Pannexin-1半通道调控NLRP3/Caspase-1介导高血压患者外周血单核细胞焦亡  

Pannexin⁃1 hemi⁃channels mediate pyroptosis on peripheral blood monocytes in patients with essential hypertension via regulating NLRP3/Caspase⁃1 pathway

作　　者：朱建[1] 杨燕[2] 徐淑楠 高娜 康品方[1] 王洪巨[1] ZHU Jian;YANG Yan;XU Shu-nan;GAO Na;KANG Pin-fang;WANG Hong-ju(Department of Cardiology the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Department of Medical Oncology the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Department of Clinical Laboratory the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China)

机构地区：[1]蚌埠医学院第一附属医院心血管内科,安徽蚌埠233004 [2]蚌埠医学院第一附属医院肿瘤内科,安徽蚌埠233004 [3]蚌埠医学院第一附属医院检验科,安徽蚌埠233004

出　　处：《海南医学院学报》2023年第10期738-745,共8页Journal of Hainan Medical University

基　　金：国家自然科学基金面上项目(81970313);安徽省自然科学基金面上项目(2008085MH238);蚌埠医学院“512人才培育计划”项目(by51201321);蚌埠医学院自然科学基金重点项目(2020byzd056)。

摘　　要：目的:探索原发性高血压(EH)患者外周血单核细胞上NLRP3/Caspase-1介导的细胞焦亡与疾病的关系及Pannexin-1(Panx-1)半通道对其的调控。方法:采集EH患者及健康受试者外周血,收集外周血浆,ELISA法检测外周血浆中IL-1β含量;免疫磁珠法分选单核细胞,并通过RT-qPCR、Western blot法测定外周血单核细胞上Panx-1、NLRP3炎症体相关分子、效应分子IL-1β及焦亡蛋白GSDMD的表达。体外培养两组人外周血单核细胞,使用免疫荧光法检测Panx-1的表达及定位。在体外培养的EH患者单核细胞上,使用Panx-1半通道抑制剂丙磺舒及特异性siRNA进行预处理,再给予NLRP3炎症体通路激活剂脂多糖(LPS)活化细胞,CCK-8法检测细胞活力,ELISA法分析培养上清中IL-1β含量,Western blot法检测单核细胞上各目的蛋白表达变化。结果:与健康受试组相比,EH组患者外周血浆中IL-1β含量升高;外周血单核细胞上Panx-1、NLRP3炎症体相关分子及GSDMD的mRNA及蛋白表达量上调;单核细胞上Panx-1蛋白表达增强且主要定位于细胞膜。在体外培养的EH患者单核细胞上,不同干预处理对细胞活力无显著影响。LPS可致细胞培养上清中IL-1β含量升高,单核细胞上NLRP3炎症体相关分子及GSDMD的蛋白表达量上调;而经丙磺舒及Panx-1 siRNA预处理细胞后,LPS上述活化效应被拮抗。结论:EH患者外周血单核细胞上NLRP3/Caspase-1的活化介导了单核细胞焦亡,且其活化受Panx-1半通道调控。Objective:To explore the role of NLRP3/Caspase-1-mediated pyroptosis on peripheral blood monocytes in patients with essential hypertension(EH),and its regulation by pannexin-1(panx-1)hemi-channels.Methods:The peripheral blood of EH patients and healthy subjects was collected,peripheral plasma of the two groups was subsequently separated,and IL-1βcontent in peripheral plasma was further measured by ELISA.In the meantime,the monocytes from peripheral blood were sorted by immunomagnetic beads,and mRNA and protein expression of Panx-1,NLRP3 inflammasome related molecules(NLRP3,ASC,Caspase-1),downstream effector IL-1β,and pyroptosis-related protein GSDMD,were analyzed by RT-qPCR and Western blot,respectively.Subsequently,human primary monocytes in two group were cultured in vitro.Immunofluorescence assay was performed to detect the expression and location of Panx-1 on monocytes.Finally,the cultured monocytes from EH patients were exposed to NLRP3 inflammasome activator lipopolysaccharide(LPS),and also to LPS pretreated with Panx-1 hemi-channel inhibitor probenecid or specific Panx-1 siRNA,followed by collection of the cell culture supernatant and monocytes.The cell viability of each group was detected by CCK-8 assay,IL-1βcontent in the culture supernatant was analyzed by ELISA,and the expression of target proteins on monocytes was determined by Western blot assay.Results:Compared with healthy subjects,EH patients showed higher IL-1βcontent in peripheral plasma,and increased expression in both mRNA and protein levels for Panx-1,NLRP3 inflammasome related molecules,IL-1βand GSDMD on peripheral monocytes.Furthermore,Panx-1 protein in EH patients was significantly higher than that in healthy subjects and localized on monocyte membrane as evidenced by immunofluorescence assay.In the cultured human primary monocytes from EH patients,the cell viability in each group showed no significant difference.LPS stimulation can induce NLRP3-dependent pyroptosis,with an increase in IL-1βcontent in cell culture supernatant and pro

关 键 词：原发性高血压 Pannexin-1 NLRP3炎症体 IL-1Β GSDMD 

分 类 号：R544.1[医药卫生—心血管疾病]