高迁移率蛋白B1对血管平滑肌细胞迁移的影响及分子机制研究

Effect of HMGB1 on the migration of vascular smooth muscle cells and its molecular mechanism

目的探讨高迁移率蛋白B1（HMGB1）对血管平滑肌细胞（VSMCs）迁移的影响及TLR4依赖的TLR4/PI3K/Akt信号通路介导的分子机制。方法体外分离培养大鼠胸主动脉VSMCs,采用不同浓度HMGB1（0.1-1 000.0ng/mL）处理,分为对照组（未经任何处理）、HMGB1组、HMGB1＋TLR4siRNA转染组、Control siRNA转染组和磷脂酰肌醇3-激酶（PI3K）抑制剂（LY294002）干预组,观察各组细胞活性及HMGB1对VSMCs迁移的影响;实时定量RT-PCR与Western blot分别检测TLR4、Akt、p-Akt、PI3K mRNA和蛋白的表达;ELISA测定PI3K的活性。结果 HMGB1（0.1-1 000.0ng/mL）呈剂量依赖性促进VSMCs迁移（P〈0.05）;经细胞活性测定,HMGB1在使用的浓度范围内对VSMCs未造成细胞毒性作用（P〈0.05）;HMGB1（100ng/mL）处理的VSMCs细胞组PI3K活性及Akt磷酸化水平明显增加（P〈0.05）;经TLR4siRNA转染发现,HMGB1引起的VSMCs迁移明显减弱（P〈0.05）,同样在PI3k抑制剂干预组,PI3K/Akt途径活化和HMGB1介导的VSMCs迁移也被明显抑制（P〈0.05）。结论 HMGB1呈剂量依赖性促进VSMCs迁移,TLR4依赖的TLR4/PI3K/Akt信号通路参与了此过程,提示以TLR4依赖的PI3K/Akt途径为靶点,可为阻塞性血管疾病的治疗提供新思路。

Objective To investigate the effect of high mobility group box-1（HMGB1）on the migration of vascular smooth cells（VSMCs）and the role of TLR4-dependent PI3K/Akt pathway in the process.Methods Primary VSMCs were isolated from the thoracic aorta of male SD rats and cultured in vitro.Control group,TLR4 siRNA transfected group,control siRNA transfected group and PI3kinhibitor（LY294002）intervention group were stimulated by HMGB1（0.1-1 000.0ng/mL）.Expression of TLR4 mRNA was detected by RT-PCR,protein expression of TLR4,Akt,pAkt,PI3 K were detected by Western blot.Activity of the immunoprecipitated PI3 Kenzyme was assessed in a competitive ELISA.The migration and cell viability of every groups were observed.Results HMGB1（0.1-1 000.0ng/mL）stimulated VSMCs migration in a dose-dependent manner and incubation of VSMCs with 100ng/mL caused a rapid migration（P〈0.05）.At the concentrations used,HMGB1 did not cause any cytotoxic effects（P〈0.05）.Migration of VSMCs toward HMGB1 was significantly inhibited by silencing of TLR4（P〈0.05）.Pretreated cells with TLR4 siRNA or the PI3 Kinhibitor LY294002could markedly block PI3K/Akt pathway activation and VSMCs migration mediated by HMGB1（both P〈0.05）.Conclusion HMGB1 stimulated VSMCs migration in a dose-dependent manner and TLR4-dependent PI3K/Akt signaling pathway played an important role in the migration of VSMCs mediated by HMGB1.This research indicates that TLR4-dependent TLR4/PI3K/Akt signaling pathway could be the target in the treatment of obstructive cardiovascular disease.