耐受
炎症因子
自噬
钙离子
LPS tolerance
Inflammatory cytokine
Autophagy
Calcium
