氧化苦参碱通过抑制NF-κB p65活性改善肝硬化大鼠肠粘膜屏障功能

发布时间：2018-01-16 02:15

  本文关键词：氧化苦参碱通过抑制NF-κB p65活性改善肝硬化大鼠肠粘膜屏障功能　出处：《南方医科大学》2014年硕士论文　论文类型：学位论文

  更多相关文章： 氧化苦参碱 肝硬化 肠粘膜屏障 核因子-κB 细胞因子 内毒素血症

【摘要】：背景目的：肝硬化是临床常见的慢性进展性肝病,由一种或多种病因长期或反复作用形成的弥漫性肝损害。早期由于肝脏代偿功能较强可无明显症状,后期则以肝功能损害和门脉高压为主要表现,并有多系统受累,晚期常出现各种并发症。肝硬化的患者容易发生细菌移位从而引起各种感染,最常见的是自发性细菌性腹膜炎和自发性菌血症。肠道细菌移位通常有多种途径,最常见的包括淋巴途径、门脉途径、腹膜途径。肠道细菌移位发生的三种途径中,肠粘膜屏障功能障碍在其中起了重要作用,因此肠粘膜屏障功能是肝硬化预后及并发症发生的一个重要预警,保护肠粘膜屏障在预防肝硬化肠道细菌移位等并发症的发生中显得尤为重要。肠粘膜屏障是指肠道能够防止肠内的有害物质如细菌和毒素穿过肠粘膜进入人体内其他组织、器官和血液循环的结构和功能的总和。因此肠粘膜屏障是防止细菌移位的重要基础和主要屏障。肠粘膜屏障主要包括机械屏障、生物屏障、化学屏障及免疫屏障。目前,关于肝硬化肠粘膜屏障损伤的机制存在不同的学说,但任何能够破坏肠粘膜屏障的因素都将引起粘膜屏障功能损伤。肝硬化肠粘膜屏障损伤的主要机制如下：缺氧及氧自由基的损伤、肠道细菌及内毒素的损伤、细胞因子的损伤以及肠道免疫功能受损等。肝硬化时可由各种因素通过多途径、多环节、多机制造成肠粘膜屏障的损伤,引起一系列的并发症。 肝硬化肠粘膜屏障损伤各种因素中,细胞因子在损伤中起了重要作用。NF-κB是调节一系列基因的转录因子,NF-κB广泛存在于各种组织中,能与许多细胞基因的启动子和增强子中的κB转录序列位发生特异性结合,调控免疫反应和炎症反应中多种蛋白的基因转录,在免疫反应和炎症反应中起着重要的作用。炎症反应中,NF-κB转录家族的激活起了关键作用,它将诱导促炎因子基因转录,从而合成细胞因子。这与文献报道相符,在坏死性小肠结肠炎大鼠模型中,抑制NF-κB活性能减少肠道促炎细胞因子的水平。同时,肠道是TNF-α,IL-1,6,8和NO等细胞因子的重要来源,肠粘膜屏障损伤导致肠道炎症,引起细胞因子释放,细胞因子激活NF-κB,这将促使炎细胞聚集并产生更多促炎细胞因子。这些细胞因子对于炎症通路和过程通常具有协同效益,这将诱导诸如趋化因子、前列腺素、血小板活化因子等继发性性介质的释放,从而加重炎症反应。如此便形成炎症瀑布样反应,加重肠粘膜屏障损伤。此外,肠道来源的促炎因子介质将诱发肝脏合成TNF-α、IL-6和激活iNOS活性。肝脏中产生的TNF-α、IL-6、NO和CO等因子将通过循环及胆道系统进入肠道,并作用于肠道,形成瀑布式炎症反应。如此,连锁的炎症反应将导致严重的肠粘膜屏障损伤。NF-κB p65是NF-κB家族5个成员之一,p65是NF-κB最多见的二聚体形式之一,有研究提示其中具有显著促炎活性的是含p65的二聚体。因此,在控制肠道炎症反应中,通过抑制NF-κB p65的活性从而减少细胞因子的释放起着至关重要的作用。这可能是保护肝硬化大鼠肠粘膜屏障免受损伤的有效治疗之一。 氧化苦参碱(OMT)是传统中药草本苦参中的提取物,它被广泛用来治疗慢性肝炎。OMT有多种药理作用,包括抗炎、抗内毒素、抗凋亡、抗肝纤维化及抗心律失常。因其抗炎作用,我们假设OMT可能对保护肠粘膜屏障损伤有效果。已有报道指出,OMT对治疗结肠炎有效,主要是通过下调NF-κB活性和抑制炎症细胞因子的释放而起效。另外,有文献证实OMT通过抑制NF-κB p65核转录从而减轻急性肠道炎症。因此,通过抑制NF-κB p65活性来减少细胞因子释放可能是最有效的方法之一。我们实验设计目的在于探究四氯化碳诱导的肝硬化大鼠模型中,OMT能否通过抑制大鼠NF-κB p65的活性及细胞因子的表达来改善肠粘膜屏障功能。 方法： 1.50只正常雄性SD大鼠,10只标记为正常对照组(A组,n=10),余40只正常大鼠采用四氯化碳皮下注射的方法进行肝硬化模型制备,采用40%四氯化碳橄榄油溶液,按照0.3mL/kg进行皮下注射,首次剂量加倍,每周2次,按个体化原则,每只大鼠根据自身体重调整剂量,共12周。最后制成肝硬化成模大鼠共30只。成模大鼠分为模型组(B组,n=15)和治疗组(C组,n=15)。12周后,C组大鼠接受5%葡萄糖溶液配置的OMT肌注(63mg/kg)共4周,1次/日；A、B两组大鼠分别肌注与体重匹配剂量的5%葡萄糖溶液4周,1次/日。与此同时,模型组与治疗组大鼠按上述方法继续接受40%四氯化碳橄榄油溶液皮下注射至16周。 2.16周实验周期结束后,所有大鼠使用3%水合氯醛进行麻醉。抽取下腔静脉.血2m1,以备内毒素检测使用。采用脊柱脱臼法处死大鼠,观察比较各组间肝脏标本情况,同时取肝右叶组织标本,10%福尔马林溶液固定,石蜡包埋,切片后行组织病理学HE染色。