黄体酮对脑缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的影响

目的探讨黄体酮对大鼠局灶性脑缺血再灌注后血脑屏障紧密连接蛋白ZO-1、occludin表达及血脑屏障通透性的影响。 方法将42只健康雄性SD大鼠按随机数字表法分为假手术组（6只）和缺血再灌注组,后者再按再灌注时间分为缺血2h再灌注3h、6h、12h、24 h、48 h及72h组（各6只）。缺血再灌注组用线栓法制备成大鼠大脑中动脉缺血再灌注模型。采用荧光分光光度法测定缺血侧脑组织中伊文氏蓝(EB)含量来评价血脑屏障的通透性,Western blotting法检测脑组织ZO-1和occludin的表达。取EB漏出最多组的时间点,增设黄体酮干预组和溶剂对照组（各6只）,与相同时间点的缺血再灌注组比较,观察黄体酮对ZO-1、occludin表达及血脑屏障通透性的影响。 结果 缺血2h再灌注3h时脑组织EB含量开始增加,再灌注24 h时达高峰;ZO-1、occludin的表达在缺血2h再灌注3h时开始下降,再灌注24 h时达最低。黄体酮干预组EB含量明显低于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。黄体酮干预组ZO-1和occludin的表达水平均明显高于缺血2h再灌注24 h组,差异有统计学意义(P＜0.05)。 结论 黄体酮町抑制缺血再灌注大鼠紧密连接蛋白ZO-1和occludin表达的降低,从而起到保护血脑屏障的作用。     

胆汁外引流对梗阻性黄疸大鼠肠黏膜紧密连接影响的实验研究

目的探讨胆汁在保护小肠黏膜屏障中的作用及其机理。方法 50只Wistar大鼠随机分为对照组(n=10)、梗阻性黄疸(阻黄)组(n=20)及外引流组(n=20)。造模10 d后测定血浆内毒素水平;取末端回肠黏膜组织,光镜下观察黏膜形态改变并测量相关指标,采用免疫组化及Western blot法检测紧密连接相关蛋白(闭锁小带蛋白-1及闭锁蛋白)的表达。结果阻黄组小肠黏膜萎缩明显,其肠绒毛高度、黏膜厚度和隐窝深度与对照组相比分别下降27.8%、21.7%和25.4%(P=0.001、0.001、0.040);外引流组各指标较对照组无明显变化(P=0.050、0.070、0.080),而明显优于阻黄组(均P=0.001)。阻黄组的血浆内毒素水平高达(1.49±0.27)EU/ml,明显高于对照组的(0.27±0.09)EU/ml(P=0.001);外引流组为(0.91±0.25)EU/ml,高于对照组而低于阻黄组(P=0.001)。免疫组化结果显示,闭锁小带蛋白-1的强阳性表达从对照组的7/10例降至阻黄组的6/20例(P=0.040),闭锁蛋白则从8/10例降至7/20例(P=0.020);而外引流组分别为8/20例(P=0.100、0.210)和9/20例(P=0.060、0.200),与前2组相比差异均没有统计学意义。Western blot分析显示,阻黄组闭锁小带蛋白-1和闭锁蛋白的表达水平较之对照组均明显下降(P=0.001、0.010);外引流组高于阻黄组(P=0.005、0.014),而闭锁小带蛋白-1的表达水平低于对照组(P=0.001),闭锁蛋白的表达与对照组比较差异无统计学意义(P=0.062)。结论肠道内胆汁缺乏会破坏肠紧密连接相关蛋白的成分,损伤肠黏膜屏障;胆道梗阻时由于存在其他因素,肠黏膜屏障损伤更严重。胆汁在保护肠黏膜屏障中具有重要作用。     

跨膜结合蛋白occludin在血吸虫病肝硬化小鼠结肠上皮细胞中的表达及其意义

目的:探讨跨膜结合蛋白occludin在血吸虫病肝硬化小鼠结肠上皮细胞中的表达变化及其意义。方法:将40只健康小鼠随机分成对照组(N组)和模型组(M组)各20只,M组采用腹部敷贴法制作血吸虫病模型;造模后120 d处死动物,用鲎试验法测定门静脉血内毒素水平;免疫组化和半定量Western印迹方法测定结肠上皮紧密连接蛋白occludin的表达;透射电镜观察结肠上皮细胞的超微结构。结果:M组门静脉血内毒素水平高于N组(P0.01);M组结肠上皮occludin蛋白的表达水平低于N组(P0.01);两者之间呈显著正相关。结论:血吸虫病肝硬化可发生肠源性内毒素易位、内毒素血症,可能与紧密连接蛋白occludin表达下降和紧密连接蛋白结构异常有关。     

Enteropathogenic Escherichia coli changes distribution of occludin and ZO-1 in tight junction membrane microdomains in vivo

