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occludin and connexin 43 expression contribute to the pathogenesis of traumatic brain edema

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Neural Regeneration Research 2013年第29期8卷 2703-2712页

作者： Wanyin Ren Guojie Jing Qin Shen Xiaoteng Yao Yingchao Jing Feng Lin Weidong Pan Guangdong Medical College Huizhou First People's Hospital

The experimental model of traumatic brain injury was established in Sprague-Dawley rats according to Feeney＇s free falling method. The brains were harvested at 2, 6 and 24 hours, and at 3 and 5 days after injury. Changes in brain water content were determined using the wet and dry weights. Our results showed that water content of tissue significantly increased after traumatic brain injury, and reached minimum at 24 hours. Hematoxylin-eosin staining revealed pathological impairment of brain tissue at each time point after injury, particularly at 3 days, with nerve cell edema, degenera- tion, and necrosis observed, and the apoptotic rate significantly increased. Immunohistochemistry and western blot analysis revealed that the expression of occludin at the injured site gradually de- creased as injury time advanced and reached a minimum at 3 days after injury; the expression of connexin 43 gradually increased as injury time advanced and reached a peak at 24 hours after in-jury. The experimental findings indicate that changes in occludin and connexin 43 expression were consistent with the development of brain edema, and may reflect the pathogenesis of brain injury.

关键词： neural regeneration traumatic brain injury brain edema connexin 43 occludin nerve cells pa-thology tight junction grants-supported paper neuroregeneration

脂多糖对绵羊输卵管上皮Claudin-1和occludin表达的影响

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核农学报 2023年第9期37卷 1775-1781页

作者：曾建林吕建树段宏伟杨帅张榕张勇胡俊杰甘肃农业大学动物医学院/甘肃省动物生殖生理及繁殖调控重点实验室甘肃兰州730070

绵羊等反刍动物瘤胃的异常代谢及炎症会造成脂多糖(LPS)异位诱发集体其他部位炎症风险,然而LPS对绵羊输卵管上皮屏障关键蛋白紧密连接蛋白封闭蛋白-1(Claudin-1)和闭合蛋白(occludin)的影响尚不清楚。为阐明LPS对绵羊输卵管上皮Claudin-... 详细信息

绵羊等反刍动物瘤胃的异常代谢及炎症会造成脂多糖(LPS)异位诱发集体其他部位炎症风险,然而LPS对绵羊输卵管上皮屏障关键蛋白紧密连接蛋白封闭蛋白-1(Claudin-1)和闭合蛋白(occludin)的影响尚不清楚。为阐明LPS对绵羊输卵管上皮Claudin-1和occludin的影响,本研究采集成年绵羊输卵管通过苏木精-伊红染色、免疫荧光、蛋白免疫印迹法(WB)检测,并体外培养原代输卵管上皮细胞通过免疫荧光、蛋白免疫印迹法及实时荧光定量PCR(qRT-PCR)进行试验。结果表明,Claudin-1和occludin表达于绵羊输卵管伞部、壶腹部和峡部上皮,Claudin-1和occludin在伞部的表达量显著高于壶腹部和峡部。采用浓度为0、10、50和100 ng·mL^(-1)的LPS诱导原代壶腹部上皮细胞,发现LPS可促进TLR4、NFκB及TNFα的mRNA表达,并影响Claudin-1和occludin的蛋白表达水平。表明LPS在短时间内可诱导输卵管炎症反应,并导致上皮细胞Claudin-1和occludin表达紊乱。进一步提示反刍动物瘤胃异常代谢及炎症导致的LPS易位,可能会诱发输卵管炎症反应,造成输卵管上皮屏障损伤及生理功能障碍,严重影响绵羊的经济价值。本研究结果为反刍动物瘤胃异常代谢及炎症疾病期间LPS易位造成全身性炎症的相关研究提供了理论基础。

关键词：绵羊输卵管脂多糖 Claudin-1 occludin

Modified Da-chai-hu Decoction regulates the expression of occludin and NF-κB to alleviate organ injury in severe acute pancreatitis rats

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Chinese Journal of Natural Medicines 2019年第5期17卷 355-362页

作者： ZHAO Guang ZHUO Yu-Zhen CUI Li-Hua LI Cai-Xia CHEN Sha-Yan LI Dan LIU Jun-Hong LI Di-Hua CUI Nai-Qiang ZHANG Shu-Kun Hepatobiliary and Pancreatic Surgery Tianjin Nankai Hospital Tianjin Hospital of Integrated Traditional Chinese and Western medicine Nankai Clinical College Tianjin Medical University Institute of Integrated Traditional Chinese and Western medicine Tianjin Nankai Hospital Tianjin Hospital of Integrated Traditional Chinese and Western medicine Department of Clinical Laboratory Tianjin Nankai Hospital Tianjin Hospital of Integrated Traditional Chinese and Western medicine