取末段回肠组织6～8cm,迅速将各组回肠组织进行观察比较后分为四等分,分别用于肠道病理组织学检查、免疫组织化学检查、肠组织中TNF-α和IL-6浓度及TNF-α和IL-6mRNA表达的检测。 3.取肠组织用0.9%生理盐水冲洗,吸干水分,10%福尔马林溶液固定,进行脱水、石蜡包埋,切片后行HE染色。置于光学显微镜下观察肠粘膜组织的病理学变化,使用Chiu氏六级评分标准进行评分。 4.使用ELISA检测试剂盒检测肠组织中TNF-α和IL-6浓度,将清洗干净的肠组织剪成碎片,在冰上的玻璃容器内研碎。用超声或者反复冻融的方法裂解细胞膜,用离心机5000r/分钟离心15分钟,取上清液作为待测标本。 5. RT-PCR的方法检测肠组织中TNF-α和IL-6mRNA的表达,按照Trizol法抽提总RNA、合成第一链cDNA、cDNA扩增等步骤进行,将得到的扩增产物进行琼脂糖凝胶电泳,使用紫外分析仪观察电泳结果,并用数码相机对电泳结果进行拍照作为半定量评估方法,并用分析软件(Glyko BandScan version5.0)分析照片结果。 6.免疫组织化学技术检测肠组织中NF-κB p65活性,严格按照NF-κB p65试剂盒说明书进行操作。操作结束后,在400倍高倍镜下选取典型视野计数100个细胞,观察阳性细胞数N1和总细胞数N,计算阳性率=N1/N×100%。 7.使用内毒素检测鲎试剂盒,运用动态浊度法对血浆样品进行内毒素检测,操作方法严格按照说明书进行。 8.统计方法：所有实验数据统计分析均采用SPSS16.0统计学软件,数据以均数±标准差(x±s)表示。组间比较经方差齐性检验示方差齐,两组间比较用t检验,两组以上比较用单因素方差分析(one-way ANO VA)。方差不齐资料比较使用非参数统计方法秩和检验中的H检验法(Kruskal-Wallis H test),以P0.05表示差异有统计学意义。 结果： 1.肠道组织病理学结果：正常组为正常大鼠肠粘膜绒毛形态；模型组大鼠肠粘膜可见绒毛萎缩、变短、断裂,可见绒毛严重水肿,上皮下间隙增大,可见大量炎细胞浸润于粘膜固有层甚至肌层,上皮层与固有层分离,粘膜层疏松；经过OMT治疗后,治疗组大鼠肠粘膜绒毛变得整齐,水肿减轻,粘膜层变得更紧密,上皮下间隙缩小。同时,浸润的炎细胞明显减少。与正常组相比,模型组评分分值明显增高(P0.05)。OMT治疗后,治疗组分值显著下降,与模型组比较有显著性差异(P0.05)。 2.肠组织中TNF-a和IL-6浓度：与正常组相比,模型组TNF-a浓度水平显著升高(48.63±18.73VS152.52±15.81, P0.05)。 OMT治疗后,治疗组TNF-a浓度水平显著下降,与模型组比较有显著性差异(152.52±15.81VS97.83±17.79,P0.05)。对比各组IL-6浓度水平变化,与TNF-a浓度水平变化具有一致性。对比正常组,模型组IL-6浓度水平明显升高(50.88±22.13VS118.82±35.71,P0.05)。OMT治疗后,治疗组IL-6浓度水平显著下降,与模型组比较差异有统计学意义(118.82±35.71VS78.07±24.94,P0.05)。 3.肠组织中TNF-a和IL-6mRNA表达水平：电泳结果示模型组中TNF-amRNA表达水平明显高于正常组；治疗组中,TNF-amRNA表达水平明显下降；模型组中IL-6mRNA表达水平明显高于正常组；治疗组中,IL-6mRNA表达水平明显下降。 4.免疫组化检测肠组织中NF-κB p65活性结果：模型组中,NF-κB p65的表达明显高于正常组中表达(78.67%±8.49%VS28.10%±9.88%)。治疗组与模型组比较,NF-κB p65表达显著下降(78.67%±8.49%VS52.20%±11.04%)。 5.血浆内毒素水平：模型组(B组)中有13只大鼠内毒素水平0.035EU/ml,而治疗组(C组)中仅为7只。模型组的内毒素水平明显高于正常组,两组比较差异有统计学意义(P0.05)。OMT治疗后,治疗组内毒素水平显著下降,与模型组比较差异有统计学意义(P0.05)。 结论： 1.肝硬化大鼠出现肠粘膜屏障功能损伤,发生了肠源性内毒素血症,OMT具有潜在保护肝硬化大鼠肠粘膜屏障功能的作用。 2.OMT具有减轻血浆内毒素水平的作用。一方面,OMT可能对内毒素有直接的清除作用,通过这项机制减轻血浆内毒素水平；更为重要的另一方面是,OMT可能通过保护肠粘膜屏障完整性来发挥降低血浆内毒素水平作用。 3.OMT保护肝硬化大鼠肠粘膜屏障功能的作用可能主要通过下调肝硬化大鼠肠道组织中NF-κB p65表达,减少促炎细胞因子TNF-a、IL-6释放。同时,OMT可能对肠道具有直接抗炎活性,通过直接抗炎作用改善肠粘膜屏障功能。
