bFGF ameliorates intestinal mucosal permeability and barrier function through tight junction proteins in burn injury rats

BackgroudTo investigate the protective effect of exogenous basic fibroblast growth factor (bFGF) treatment on the intestinal mucosa in scalded rats.MethodsThirty-six SD rats were randomly divided into 3 groups (n = 12): sham group, scald group and bFGF group (0.5 mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and Chiu’s grading system. H&E staining was used to detect the morphological changes of intestinal mucosa. Immunohistochemistry was used to observe zonula occludens-1 (ZO-1) and occludin. Western blot assay was used to detect the expression of ZO-1, Claudin-1, occludin and myosin light-chain kinase (MLCK).ResultsThe results demonstrated that following bFGF treatment, permeability of the intestinal epithelium barrier of was significantly decreased compared to scald group. H&E staining and Chiu’s grading were consistent with previous result. The expression of ZO-1, Claudin-1, occludin in bFGF group were significantly increased compared to scald group, while MLCK protein was decreased.ConclusionsbFGF ameliorates permeability of intestinal mucosa after burns. The possible mechanism may be relate to bFGF could increase the expression level of tight junction proteins (TJPs).     

重视烧伤后肠道紧密连接屏障功能障碍的研究

Severe burn injury is often accompanied by intestinal epithelial tight junction barrier dysfunction, which is believed to be closely associated with postburn shock, inflammation, hypermetabolism, infection, organ dysfunction etc. Recent studies have documented the critical role of tight junction-associated protein regulation in intestinal epithelial barrier dysfunction induced by severe burn injury. Myosin light chain (MLC) phosphorylation regulated by both myosin light chain kinase, which can phosphorylate MLC directly, and Rho-associated kinase,which can inhibit MLC phosphatase and then induce MLC phosphorylation indirectly, play a critical role in intestinal epithelial tight junction barrier dysfunction which occurs in severe burn injury. Recent advances have provided new insights into the mechanisms and the therapeutic strategies of intestinal epithelial tight junction barrier dysfunction following severe burn injury.     

不同营养方式对梗阻性黄疸大鼠肠紧密连接蛋白的影响

目的 观察肠内营养(EN)及肠外营养(PN)对阻塞性黄疸(OJ)大鼠小肠紧密连接蛋白的影响.方法 将50只Wistar大鼠随机分5组,EN组和PN组给予等热量等氮量营养.7 d后采用免疫组织化学和Western blot法检测各组末端回肠黏膜的闭锁小带-1(ZO-1)、闭锁蛋白(Occludin)与肌球蛋白轻链激酶(MLCK)的表达,并对Western blot图像进行定量分析.结果 正常回肠黏膜ZO-1和Occludin沿绒毛下方均匀连续分布,MLCK主要分布在细胞质内.阻黄时ZO-1、Occludin和MLCK分布散乱,染色稀疏.PN组的ZO-1、Occludin和MLCK的强阳性染色数由阻黄组的2、2、1例升至4、5、3例(P均>0.05),而EN组的强阳性染色数分别上升为7、6、5例(P均<0.05).通过对Western blot显影图像进行定量分析,EN组和PN组的ZO-1的灰度值较阻塞性黄疸组明显上升(均P<0.05);Occludin和MLCK的灰度值在EN组上升(P<0.05),PN组没有变化.结论 梗阻性黄疸时大鼠小肠黏膜上皮ZO-1、Occludin和MLCK分布紊乱,表达下降.肠内、外营养均能够恢复受损的紧密连接蛋白,肠内营养的作用更强.     

The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study.

The timely opening and closing of inter-Sertoli cell tight junctions in the rat testis are essential cellular events in the completion of spermatogenesis. They permit the passage of preleptotene and leptotene spermatocytes to cross the blood-testis barrier from the basal compartment to the adluminal compartment of the seminiferous epithelium so that these cells can continue their further development into spermatids. However, the mechanism by which these events is regulated remains a mystery in male reproductive physiology. As part of our long-term goal of understanding the biology of this event and its regulation, transepithelial electrical resistance (TER) across the Sertoli cell epithelia when inter-Sertoli tight junctions were being assembled in vitro was quantified to assess the effects of different inhibitors of phosphatases and kinases on the inter-Sertoli tight junction permeability barrier. It was shown that inhibitors of protein tyrosine phosphatases (PTPi) and inhibitors of protein Ser/Thr phosphatases (PPi) could perturb the assembly and maintenance of the inter-Sertoli tight junction permeability barrier. Moreover, the inhibitory effects of PTPi were abolished by pretreating Sertoli cells with protein tyrosine kinase inhibitor (PTKi), which illustrates the specificity of the PTPi treatment. A cyclic adenosine monophosphate-dependent protein kinase A (PKA) activator and inhibitors of calcium-diacylglycerol-dependent protein kinase C (PKC) can also perturb the inter-Sertoli tight junction permeability barrier, which suggests that opening and closing of the inter-Sertoli tight junctions during spermatogenesis is likely regulated, at least in part, by the PKA/PKC pathways. Needless to say, these results illustrate that the interplay of protein kinases and phosphatases, which regulate the intracellular phosphoprotein content of Sertoli cells possibly via PKA and PKC signal transduction pathways, plays a crucial role in modulating the assembly and maintenance of inter-Sertoli tight junctions in the testis.     

Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments

As the first step in understanding the physiologic functions of claudins (tight junction integral membrane proteins) in nephrons, the expression of claudin-1 to -16 in mouse kidneys was examined by Northern blotting. Among these claudins, only claudin-6, -9, -13, and -14 were not detectable. Claudin-5 and -15 were detected only in endothelial cells. Polyclonal antibodies specific for claudin-7 and -12 were not available. Therefore, the distributions of claudin-1, -2, -3, -4, -8, -10, -11, and -16 in nephron segments were examined with immunofluorescence microscopy. For identification of individual segments, antibodies specific for segment markers were used. Immunofluorescence microscopic analyses of serial frozen sections of mouse kidneys with polyclonal antibodies for claudins and segment markers revealed that claudins demonstrated very complicated, segment-specific, expression patterns in nephrons, i.e., claudin-1 and -2 in Bowman's capsule, claudin-2, -10, and -11 in the proximal tubule, claudin-2 in the thin descending limb of Henle, claudin-3, -4, and -8 in the thin ascending limb of Henle, claudin-3, -10, -11, and -16 in the thick ascending limb of Henle, claudin-3 and -8 in the distal tubule, and claudin-3, -4, and -8 in the collecting duct. These segment-specific expression patterns of claudins are discussed, with special reference to the physiologic functions of tight junctions in nephrons.     

Intestinal tight junction in allograft after small bowel transplantation

INTRODUCTION: In the intestinal physical barrier, tight junctions between intestinal epithelial cells play a central role. There is increasing evidence that rejection after small bowel transplantation promotes intestinal barrier injury to allografts. Our aim was to study the morphological changes of tight junctions in allografts during rejection. METHODS: Small bowel transplantation was performed using the F344 to Lewis rat model. Animals were divided into three groups: isogeneic controls, acute rejection group, and chronic rejection group. Allograft rejection was characterized by hemotoxylin and eosin staining of mucosal tissue sections. Tight junctions in grafts were investigated by transmission electron microscopy. RESULTS: Acutely rejected allografts showed severe mucosal injury and completely loosened tight junctions, while chronically rejected allografts revealed less mucosal injury and remained with partial integrity of their tight junctions. CONCLUSION: Our study demonstrated for the first time the morphological alterations of tight junctions in allograft mucosa during acute and chronic rejections, suggesting disruption of tight junctions was relative to the intestinal inflammatory processes.     

肠外肠内营养对腹腔感染大鼠肠上皮紧密连接和屏障功能的影响

目的　探讨肠外肠内营养对腹腔感染大鼠肠上皮紧密连接和屏障功能的影响。方法　14只存活6d的腹腔感染SD大鼠分别给予肠外营养(PN组)、肠外营养 肠内营养(PN EN组)。两组动物供给等热、等氮量。第6天处死动物,取末段回肠和结肠采用免疫组化法测定其跨膜结合蛋白(occludin)表达及肠上皮浆细胞IgA表达并定量;取腔静脉血及肺、肝、肠系膜淋巴组织匀浆后作细菌培养测细菌易位率;取门静脉血经鲎试剂法检测内毒素含量。结果　PN EN组小肠和大肠occludin及IgA表达明显优于PN组(P <0 .0 5及P <0 .0 1) ;血、肺、肝、肠系膜淋巴组织的细菌易位率和内毒素水平均低于PN组(P <0 .0 5 )。结论　肠内营养提高了肠上皮occludin表达,增加了肠道IgA的分泌,改善机械及免疫屏障,从而减少细菌易位。     