Modified Da-chai-hu Decoction(MDD), a traditional Chinese medicinal formulation, which was empirically generated from Da-chai-hu decoction, has been utilized to treat severe acute pancreatitis(SAP) for decades. The aim of the present study was to explore its potential organprotective mechanism in SAP. In the present study, rat SAP model was induced by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct, MDD(23.35 g/kg body weight, twelve times the clinical dose) were orally given at 2 h before and 10 h after injection. At 12 h after model induction, blood was taken from vena cava for analysis of amylase, diamine oxidase(DAO), pulmonary surfactant protein-A(SP-A), and C-reactive protein(CRP). Histopathological change of pancreas, ileum and lung was assayed by H&E staining, myeloperoxidase(MPO) activity were determinated using colorimetric assay, and the expressions of occludin and nuclear factor-κB(NF-κB) were detected by real-time RT-PCR and western blot, respectively. In addition, the tissue concentrations of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and monocyte chemoattractant protein-1(MCP-1) were measured by enzyme-linked immunosorbent assay(ELISA). The results showed that in SAP rats, MDD significantly alleviated histopathological damage, depressed the MPO activity and the concentrations of TNF-α, IL-1β, and MCP-1 of pancreas, ileum and lung, and reduced the serum levels of amylase [(3283.4±585.5) U·L^(-1) vs(5626.4±795.1) U·L^(-1)], DAO[(1100.1±334.3) U·L^(-1) vs(1666.4±525.3) U·L^(-1)] and CRP [(7.6±1.2) μg·mL^(-1) vs(17.8±3.8) μg·mL^(-1)]. However, the serum SP-A concentration [(106.1±16.6) pg·mL^(-1) vs(90.1±14.9) pg·mL^(-1)] was elevated when treated SAP rats with MDD. Furthermore, MDD increased the occludin expression and reduced the NF-κB expression in pancreas, ileum and lung of SAP rats. Our findings suggested that MDD administration was an effective therapeutic approach for SAP treatment. It could up-regulate occludin expression to protect intercellular tigh

关键词： Severe acute pancreatitis Modified Da-chai-hu Decoction occludin Nuclear factor-KB Ileum Lung

针刺并用补阳还五汤对MCAO大鼠血脑屏障occludin、Claudin-5的影响

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针刺并用补阳还五汤对MCAO大鼠血脑屏障Occludin、Claudin-5的影响

作者：赵小湖南中医药大学

学位级别：硕士

目的:本研究通过观察“醒脑开窍”针刺法并用补阳还五汤对MCAO模型大鼠脑梗死面积、脑组织含水量、血脑屏障通透性及紧密连接蛋白occloudin、claudin-5的影响,探究针药并用对脑梗死的部分治疗机制。方法:SPF级SD大鼠随机分为假手术组及... 详细信息

目的:本研究通过观察“醒脑开窍”针刺法并用补阳还五汤对MCAO模型大鼠脑梗死面积、脑组织含水量、血脑屏障通透性及紧密连接蛋白occloudin、claudin-5的影响,探究针药并用对脑梗死的部分治疗机制。方法:SPF级SD大鼠随机分为假手术组及手术造模组,选取大脑中动脉栓塞法造成脑梗死模型,术后成模大鼠二次随机分成模型组、针刺组、药物组、针药组。针刺组选取“人中、内关、三阴交”穴,药物组予补阳还五汤灌胃,针药组与假手术组均针刺并补阳还五汤灌胃,模型组只抓取固定不予干预,针刺、固定操作每次20min,每日2次,中药灌胃0.6ml/100g/只给药,每日 1次,持续5天。干预后,采用TTC法染色检测脑组织梗塞面积、干湿重法检测脑组织含水量、伊文思蓝(EB)法检测 BBB 通透性、Western blot 检测 occludin、claudin-5表达水平。结果:1.脑梗死面积:术后3d,模型组、针刺组、药物组、针药组组间差异不明显(P>0.05);术后5d,针药组比针刺组脑梗面积明显减小(P0.05);针药组比模型组脑组织含水量显著减少(P0.05);术后5d,模型组、针刺组、药物组、针药组组间差异不显著(P>0.05)。3.血脑屏障通透性检测:术后3d、5d两个时间点,与假手术组相比,模型组脑组织EB含量增加(P0.05),针药组比针刺组、药物组EB含量减小,差异无统计学意义(P>0.05);在5d,与模型组相比,针刺组、药物组、针药组脑组织EB含量均显著下降(P0.05)。4.血脑屏障紧密连接蛋白occludin、claudin-5表达检测:①occludin蛋白表达:在3 d,假手术组、模型组、针刺组、药物组、针药组组间差异不明显(P>0.05);在5d,与假手术组相比,模型组occludin表达量显著减少(Poccludin蛋白表达增加(P0.05),与针刺组、药物组相比,针药组occludin蛋白表达水平明显上升(P0.05);术后3d,模型组、针刺组、药物组、针药组组间差异无统计学意义(P>0.05);术后5d,与针刺组、药物组相比,针药组claudin-5蛋白表达上升,差异无统计学意义(P>0.05)。结论:1.醒脑开窍针法并用补阳还五汤可有效缩小大鼠脑梗死面积。2.醒脑开窍针法并用补阳还五汤可减轻大鼠脑水肿。3.醒脑开窍针法并用补阳还五汤可降低血脑屏障通透性。4.醒脑开窍针法并用补阳还五汤可能通过促进血脑屏障紧密连接蛋白occludin的表达来减轻BBB损伤。

