刺槐素对小鼠脑缺血再灌注损伤后血脑屏障的影响

目的 探讨刺槐素对小鼠脑缺血再灌注损伤后血脑屏障紧密连接主要结构蛋白ZO-1,Occludin和Claudin-5表达水平的影响。方法 将18只雄性C57BL/6小鼠采用随机数字法分为假手术组、缺血再灌注组和刺槐素治疗组,每组各6只; 刺槐素治疗组再灌注时腹腔注射刺槐素(Acacetin)(25 mg/kg); 采用改良线栓法制作大脑中动脉闭塞模型,再灌注24 h后免疫组化测定Occludin,Claudin-5及ZO-1染色阳性细胞数。结果 免疫组化染色显示,与假手术组比较,缺血再灌注组无论是皮层还海马,紧密连接蛋白Occludin,Claudin-5及ZO-1染色阳性细胞数显著减少(P<0.01); 与缺血再灌注组比较,刺槐素治疗组皮层和海马紧密连接蛋白Occludin,Claudin-5及ZO-1染色阳性细胞数显著增加(P<0.01)。结论 刺槐素可能通过增加Occludin,Claudin-5及ZO-1蛋白表达来降低血脑屏障的通透性,从而在脑缺血再灌注损伤中发挥神经保护作用。     

缺血性脑卒中对犬肠屏障功能的影响

目的 研究缺血性脑卒中对犬肠黏膜屏障功能的影响以及细胞凋亡和紧密连接蛋白在其中的可能机制.方法 杂犬20只,按随机数字表法分为梗死组和假手术组,每组10只.梗死组采用双硅柱置入法制作缺血性脑卒中模型,假手术组动物不置入栓子,其他同梗死组.光镜下观察肠黏膜形态;免疫组织化学法分析肠黏膜紧密连接蛋白闭锁蛋白和闭锁小带蛋白1的表达;采用TdT介导的dUTP缺口末端标记技术检测肠上皮细胞凋亡.结果 梗死组动物均形成脑梗死.和假手术组相比,梗死组紧密连接蛋白闭锁蛋白和闭锁小带蛋白1表达均明显降低(闭锁蛋白平均光密度值:0.20±0.01比0.22±0.01,P=0.007;闭锁小带蛋白1平均光密度值:0.20±0.01比0.22±0.02,P=0.008);梗死组凋亡指数明显增高(29.04 ±3.79比6.44±1.24,P=0.002);闭锁蛋白与闭锁小带蛋白1呈正相关(R=0.71,P=0.02);凋亡指数与闭锁蛋白和闭锁小带蛋白1均呈负相关(R=-0.91,P=0.00;R=-0.77,P=0.01).光镜下梗死组存在小肠病理损伤,假手术组小肠结构正常.结论 缺血性脑卒中时,肠道屏障功能发生损害.紧密连接蛋白闭锁蛋白和闭锁小带蛋白1表达降低,从而导致紧密连接破坏.肠黏膜细胞凋亡上调是肠屏障障碍的细胞学基础,凋亡是缺血性脑卒中时肠上皮细胞死亡的重要形式.     