Diarrhea is a disease caused by enteropathogenic Escherichia coli (EPEC) infection, which caused the deaths of several hundred thousand children each year. However, the molecular mechanisms underlying EPEC infection in vivo are not fully understood. In the present study, we used the C57BL/6J mouse as an in vivo model of EPEC infection and investigated the effect of EPEC on tight junction (TJ) structure and barrier function. TJ ultrastructure was studied by transmission electron microscopy and a small molecule tracer biotin was used to examine the paracellular permeability of the colon. The distribution of TJ proteins occludin and ZO-1 in the epithelium was investigated by immunofluorescence microscopy. Our results demonstrated that TJ structure was disrupted following EPEC infection. And the morphological changes of TJ were accompanied by increased paracellular permeability which led to impairment of TJ barrier function. Immunofluorescency analysis revealed that occludin and ZO-1 were translocated from villous membrane to the cytoplasm in intestinal epithelial cells during EPEC invasion. Moreover, wild-type EPEC and the mutant EPEC strain, ΔespF, had similar effects on barrier function and TJ protein localization at 5 days postinfection. Our findings demonstrate that EPEC infection in vivo led to disruption of tight junction barrier function. These results may provide insights into the molecular mechanism of the pathogenesis of EPEC infection.     

Emodin enhances alveolar epithelial barrier function in rats with experimental acute pancreatitis

AIM: To investigate the effect of emodin on expression of claudin4, claudin5 and occludin, as well as the alveolar epithelial barrier in rats with pancreatitis induced by sodium taurocholate. METHODS: Experimental pancreatitis was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Emodin was injected via the external jugular vein 3 h after induction of acute pancreatitis. Rats from sham operation group and acute pancreatitis group were injected with normal saline (an eq...     

Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure

AIM:To determine the effect of tumor necrosis factor alpha(TNF-α) on intestinal permeability(IP) in mice with fulminant hepatic failure(FHF),and the expression of tight junction proteins.METHODS:We selected D-lactate as an index of IP,induced FHF using D-galactosamine/lipopolysaccharide and D-galactosamine/TNF-α,assessed the results using an enzymatic-spectrophotometric method,transmission electron microscopy,immunohistochemistry,Western blotting and real-time quantitative polymerase chain reaction.The effect of the administration of antiTNF-α immunoglobulin G(IgG) antibody,before the administration of D-galactosamine/lipopolysaccharide,on TNF-α was also assessed.RESULTS:IP was significantly increased in the mouse model of FHF 6 h after injection(13.57 ± 1.70 mg/L,13.02 ± 1.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.001).Electron microscopic analysis revealed tight junction(TJ) disruptions,epithelial cell swelling,and atrophy of intestinal villi.Expression of occludin and claudin-1 mRNA was significantly decreased in both FHF models(occludin:0.57 ± 0.159 fold vs baseline,P = 0.000;claudin-1:0.3067 ± 0.1291 fold vs baseline,P = 0.003),as were the distribution density of proteins in the intestinal mucosa and the levels of occludin and claudin-1 protein(occludin:0.61 ± 0.0473 fold vs baseline,P = 0.000;claudin-1:0.6633 ± 0.0328 fold vs baseline,P = 0.000).Prophylactic treatment with antiTNF-α IgG antibody prevented changes in IP(4.50 ± 0.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.791),intestinal tissue ultrastructure,and the mRNA levels of occludin and claudin-1 expression(occludin:0.8865 ± 0.0274 fold vs baseline,P = 0.505;claudin-1:0.85 ± 0.1437 fold vs baseline,P = 0.1),and in the protein levels(occludin:0.9467 ± 0.0285 fold vs baseline,P 0.05;claudin-1:0.9533 ± 0.0186 fold vs baseline,P = 0.148).CONCLUSION:Increased in IP stemmed from the downregulation of the TJ proteins occludin and claudin-1,and destruction of the TJ in the colon,which were induced by TNF-α in FHF mice.     

小檗碱对酒精性肝炎小鼠“肠漏”的保护作用*

目的 探讨小檗碱（BBR）对酒精性肝炎小鼠“肠漏”的保护作用。方法 将40只雄性C57BL/6小鼠随机分为对照组、酒精组、BBR组和酒精联合BBR组,饲养8 w。采用ELISA法检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和内毒素（LPS）水平。常规行组织病理学和透射电镜检查肝组织学改变和肠上皮细胞间紧密连接（TJ）超微结构。体外培养肠上皮细胞Caco-2,采用免疫荧光法检测经酒精和BBR处理后的细胞间紧密连接主要结构蛋白occludin表达。结果 酒精处理组小鼠血清ALT、AST和LPS水平分别为（46.5± 5.9） U/L、（57.0± 6.1） U/L和（0.40± 0.05） ng/ml,显著高于对照组（P< 0.01）;酒精联合BBR组血清ALT、AST和LPS水平分别为（27.6± 4.2） U/L、（31.8± 4.1） U/L和（0.24± 0.03） ng/ml,均显著低于酒精组（P< 0.05）,而与对照组无显著性差异（P> 0.05）;酒精联合BBR组肝组织细胞变性和炎细胞浸润较酒精干预组明显改善;电镜下,可见酒精处理组小鼠肠上皮细胞间紧密连接结构模糊、缝隙明显增宽,酒精联合BBR组以上改变明显减轻;酒精处理组Caco-2细胞膜occludin分布减少、中断,部分进入胞浆内,酒精联合BBR组分布虽也减少,但仍连续,无中断。结论 小檗碱能改善慢性乙醇摄入诱导的细胞膜occludin减少和上皮细胞间紧密连接结构破坏,改善“肠漏”而具有保护肝脏作用。*基金项目：北京市自然科学基金资助项目(编号：7154194）;国家自然科学基金资助项目（编号：81570536）作者单位：150086 哈尔滨市 哈尔滨医科大学附属第二医院体检中心（王洪岩）;北京市首都医科大学附属北京天坛医院消化内科（李鑫,迟程,徐有青）第一作者：王洪岩,女,31岁,医学博士,住院医师。主要从事酒精性肝病的防治研究。E-mail: xinglin_freedom@126.com     

