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

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（0．1～1000．0 ng ／mL）处理，分为对照组（未经任何处理）、HMGB1组、HMGB1＋ TLR4 siRNA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（0．1～1000．0 ng／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（100 ng／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 TLR4 siRNA 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。

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

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 PI3k inhibitor (LY294002) intervention group were stimulated by HMGB1 (0 .1-1 000 .0 ng/mL) .Expression of TLR4 mRNA was detected by RT‐PCR ,protein expression of TLR4 ,Akt ,pAkt ,PI3K were detected by Western blot .Activity of the im‐munoprecipitated PI3K enzyme was assessed in a competitive ELISA .The migration and cell viability of every groups were ob‐served .Results HMGB1 (0 .1 -1 000 .0 ng/mL) stimulated VSMCs migration in a dose‐dependent manner and incubation of VSMCs with 100 ng/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 PI3K inhibitor LY294002 could 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 cardiovascu‐lar disease .