麝香黄芪复方滴丸对体外缺血缺氧血脑屏障通透性及相关调节蛋白的影响

目的探究麝香黄芪复方滴丸调控体外缺血缺氧性血脑屏障(blood-brain barrier,BBB)模型的作用机制。方法利用大鼠脑微血管内皮细胞(brain microvessel endothelial cells,BMEC)与星形胶质细胞(astrocytes,AS)共培养建立体外BBB模型及缺血缺氧BBB模型。随机分为正常对照组、模型组、麝香黄芪复方滴丸高剂量组(500μg/mL)、中剂量组(100μg/mL)、低剂量组(10μg/mL)及依达拉奉对照组。通过跨内膜电阻(TEER)、辣根过氧化物酶法(HRP)检测BBB通透性变化情况。Western Blot法检测紧密连接(tight junction,TJ)相关蛋白闭合蛋白-5(Claudin-5)、连接黏附分子-A(junctional adhesion molecule,JAM-A)、跨膜蛋白的咬合蛋白(Occludin)和闭锁小带蛋白-1(zonula Occludens,ZO-1)表达水平。结果TEER及HRP实验显示,与正常对照组相比,模型组血脑屏障通透性增加(P<0.05);麝香黄芪复方滴丸高、中、低剂量组和依达拉奉组1 d、2 d、3 d各组通透性明显降低(P<0.05);WB结果显示,模型组Claudin-5、JAM-A、Occludin、ZO-1蛋白表达较正常组显著降低(P<0.05);麝香黄芪复方滴丸高、中剂量组较模型组Claudin-5、JAM-A、Occludin、ZO-1蛋白表达升高(P<0.05)。结论麝香黄芪复方滴丸能降低缺血缺氧性BBB模型的通透性,其作用机制与调控紧密连接蛋白而达到修复BBB的作用有关。     

回药扎里奴思方联合BMSCs移植对脑缺血再灌注大鼠BBB紧密连接蛋白的影响

目的观察扎里奴思方联合BMSCs移植对MCAO模型大鼠BBB上Occludin和Claudin mRNA表达的影响。方法 250只SD大鼠随机分为假手术组、模型组、扎方组、移植组和联合组,除假手术组10只外,其余各组15只;线栓法制备MCAO模型,体外全骨髓贴壁法培养及扩增BMSCs;大鼠灌胃给药[14.6 g/(kg·d)],BMSCs悬浮液经颈内动脉移植入脑(2×106/200μl);移植后1 d、3 d、7 d、14 d取材,干湿重法检测脑含水量,Real timePCR技术检测Occludin和Claudin mRNA表达。结果模型大鼠脑含水量较假手术组增加(P0.01),Occludin和Claudin mRNA表达降低(P0.01);与模型组比较,扎方、移植及联合各3 d、7 d、14 d组脑含水量降低(P0.01),各组各时间点Occludin和Claudin mRNA表达增高(P0.01);与移植组比较,扎方1 d组脑含水量降低(P0.05),3 d组Occludin mRNA表达增高(P0.01),7 d组表达降低(P0.01),扎方1 d组Claudin mRNA表达增高(P0.05),7 d、14 d组表达降低(P0.01),联合各组脑含水量均降低,以1 d、14 d明显(P0.01),各组Occludin和Claudin mRNA表达均增高(P0.01);扎方与联合组比较,联合各组脑含水量降低,以7 d、14 d组明显(P0.01,P0.05),Occludin和Claudin mRNA表达增高(P0.01);同组间比较,均以7 d组变化显著,脑含水量呈先增后减趋势,Occludin和Claudin mRNA表达呈先减后增趋势,14 d有明显改善(P0.01)。结论脑缺血再灌注损伤后BBB受损,通透性改变,脑水肿形成,以7 d最为明显;扎里奴思方和BMSCs移植均可不同程度改善脑缺血后脑水肿程度,以二者联合应用作用显著,其机制可能与干预Occludin和Claudin mRNA动态表达有关。     

JCOG trials of systemic chemotherapy for unresectable or recurrent gastric cancer

