本文關(guān)鍵詞：氧化苦參堿通過(guò)抑制NF-κB p65活性改善肝硬化大鼠腸粘膜屏障功能　出處：《南方醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 氧化苦參堿 肝硬化 腸粘膜屏障 核因子-κB 細(xì)胞因子 內(nèi)毒素血癥

【摘要】：背景目的：肝硬化是臨床常見(jiàn)的慢性進(jìn)展性肝病,由一種或多種病因長(zhǎng)期或反復(fù)作用形成的彌漫性肝損害。早期由于肝臟代償功能較強(qiáng)可無(wú)明顯癥狀,后期則以肝功能損害和門(mén)脈高壓為主要表現(xiàn),并有多系統(tǒng)受累,晚期常出現(xiàn)各種并發(fā)癥。肝硬化的患者容易發(fā)生細(xì)菌移位從而引起各種感染,最常見(jiàn)的是自發(fā)性細(xì)菌性腹膜炎和自發(fā)性菌血癥。腸道細(xì)菌移位通常有多種途徑,最常見(jiàn)的包括淋巴途徑、門(mén)脈途徑、腹膜途徑。腸道細(xì)菌移位發(fā)生的三種途徑中,腸粘膜屏障功能障礙在其中起了重要作用,因此腸粘膜屏障功能是肝硬化預(yù)后及并發(fā)癥發(fā)生的一個(gè)重要預(yù)警,保護(hù)腸粘膜屏障在預(yù)防肝硬化腸道細(xì)菌移位等并發(fā)癥的發(fā)生中顯得尤為重要。腸粘膜屏障是指腸道能夠防止腸內(nèi)的有害物質(zhì)如細(xì)菌和毒素穿過(guò)腸粘膜進(jìn)入人體內(nèi)其他組織、器官和血液循環(huán)的結(jié)構(gòu)和功能的總和。因此腸粘膜屏障是防止細(xì)菌移位的重要基礎(chǔ)和主要屏障。腸粘膜屏障主要包括機(jī)械屏障、生物屏障、化學(xué)屏障及免疫屏障。目前,關(guān)于肝硬化腸粘膜屏障損傷的機(jī)制存在不同的學(xué)說(shuō),但任何能夠破壞腸粘膜屏障的因素都將引起粘膜屏障功能損傷。肝硬化腸粘膜屏障損傷的主要機(jī)制如下：缺氧及氧自由基的損傷、腸道細(xì)菌及內(nèi)毒素的損傷、細(xì)胞因子的損傷以及腸道免疫功能受損等。肝硬化時(shí)可由各種因素通過(guò)多途徑、多環(huán)節(jié)、多機(jī)制造成腸粘膜屏障的損傷,引起一系列的并發(fā)癥。 肝硬化腸粘膜屏障損傷各種因素中,細(xì)胞因子在損傷中起了重要作用。NF-κB是調(diào)節(jié)一系列基因的轉(zhuǎn)錄因子,NF-κB廣泛存在于各種組織中,能與許多細(xì)胞基因的啟動(dòng)子和增強(qiáng)子中的κB轉(zhuǎn)錄序列位發(fā)生特異性結(jié)合,調(diào)控免疫反應(yīng)和炎癥反應(yīng)中多種蛋白的基因轉(zhuǎn)錄,在免疫反應(yīng)和炎癥反應(yīng)中起著重要的作用。炎癥反應(yīng)中,NF-κB轉(zhuǎn)錄家族的激活起了關(guān)鍵作用,它將誘導(dǎo)促炎因子基因轉(zhuǎn)錄,從而合成細(xì)胞因子。這與文獻(xiàn)報(bào)道相符,在壞死性小腸結(jié)腸炎大鼠模型中,抑制NF-κB活性能減少腸道促炎細(xì)胞因子的水平。同時(shí),腸道是TNF-α,IL-1,6,8和NO等細(xì)胞因子的重要來(lái)源,腸粘膜屏障損傷導(dǎo)致腸道炎癥,引起細(xì)胞因子釋放,細(xì)胞因子激活NF-κB,這將促使炎細(xì)胞聚集并產(chǎn)生更多促炎細(xì)胞因子。這些細(xì)胞因子對(duì)于炎癥通路和過(guò)程通常具有協(xié)同效益,這將誘導(dǎo)諸如趨化因子、前列腺素、血小板活化因子等繼發(fā)性性介質(zhì)的釋放,從而加重炎癥反應(yīng)。如此便形成炎癥瀑布樣反應(yīng),加重腸粘膜屏障損傷。此外,腸道來(lái)源的促炎因子介質(zhì)將誘發(fā)肝臟合成TNF-α、IL-6和激活iNOS活性。肝臟中產(chǎn)生的TNF-α、IL-6、NO和CO等因子將通過(guò)循環(huán)及膽道系統(tǒng)進(jìn)入腸道,并作用于腸道,形成瀑布式炎癥反應(yīng)。如此,連鎖的炎癥反應(yīng)將導(dǎo)致嚴(yán)重的腸粘膜屏障損傷。NF-κB p65是NF-κB家族5個(gè)成員之一,p65是NF-κB最多見(jiàn)的二聚體形式之一,有研究提示其中具有顯著促炎活性的是含p65的二聚體。因此,在控制腸道炎癥反應(yīng)中,通過(guò)抑制NF-κB p65的活性從而減少細(xì)胞因子的釋放起著至關(guān)重要的作用。