摘要
目的探讨Vaspin对TNF-α介导的人脐静脉内皮细胞（HUVECs）NF-κB和P13K／Akt信号通路的影响。方法体外分离并培养HUVECs。NF-κB荧光酶报告质粒瞬时转染HUVECs，用不同浓度（0～320wg／L）Vaspin预培养，随后用lO斗∥L的TNF．仪作用于HUVECs，荧光酶报告分析法测定NF-κB的转录活性。Westernblot测定磷酸化的Akt水平。ELISA测定细胞上清液中IL-1及IL-6的浓度。Real．timePCR、Westernblot检测细胞问黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）及单核细胞趋化蛋白-1（MCP-1）的mRNA和蛋白表达水平。结果在HUVECs中，Vaspin抑制TNF．仅介导的NF-κB转录活性（P〈0．05），并且NF-κB下游因子IL-1、IL-6、ICAM-1、VCAM-1和MCP-1的表达降低（P〈0．05）。Vaspin增加了炎性因子TNF-d介导的磷酸化的Akt水平（P〈0．05）。结论Vaspin抑制HUVECs中TNF-Ⅱ介导的NF-κB信号通路，增强TNF-α介导的P13K／Akt信号通路的传导。
Objective To investigate the effects of visceral adipose tissue-derived serpin （Vaspin） on nuclear fac- tor-kappa B （NF-κB） and phosphatidylinositol 3-kinase （PI3K）/Akt signal pathways in human umbilical vein en- dothelial cells （HUVECs） stimulated by tumor necrosis factor-α （TNF-α） to elucidate the role of Vaspin in human endothelial cells of inflammation and insulin resistance. Methods HUVECs were isolated and cultured in vitro. A NF-κB luciferase reporter system was constructed and transiently transfected into HUVECs. Following transfection, HUVECs were pretreated with various concentrations of Vaspin （0 -320 μg/L） before 10 μg/L TNF-α stimulation.The transcription activity of NF-κB was determined using luciferase reporter assay. The level of Akt phosphoryla- tion, expression of NF-κB downstream inflammatory cytokines IL-1 and IL-6, and mRNA and protein expression of intercellular cell adhesion molecule-1 （ ICAM- 1 ）, vascular cell adhesion molecule- 1 （ VCAM- 1 ） and monocyte chemotactic protein-1 （MCP-1） were determined by routin technology. Results Vaspin inhibited TNF-α mediated activation of NF-κB and its downstream molecules （P 〈 0. 05）. Vaspin significantly increased Akt phosphorylation in TNF-α stimulated endothelial cells （P 〈 0.05 ）. Conclusions Vaspin protects endothelial cells from TNF-α in- duced inflammation and insulin resistance by combination with inhibition of NF-κB, its downstream molecules and the upregulation of the PI3-kinase/Akt signal pathway.
出处
《基础医学与临床》
CSCD
2015年第8期1020-1024,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(81200591
81471025)
山西省留学回国人员科技活动择优资助项目(2013-251)