关键词：针刺并用补阳还五汤 MCAO大鼠血脑屏障 occludin claudin-5

Claudin-11 and occludin are major contributors to Sertoli cell tight junction function, in vitro

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Asian Journal of Andrology 2016年第4期18卷 620-626页

作者： Mark J McCabe Caroline FH Foo Marcel E Dinger Peter M Smooker Peter G Stanton Male Fertility Regulation Laboratory Hudson Institute of Medical Research Monash Medical Centre Clayton Victoria 3168 Australia School of Applied Sciences Royal Melbourne Institute of Technology University Bundoora Victoria 3088 Australia Kinghorn Centre for Clinical Genomics Garvan Institute of Medical Research Darlinghurst New South Wales 2010 Australia St Vincent's Clinical School UNSW Sydney New South Wales 2052 Australia.

The Sertoli cell tight junction （T J） is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% （P 〈 0.01）, 51% （P 〈 0.01）, and 62% （P 〈 0.01）, respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly （P 〈 0.01） to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant （P 〈 0.01） contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function.

关键词： blood-testis barrier claudin-11 occludin Sertoli cell tight junction siRNA

鸭坦布苏病毒感染对occludin和claudin-1基因mRNA表达的影响

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山东农业科学 2022年第10期54卷 122-126,172页

作者：逯璐苏兴海马世宝张琳山东管理学院新兴业态发展研究所山东济南250357 招远市动物疫病预防控制中心山东招远265400 德州市陵城区畜牧兽医服务中心山东德州253599 山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室山东济南250100

紧密连接蛋白不仅是维持上皮屏障功能的重要蛋白,更是多种病毒感染细胞的受体与辅助分子。为了明确紧密连接主要组成蛋白occludin和claudin-1在鸭坦布苏病毒(Duck Tembusu virus, DTMUV)入侵细胞中的作用,本试验利用荧光定量PCR方法对... 详细信息

紧密连接蛋白不仅是维持上皮屏障功能的重要蛋白,更是多种病毒感染细胞的受体与辅助分子。为了明确紧密连接主要组成蛋白occludin和claudin-1在鸭坦布苏病毒(Duck Tembusu virus, DTMUV)入侵细胞中的作用,本试验利用荧光定量PCR方法对病毒感染的非洲绿猴肾(vero)细胞两基因mRNA的表达量进行动态检测。结果表明,occludin和claudin-1的表达量与病毒含量具有相关性,感染后48 h内随着病毒含量的增加,occludin和claudin-1的mRNA上调表达,当病毒拷贝数下降且细胞出现明显病变后,两基因变为下调表达。试验结果有助于明确occludin和claudin-1在介导DTMUV入侵细胞中的作用,为后续功能受体的筛选和抗病毒制剂的研发提供依据。

关键词：鸭坦布苏病毒 occludin claudin-1 定量检测

珍珠龙胆石斑鱼闭锁小带蛋白-1、闭锁蛋白和跨膜蛋白-15a基因的克隆及其在精氨酸干预下的肠道组织表达

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动物营养学报 2021年第8期33卷 4645-4661页

