川芎嗪通过Rac1/LIMK1通路改善脂多糖诱导的人脐静脉内皮细胞骨架重构的研究  

Tetramethylpyrazine ameliorates lipopolysaccharide-induced cytoskeleton remodeling of human umbilical vein endothelial cells through the Rac1/LIMK1 pathway

作　　者：丁渡山 高可飞 闵思敏 刘赛赛 刘小碣 仝洁 张铭 张小楠 赵士弟[1] 李言[1] DING Dushan;GAO Kefei;MIN Simin;LIU Saisai;LIU Xiaojie;TONG Jie;ZHANG Ming;ZHANG Xiaonan;ZHAO Shidi;LI Yan(Key Laboratory of Cardiovascular and Cerebrovascular Diseases,Bengbu Medical College,Bengbu 233030,China;Department of Respiratory Physicians,The First Affiliated Hospital of Bengbu Medical College,Bengbu 233000,China)

机构地区：[1]蚌埠医学院心脑血管疾病基础与临床重点实验室,蚌埠233030 [2]蚌埠医学院第一附属医院呼吸内科,蚌埠233000

出　　处：《生命的化学》2022年第3期587-596,共10页Chemistry of Life

基　　金：国家自然科学基金项目(81673791);蚌埠医学院研究生创新计划项目(Byycx20002)。

摘　　要：该文旨在从Ras相关的C3肉毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)/LIM激酶1(LIM kinase 1,LIMK1)通路探讨川芎嗪(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)骨架重构的保护作用。采用Ⅷ因子免疫荧光鉴定原代内皮细胞;分别用0、50、100、200、300 ng/mL的LPS处理对数生长期的原代HUVECs;采用低、中、高3个剂量的TMP对HUVECs预处理12 h;利用F-actin免疫荧光染色观察细胞骨架变化;通过跨膜电阻值(transepithelial electrical resistance,TEER)和FITC-葡聚糖Transwell实验检测人脐静脉内皮细胞通透性;Western blot检测Rac1、LIMK1的表达以及相应磷酸化水平;免疫荧光观察磷酸化LIMK1蛋白的细胞定位和表达水平变化;实时荧光定量PCR(quantitative real-time PCR,qPCR)检测Rac1和LIMK1的mRNA水平。结果显示,Ⅷ因子免疫荧光鉴定原代提取的内皮细胞符合要求;HUVECs经LPS诱导后,镜下形态由卵圆形向长梭形改变,纤维状肌动蛋白(filament actin,F-actin)增多增粗,TEER降低及单层内皮细胞通透性增加;Rac1、LIMK1的表达及磷酸化水平呈LPS梯度依赖性升高(P<0.05);低浓度的TMP降低LPS诱导的F-actin增多增粗作用,并降低Rac1、LIMK1的表达及其磷酸化水平(P<0.05);高浓度的TMP可以抑制Rac1激活(P<0.05),但对LPS诱导的HUVECs细胞骨架重构和通透性增加没有保护作用(P>0.05)。该研究结果表明,TMP通过Rac1/LIMK1通路保护LPS诱导的HUVECs骨架重构,抑制由LPS诱导的HUVECs通透性增大,对临床运用TMP改善由内皮细胞高通透性引起的急性肺损伤提供理论依据。The aim of this study was to investigate protective effects of tetramethylpyrazine(TMP) on lipopolysaccharide(LPS)-induced cytoskeleton remodeling of human umbilical vein endothelial cells(HUVECs) through the Ras-related C3 botulinum toxin substrate 1(Rac1)/LIM kinase 1(LIMK1) pathway.Primary HUVECs were identified by Ⅷ factor immunofluorescence and following treatment with 0, 50, 100,200 and 300 ng/mL LPS for 12 h, respectively, at logarithmic growth stage. HUVECs were pretreated with low, medium and high doses of TMP for 12 h. Cytoskeleton changes were observed by immunofluorescence staining with F-actin. HUVECs permeability was evaluated by transepithelial electrical resistance(TEER) and FITC-dextran Transwell test. Western blot was used to detect the expression levels of Rac1, LIMK1 and its phosphorylated form. The localization and expression levels of LIMK1 phosphorylated protein were observed by immunofluorescence. The mRNA expressions of Rac1 and LIMK1 were detected by qPCR. The results showed that Ⅷ factor immunofluorescence identification confirmed that the primary extracted endothelial cells met the requirements. After LPS induction, HUVECs changed from oval shape to long spindle shape,increased F-actin and thickened, decreased TEER and increased permeability of monolayer endothelial cells.The phosphorylation of Rac1, LIMK1 and their expressions increased in a LPS-dependent manner(P<0.05).At low concentrations, TMP reduced LPS-induced increase and coarsening of filament actin(F-actin), and decreased Rac1, LIMK1 and their phosphorylation expressions(P<0.05). However, high concentrations of TMP inhibited Rac1 activation(P<0.05), but did not protect against LPS-induced cytoskeletal remodeling and increased permeability in HUVECs(P>0.05). The results of this study indicate that TMP protects LPS-induced HUVECs cytoskeleton remodeling and Inhibition of LPS-induced permeability increase of HUVECs through Rac1/LIMK1 pathway. The results of this work provide theoretical basis for clinical application of TMP

关 键 词：脂多糖 脐静脉内皮细胞 川芎嗪 细胞骨架重构 

分 类 号：R285[医药卫生—中药学]