Background  Claudin, occludin, and zonula occludens (ZO)-1 are known as tight-junction-associated proteins. The aim of this study was to examine the expression of these proteins in gastric carcinoma. Methods  Gastric cancer tissues (n = 124) were obtained from 124 patients who underwent gastrectomy at our hospital between January 2000 and December 2004. The expression of the above tight-junction-associated proteins in carcinoma, normal mucosa, and metaplastic epithelium was examined using immunohistochemistry. In addition, the expression of claudin-4 mRNA was examined in fresh frozen tissue obtained from 34 patients. Results  Significant correlations were seen between the expression of claudin-4, occludin, and ZO-1. In regard to claudin-4, significant correlations were seen between the expression of claudin-4 evaluated by immunohistochemistry and the expression of claudin-4 mRNA. Claudin-4 expression was significantly decreased in tumors with undifferentiated-type adenocarcinoma, advanced T stage, lymph node metastasis, and peritoneal metastasis. Occludin and ZO-1 expression was significantly decreased in tumors with undifferentiated-type adenocarcinoma. Overall survival was significantly shorter in patients with low claudin-4 expression. Cox multivariate analysis revealed that low claudin-4 expression was independently associated with significantly decreased overall survival. Conclusion  Tight-junction-associated proteins, particularly claudin-4, may play important roles in determining invasiveness, metastatic potential, and survival in gastric cancer.     

The loss of αSNAP downregulates the expression of occludin in the intestinal epithelial cell of acute pancreatitis model

Background and objectiveIntestinal barrier damage is an important event during the occurrence and progression of severe acute pancreatitis. The expression of occludin, one of the main components of the intestinal barrier proteins, is regulated by various factors related to intestinal barrier formation and the remodeling process. The αSNAP, as a novel membrane protein, is ubiquitously expressed in intestinal epithelial cells. This study aimed to investigate the role of αSNAP in acute pancreatitis and the relationship between occludin and αSNAP.MethodsMild and severe acute pancreatitis models were established by retrograde injections of 0.5% and 3.8% sodium taurocholate solutions, respectively, into rat pancreaticobiliary ducts. The animals were killed at 1, 2, and 3 days after the injection, and the pathological changes of the pancreas and intestinal mucosa, the changes in intestinal permeability, and the protein expression of occludin and αSNAP were assessed. Cultured epithelial IEC-6 cells were further infected with lentiviral αSNAP shRNA, cell apoptosis was determined with flow cytometry (FCM), and any changes in occludin expression were detected by Western blotting and immunofluorescent staining.ResultsThis pathologic study of a rat acute pancreatitis model indicated pancreatic tissue necrosis and inflammatory cell infiltration; the intestinal villi in the severe acute pancreatitis (SAP) group demonstrated edema, lodging, atrophy, and intestinal epithelial cell necrosis, and shedding. The intestinal permeability in rats with pancreatitis increased significantly. The SAP group showed significantly increased levels of serum TNF-α and endotoxins. The results of immunofluorescent staining and Western blotting revealed that compared with the SO (sham operation) and MAP (mild acute pancreatitis) groups, the SAP group displayed significantly downregulated protein expressions of αSNAP and occludin in the intestinal epithelial cells. After the lentiviral transduction of αSNAP shRNA, apoptosis in IEC-6 cells was drastically increased, whereas the expression of occludin was decreased significantly.ConclusionThe downregulated expression of αSNAP in intestinal epithelial cells leads to reduced occludin expression and enhanced apoptosis of intestinal epithelial cells. Hence, the permeability of the intestinal barrier may be increased in a severe acute pancreatitis model.     

急性肝衰竭小鼠Occludin表达与血脑屏障通透性的关系

目的探讨乙酰氨基酚/扑热息痛(acetam inophen/paracetam ol,APAP)导致的急性肝衰竭(acute liverfailure,ALF)小鼠模型中血脑屏障(blood brain barrier,BBB)通透性与脑组织紧密连接(tight junction,TJ)蛋白O ccludin表达的关系。方法应用APAP复制ALF小鼠模型,检测脑中伊文思蓝(evans blue,EB)的含量反映BBB通透性,电镜下观察TJ的结构变化。应用W estern blotting方法检测脑组织中O ccludin蛋白的表达。结果小鼠脑组织EB含量于2 h开始升高(2.29±0.42)μg/g,6 h达到峰值(3.61±0.33)μg/g,脑组织中O ccludin蛋白表达2 h开始下降,6 h表达最低,同时电镜下观察到TJ结构出现破坏。结论在APAP所致的ALF动物模型中,BBB通透性增加,表明血管源性脑水肿机制参与脑水肿形成,与其相反,O ccludin表达呈下降趋势,提示脑水肿的发生与O ccludin表达下降引起TJ结构破坏有关。     

