Increased caveolin-1 expression precedes decreased expression of occludin and claudin-5 during blood–brain barrier breakdown

The significance of caveolin-1, a major constituent of caveolae, and the tight junction proteins occludin and claudin-5 in early blood–brain barrier (BBB) breakdown was assessed by sequential demonstration of the expression of these proteins over a period of 12 h to 6 days post-lesion in the rat cortical cold injury model. Pial and intracerebral vessels of control rats showed punctuate endothelial immunoreactivity for caveolin-1 and caveolin-2, while claudin-5 and occludin were localized as longitudinal strands in endothelium. During the early phase of BBB breakdown following injury at 12 h and on day 2, western blot analyses detected a significant increase in caveolin-1 expression at the lesion site while immunohistochemistry showed that the caveolin-1 increase was localized to the endothelium of lesion vessels. Decreased expression of occludin occurred at the lesion site only on days 2 and 4 post-lesion while claudin-5 expression was decreased only on day 2. Dual labeling for fibronectin, a marker of BBB breakdown, and caveolin-1 or the tight junction proteins demonstrated that only lesion vessels with BBB breakdown showed a marked increase of caveolin-1, loss of occludin and reduced localization of claudin-5. The issue whether these alterations precede or follow BBB breakdown is uncertain; however, increased expression of caveolin-1 preceded the decreased expression of occludin and claudin-5. Thus caveolae and caveolin-1 have an important role in early BBB breakdown and could be potential therapeutic targets in the control of early brain edema.     

Biphasic cytoarchitecture and functional changes in the BBB induced by chronic inflammatory pain

The blood-brain barrier (BBB) is a dynamic system which maintains brain homeostasis and limits CNS penetration via interactions of transmembrane and intracellular proteins. Inflammatory pain (IP) is a condition underlying several diseases with known BBB perturbations, including stroke, Parkinson's, multiple sclerosis and Alzheimer's. Exploring the underlying pathology of chronic IP, we demonstrated alterations in BBB paracellular permeability with correlating changes in tight junction (TJ) proteins: occludin and claudin-5. The present study examines the IP-induced molecular changes leading to a loss in functional BBB integrity. IP was induced by injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the right hindpaw of female Sprague-Dawley rats. Inflammation and hyperalgesia were confirmed, and BBB paracellular permeability was assessed by in situ brain perfusion of [14C]sucrose (paracellular diffusion marker). The permeability of the BBB was significantly increased at 24 and 72 h post-CFA. Analysis of the TJ proteins, which control the paracellular pathway, demonstrated decreased claudin-5 expression at 24 h, and an increase at 48 and 72 h post-injection. Occludin expression was significantly decreased 72 h post-CFA. Expression of junction adhesion molecule-1 (JAM-1) increased 48 h and decreased by 72 h post-CFA. Confocal microscopy demonstrated continuous expression of both occludin and JAM-1, each co-localizing with ZO-1. The increased claudin-5 expression was not limited to the junction. These results provide evidence that chronic IP causes dramatic alterations in specific cytoarchitectural proteins and demonstrate alterations in molecular properties during CFA, resulting in significant changes in BBB paracellular permeability.     

深低温停循环后家兔脑血管内皮细胞紧密连接蛋白Occludin表达变化的实验研究

目的研究深低温停循环后家兔脑血管内皮细胞紧密连接蛋白Occludin表达变化,探讨Occludin蛋白表达异常在深低温停循环后家兔血脑屏障通透性升高过程中的作用。方法健康新西兰兔16只,雌雄不限,随机分成体外循环组和深低温停循环组2组。其中深低温持续时间为60分钟。用RT-PCR和Western Blot方法观察MRNA和蛋白水平的变化。结果与体外循环组相比,深低温停循环组家兔脑血管内皮细胞Occludin的mRNA和蛋白表达水平下调（P〈0.05）。结论occludin蛋白表达减少是血脑屏障通透性升高的重要环节。对Occludin蛋白变化的研究有助于更深入地研究血脑屏障的破坏与脑损伤的关系。     

