CXCL1通过促进蛋白激酶B磷酸化导致bEND.3小鼠脑微血管内皮细胞骨架收缩和通透性增加  

CXCL1 induces the contraction of endothelial cytoskeleton and increases permeability in mouse cerebral endothelium bEND.3 cells by promoting protein kinase B phosphorylation

作　　者：韩雨红 董逸舒 李雪蒙 熊倩倩 陈晓芬 李柏青 汪洪涛 宋传旺 吴凤娇 HAN Yuhong;DONG Yishu;LI Xuemeng;XIONC Qianqian;CHEN Xiaofen;LI Baiqing;WANG Hongtao;SONG Chuanwang;WU Fengjiao(Anhui Province Key Laboratory of Immunology in Chronic Diseases,Bengbu Medical College,Bengbu 233030,China;Department of Immunology,School of Clinical Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]慢性疾病免疫学基础与临床安徽省重点实验室,安徽蚌埠233030 [2]蚌埠医学院检验医学院免疫学教研室,安徽蚌埠233030

出　　处：《细胞与分子免疫学杂志》2023年第2期117-123,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81801573,82071849);安徽省青年科学基金(1808085QH253)。

摘　　要：目的探讨脓毒症脑病炎症中CXC趋化因子配体1(CXCL1)及其受体CXC趋化因子受体2(CXCR2)对脑内皮细胞骨架重排和通透性的影响及其相关机制。方法野生型小鼠腹腔注射脂多糖(LPS)(10 mg/kg)建立脓毒症脑病模型,ELISA检测全脑组织肿瘤坏死因子α(TNF-α)和CXCL1含量。使用500 ng/mL LPS和200 ng/mL TNF-α刺激bEND.3脑微血管内皮细胞后,Western blot法检测细胞CXCR2的表达。以150 ng/mL CXCL1刺激bEND.3细胞,免疫荧光染色观察脑内皮细胞骨架纤维型肌动蛋白(F-actin)重排的变化。脑内皮细胞通透性实验中,将bEND.3细胞随机分为PBS对照组、CXCL1组、CXCL1联合选择性CXCR2拮抗剂SB225002组,Transwell^(TM)小室检测各组通透性变化情况。CXCL1刺激bEND.3细胞后,Western blot法检测细胞中蛋白激酶B(AKT)和磷酸化AKT(p-AKT)蛋白表达。结果腹腔注射LPS导致小鼠全脑组织中TNF-α和CXCL1水平显著升高;LPS和TNF-α刺激可增高bEND.3细胞CXCR2蛋白的表达。CXCL1刺激bEND.3细胞使其细胞骨架收缩,细胞间隙形成增加,且细胞渗透性增加,而CXCR2拮抗剂SB225002预处理可显著抑制CXCL1引起的脑内皮细胞肌动蛋白应力纤维和细胞间隙的形成、以及通透性的增高,同时CXCL1(150 ng/mL)刺激使得脑内皮细胞AKT的磷酸化水平明显升高。结论CXCL1通过AKT磷酸化激活引起脑内皮细胞骨架收缩、细胞通透性增加,CXCR2拮抗剂SB225002可有效抑制CXCL1诱导的细胞骨架收缩和通透性升高。Objective To investigate the effects of C-X-C motif chemokine ligand 1(CXCL1)and its receptor CXCR2 on the cerebral endothelial cytoskeleton rearrangement and permeability in the inflammation of septic encephalopathy.Methods The murine model of septic encephalopathy was established by intraperitoneal injection of LPS(10 mg/kg).The levels of TNF-αand CXCL1 in the whole brain tissue were detected by ELISA.The expression of CXCR2 was detected by Western blot analysis after bEND.3 cells were stimulated with 500 ng/mL LPS and 200 ng/mL TNF-α.After treated with CXCL1(150 ng/mL),the changes of endothelial filamentous actin(F-actin)rearrangement in bEND.3 cells were observed by immunofluorescence staining.In the cerebral endothelial permeability test,bEND.3 cells were randomly divided into PBS control group,CXCL1 group,and CXCL1 combined with CXCR2 antagonist SB25002 group.Then endothelial transwell permeabilt assay kit was used to detect the endothelial permeability changes.After stimulated with CXCL1 in bEND.3 cells,Western blot analysis was used to detect the expression of protein kinase B(AKT)and phosphorylated-AKT(p-AKT).Results Intraperitoneal injection of LPS significantly increased the levels of TNF-αand CXCL1 in the whole brain.LPS and TNF-αboth upregulated the expression of CXCR2 protein in bEND.3 cells.CXCL1 stimulation induced the endothelial cytoskeleton contraction,increased paracellular gap formation and elevated endothelial permeability in bEND.3 cells,which was inhibited by the pretreatment with SB225002(CXCR2 antagonist).Furthermore,CXCL1 stimulation also enhanced the phosphorylation of AKT in bEND.3 cells.Conclusion CXCL1 induces the cytoskeleton contraction and increased permeability through AKT phosphorylation in bEND.3 cells,which can be effectively inhibited by CXCR2 antagonist SB225002.

关 键 词：脂多糖 bEND.3脑微血管内皮细胞 CXC趋化因子配体1(CXCL1) CXC趋化因子受体2(CXCR2) 脑内皮细胞 细胞骨架重排 

分 类 号：S853.54[农业科学—临床兽医学] R714.143[农业科学—兽医学] R364.5[农业科学—畜牧兽医] Q245[医药卫生—妇产科学]