阿司匹林对人胃黏膜上皮细胞生长和occludin蛋白表达的影响及其机制研究

背景：研究显示ERK信号通路激活可促进细胞生长,上调紧密连接蛋白oceludin表达,而阿司匹林可抑制ERK的磷酸化激活。目的：探讨阿司匹林对人胃黏膜上皮细胞生长和occludin蛋白表达的影响及其可能机制。方法：建立人胃黏膜上皮细胞株GES-1单层细胞模型．M1Tr法检测阿司匹林（5-20mmol／L）和MEK抑制剂U0126（10～40μmol／L）对GES-1细胞的生长抑制作用。将GES-1细胞分为阿司匹林（8．78mmol／L）组、U0126（14．91μmol／L）组、联合组（U0126＋阿司匹林）和阴性对照组,倒置相差显微镜下观察细胞形态学变化,流式细胞术检测细胞凋亡情况,蛋白质印迹法检测P-ERK1／2、occludin蛋白表达。结果：阿司匹林和U0126均能剂量依赖性地抑制GES．1细胞生长,联合组细胞凋亡较两药单用更为显著,贴壁细胞数量减少更为明显．细胞变圆、悬浮。阿司匹林组、U0126组和联合组P-ERK1／2、occludin蛋白表达量均显著低于阴性对照组,U0126组和联合组降低更为明显。结论：阿司匹林可抑制人胃黏膜上皮细胞生长．下调occludin蛋白表达,其机制至少部分与抑制ERK信号通路激活有关。     

益气托毒活血中药复方对溃疡性结肠炎大鼠肠道黏膜屏障的影响

目的:探讨益气托毒活血中药复方对三硝基苯磺酸(TNBS)诱导的大鼠结肠炎肠黏膜上皮细胞紧密连接蛋白的调控,及对肠黏膜屏障可能的保护机制.方法:健康成年SD大鼠52只,7只作为正常对照组,剩余45只大鼠采用TNBS法制作溃疡性结肠炎(UC)大鼠模型,第3天随机抽取2只解剖,观察造模是否成功,余下的43只大鼠分为模型组13只,柳氮磺吡啶片(SASP)组l5只,中药组15只,灌胃治疗10 d后观察病理形态学并进行疾病活动指数(DAI)、结肠黏膜损伤指数(CMDI)评分,用EILSA法测定血清内毒素,用免疫组化法测定紧密连接(tight junction,TJ)相关蛋白咬合蛋白(Occludin)表达.结果:TNBS法诱导大鼠结肠炎后,DAI和CM-DI评分增高,血清内毒素(LPS)水平增高,结肠黏膜Occludin蛋白下降;SASP及中药均能降低UC大鼠DAI和CMDI评分,降低血清LPS水平,上调结肠黏膜Occludin蛋白表达,差异均有统计学意义(P＜0.05);与SASP组相比,中药组DAI评分较低,差异有统计学意义(P＜0.05).结论:益气托毒活血复方是治疗UC的有效方法,其可能通过上调肠黏膜Occludin蛋白蛋白表达,改善肠黏膜通透性,抑制内毒素通过紧密连接进入体循环,从而起到对UC大鼠肠黏膜屏障的保护作用.     

Viability of microvascular endothelial cells to direct exposure of formalin,lambda-carrageenan,and complete Freund's adjuvant

We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood–brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, λ-carrageenan, or complete Freund's adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (34%) and a significant increase in cell permeability to [¹⁴C]sucrose at 120 min (137%). Brain perfusion using female Sprague–Dawley rats showed no difference in paracellular permeability to [¹⁴C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood–brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood–brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.