作者：陈东鸿崔晓李广何凯韩煜轩陈冠霖杨奇慧迟淑艳广东海洋大学水产学院湛江524088 南方海洋科学与工程广东省实验室(湛江) 湛江524025

本试验旨在研究珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus)闭锁小带蛋白-1(ZO-1)、闭锁蛋白(occludin)和跨膜蛋白-15a(Claudin-15 a)基因的克隆及其在精氨酸干预下的肠道组织表达。通过cDNA末端快速扩增(RACE)-PCR技术克隆珍珠龙胆石斑鱼ZO-1、occludin和Claudin-15 a的cDNA全长,运用实时荧光定量PCR方法分析其在健康珍珠龙胆石斑鱼脑、鳃、皮肤、肌肉、肝脏、胃、前肠、中肠、后肠、头肾、体肾、脾脏和心脏13种组织中的mRNA相对表达量。选择375尾珍珠龙胆石斑鱼,初重为(20.79±0.09)g,随机分为3组,每组5个重复,每个重复25尾鱼。各组饲料精氨酸水平分别为1.96%、3.06%和3.74%。养殖8周,测定肠道中ZO-1、occludin和Claudin-15 a mRNA相对表达量。结果表明:1)ZO-1的cDNA全长为6355 bp,其中开放阅读框(ORF)为5106 bp,可编码1701个氨基酸,5′-非翻译区(UTR)和3′-UTR分别为105、1144 bp,无跨膜区域。occludin的cDNA全长为2222 bp,其中ORF为1515 bp,可编码504个氨基酸,5′-UTR和3′-UTR分别为69、638 bp,有4个跨膜区域。Claudin-15 a的cDNA全长为1584 bp,其中ORF为669 bp,可编码222个氨基酸,5′-UTR和3′-UTR分别为387、528 bp,有5个跨膜区域。2)系统发育树和氨基酸序列比对结果表明,珍珠龙胆石斑鱼ZO-1与高体鰤的同源性最高(94%),occludin与大黄鱼的同源性最高(84%),Claudin-15 a与环纹圆天竺鲷的同源性最高(92%)。3)后肠中ZO-1 mRNA相对表达量极显著高于其他组织(P0.05);前肠、中肠中Claudin-15 a mRNA相对表达量显著高于其他各组织(Poccludin mRNA相对表达量显著高于其他各组织(Poccludin、Clauin-15 a mRNA相对表达量显著高于1.96%和3.74%精氨酸水平组(Poccludin的cDNA序列长度。饲料适宜精氨酸水平可显著上调珍珠龙胆石斑鱼肠道ZO-1、occludin、Claudin-15 a mRNA相对表达量,维护肠道黏膜屏障完整,促进鱼健康生长。

关键词：珍珠龙胆石斑鱼 ZO-1 occludin Claudin-15 a 精氨酸

从miR-122a研究淫羊藿苷对TNF-α诱导的肠上皮细胞紧密连接occludin减少的改善作用

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从miR-122a研究淫羊藿苷对TNF-α诱导的肠上皮细胞紧密连接Occlud...

作者：李艳丽三峡大学

学位级别：硕士

研究背景:肠腔是有大量致病细菌存在的复杂微环境。肠道屏障功能破坏会引起肠腔细菌移位,引起多种疾病。因此,良好的肠道粘膜屏障功能对维持机体的健康至关重要。肠上皮细胞之间的紧密连接occludin参与构成肠粘膜屏障的核心—机械屏障... 详细信息

