千层纸素A调控胆碱能抗炎通路抑制结核分枝杆菌致巨噬细胞焦亡  

Oroxylin A regulates cholinergic anti-inflammatory pathway to inhibit Mycobacterium tuberculosis-induced macrophage pyroptosis

作　　者：张贺敏 沈晶晶 刘芳琳 吴鉴超 刘亚奇 姜昕[1] Zhang Hemin;Shen Jingjing;Liu Fanglin;Wu Jianchao;Liu Yaqi;Jiang Xin(Department of Immunology and Pathogenic Biology,School of Integrative Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学中西医结合学院免疫学与病原生物学教研室,上海201203

出　　处：《中华微生物学和免疫学杂志》2024年第5期423-430,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(81873069)。

摘　　要：目的探讨千层纸素A(oroxylin A,OrA)对结核分枝杆菌感染诱导的巨噬细胞焦亡的作用及分子机制。方法构建结核分枝杆菌感染J774A.1细胞体外模型;MTT法检测OrA对J774A.1细胞活力的影响;乳酸脱氢酶(lactate dehydrogenase,LDH)检测试剂盒检测细胞LDH释放水平;Western blot检测细胞焦亡相关蛋白NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、GSDMD-N、caspase1-p20、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、α7烟碱乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7nAChR)蛋白质表达水平及NF-κB p65的磷酸化。ELISA检测各组细胞IL-1β表达水平;免疫荧光检测α7nAChR蛋白及定位。结果MTT结果表明OrA在80μmol/L浓度范围内对J774A.1细胞无细胞毒性;OrA抑制J774A.1细胞LDH的释放,降低细胞焦亡相关蛋白NLRP3、GSDMD-N、caspase1-p20的蛋白质表达水平以及ASC寡聚化,促进α7nAChR蛋白表达,并抑制NF-κB p65磷酸化和IL-1β的释放;加入α7nAChR激动剂PNU282987可降低GSDMD-N并抑制NF-κB p65磷酸化。结论OrA可能通过调控胆碱能抗炎通路抑制结核分枝杆菌感染诱导的巨噬细胞焦亡。Objective To investigate the effect of oroxylin A(OrA)on macrophage pyroptosis induced by Mycobacterium tuberculosis(Mtb)infection and the underlying molecular mechanism.Methods An in vitro infection model was constructed by infecting J774A.1 cells with Mtb.The MTT method was used to detect the effect of OrA on the viability of J774A.1 cells.Lactate dehydrogenase(LDH)assay kit was used to detect the release of LDH by J774A.1 cells.Western blot was used to detect the protein levels of pyroptosis-related proteins such as NOD-like receptor thermal protein domain associated protein 3(NLRP3),GSDMD-N,caspase1-p20,apoptosis-associated speck-like protein containing a CARD(ASC),andα7 nicotinic acetylcholine receptor(α7nAChR)as well as the phosphorylation of NF-κB p65.ELISA was used to detect the levels of IL-1βin each group.Immunofluorescence was performed to detect the expression and localization ofα7nAChR.Results MTT results showed that OrA had no cytotoxicity to J774A.1 cells at concentrations below 80μmol/L.OrA reduced the release of IL-1β,down-regulated the expression of NLRP3,GSDMD-N and caspase1-p20 at protein level,inhibited ASC oligomerization,promoted the expression ofα7nAChR at protein level,and inhibited the phosphorylation of NF-κB p65.In the presence ofα7nAChR agonist PNU282987,the protein levels of GSDMD-N decreased and the phosphorylation of NF-κB p65 was inhibited.Conclusions OrA may inhibits Mtb infection-induced macrophage pyroptosis through regulating cholinergic anti-inflammatory pathway.

关 键 词：细胞焦亡 千层纸素A 结核分枝杆菌 宿主导向疗法 α7烟碱乙酰胆碱受体 

分 类 号：R515[医药卫生—内科学]