Bile acids permeabilize the blood brain barrier after bile duct ligation in rats via Rac1-dependent mechanisms

BackgroundThe blood brain barrier tightly regulates the passage of molecules into the brain and becomes leaky following obstructive cholestasis. The aim of this study was to determine if increased serum bile acids observed during cholestasis permeabilize the blood brain barrier.MethodsRats underwent bile duct ligation or deoxycholic or chenodeoxycholic acid injections and blood brain barrier permeability assessed. In vitro, the permeability of rat brain microvessel endothelial cell monolayers, the expression and phosphorylation of occludin, ZO-1 and ZO-2 as well as the activity of Rac1 was assessed after treatment with plasma from cholestatic rats, or bile acid treatment, in the presence of a Rac1 inhibitor.ResultsBlood brain barrier permeability was increased in vivo and in vitro following bile duct ligation or treatment with bile acids. Associated with the bile acid-stimulated increase in endothelial cell monolayer permeability was elevated Rac1 activity and increased phosphorylation of occludin. Pretreatment of endothelial cell monolayers with a Rac1 inhibitor prevented the effects of bile acid treatment on occludin phosphorylation and monolayer permeability.ConclusionsThese data suggest that increased circulating serum bile acids may contribute to the increased permeability of the blood brain barrier seen during obstructive cholestasis via disruption of tight junctions.     

中药清肠栓对溃疡性结肠炎大鼠结肠黏膜紧密连接蛋白ZO-1、occludin的影响

目的:探讨复方中药清肠栓对三硝基苯磺酸(TNBS)诱导的UC大鼠结肠黏膜上皮紧密连接相关蛋白-l(ZO-1)、闭锁蛋白(occludin)的修复作用.方法:清洁级♂SD大鼠36只,随机分为正常组、模型组、SASP组、清肠栓高、中、低剂量组,每组6只.选用TNBS诱导的UC大鼠模型.采用免疫荧光的方法观察各组大鼠结肠黏膜...     

Correlation between the destruction of tight junction by patulin treatment and increase of phosphorylation of ZO-1 in Caco-2 human colon cancer cells

Patulin is a mycotoxin and its contamination of food has been reported to cause gastrointestinal inflammation, ulcers, and bleeding. The toxicity of patulin is thought to be due to the destruction of tight junctions (TJs) in gastrointestinal tissues. However, the precise mechanism has not been clarified. Here, we investigated the phosphorylation of TJ components. The transepithelial electrical resistance (TER) of Caco-2 human colon cancer cells decreased gradually during the first 24 h of treatment with 50 μM patulin. Immunofluorescence microscopy showed that the TJ proteins ZO-1 and claudin-4, but not occludin, had decreased after 24 h and decreased from the cell-cell contact regions of TJs after 48 h of patulin treatment. Western blotting showed that the level of ZO-1 decreased after 48 h of patulin treatment, but the levels of claudin-4 and occludin remained at the initial level until 72 h. Phosphorylation of ZO-1 was detected by 24 h and increased markedly after 72 h of patulin treatment. However, phosphorylation of claudin-4 and occludin was not detected by probing with anti-phosphotyrosine antibody. Immunoprecipitation showed that interaction of ZO-1 with claudin-4 had decreased after 48 h and was completely absent after 72 h. These results suggest that phosphorylation caused the degradation of ZO-1 protein and the decrease in TER induced by patulin treatment of Caco-2 cells.     

枸杞多糖对糖尿病大鼠血-视网膜屏障保护的研究

目的 探讨链脲佐菌素(streptozotocin, STZ)诱导的糖尿病(diabetesmellitus,DM)大鼠血-视网膜屏障的渗漏与视网膜Occludin的表达,观察枸杞多糖(lycium bararum polysaccharides, LBP)对Occludin表达的影响,探索枸杞多糖对糖尿病视网膜病变的保护作用及机理.方法 SD大鼠63只,随机分为正常对照组(CON组)、LBP治疗组(LBP组)、DM生理盐水组(DM组).尾静脉注射STZ建立DM模型,成模后LBP组予以LBP灌胃(200 mg·kg-1·d-1),DM组等量生理盐水灌胃,4、8、12w处死动物.用免疫组织化学染色及灰度值、Western blotting及检测各组大鼠视网膜Occludin的表达情况;用伊凡思蓝方法检测视网膜血管渗透性.结果 免疫组织化学分析证实视网膜Occludin主要表达在视网膜神经纤维层、神经节细胞层、内网状层、及内颗粒层上.Western blotting分析证明随着糖尿病视网膜病变的发生发展, STZ诱导的DM大鼠视网膜Occludin表达量显著减少,DM组与正常对照组和LBP组差异均有统计学意义(P<0.05).造模后4w、8w和12w大鼠视网膜血管渗透性增加,DM组伊凡思蓝含量分别为(26.23±2.00)μg·-1、(29.78±1.78) μg·-1、 (34.08±3.03) μg·-1, LBP能够改变这种变化, LBP组伊凡思蓝含量分别为(13.27±0.77) μg·-1、 (18.01±1.77) μg·-1、 (25.05±1.50) μg·-1.结论 STZ诱导的DM大鼠Occludin表达量减低, LBP可显著抑制这种改变,改善DM大鼠血-视网膜屏障的渗漏.     

