基于NF-κB炎性通路探讨纤维连接蛋白在冠心病血瘀证的作用  

Exploration of Fibronectin Role in Coronary Heart Disease with Blood Stasis Syndrome Based on NF-κB Inflammatory Pathway

作　　者：罗晓欣 肖隋熙 马至言 胡伊蕾 高翎薇 简维雄 LUO Xiaoxin;XIAO Suixi;MA Zhiyan;HU Yilei;GAO Lingwei;JIAN Weixiong(Hunan university of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中医学院,长沙410208

出　　处：《中国中医基础医学杂志》2024年第9期1545-1553,共9页JOURNAL OF BASIC CHINESE MEDICINE

基　　金：国家自然科学基金项目(81973753,82374334);湖南省自然科学基金项目(2022JJ30433);湖南省研究生科研创新项目(CX20230794);湖南中医药大学研究生科研创新课题(2023CX83)。

摘　　要：目的利用蛋白质组学寻找抑制冠心病血瘀证的关键蛋白,并基于核因子(nuclear factoer,NF)-κB炎性信号通路和内皮细胞活化模型验证关键蛋白纤维连接蛋白(fibronectin 1,Fn)对冠心病血瘀证的作用。方法将冠心病血瘀证组患者和健康组受试者的血清进行相对定量和绝对定量的等比标记(isobaic tag for relative and absolute quantitation,ITRAQ)蛋白质组学分析,将差异蛋白进行GO和KEGG功能富集,从与冠心病密切相关的富集通路中筛选出关键蛋白Fn。利用60μg/mL的氧化低密度脂蛋白(oxidized low density lipoprotein,oxLDL)构建内皮细胞活化模型,在内皮细胞活化模型中加入5μg/cm 2、10μg/cm 2、20μg/cm 23个浓度的血浆Fn或敲减内皮细胞来源Fn的方法进行干预。分组为空白组、模型组、对照组、Fn EC-KD组、Fn5组、Fn10组、Fn20组。细胞免疫荧光法和免疫印迹法检测各组NF-κB核位移和NF-κB蛋白表达水平,ELISA检测培养上清的细胞间黏附分子(intercellular cell adhesion molecule,ICAM)-1、血管细胞黏附分子(vascular cell adhesion molecule,VCAM)-1、前列环素I-2(Proataglandin-I-2,PGI2)、内皮素的蛋白含量。结果血清蛋白质组学结果显示健康组和冠心病血瘀证组存在121种差异蛋白,51种血瘀证组下调蛋白集中在补体与凝血级联、肌动蛋白细胞骨架的调节、细胞外基质-受体相互作用途径,70种血瘀证组上调蛋白集中在补体与凝血级联、肌动蛋白细胞骨架的调节、血小板活化、吞噬体途径。Fn与冠心病密切相关且在血瘀证患者中下调。细胞实验发现各组NF-κB总蛋白表达差异无统计学意义(P>0.05);与空白组比较模型组NF-κB核位移增强,ICAM-1、VCAM-1、内皮素表达上调,PGI2分泌下调(P<0.05);与模型组比较,Fn5组、Fn10组、Fn20组NF-κB核位移减弱,VCAM-1、ICAM-1、内皮素分泌下调(P<0.05),PGI2分泌显著上调(P<0.05);与模型组比较,Fn EC-KD组NF-κB核位移减弱,VCAMObjective To search for the key protein of inhibiting blood stasis syndrome of coronary heart disease by proteomics,and to verify the effect of fibronectin 1(Fn)on blood stasis syndrome of coronary heart disease based on nuclear factoer kappa-B(NF-κB)inflammatory signal pathway and endothelial cell injury model.Methods the serum of patients with coronary heart disease of blood stasis syndrome and healthy subjects were analyzed by ITRAQ marker proteomics,and the differential proteins were enriched by GO and KEGG functions.Fn,a key protein,was screened from the enrichment pathway closely related to coronary heart disease.A model of endothelial cell activation was established using 60μg/mL of oxidized Low-density lipoprotein(oxLDL),three concentrations of plasma Fn(5μg/cm 2,10μg/cm 2,20μg/cm 2)or knockdown of endothelial cell-derived Fn were added to the endothelial cell activation model.The nuclear translocation of NF-κB and the expression of NF-κB protein were detected by immunofluorescence and western blotting in Blank group,oxLDL Group,control group,Fn EC-KD Group,Fn5 Group,Fn10 Group and Fn20 group,the levels of ICAM-1,VCAM-1,Prostacyclin-I-2 and endothelin in the culture supernatant were detected by ELISA.Results The results of serum proteomics showed that there were 121 different proteins in the healthy group and the coronary heart disease with blood stasis syndrome group,the 51 down-regulated proteins in the blood stasis syndrome groups were concentrated in the complement-coagulation cascade,the regulation of actin cytoskeleton,and the ECM-receptor interaction pathway,the 70 up-regulated proteins in the blood stasis syndrome groups were concentrated in the complement and coagulation cascade,the regulation of actin cytoskeleton,platelet activation and phagocytic pathway.Fn is closely related to coronary heart disease and down-regulated in patients with blood stasis syndrome.There was no significant difference in the total protein expression of NF-κB among the three groups(P>0.05).Compared with the con

关 键 词：血浆蛋白质组学 冠心病血瘀证 血浆纤维连接蛋白 内皮细胞活化 

分 类 号：R259[医药卫生—中西医结合]