病灶区灌注罗格列酮对脑出血家兔血肿周围咬合蛋白和闭合小环蛋白1 mRNA表达水平及血-脑屏障通透性的影响

目的观察病灶区灌注罗格列酮(RSG)对家兔脑出血(ICH)模型血肿周围咬合蛋白和闭合小环蛋白1(ZO-1)mRNA表达水平、血-脑屏障(BBB)通透性及神经功能评分的影响。方法选择健康雄性家兔45只,体质量2.0~2.5 kg,按随机数字表法分为对照组、ICH模型组和罗格列酮(RSG)治疗组,每组15只(其中5只用于BBB测定)。对照组模拟制作颅内血肿的过程,穿刺成功后对兔靶点注射等渗盐水0.3 ml,6 h后再次注入等渗盐水0.1 ml;ICH模型组穿刺成功后靶点注射自体非抗凝动脉血0.3 ml,6 h后靶点再次注入等渗盐水0.1 ml;RSG治疗组在ICH模型制作成功后6 h,血肿区灌注RSG 0.5 mg(溶于0.1 ml等渗盐水中)。继续饲养至第7天,对各组家兔进行神经功能缺损评分(Purdy评分)后处死,逆转录聚合酶链反应(RT-PCR)法检测血肿周围脑组织咬合蛋白及ZO-1 mRNA的表达水平;采用甲酰胺方法测量血肿周围脑组织伊文思蓝(EB)含量以评估BBB通透性。结果 (1)神经功能评分:对照组、ICH模型组、RSG治疗组Purdy评分分别为(2.53±0.05)、(8.13±0.06)、(6.67±0.08)分,组间差异有统计学意义(F=459.116,P0.01);与对照组比较,ICH模型组、RSG治疗组的Purdy评分均明显增加(均P0.01);与ICH模型组比较,RSG治疗组的Purdy评分降低(P0.05);(2)咬合蛋白及ZO-1 mRNA表达水平:对照组、ICH模型组、RSG治疗组咬合蛋白及ZO-1 mRNA差异均有统计学意义(每组分别为1.013±0.051、1.001±0.045;0.221±0.017、0.247±0.019;0.498±0.041、0.613±0.045;F值分别为443.924、381.929,均P0.01),与对照组比较,ICH模型组、RSG治疗组咬合蛋白及ZO-1 mRNA表达均显著减少(均P0.01);与ICH模型组比较,RSG治疗组咬合蛋白及ZO-1 mRNA的表达增加(均P0.05);(3)BBB通透性:对照组、ICH模型组、RSG治疗组EB含量分别为(12.0±1.0)、(51.6±0.9)、(36.4±1.0)μg/g,组间差异有统计学意义(F=223.516,P0.01);与对照组比较,ICH模型组、RSG治疗组的EB含量明显增加(均P0.01);与ICH模型组比较,RSG治疗组EB含量明显降低(P0.01)。结论病灶区灌注RSG可明显改善家兔ICH后的神经功能,增加血肿周围脑组织咬合蛋白和ZO-1mRNA的表达水平,降低BBB通透性。     

脓毒症诱导血脑屏障通透性增加的机制研究

目的:探究脓毒症对血脑屏障通透性的影响。方法:采用盲肠结扎穿孔术建立脓毒症大鼠模型。根据干预时间随机（随机数字法）分组：假手术组、脓毒症1 d组、脓毒症4 d组、脓毒症7 d组。采用荧光素钠检测血脑屏障的通透性变化;采用Western blot和免疫荧光方法检测脑微血管内皮细胞紧密连接蛋白的表达变化。结果:和假手术组大...     

Ammonium affects tight junctions and the cytoskeleton in MDCK cells

In the kidney medulla, tubule cells are exposed not only to elevated NaCl but also to high NH₄Cl concentrations. Although it is well known that long-term exposure to high NaCl concentrations leads to reorganization of the actin-based cytoskeleton and to altered transport properties of renal epithelial cells, there have been no comparable studies on the effects of elevated extracellular NH₄Cl concentrations. We therefore examined the effect of prolonged (up to 72 h) exposure of Madin-Darby canine kidney (MDCK) cells to increased NH₄Cl concentrations on the actin-based cytoskeleton, the transepithelial electrical resistance (TER) and the expression and intracellular distribution of the tight junction protein occludin. NH₄Cl exposure resulted in rarefaction of cytoplasmic stress fibres, formation of intense peripheral actin bands and reduced abundance of both F- and G-actin. While under control conditions occludin staining was restricted to the tight junction region, ample dot-like intracellular staining was apparent after NH₄Cl exposure. These changes in cell structure were associated with an increase in TER and the enhanced expression of an additional putative, 40-kDa occludin isoform. Exposure to elevated extracellular NH₄Cl concentrations thus leads to distinct alterations in the architecture and transepithelial transport properties of MDCK cells that may also be relevant for the tubule cells of the renal inner medulla.     

