前蛋白转化酶枯草溶菌素9通过肝源性巨噬细胞移动抑制因子调控动脉粥样硬化炎症反应的研究  

Modulation of atherosclerotic inflammatory response by the preprotein convertase chymotrypsin 9 via hepatic-derived macrophage mobile inhibitory factor

作　　者：李文玲 刘芬[1] 张雪鹤 余小林[2] 张冀鑫 房彬彬[1] 张志扬 杨毅宁 高晓明[1] 李晓梅[1] LI Wen-ling;LIU Fen;ZHANG Xue-he;YU Xiao-lin;ZHANG Ji-xin;FANG Bin-bin;ZHANG Zhi-yang;YANG Yining;GAO Xiao-ming;LI Xiao-mei(Heart Center,the First Affiliated Hospital of Xinjiang Medical UniverSity,Urumqi,830054,China;Xinjiang Uygur Autonomous Region People’s Hospital cardiology department,Urumqi 830001)

机构地区：[1]新疆医科大学第一附属医院心脏中心,新疆维吾尔自治区乌鲁木齐市830054 [2]新疆维吾尔自治区人民医院心内科

出　　处：《中国心血管病研究》2023年第9期815-821,共7页Chinese Journal of Cardiovascular Research

基　　金：国家自然科学基金(82070368);新疆维吾尔自治区重点研发项目(2020B03002);新疆维吾尔自治区高校科研计划项目(XJEDU2021I015)。

摘　　要：目的利用人细胞株,系统研究PCSK9是否通过肝源性MIF调控单核/巨噬细胞炎症反应,进而为动脉粥样硬化(Atherosclerosis)等炎症性疾病的临床防控提供新的思路和依据。方法利用小干扰RNA(Small interfering RNA,SiRNA)介导的MIF基因转染THLE-3细胞,将实验分为MIF低表达组(Si-MIF-THLE-3)和MIF正常表达组(Con-THLE-3),通过实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)检测肝细胞中PCSK9、MIF mRNA表达。在Si-MIF-THLE-3组和Con-THLE-3组中分别给与PCSK9重组蛋白进行干预,通过Western Blot检测肝细胞中PCSK9、MIF蛋白表达,并用ELISA测定培养液中PCSK9及MIF的含量,并收集上述肝细胞的培养液,-80℃冻存。利用SiRNA介导的MIF基因转染THP-1细胞,将实验分为MIF低表达组(Si-MIF-THP-1)和MIF正常表达组(Con-THP-1),通过Western Blot检测MIF的蛋白表达。将收集的肝细胞培养液分别加入Si-MIF-THP-1和Con-THP-1组共培养48 h,其中PCSK9干预组另设TLR4特异性抑制剂TAK-242(1μM)(P+T)。通过ELISA检测炎症因子表达;Western Blot检测THP-1细胞株中TLR4—NF-κB信号通路的表达。结果①Si-MIF-THLE-3组较Con-THLE-3组的MIF mRNA表达显著降低,而PCSK9的mRNA表达升高(P均<0.05)。②PCSK9干预后肝细胞MIF蛋白的表达以及分泌至培养液中的含量升高,而PCSK9蛋白的表达以及培养液中的含量不变(P均<0.05)。③Si-MIF-THP-1组较Con-THP-1组的MIF蛋白表达显著降低(P均<0.05)。④Si-MIF-THP-1组较Con-THP-1组炎症因子的表达低,PCSK9干预组较正常组和P+T组的炎症因子表达升高;PCSK9干预组较正常组和P+T组的TLR4蛋白表达升高;Si-MIF-THP-1组较Con-THP-1组的IKBα蛋白表达高,PCSK9干预组较正常组和P+T组的IKBα蛋白表达低;PCSK9干预组较正常组和P+T组的P65/P50蛋白表达高(P均<0.05)。结论PCSK9可诱导肝细胞MIF的合成与分泌,肝细胞分泌的MIF可与巨噬细胞表面受体TLR4结合,激活下游NF-κB炎症信号通路,促进TNF-α、白介-6、Objective To systematically investigate whether proprotein convertase subtiliSin/kexin type 9(PCSK9)regulates monocyte/macrophage inflammatory response through liver-derived MIF using human cell translines,and then provide new ideas and basis for clinical prevention and control of inflammatory diseases such as Atherosclerosis.Methods Using small interfering RNA(SiRNA)-mediated MIF gene transfection in THLE-3 cells,the experiment was divided into MIF low expression group(Si-MIF-THLE-3)and MIF normal expression group(Con-THLE-3),and PCSK9 and MIF mRNA expression in hepatocytes was detected by real-time fluorescence quantitative PCR(quantitative real-time PCR(RT-qPCR)to detect PCSK9 and MIF mRNA expression in hepatocytes.PCSK9 recombinant protein was administered for intervention in Si-MIF-THLE-3 and Con-THLE-3 groups,respectively,and PCSK9 and MIF protein expression in hepatocytes were detected by Western Blot,and the contents of PCSK9 and MIF in culture medium were determined by ELISA,and the culture medium of the above hepatocytes were collected.USing SiRNA-mediated MIF gene transfection of THP-1 cells,the experiment was divided into MIF low expression group(Si-MIF-THP-1)and MIF normal expression group(Con-THP-1),and the protein expression of MIF was detected by Western Blot.The collected hepatocyte cultures were added to Si-MIF-THP-1 and Con-THP-1 groups for a total of 48 h.The PCSK9 intervention group was additionally equipped with the TLR4-specific inhibitor TAK-242(1μM)(P+T).The expression of inflammatory factors was detected by ELISA;the expression of TLR4-NF-κB Signaling pathway in THP-1 cell lines was detected by Western Blot.Results①MIF mRNA expression was Significantly lower in the Si-MIF-THLE-3 group compared to the Con-THLE-3 group,while PCSK9 mRNA expression was elevated(both P<0.05).②The expression of MIF protein and its secretion into the culture medium were elevated in hepatocytes after PCSK9 intervention,while the expression of PCSK9 protein and its content in the culture medium were unchang

关 键 词：前蛋白转化酶枯草溶菌素-9 巨噬细胞移动抑制因子 巨噬细胞 炎症反应 

分 类 号：R541.4[医药卫生—心血管疾病]