Increased blood–brain barrier permeability and altered tight junctions in experimental diabetes in the rat: contribution of hyperglycaemia and matrix metalloproteinases

Aims/hypothesis Although diabetes mellitus is associated with peripheral microvascular complications and increased risk of neurological events, the mechanisms by which diabetes disrupts the blood–brain barrier (BBB) are not known. Matrix metalloproteinase (MMP) activity is increased in diabetic patients, is associated with degradation of tight junction proteins, and is a known mediator of BBB compromise. We hypothesise that diabetes leads to compromise of BBB tight junctions via stimulation of MMP activity. Materials and methods Diabetes was induced in the rat with streptozotocin. At 14 days after injection, BBB function was assessed by in situ brain perfusion. Tight junction proteins were assessed by immunoblot and immunofluorescence. Plasma MMP activity was quantified by fluorometric gelatinase assay and gel zymography. Results In streptozotocin-treated animals, permeability to [¹⁴C]sucrose increased concurrently with decreased production of BBB tight junction proteins occludin (also known as OCLN) and zona occludens 1 (ZO-1, also known as tight junction protein 1 or TJP1). Insulin treatment, begun on day 7, normalised blood glucose levels and attenuated BBB hyperpermeability to [¹⁴C]sucrose. Neither acute hyperglycaemia in naive animals nor acute normalisation of blood glucose in streptozotocin-treated animals altered BBB permeability to [¹⁴C]sucrose. Plasma MMP activity was increased in streptozotocin-treated animals. Conclusions/interpretation These data indicate that diabetes increases BBB permeability via a loss of tight junction proteins, and that increased BBB permeability in diabetes does not result from hyperglycaemia alone. Increased plasma MMP activity is implicated in degradation of BBB tight junction proteins and increased BBB permeability in diabetes. Peripheral MMP activity may present a novel target for protection of the BBB and prevention of neurological complications in diabetes. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users.     

Pathophysiology of increased intestinal permeability in obstructive jaundice

Despite advances in preoperative evaluation and postoperative care, intervention, especially surgery, for relief of obstructive jaundice still carries high morbidity and mortality rates, mainly due to sepsis and renal dysfunction. The key event in the pathophysiology of obstructive jaundice-associated complications is endotoxemia of gut origin because of intestinal barrier failure. This breakage of the gut barrier in obstructive jaundice is multi-factorial, involving disruption of the immunologic, biological and mechanical barrier. Experimental and clinical studies have shown that obstructive jaundice results in increased intestinal permeability. The mechanisms implicated in this phenomenon remain unresolved, but growing research interest during the last decade has shed light in our knowledge in the field. This review summarizes the current concepts in the pathophysiology of obstructive jaundice-induced gut barrier dysfunction, analyzing pivotal factors, such as altered intestinal tight junctions expression, oxidative stress and imbalance of enterocyte proliferation and apoptosis. Clinicians handling patients with obstructive jaundice should not neglect protecting the intestinal barrier function before, during and after intervention for the relief of this condition, which may improve their patients’ outcome.     

紧密连接相关蛋白Occludin的研究进展

Occludin是紧密连接相关蛋白中最具有代表性的蛋白之一,它封闭细胞旁路,形成紧密连接的基础结构.Occludin的生理功能主要包括栅栏功能和细胞旁屏障功能,其表达低下可导致肺上皮屏障、血脑屏障、肠上皮屏障及血睾屏障的破坏,与多种疾病的发生、发展密切相关.因此,有关Occludin蛋白的调控机制及其临床意义已成为国内外研究的热点.     

谷氨酰胺对体外肠上皮细胞缺血再灌注损伤后occludin蛋白表达的影响

目的 探讨谷氨酰胺(GLN)对体外肠缺血再灌注损伤后肠上皮细胞活性及紧密连接蛋白occludin表达的影响,以研究GLN对肠黏膜保护作用的机制.方法 利用Caco-2细胞建立缺氧/复氧模型,模仿肠缺血再灌注,然后用含有不同浓度GLN的培养液继续培养.利用MTT检测细胞活性,利用RT-PCR和Western blot检测occludin蛋白的表达情况.结果 MTT结果显示,与0 mmol/L GLN组(0.635±0.041、0.690±0.032)相比,补充GLN后细胞OD值明显增加(4 mmol/L GLN组:0.716±0.040、0.904±0.089,8 mmol/L GLN组:0.768±0.040、0.856±0.073,P均<0.05).RT-PCR结果显示,与0 mmol/L GLN组(0.244±0.037、0.591±0.031)相比,GLN影响occludin蛋白mRNA的相对表达量(4 mmol/L GLN组:0.371±0.057、0.799±0.054,8 mmol/L GLN组:0.714±0.030、0.547±0.064,P均<0.05).Western blot结果显示,与0 mmol/L GLN组(35.056±2.313、74.309±1.528)相比,GLN可以增加紧密连接蛋白occludin表达(4 mmol/L GLN组:63.432±0.649、101.759±1.214,8 mmol/L GLN组:99.453±0.526、99.573±2.082,P均<0.05).结论 GLN促进肠上皮的增殖,同时在基因和蛋白水平上影响紧密连接蛋白occludin的表达.     

Pathology and new players in the pathogenesis of brain edema

Brain edema continues to be a major cause of mortality after diverse types of brain pathologies such as major cerebral infarcts, hemorrhages, trauma, infections and tumors. The classification of edema into vasogenic, cytotoxic, hydrocephalic and osmotic has stood the test of time although it is recognized that in most clinical situations there is a combination of different types of edema during the course of the disease. Basic information about the types of edema is provided for better understanding of the expression pattern of some of the newer molecules implicated in the pathogenesis of brain edema. These molecules include the aquaporins, matrix metalloproteinases and growth factors such as vascular endothelial growth factors A and B and the angiopoietins. The potential of these agents in the treatment of edema is discussed. Since many molecules are involved in the pathogenesis of brain edema, effective treatment cannot be achieved by a single agent but will require the administration of a “magic bullet” containing a variety of agents released at different times during the course of edema in order to be successful.