麝香配伍乳香对大鼠前列腺上皮细胞紧密连接结构相关蛋白表达的影响

目的:探讨麝香-乳香配伍处理对正常及慢性前列腺炎模型大鼠前列腺上皮细胞紧密连接结构相关蛋白表达的影响。方法:80只雄性SD大鼠随机分为8组:正常空白对照组、正常麝香-乳香组、正常麝香组、正常乳香组、模型空白组、模型麝香-乳香组、模型麝香组、模型乳香组。制备慢性前列腺炎大鼠模型,造模60 d时根据组别分别给予麝香0.021 g/(kg·d)、乳香1.05 g/(kg·d)剂量灌胃,麝香-乳香组给予联合剂量,空白组给予生理盐水灌胃。各组别连续灌胃3 d,处死大鼠,取前列腺组织,固定后以免疫组化法检测大鼠前列腺上皮屏障紧密连接功能相关蛋白紧密连接蛋白1、紧密连接蛋白3、闭锁蛋白、胞质附着蛋白1(ZO-1)的表达情况。结果:在病理状态下,仅紧密连接蛋白1的表达增高有统计学意义。麝香、乳香处理后,在生理/病理状态下对4种蛋白的调节作用不同:生理状态下,单用麝香(824.6±393.3)、乳香(982.0±334.0)或配伍处理(1 088.1±640.2)均可大幅度增高紧密连接蛋白1的表达(P0.05,P0.01);对于紧密连接蛋白3,单用乳香表达上调(1 009.5±243.6,P0.05),单用麝香(597.5±80.7)差异无统计学意义,配伍麝香(678.4±255.1)可降低乳香上调表达作用;单用麝香(693.0±424.8)、乳香(732.1±302.0)或配伍处理(560.2±202.3)对闭锁蛋白表达影响差异无统计学意义;单用麝香(290.0±166.8)、乳香(419.7±108.1)对ZO-1表达影响差异无统计学意义,配伍处理后表达明显下调(197.7±98.2,P0.05)。慢性前列腺炎病理状态下,大鼠前列腺组织紧密连接蛋白1(823.0±100.1)、闭锁蛋白(1 160.0±32.2)表达显著增高(P0.01,P0.05);单用麝香(764.9±179.0)、乳香(468.4±220.4)或配伍(335.1±204.0)使用可下调紧密连接蛋白1表达(P0.05);单用麝香(700.1±223.7)或乳香(744.6±94.5)可上调紧密连接蛋白3表达(P0.05);单用麝香(749.6±321.7)、乳香(615.0±221.0)或配伍处理(505.8±523.7)均可下调闭锁蛋白表达;单用乳香可上调ZO-1表达(443.2±144.9),与麝香配伍处理后表达下调(213.5±24.9),与单用乳香组比较差异有统计学意义(P0.05)。结论:麝香、乳香对生理/病理状态下前列腺上皮屏障结构紧密连接相关蛋白的调节作用具有选择性和双向性,主要通过ZO-1蛋白的下调实现促通透性作用,而通过调节紧密连接蛋白1、紧密连接蛋白3、闭锁蛋白表达以维持结构稳定性。     

Tumor necrosis factor alpha disrupts tight junction assembly

BACKGROUND: We have previously shown an increase in intestinal permeability and a corresponding decrease in the expression of tight junction (TJ) proteins in the in testines of patients with Crohn's disease (CD). Tumor necrosis factor-alpha (TNFalpha) has been implicated in the inflammatory process of CD and its suppression has therapeutic benefit. ZO-1, occludin, and the claudins are key proteins in the TJ. Hypothesis: TNFalpha disrupts the TJ. METHODS: MDCK cells were incubated with TNFalpha (0-100 ng/ml) for 5 days. Qualitative evaluation of the TJ was done with monoclonal antibody to ZO-1 detected by an immunofluorescence. Duplicate cells were lysed and ZO-1, occludin, and claudin-1 amount determined by western blot. RESULTS: Immunofluorescent staining of MDCK cells for ZO-1 showed TJ structural disruption with increasing amount of TNFalpha characterized by fragmented staining of ZO-1. There were no significant differences in quantitation of ZO-1 or occludin in the MDCK cells for all TNFalpha concentrations. There was a significant decrease in the amount of claudin-1 with increasing concentration of TNFalpha. CONCLUSIONS: (1) MDCK TJs are qualitatively disrupted by TNFalpha. (2) This disruption is not because of a decrease in cell number, lack of cell layer confluency, or a decrease in the amount of ZO-1 or occludin. (3) The amount of claudin-1 present in the cell is decreased with increasing amounts of TNFalpha suggesting that the lack of claudin-1 may cause a relocation of ZO-1 away from the TJ. (4) This rearrangement may play a role in the increased intestinal permeability seen in CD and other diseases.     