這可能是保護(hù)肝硬化大鼠腸粘膜屏障免受損傷的有效治療之一。 氧化苦參堿(OMT)是傳統(tǒng)中藥草本苦參中的提取物,它被廣泛用來(lái)治療慢性肝炎。OMT有多種藥理作用,包括抗炎、抗內(nèi)毒素、抗凋亡、抗肝纖維化及抗心律失常。因其抗炎作用,我們假設(shè)OMT可能對(duì)保護(hù)腸粘膜屏障損傷有效果。已有報(bào)道指出,OMT對(duì)治療結(jié)腸炎有效,主要是通過(guò)下調(diào)NF-κB活性和抑制炎癥細(xì)胞因子的釋放而起效。另外,有文獻(xiàn)證實(shí)OMT通過(guò)抑制NF-κB p65核轉(zhuǎn)錄從而減輕急性腸道炎癥。因此,通過(guò)抑制NF-κB p65活性來(lái)減少細(xì)胞因子釋放可能是最有效的方法之一。我們實(shí)驗(yàn)設(shè)計(jì)目的在于探究四氯化碳誘導(dǎo)的肝硬化大鼠模型中,OMT能否通過(guò)抑制大鼠NF-κB p65的活性及細(xì)胞因子的表達(dá)來(lái)改善腸粘膜屏障功能。 方法： 1.50只正常雄性SD大鼠,10只標(biāo)記為正常對(duì)照組(A組,n=10),余40只正常大鼠采用四氯化碳皮下注射的方法進(jìn)行肝硬化模型制備,采用40%四氯化碳橄欖油溶液,按照0.3mL/kg進(jìn)行皮下注射,首次劑量加倍,每周2次,按個(gè)體化原則,每只大鼠根據(jù)自身體重調(diào)整劑量,共12周。最后制成肝硬化成模大鼠共30只。成模大鼠分為模型組(B組,n=15)和治療組(C組,n=15)。12周后,C組大鼠接受5%葡萄糖溶液配置的OMT肌注(63mg/kg)共4周,1次/日；A、B兩組大鼠分別肌注與體重匹配劑量的5%葡萄糖溶液4周,1次/日。與此同時(shí),模型組與治療組大鼠按上述方法繼續(xù)接受40%四氯化碳橄欖油溶液皮下注射至16周。 2.16周實(shí)驗(yàn)周期結(jié)束后,所有大鼠使用3%水合氯醛進(jìn)行麻醉。抽取下腔靜脈.血2m1,以備內(nèi)毒素檢測(cè)使用。采用脊柱脫臼法處死大鼠,觀察比較各組間肝臟標(biāo)本情況,同時(shí)取肝右葉組織標(biāo)本,10%福爾馬林溶液固定,石蠟包埋,切片后行組織病理學(xué)HE染色。取末段回腸組織6～8cm,迅速將各組回腸組織進(jìn)行觀察比較后分為四等分,分別用于腸道病理組織學(xué)檢查、免疫組織化學(xué)檢查、腸組織中TNF-α和IL-6濃度及TNF-α和IL-6mRNA表達(dá)的檢測(cè)。 3.取腸組織用0.9%生理鹽水沖洗,吸干水分,10%福爾馬林溶液固定,進(jìn)行脫水、石蠟包埋,切片后行HE染色。置于光學(xué)顯微鏡下觀察腸粘膜組織的病理學(xué)變化,使用Chiu氏六級(jí)評(píng)分標(biāo)準(zhǔn)進(jìn)行評(píng)分。 4.使用ELISA檢測(cè)試劑盒檢測(cè)腸組織中TNF-α和IL-6濃度,將清洗干凈的腸組織剪成碎片,在冰上的玻璃容器內(nèi)研碎。用超聲或者反復(fù)凍融的方法裂解細(xì)胞膜,用離心機(jī)5000r/分鐘離心15分鐘,取上清液作為待測(cè)標(biāo)本。 5. RT-PCR的方法檢測(cè)腸組織中TNF-α和IL-6mRNA的表達(dá),按照Trizol法抽提總RNA、合成第一鏈cDNA、cDNA擴(kuò)增等步驟進(jìn)行,將得到的擴(kuò)增產(chǎn)物進(jìn)行瓊脂糖凝膠電泳,使用紫外分析儀觀察電泳結(jié)果,并用數(shù)碼相機(jī)對(duì)電泳結(jié)果進(jìn)行拍照作為半定量評(píng)估方法,并用分析軟件(Glyko BandScan version5.0)分析照片結(jié)果。 6.免疫組織化學(xué)技術(shù)檢測(cè)腸組織中NF-κB p65活性,嚴(yán)格按照NF-κB p65試劑盒說(shuō)明書(shū)進(jìn)行操作。操作結(jié)束后,在400倍高倍鏡下選取典型視野計(jì)數(shù)100個(gè)細(xì)胞,觀察陽(yáng)性細(xì)胞數(shù)N1和總細(xì)胞數(shù)N,計(jì)算陽(yáng)性率=N1/N×100%。 7.使用內(nèi)毒素檢測(cè)鱟試劑盒,運(yùn)用動(dòng)態(tài)濁度法對(duì)血漿樣品進(jìn)行內(nèi)毒素檢測(cè),操作方法嚴(yán)格按照說(shuō)明書(shū)進(jìn)行。 8.