[Abstract]:Background and objective: liver cirrhosis is a common clinical progression of chronic liver disease, diffuse liver damage or long-term repeated action formed by one or more causes. Early because the liver compensatory function is strong without obvious symptoms, the damage to the liver function and portal hypertension as the main manifestation, and multi system involvement, often appear late all kinds of complications. Liver cirrhosis patients susceptible to bacterial translocation and cause infection, the most common is the spontaneous bacterial peritonitis and bacteremia. Spontaneous bacterial translocation usually have a variety of ways, including the most common lymphatic pathway, portal vein pathway, peritoneal ways. Three ways of occurrence of intestinal bacterial translocation, intestinal mucosal barrier dysfunction in which played an important role. Therefore, the intestinal mucosal barrier function is an important early warning of cirrhosis prognosis and complications, protect the intestinal mucosal barrier in the prevention of liver It is particularly important to the hardening of intestinal bacterial translocation and other complications. Intestinal mucosal barrier refers to the intestinal tract can prevent intestinal harmful substances such as bacteria and toxins through the intestinal mucosa into other tissues in the body, the sum of organs and blood circulation. So the structure and function of the intestinal mucosal barrier is the main barrier and an important basis for preventing bacterial translocation the intestinal mucosal barrier. Including mechanical barrier, biologic barrier, chemical barrier and immune barrier. At present, the mechanism of liver cirrhosis on intestinal mucosal barrier injury in different theories, but any damage to the intestinal mucosa barrier factors will cause mucosal barrier function following injury. The mechanism of intestinal mucosal barrier injury in cirrhosis: hypoxia and oxygen free radical damage, intestinal bacteria and endotoxin injury, cytokines damage and intestinal immune function damage in liver cirrhosis by various. A series of complications are caused by the damage of the intestinal mucosal barrier by multiple routes, multiple links and multiple machines.
