川芎嗪对脂多糖诱导的人脐静脉内皮细胞高通透性的保护作用研究

目的观察川芎嗪（tetramethylpyrazine, TMP）对脂多糖（lipopolysaccharide, LPS）诱导的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）通透性变化和细胞骨架相关蛋白表达影响。方法以体外培养的HUVECs为实验对象，将细胞随机分6组:正常对照组, LPS组（1μg/mL LPS）, TMP低、中、高剂量防治组（60μg/mL TMP+1μg/mL LPS、120μg/mL TMP+1μg/mL LPS、240μg/mL TMP+1μg/mL LPS）, 纯TMP组（240μg/mL TMP）。Transwell小室内建立内皮细胞通透性模型；细胞电压电阻仪测定各组细胞跨内皮细胞电阻值（TEER）；FITC-葡聚糖法检测各组细胞通透性变化，Pull-Down实验拉下Rac1-GTPase活性蛋白，Western blotting检测各组细胞Rac1-GTPase、Rac1、LIMK1和p-LIMK1的蛋白表达。结果3组不同浓度的TMP均可以抑制LPS诱导的内皮细胞TEER降低以及降低LPS导致的内皮细胞通透性的增加（P < 0.05~P < 0.01）；Western blotting实验显示TMP可以有效抑制LPS诱导的HUVECs中的Rac1-GTPase、p-LIMK1、Rac1、LIMK1表达（P < 0.05~P < 0.01）。结论TMP可以有效抑制LPS诱导的HUVECs通透性升高，其机制可能与其下调Rac1/LIMK1信号通路中Rac1-GTPase活性蛋白、LIMK1磷酸化蛋白表达有关。

Protective effect of tetramethylpyrazine on lipopolysaccharide-induced high permeability of human umbilical vein endothelial cells

ObjectiveTo investigate the effect of tetramethylpyrazine (TMP) on the permeability of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) and the expression of cytoskeleton-related proteins.MethodsHUVECs cultured in vitro were randomly divide into normal control group, LPS group (1 μg/mL LPS), TMP low, medium and high dose group (60 μg/mL TMP+1 μg/mL LPS, 120 μg/mL TMP+1 μg/mL LPS, 240 μg/mL TMP+1 μg/mL LPS), pure TMP group (240 μg/mL TMP).Endothelial cell permeability model was established in Transwell chamber; cell voltage resistance meter was used to measure the trans-endothelial electrical resistance in each group; FITC-dextran method was used to detect the permeability change in each group, and Pull-Down experiment was used to collect Rac1-GTPase active protein, Western blotting was used to detect the protein expression of Rac1-GTPase, Rac1, LIMK1 and p-LIMK1 in each group.ResultsTEER and permeability coefficient showed that three different concentrations of TMP inhibited the decrease of TEER induced by LPS in endothelial cells and decreased LPS-induced endothelial cell permeability(P < 0.05 to P < 0.01);Western blotting analysis showed that TMP could effectively inhibit the LPS-induced up-regulation of Rac1-GTPase, p-LIMK1, Rac1 and LIMK1 in HUVECs(P < 0.05 to P < 0.01).ConclusionsTMP can effectively inhibit the increase of permeability of HUVECs induced by LPS, which may be related to the down-regulation of Rac1-GTPase active protein and LIMK1 phosphorylation protein expression in Rac1/LIMK1 signaling pathway.