統(tǒng)計(jì)方法：所有實(shí)驗(yàn)數(shù)據(jù)統(tǒng)計(jì)分析均采用SPSS16.0統(tǒng)計(jì)學(xué)軟件,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示。組間比較經(jīng)方差齊性檢驗(yàn)示方差齊,兩組間比較用t檢驗(yàn),兩組以上比較用單因素方差分析(one-way ANO VA)。方差不齊資料比較使用非參數(shù)統(tǒng)計(jì)方法秩和檢驗(yàn)中的H檢驗(yàn)法(Kruskal-Wallis H test),以P0.05表示差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.腸道組織病理學(xué)結(jié)果：正常組為正常大鼠腸粘膜絨毛形態(tài)；模型組大鼠腸粘膜可見(jiàn)絨毛萎縮、變短、斷裂,可見(jiàn)絨毛嚴(yán)重水腫,上皮下間隙增大,可見(jiàn)大量炎細(xì)胞浸潤(rùn)于粘膜固有層甚至肌層,上皮層與固有層分離,粘膜層疏松；經(jīng)過(guò)OMT治療后,治療組大鼠腸粘膜絨毛變得整齊,水腫減輕,粘膜層變得更緊密,上皮下間隙縮小。同時(shí),浸潤(rùn)的炎細(xì)胞明顯減少。與正常組相比,模型組評(píng)分分值明顯增高(P0.05)。OMT治療后,治療組分值顯著下降,與模型組比較有顯著性差異(P0.05)。 2.腸組織中TNF-a和IL-6濃度：與正常組相比,模型組TNF-a濃度水平顯著升高(48.63±18.73VS152.52±15.81, P0.05)。 OMT治療后,治療組TNF-a濃度水平顯著下降,與模型組比較有顯著性差異(152.52±15.81VS97.83±17.79,P0.05)。對(duì)比各組IL-6濃度水平變化,與TNF-a濃度水平變化具有一致性。對(duì)比正常組,模型組IL-6濃度水平明顯升高(50.88±22.13VS118.82±35.71,P0.05)。OMT治療后,治療組IL-6濃度水平顯著下降,與模型組比較差異有統(tǒng)計(jì)學(xué)意義(118.82±35.71VS78.07±24.94,P0.05)。 3.腸組織中TNF-a和IL-6mRNA表達(dá)水平：電泳結(jié)果示模型組中TNF-amRNA表達(dá)水平明顯高于正常組；治療組中,TNF-amRNA表達(dá)水平明顯下降；模型組中IL-6mRNA表達(dá)水平明顯高于正常組；治療組中,IL-6mRNA表達(dá)水平明顯下降。 4.免疫組化檢測(cè)腸組織中NF-κB p65活性結(jié)果：模型組中,NF-κB p65的表達(dá)明顯高于正常組中表達(dá)(78.67%±8.49%VS28.10%±9.88%)。治療組與模型組比較,NF-κB p65表達(dá)顯著下降(78.67%±8.49%VS52.20%±11.04%)。 5.血漿內(nèi)毒素水平：模型組(B組)中有13只大鼠內(nèi)毒素水平0.035EU/ml,而治療組(C組)中僅為7只。模型組的內(nèi)毒素水平明顯高于正常組,兩組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。OMT治療后,治療組內(nèi)毒素水平顯著下降,與模型組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1.肝硬化大鼠出現(xiàn)腸粘膜屏障功能損傷,發(fā)生了腸源性?xún)?nèi)毒素血癥,OMT具有潛在保護(hù)肝硬化大鼠腸粘膜屏障功能的作用。 2.OMT具有減輕血漿內(nèi)毒素水平的作用。一方面,OMT可能對(duì)內(nèi)毒素有直接的清除作用,通過(guò)這項(xiàng)機(jī)制減輕血漿內(nèi)毒素水平；更為重要的另一方面是,OMT可能通過(guò)保護(hù)腸粘膜屏障完整性來(lái)發(fā)揮降低血漿內(nèi)毒素水平作用。 3.OMT保護(hù)肝硬化大鼠腸粘膜屏障功能的作用可能主要通過(guò)下調(diào)肝硬化大鼠腸道組織中NF-κB p65表達(dá),減少促炎細(xì)胞因子TNF-a、IL-6釋放。同時(shí),OMT可能對(duì)腸道具有直接抗炎活性,通過(guò)直接抗炎作用改善腸粘膜屏障功能。
[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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本文編號(hào)：1431098