Irinotecan injures tight junction and causes bacterial translocation in rat

BackgroundThe objective of this study was to evaluate the efficacy of resuscitative endovascular aortic balloon occlusion (REBOA) of the distal aorta in a porcine model of pelvic hemorrhage.MethodsSwine were entered into three phases of study: injury (iliac artery), hemorrhage (45 s), and intervention (180 min). Three groups were studied: no intervention (NI, n = 7), a kaolin-impregnated gauze (Combat Gauze) (CG, n = 7), or REBOA (n = 7). The protocol was repeated with a dilutional coagulopathy (CG-C, n = 7, and REBOA-C, n = 7). Measures of physiology, rates of hemorrhage, and mortality were recorded.ResultsRate of hemorrhage was greatest in the NI group, followed by the REBOA and CG groups (822 ± 415 mL/min versus 11 ± 13 and 0.2 ± 0.4 mL/min respectively; P < 0.001). MAP following intervention (at 15 min) was the same in the CG and REBOA groups and higher than in the NI group (70 ± 4 and 70 ± 11 mm Hg versus 5 ± 13 mm Hg respectively; P < 0.001). There was 100% mortality in the NI group, with no deaths in the CG or REBOA group. In the setting of coagulopathy, the rate of bleeding was higher in the CG-C versus the REBOA-C group (229 ± 295 mL/min versus 20 ± 7 mL/min, P = 0.085). MAP following intervention (15 min) was higher in the REBOA-C than the CG-C group (71 ± 12 mm Hg versus 28 ± 31 mm Hg; P = 0.005). There were 5 deaths (71.4%) in the CG-C group, but none in the REBOA-C group (P = 0.010).ConclusionBalloon occlusion of the aorta is an effective method to control pelvic arterial hemorrhage. This technique should be further developed as an adjunct to manage noncompressible pelvic hemorrhage.     

梗阻性黄疸大鼠肠黏膜上皮紧密连接蛋白和MLCK的研究

目的:探讨梗阻性黄疸肠黏膜屏障破坏的机制。方法:建立梗阻性黄疸大鼠的动物模型,分别于胆管结扎10 d和20 d后，采用免疫组化、Western blot方法检测末端回肠黏膜的紧密连接蛋白成员ZO-1和Occludin与肌球蛋白轻链激酶（MLCK）的分布和表达。结果:正常回肠黏膜层ZO-1和Occludin的分布相似，主要位于上皮细胞的边缘，细胞膜顶端，沿绒毛下方均匀连续分布；MLCK主要分布在细胞浆内。梗阻性黄疸时ZO-1和Occludin分布不均, 染色变淡，线条模糊，边缘粗糙有毛刺状突起；MLCK分布散乱，染色稀疏。与对照组相比20 d组和10 d组的ZO-1，Occludin，MLCK数量明显减少，强阳性表达率分别从70.0 %，80.0 %，70.0 %降至10 d组的28.6 %，28.6 %，28.6 % (均P＜0.05)和20 d组的ZO-1从10 d 组的28.6 %降至14.3 %, 35.7 %, 21.4 % (均P＜0.05)。20 d 组的ZO-1下降较10 d组更为明显（P<0.05）, 而Occludin和MLCK变化不明显。Western blot检测的结果与之相似。结论:梗阻性黄疸时大鼠小肠黏膜上皮ZO-1，Occludin，MLCK分布紊乱，表达下降，小肠黏膜上皮屏障的完整性破坏。     

Regulation of tight junction proteins and bladder epithelial paracellular permeability by an antiproliferative factor from patients with interstitial cystitis

PURPOSE: Previous reports have suggested that the bladder epithelial barrier may be compromised in interstitial cystitis (IC). Antiproliferative factor (APF) is a small glycoprotein made specifically by bladder epithelial cells in patients with IC that induces changes in expression of certain epithelial cell proteins and profoundly inhibits cell growth. Therefore, we confirmed the increased permeability and decreased tight junction formation of bladder epithelial cell monolayers grown from biopsies in patients with IC compared to cells from normal controls. We then determined the effect of APF on the permeability of normal bladder epithelial cell monolayers and the expression of tight junction proteins. MATERIALS AND METHODS: Permeability was determined by measuring the C-mannitol and H-inulin flux between cells in confluent monolayers on Transwell culture plates (Corning, Corning, New York). Tight junction formation was assessed by immunofluorescence microscopy and the expression of specific proteins was determined by Western blot. RESULTS: APF treatment caused significant increases in the paracellular permeability of normal bladder epithelial cell monolayers and the attenuation of tight junctions compared to mock APF, similar to changes seen in IC cells. APF treatment also decreased expression of the tight junction proteins zonula occludens-1 and occludin. CONCLUSIONS: Because of its apparent effects on bladder epithelial cell tight junctions and paracellular permeability in vitro, APF may contribute to the leakiness of the bladder epithelial barrier seen in IC.