The intestinal mucosal barrier injury of liver cirrhosis factors, cytokines in injury plays an important role in the.NF- kappa B transcription factors regulate a series of genes, NF- kappa B widely exist in various tissues, can kappa B transcribed sequences with many cell gene promoter and enhancer in the occurrence of specific binding, gene a variety of transcription protein regulate the immune response and inflammation, plays an important role in the immune response and inflammation. Inflammatory reaction, NF- kappa B family transcription activation plays a key role, it will induce proinflammatory cytokine gene transcription and synthesis of cytokines. This is consistent with the literature, in necrotizing enterocolitis rat model, inhibition of NF- kappa B activity can reduce intestinal proinflammatory cytokine levels. At the same time, the intestinal tract is an important source of TNF- alpha, IL-1,6,8 and NO and other cytokines, leading to intestinal tract inflammation of the intestinal mucosal barrier injury induced Since the release of cytokines and cytokine activation of NF- kappa B, which will lead to the inflammatory cells and produce more proinflammatory cytokines. These cytokines usually have synergistic benefits for inflammatory pathways and processes, such as this will induce chemokine release, prostaglandin, platelet activating factor and other secondary medium, thereby increasing the inflammatory reaction. So they formed inflammatory reaction, enhance intestinal mucosal barrier injury. In addition, gut derived proinflammatory cytokines induced by medium hepatic synthesis of TNF- alpha, IL-6 and activation of iNOS in the liver. Produced by IL-6, NO and TNF- alpha, CO and other factors will enter the intestinal tract through the circulation and biliary system, and its function in the gut and form a cascade of inflammatory reactions. So, inflammatory reaction chain will cause severe damage to intestinal mucosal barrier of.NF- kappa B p65 NF- is one of the 5 members of kappa B family, p65 is one of the two dimer form of NF- kappa B the most common, Some studies suggest that the significant pro-inflammatory activity is two dimer containing p65. Therefore, in the control of intestinal inflammation in NF- by inhibiting the activity of p65 K B and reduce the release of cytokines plays a vital role. This is probably one of the effective treatment of intestinal mucosal barrier protection against injury in cirrhotic rats.
Oxymatrine (OMT) is a traditional Chinese herbal extract from Sophora flavescens, it is widely used to treat chronic hepatitis.OMT has a variety of pharmacological effects, including anti-inflammatory, anti endotoxin, anti apoptosis, anti fibrosis and anti arrhythmia. Because of its anti-inflammatory effects, we hypothesized that OMT may have the effect to protect the intestinal mucosal barrier injury. The report pointed out that OMT is effective in the treatment of ulcerative colitis, is the main work by down-regulation of NF- kappa B activity and inhibit the release of inflammatory cytokines. In addition, it is reported that OMT can relieve acute intestinal inflammation by inhibiting NF- kappa B p65 nuclear transcription. Therefore, to reduce cytokine NF- by inhibiting the activity of p65 K B release may be one of the the most effective way. Our purpose is to explore the design of experimental cirrhosis rats induced by carbon tetrachloride, whether through the expression of OMT activity and cytokine inhibition of rat NF- kappa B p65 to improve the intestinal Mucosal barrier function.
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