心脑舒通胶囊及有效成分对氧糖剥夺/复氧损伤脑微血管内皮细胞屏障完整性的影响及机制研究  

Effect of Xinnao Shutong capsule and its active ingredients on damage of cerebral microvascular endothelial cell barrier integrity caused by oxygen glucose deprivation/reoxygenation and its mechanism

作　　者：王萌[1] 曹玉爽 郭莉琛 杜鑫苑 柴丽娟[1] 袁庆 胡利民[1] WANG Meng;CAO Yu-shuang;GUO Li-chen;DU Xin-yuan;CHAI Li-juan;YUAN Qing;HU Li-min(State Key Laboratory of Component-based Chinese Medicine,Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae,Ministry of Education,Key Laboratory of Chinese Medicine Pharmacology,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China)

机构地区：[1]天津中医药大学组分中药国家重点实验室、方剂学教育部重点实验室、天津市中药药理学重点实验室,天津301617

出　　处：《中国药理学通报》2023年第12期2378-2384,共7页Chinese Pharmacological Bulletin

基　　金：国家“重大新药创制”科技重大专项基金资助项目(No 2011ZX09201-201);国家自然科学基金资助项目(No 81573644)。

摘　　要：目的探讨心脑舒通胶囊(Xinnao shutong,XNST)及其单体成分对氧糖剥夺/复氧(OGD/R)损伤脑微血管内皮细胞屏障结构及紧密连接蛋白表达的影响及机制。方法将小鼠脑微血管内皮细胞株bEnd.3接种于Transwell小室上层,建立OGD/R损伤模型,通过细胞跨膜电阻值及荧光素-钠透过率考察药物对内皮细胞屏障完整性的影响;采用Claudin-5免疫荧光染色观察内皮细胞间紧密连接结构变化;RT-PCR和Western blot法检测紧密连接蛋白Claudin-5、Occludin、ZO-1的mRNA和蛋白表达水平。Western blot法检测MAPKs(JNK、p38、ERK)、I kappa Bα、I kappa B激酶磷酸化蛋白表达水平,Western blot及免疫荧光法检测NF-κB/p65的入核表达。结果XNST及其3个单体成分均能明显增加内皮细胞电阻值,降低荧光素-钠透过率;Claudin-5荧光染色结果显示,模型组细胞间紧密连接明显破坏,而XNST及其单体成分能明显改善其紧密连接结构。RT-PCR及Western blot结果显示其能明显上调Claudin-5、Occludin和ZO-1的mRNA及蛋白表达,进一步对其机制研究表明,XNST及其单体成分能明显抑制JNK、p38、ERK的磷酸化,抑制I kappa Bα、I kappa B激酶磷酸化,并明显抑制NF-κB/p65的核转位。结论XNST及单体成分均可通过增加细胞间紧密连接结构促进屏障完整性发挥脑保护作用,其机制可能与抑制NF-κB和MAPKs信号通路激活有关。Aim To investigate the effect of XNST and its monomeric components on the barrier structure and tight junction protein expression of brain microvascular endothelial cells damaged by oxygen glucose deprivation/reoxygenation(OGD/R)and the possible mechanism.Methods The mouse brain microvascular endothelial cell line bEnd.3 was inoculated in the upper layer of the Transwell chamber to establish an OGD/R damage model,and the effect of the drug on the integrity of the endothelial cell barrier was investigated by the transmembrane resistance value and fluorescein-sodium transmittance.Claudin-5 immunofluorescence staining was used to observe the changes of tight junction structure between endothelial cells.RT-PCR and Western blot were employed to detect mRNA and protein expression levels of tightly linked proteins Claudin-5,Occludin,ZO-1.Western blot was applied to detect the expression levels of MAPKs(JNK,p38,ERK),I kappa Bα,I kappa B kinase phosphorylated protein expression,and Western blot and immunofluorescence were utilized to detect NF-κB/p65 nucleation expression.Results XNST and its three monomers could significantly increase endothelial cell resistance and decrease fluorescein-sodium transmittance.Claudin-5 fluorescence staining showed that the tight junction between cells in the model group was significantly damaged,while XNST and its monomer components could significantly improve its tight structure.RT-PCR and Western blot results showed that it could significantly upregulate the expression of mRNA and protein of Claudin-5,Occludin and ZO-1,and further study on the mechanism showed that XNST and its monomer components could significantly inhibit the phosphorylation of JNK,p38 and ERK,inhibit the phosphorylation of I kappa Bαand I kappa B kinases,and significantly inhibit the nuclear translocation of NF-κB/p65.Conclusion Both XNST and its monomeric components can exert cerebroprotective effects by increasing the tight junction structure between cells to promote barrier integrity,and the mechanism may be re

关 键 词：心脑舒通胶囊 OGD/R 脑微血管内皮细胞 紧密连接 MAPKs通路 NF-ΚB通路 

分 类 号：R-332[医药卫生] R282.71R322.81R329.24R743.31