四君子汤对UC模型大鼠的治疗作用及其对紧密连接蛋白Occludin表达的影响

目的:观察四君子汤对2,4,6-三硝基苯磺酸(TNBS)诱导的溃疡性结肠炎大鼠治疗作用及其对黏膜上皮细胞紧密连接蛋白闭合蛋白(Occludin)表达的影响。方法:SD大鼠实验分为正常组,模型组,阳性药组(柳氮磺胺吡啶肠溶片,SASP,0.4 g·kg~(-1)),四君子汤高、中、低剂量给药组(生药5.6,2.8,1.4 g·kg~(-1)),每组8只。除正常组外,其余各组采用TNBS灌肠法制作溃疡性结肠炎模型,造模24 h后各组分别按设计剂量连续ig给药10 d。观察大鼠饮食、精神状态、疾病活动指数、大便次数与质量等情况,末次给药24 h后,处死动物取结肠,肉眼观察结肠黏膜的病变并进行结肠大体形态损伤评分,取病变部位进行HE染色,光镜下评价黏膜病理损伤指数,免疫组织化学法检测大鼠结肠Occludin蛋白的表达。结果:模型组大鼠DAI评分、结肠形态与组织学损伤评分等均比正常组高,Occludin蛋白表达较正常组低(P0.05),四君子汤与SASP治疗能不同程度减轻TNBS诱导的UC模型对黏膜的破坏,使紧密连接蛋白Occludin表达升高,抑制结肠通透性的增加,保护结肠黏膜屏障功能(P0.05)。结论:四君子汤能有效增加Occludin蛋白表达,促进实验性UC大鼠结肠黏膜屏障功能修复,这可能是其治疗结肠炎的机制之一。     

桃红四物汤抗癫痫作用机制研究

目的研究桃红四物汤的抗癫痫作用机制。方法建立戊四氮(PTZ)点燃大鼠癫痫模型,随机分为对照组、模型组和桃红四物汤低、中、高剂量组。桃红四物汤剂量分别为0.7、1.4、2.1 g/kg,给药体积为10 m L/kg,每日ig给药1次,连续给药2周。给药第0、14天观察大鼠癫痫发作程度。给药结束处死大鼠,伊文思蓝(EB)法测定血脑屏障通透性,Western blotting法和免疫荧光法测定脑组织及不同部位3种紧密连接蛋白:闭锁连接蛋白-1(ZO-1)、基质金属蛋白酶-9(MMP-9)、Occludin蛋白表达水平。结果高、中、低剂量桃红四物汤可显著降低大鼠癫痫发作程度和血脑屏障通透性(P0.01)。低剂量桃红四物汤可显著升高大鼠脑组织皮层、海马及纹状体的MMP-9表达水平(P0.05);中、高剂量桃红四物汤可显著升高大鼠脑组织ZO-1、MMP-9、Occludin表达水平(P0.05、0.01)。结论桃红四物汤可显著减轻癫痫发作程度,作用机制可能与降低血脑屏障通透性及升高脑组织紧密连接蛋白表达水平有关。     

氯吡格雷对小鼠小肠黏膜的损伤及其机制研究

目的 初探氯吡格雷对小鼠小肠黏膜的损伤及可能的机制。方法 20 只无特定病原体（SPF）级 BALB/c 雄性小鼠,随机分为2 组：空白对照组、氯吡格雷组,空白对照组灌服生理盐水,氯吡格雷组灌服氯吡 格雷,2 周后处死小鼠,观察小鼠小肠黏膜大体变化及组织学变化,免疫组织化学（简称免疫组化）法检测小 肠黏膜组织中Occludin 蛋白的表达情况。结果 氯吡格雷组小鼠小肠黏膜大体损伤、组织病理学损伤高于空 白对照组（P <0.05）;免疫组化测得氯吡格雷组小鼠小肠黏膜Occludin 蛋白表达低于空白对照组（P <0.05）。 结论 氯吡格雷可造成小鼠小肠黏膜损伤;可能通过降低Occludin 蛋白表达造成小肠黏膜损伤。