研究背景:肠腔是有大量致病细菌存在的复杂微环境。肠道屏障功能破坏会引起肠腔细菌移位,引起多种疾病。因此,良好的肠道粘膜屏障功能对维持机体的健康至关重要。肠上皮细胞之间的紧密连接occludin参与构成肠粘膜屏障的核心—机械屏障。多种炎性因子能通过诱导靶向occludin m RNA的miRNA表达上调,导致肠上皮紧密连接occludin减少以及机械屏障功能降低,尤其是肿瘤坏死因子-α(Tumor necrosis factorα,TNF-α)。TNF-α能通过诱导靶向occludin m RNA的miRNA-122a导致occludin表达减少、肠道通透性升高。淫羊藿苷(icariin,ICA)有广泛的药理作用,包括抗炎、抗氧化、抑制miRNA的表达。淫羊藿苷在体内能上调肠道occludin蛋白的表达水平。然而,药物ICA是否通过miRNA发挥抗炎和屏障保护作用目前尚不清楚。目的:研究ICA对TNF-α诱导的Caco-2单层细胞紧密连接蛋白occludin减少的调节作用,并探讨其可能的机制与ICA下调相关的miRNA表达有关。方法:(1)Transwell培养Caco-2细胞21天建立肠上皮单层屏障,通过肠道碱性磷酸酶(Intestinal alkaline phosphatase,IAP)的活性和跨上皮细胞电阻(Transepithelial Electrical Resistance,TEER)评估单层细胞是否建立。MTT、免疫荧光检测10 ng/m L或20 ng/m L TNF-α对单层Caco-2细胞活力以及紧密连接蛋白occludin蛋白的影响。(2)免疫荧光、Western blot、q PCR检测ICA对TNF-α刺激的Caco-2单层细胞紧密连接occludin蛋白和m RNA表达影响。TEER、4-KD FITC-葡聚糖通过率检测ICA对TNF-α刺激的Caco-2单层细胞通透性的影响。(3)q PCR检测ICA对TNF-α刺激的Caco-2单层细胞相关的miRNA的表达水平。(4)转染miRNA-122a mimics,q PCR检测Caco-2细胞和单层细胞occludin m RNA和miRNA-122a表达的变化。(5)转染miRNA-122a mimics,Western blot、免疫荧光检测ICA对TNF-α刺激的Caco-2单层细胞紧密连接occludin蛋白和m RNA表达影响。TEER、4-KD FITC-葡聚糖通过率检测ICA对TNF-α刺激的Caco-2单层细胞通透性的影响。结果:(1)IAP结果显示,生长21天的Caco-2单层细胞的IAP基底侧(apical,AP)/肠腔侧(basolateral,BP)活力比值达到4.5,TEER达到500Ω/cm,表明Caco-2单层细胞模型成功建立。MTT结果显示,TNF-α对Caco-2细胞活力无明显影响。免疫荧光结果显示10 ng/m L TNF-α刺激Caco-2单层细胞48 h后,紧密连接occludin蛋白铺路石样线性荧光断裂,20 ng/m L TNF-α刺激Caco-2单层细胞24 h或48 h后,occludin蛋白荧光强度进一步降低。因此,以20 ng/m L TNF-α刺激Caco-2单层细胞24 h作为模型组给药方式。(2)免疫组化、Western blot、q PCR结果显示,Caco-2单层细胞occludin蛋白和m RNA的表达在TNF-α的刺激下明显降低,然而,ICA能够缓解TNF-α诱导的occludin蛋白和m RNA的降低。同时,在TNF-α的刺激下,Caco-2单层细胞TEER明显降低、4-KD FITC-葡聚糖通过率明显升高。ICA能够逆转TNF-α导致的变化。这些结果表明ICA能够缓解TNF-α诱导的occludin表达下调、肠屏障功能破坏。(3)q PCR结果显示,miRNA-122a的表达在TNF-α的诱导明显增加,ICA能降低TNF-α诱导的miRNA-122a表达上调。(4)q PCR结果显示,转染miR-122a mimics后,Caco-2细胞miRNA-122a的表达明显升高,occludin m RNA的表达明显降低。(5)与ICA组相比,免疫组化、Western blot结果显示miR-122a mimics组occludin蛋白表达明显下调,荧光信号减弱,同时TEER降低,4-KD FITC-葡聚糖通过率明显升高。结果表明转染miR-122a mimics后,ICA对TNF-α诱导的occludin蛋白的破坏和肠屏障功能破坏的保护作用明显减弱。结论:ICA能缓解TNF-α诱导的Caco-2单层细胞occludin表达减少和肠屏障功能受损,这种保护作用通过下调miR-122a的表达介导的。

关键词： ICA TNF-α occludin 肠屏障功能 miRNA-122a

occludin表达与胃癌发生发展的相关性

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细胞与分子免疫学杂志 2010年第10期26卷 1008-1011页

作者：陈虹张声郑珂王行富福建医科大学附属第一医院病理科福建福州350005

目的:探讨occludin mRNA、蛋白表达水平与胃癌发生发展、侵袭转移的关系。方法:应用组织芯片免疫组化染色和实时荧光定量PCR的方法,检测occludin在胃腺癌组织中的表达并分析其与临床特征的相关性。结果:免疫组织化学检测显示,occludin... 详细信息

目的:探讨occludin mRNA、蛋白表达水平与胃癌发生发展、侵袭转移的关系。方法:应用组织芯片免疫组化染色和实时荧光定量PCR的方法,检测occludin在胃腺癌组织中的表达并分析其与临床特征的相关性。结果:免疫组织化学检测显示,occludin在癌旁正常胃黏膜上皮中阳性表达率高于胃腺癌组织(96.9%vs85.4%,84.6%,89.2%,78.5%)(Poccludin的表达水平与胃腺癌TNM分期和分化程度有关(Poccludin mRNA在胃癌组织和癌旁正常胃组织中的表达无明显差异(P>0.05)。结论:occludin蛋白的表达水平与胃腺癌的发生发展和侵袭转移呈负相关。

关键词：组织芯片实时荧光定量PCR occludin 胃肿瘤