恩格列净通过转录因子EB调控溶酶体自噬改善人原代脐静脉内皮细胞损伤  

Empagliflozin regulating lysosomal autophagy through transcription factor EB to alleviate human umbilical vein endothelial cells injury

作　　者：吴春慧 吴慧洋 吴懋 牛超[1] 朱进顺 褚茂平[1] WU Chunhui;WU Huiyang;WU Mao;NIU Chao;ZHU Jinshun;CHU Maoping(Department of Children’s Heart Cardiovascular,the Second Afffliated Hospital&Yuying Children’s Hospital of Wenzhou Medical University,Wenzhou 325027,China;Department of Oncology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第二医院儿童心血管内科,浙江温州325027 [2]温州医科大学附属第一医院肿瘤内科,浙江温州325015

出　　处：《温州医科大学学报》2024年第7期517-527,共11页Journal of Wenzhou Medical University

基　　金：国家自然科学基金项目(82370509,82100523)。

摘　　要：目的:探究恩格列净(EMPA)改善人原代脐静脉内皮细胞(HUVECs)内皮损伤的机制。方法:用TNFα对HUVECs进行刺激,分为5组:①对照组:PBS处理;②TNFα组:TNFα刺激;③EMPA组:TNFα+EMPA;④BAF组:巴夫洛霉素A1(BafA1)+TNFα+EMPA;⑤siTFEB组:细胞造模前预干扰转录因子EB(TFEB),余同EMPA组。造模24h后收集细胞。分别进行Westernblot检测血管内皮钙黏蛋白(VE-cadherin)、细胞间黏附分子(ICAM)、血管细胞黏附分子(VCAM)、溶酶体相关膜糖蛋白1(LAMP1)、P62、LC3、TFEB、pTFEB等蛋白的表达情况;细胞免疫荧光检测血管内皮VE-cadherin、LAMP1、TFEB等指标;单核细胞黏附、细胞渗漏、RNA测序检测、腺病毒转染GFP-LC3检测细胞内的LC3堆积量;慢病毒转染RFP-GFP-LC3检测细胞内的自噬流。结果:Westernblot结果显示,TNFα组较对照组的P62、LC3、pTFEB表达水平升高(P<0.01),LAMP1、TFEB下降(P<0.01);EMPA组较TNFα组P62、LC3、pTFEB表达水平下降(P<0.05),LAMP1、TFEB升高(P<0.05);BAF组较EMPA组的P62、LC3表达量升高(P<0.01);干扰TFEB后,siTFEB组较EMPA组的P62、LC3、表达量升高(P<0.01),LAMP1下降(P<0.01),免疫荧光结果显示TNFα组较对照组的TFEB在胞质堆积增加(P<0.01),EMPA组较TNFα组TFEB在胞质堆积减少(P<0.01),RNA测结果提示EMPA激活自噬通路;RFP-GFP-LC3双荧光结果显示EMPA组较TNFα组自噬溶酶体形成增多(P<0.01)。结论:EMPA通过TFEB增强溶酶体自噬,改善HUVECs内皮损伤。Objective:To investigate the mechanism of how Empagliflozin(EMPA)improves endothelial injury in human primary umbilical vein endothelial cells(HUVECs).Methods:HUVECs were stimulated with were stimulated with TNFα,and divided into four groups:①Control group:treated with PBS;②TNFαgroup:stimulated with TNFα;③EMPA group:TNFα+EMPA;④BAF group:Bafilomycin A1(Baf A1)+TNFα+EMPA;⑤siTFEB group:transcription factor EB(TFEB)was pre-interfered before cell modeling,and the rest processing was the same as the EMPA group.After 24 hours of modeling,cells were collected.Western blot(detecting the expression of proteins such as lysosome-associated membrane protein 1(LAMP1),P62,LC3,VE-cadherin,TFEB,pTFEB,etc.),cell immunofluorescence(VE-cadherin,LAMP1,TFEB,etc.),monocyte adhesion,cell leakage,RNA sequencing,adenovirus transfection GFP-LC3(detecting the amount of LC3 accumulation in cells),lentivirus transfection RFP-GFP-LC3(detecting autophagy flow in cells)were performed.Results:Western blot results showed that,compared with the control group,the TNFαgroup significantly increased levels of P62,LC3,and pTFEB(P<0.01),while LAMP1 and TFEB levels decreased significantly(P<0.01).Compared with the TNFαgroup,the EMPA group showed decreased P62,LC3,and pTFEB levels(P<0.05)but increased LAMP1 and TFEB levels(P<0.05).The BAF group demonstrated higher P62 and LC3 expression than the EMPA group(P<0.01).After TFEB interference,the siTFEB group had increased P62 and LC3 expression compared with the EMPA group(P<0.01),with LAMP1 level decreased(P<0.01).Immunofluorescence results indicated that TNFαgroup had increased TFEB accumulation in the cytoplasm,compared with the control group(P<0.01),and the EMPA group had less TFEB cytoplasmic accumulation than the TNFαgroup(P<0.01).RNA measurements suggested that EMPA activated the autophagy pathway.The RFP-GFP-LC3 dual fluorescence results showed that the EMPA group had more autolysosome formation than the TNFαgroup(P<0.01).Conclusion:Empagliflozin enhances lysosomal autophagy through

关 键 词：川崎病 人脐静脉内皮细胞 自噬 内皮细胞损伤 转录因子EB 钠-葡萄糖共转运体2 

分 类 号：R725.4[医药卫生—儿科] R725.9[医药卫生—临床医学] R543.3