瞬时受体电位通道香草醛亚型-5通过调控NF-κB通路改善免疫微环境治疗小鼠动脉粥样硬化的作用机制  

Mechanism of transient receptor potential channel vanillin subtype-5 on improvement of immune microenvironment by regulating NF-κB pathway in the treatment of atherosclerosis in mice

作　　者：王兴 高爱宝 刘洪波 WANG Xing;GAO Aibao;LIU Hongbo(Department of General Medicine,Yan’an People’s Hospital,Yan’an Shaanxi 716000;Department of Critical Care Medicine,Xi’an International Medical Center Hospital,Xi’an 710016,China)

机构地区：[1]延安市人民医院综合内科,陕西延安716000 [2]西安国际医学中心医院重症医学科,西安710016

出　　处：《临床与病理杂志》2023年第6期1063-1073,共11页Journal of Clinical and Pathological Research

基　　金：国家自然科学基金(81700314)。

摘　　要：目的:分析瞬时受体电位通道香草醛亚型-5(transient receptor potential vanilloid type 5,TRPV5)对载脂蛋白E基因敲除(apolipoprotein E gene knockout,ApoE^(−/−))小鼠动脉粥样硬化发病机制的影响。方法:构建过表达TRPV5的携带绿色荧光蛋白重组腺病毒(adeno-associated virus-TRPV5-green fluorescent protein,AAV-TRPV5-GFP),将其转染RAW264.7细胞以及经静脉注射到ApoE^(−/−)小鼠体内。ApoE^(−/−)小鼠喂食含21%脂肪和0.15%胆固醇的西方式高脂食物8周后,随机分为2组:AAV-TRPV5-GFP组(n=10)和AAV-GFP组(n=10)。采用油红O染色检测主动脉病变大小,ELISA试剂盒法测定血脂、炎症因子水平,高效液相色谱法测定细胞的胆固醇酯,液体闪烁光谱法检测细胞的3H-胆固醇放射性,以及蛋白质印迹法分析胆固醇转运、炎症调节蛋白表达。结果:与0周相比,ApoE^(−/−)小鼠血管组织中的TRPV5水平随着西方饮食喂养时间延长持续降低。在西方饮食喂养第14周时,与AAV-GFP组小鼠相比,AAV-TRPV5-GFP组小鼠整个主动脉的油红O阳性区域减少,主动脉根部病变显著减小,并且小鼠血浆甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)、白细胞介素(interleukin,IL)-1β和IL-6水平显著降低,血浆高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)水平显著提高并改善了HDL功能。此外,注射AAV-TRPV5-GFP的ApoE^(−/−)小鼠的腹腔巨噬细胞中磷酸化β-连环素(phosphoβ-catenin,P-β-catenin)和磷酸化核因子κB(phospho nuclear factor kappa-B,P-NF-κB)的表达水平低于AAV-GFP小鼠。体外实验中,与转染AAV-GFP的细胞相比,转染AAV-TRPV5-GFP的RAW264.7细胞的油红O染色降低了34.4%,细胞胆固醇含量降低43.9%,胆固醇外流至载脂蛋白AI(apolipoprotein AI,apoAI)增加了38.3%。蛋白质印迹法显示:转染AAVTRPV5-GFP�Objective:To analyze the effect of transient receptor potential vanilloid type 5(TRPV5)on the pathogenesis of atherosclerosis in apolipoprotein E gene knockout(ApoE^(−/−))mice.Methods:A adeno-associated virus-TRPV5-green fluorescent protein(AAV-TRPV5-GFP)was constructed and transfected in RAW264.7 cells as well as injected intravenously into ApoE^(−/−)mice.ApoE^(−/−)mice were fed western-style high-fat food containing 21%fat and 0.15%cholesterol for 8 weeks,and were randomly divided into 2 groups:AAV-TRPV5-GFP group(n=10)and AAVGFP group(n=10).The size of aortic lesions was detected by oil red O staining,the levels of blood lipids and inflammatory cytokines were determined by ELISA kit method,the cholesterol esters were determined by high performance liquid chromatography,the 3H cholesterol radioactivity was detected by liquid scintillation spectrometry,and the expression of cholesterol transport and inflammatory regulatory proteins were analyzed by Western blotting.Results:Compared with 0 week,the levels of TRPV5 in vascular tissue were consistently decreased with the prolongation of Western diet in ApoE^(−/−)mice.At the 14th week of Western diet feeding,compared with the AAV-GFP group,the positive area of oil red O in the whole aorta and the size of aortic root lesion in the AAV-TRPV5-GFP group were significantly reduced,and the plasma levels of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),tumor necrosis factorα(TNF-α),interleukin(IL)-1β,and IL-6 were significantly decreased,while the plasma level of high-density lipoprotein cholesterol(HDL-C)was significantly increased and HDL function was improved.In addition,the expression levels of phosphoβ-catenin(P-β-catenin)and phospho nuclear factor kappa-B(P-NF-κB)in peritoneal macrophages of ApoE^(−/−)mice injected with AAV-TRPV5-GFP were lower than those of AAVGFP mice.In vitro,compared with AAV-GFP transfected cells,oil red O staining of RAW264.7 cells transfected with AAV-TRPV5-GFP decreased by 34.4%,ch

关 键 词：瞬时受体电位通道香草醛亚型-5 ApoE–/–小鼠 动脉粥样硬化 胆固醇转运 炎症因子 

分 类 号：R54[医药卫生—心血管疾病]