Hypoxia-induced changes in tight junction permeability of brain capillary endothelial cells are associated with IL-1beta and nitric oxide

We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.     

紧密连接蛋白ZO-1、occludin和actin参与缺氧缺血诱导的血脑屏障通透性增加

目的：探讨血脑屏障紧密连接（blood-brain barrier-tight junction,BBB-TJ）蛋白ZO-1、occludin和actin在缺氧缺血诱导的血脑屏障（blood-brain barrier,BBB）通透性增加中的变化及其机制。方法：利用人脐静脉内皮细胞系ECV304与星形胶质细胞（astrocytes, AS）共培养建立体外BBB模型,模型随机分为正常对照组和缺氧缺血两组。透射电镜观察两组间BBB-TJ的变化,直接免疫荧光观察细胞骨架蛋白actin分布的改变。γ计数仪检测大分子物质 125I-牛血清白蛋白（125I-BSA）通透曲线观察BBB通透性的改变, Western blot检测细胞骨架蛋白actin,胞浆附着蛋白ZO-1,跨膜蛋白occludin的表达量的改变。结果：透射电镜观察培养第10天的体外BBB模型,可见内皮细胞连接紧密,细胞间形成光滑、连续、较高密度的紧密连接。缺氧缺血后5 h,内皮细胞间连接开放,形成裂隙。直接免疫荧光下检测可见周边Actin丝带模糊,部分断裂,形成细胞间裂隙。缺氧缺血组 125I-BSA的通透量增加,与对照组比较差异有统计学意义（P<0.01）。同时ZO-1的表达量显著减少,而occludin和actin的表达量无明显改变。结论： 缺氧缺血诱导occludin的位置分布改变和ZO-1的表达量减少进而促使actin蛋白发生重排,是导致缺氧缺血后BBB通透性增加的可能机制之一。     

紧密连接蛋白occludin与肺部疾病关系的研究进展

紧密连接(tight junction,TJ)是正常上皮细胞或内皮细胞间机械屏障的重要结构基础,由多种蛋白质组成,呈连续带状分布,连接相邻的细胞,在维持机体内环境的稳定和发挥机体正常生理功能等方面具有重要作用.Occludin蛋白是紧密连接中重要的结构蛋白,本文就occludin蛋白的结构与功能、调控机制及其与肺部疾病关系的研究进展作一综述.     

右美托咪定对脓毒血症大鼠肾功能及紧密连接蛋白表达的影响

目的探讨右美托咪定对脓毒血症大鼠肾功能及紧密连接蛋白表达的影响。方法 SD大鼠30只,随机分为右美托咪定处理组(Dex组)、脓毒血症模型组(M组)、生理盐水对照组(C组),每组10只。Dex组大鼠静脉注射脂多糖(LPS)5 mg/kg,之后泵注Dex 7μg/kg,15 min后调整剂量为5μg/kg·h,持续泵注30 min;模型组按同样方法给予LPS后泵注生理盐水;C组始终采用上述方法给予生理盐水。24 h后取血液分离血浆,多功能生化仪检测肌酐(Scr)和尿素氮(BUN)含量;ELISA检测IL-1β和TNF-α含量;Western blot检测肾组织中紧密连接蛋白ZO-1和occludin的表达变化。结果与C组相比,模型组损伤较重,可见大量炎性细胞浸润,有肾小球肾炎表现,而Dex组未见明显的炎症细胞,细胞异常较少,与模型组相比,损伤减轻。肾功能及炎性因子检测结果显示,与C组比较,各组血浆中Scr、BUN、IL-1β和TNF-α含量均显著升高(P0.05),ZO-1和occludin蛋白表达水平均下降(P0.05);与模型组比较,Dex组各项指标均明显降低(P0.05),ZO-1和occludin蛋白表达水平均升高(P0.05)。结论右美托咪定可改善脓毒血症所致的急性肾损伤的肾功能,抑制炎症反应,提升紧密连接蛋白,对肾脏有一定的保护作用。