【作者】 陈焰；

【导师】 姒健敏；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 溃疡性结肠炎(Ulcerative Colitis,UC)主要临床表现为反复发作的腹痛、腹泻、粘液脓血便等,病变多累及直肠和乙状结肠。UC在欧美国家发病率高,我国的发病率正急剧上升。由于UC病因不明且治疗困难,给社会生产力和个人生活质量带来极大影响,引起了各国高度重视,是当今国际消化病诊治研究的热点。有报道显示在我国70.2%UC患者为局限性的直肠乙状结肠炎,对该类患者的治疗主要以灌肠为主。灌肠可以使药物直接和病变部位接触并减少药物的全身副作用。目前用于灌肠的药物主要有5-氨基水杨酸(5-Aminosalicylic Acid,5-ASA)和各种糖皮质激素,但5-ASA价格昂贵,且目前国内还未开发灌肠剂型;糖皮质激素灌肠后仍有较多患者出现全身副作用,研究高效安全且价格合适的灌肠成品有极大的意义。云母是硅酸盐类矿物药之一,古代医家认为云母有“生肌敛疮”、“补中”、“主下痢”等功用,其药用价值在我国历史悠久,很可能云母灌肠对UC有效。选择和建立合适的动物模型是研究UC药物治疗不可缺少的重要方法。UC的动物模型虽然有众多研究,但至今尚不理想。化学诱导性结肠炎是比较广泛采用的UC模型,包括葡聚糖硫酸钠(Dextran Sulfate soduium,DSS)及2,4,6-三硝基苯磺酸(Trinitrobenzene Sulfonic Acid,TNBS)诱导性结肠炎模型。DSS诱导性结肠炎主要在结肠远端,其病理表现符合UC的特点,但是这个模型的一个很大的障碍是DSS非常昂贵。TNBS诱导性结肠炎模型是一种经典的炎症性肠病模型,诱导后产生的远端结肠病变持续存在,有许多学者把TNBS诱导性结肠炎模型作为UC的模型。为了验证云母灌肠对结肠炎的治疗作用,我们首先建立起两种动物模型并对其疗效进行双重验证。我们重复了经典的TNBS诱导性结肠炎,并尝试建立了一种新的复合型大鼠DSS/乙醇化学诱导性结肠炎模型。将雌性SD大鼠随机分为4组,A组为模型组,B组和C组为阳性对照组,D组为正常对照组。A组和B组的大鼠自由饮用2%DSS 3天,C组和D组的大鼠自由饮用消毒后的自来水3天。第4天,A组和B组的大鼠分别灌肠内注入0.5ml 30%乙醇或生理盐水,C组和D组分别结肠内灌注0.5ml 30%乙醇或生理盐水。各组进行疾病活动指数的评分,同时在结肠注入乙醇或生理盐水后24小时、3天、7天、14天和21天随机处死每组大鼠各5只进行大体评分和病理评分;结果显示模型组大鼠自由饮用2%DSS 3天后灌肠注入30%乙醇可以诱导出现类似UC的体重下降、腹泻和血便。大体可见远端结肠有溃疡和炎症,病理显示炎症细胞侵润、隐窝脓肿和异型增生等UC的的病理特点。和正常对照组相比,模型组大鼠的体重明显减少,结肠的大体和病理积分增加;该模型和人类UC具有类似的临床和形态特点,且相对价格便宜、制作简单。我们先以云母灌肠治疗大鼠DSS/乙醇化学诱导性结肠炎来确认云母的治疗作用。正常对照组、DSS/乙醇模型组、云母治疗组、5-ASA治疗组在制模的当天开始灌肠用药,连续用药10天。观察治疗前后大鼠体重的改变和治疗后大鼠结肠的大体和病理积分。云母或5-ASA灌肠治疗DSS/乙醇结肠炎后大鼠体重减少的情况有明显改善,同时大体积分明显减少,病理积分也同样明显减少。通过该部分研究明确了云母灌肠治疗DSS/乙醇化学诱导性结肠炎有效。然后进一步在TNBS诱导性结肠炎模型上进行量效研究。TNBS诱导性结肠炎模型建立的当天分别给予低剂量云母(240mg/kg/d)、中剂量云母(480mg/kg/d)、高剂量云母(960mg/kg/d)和5-ASA(360mg/kg/d)灌肠。用药14天后,比较大鼠治疗前后体重改变情况和结肠的大体、病理积分;结肠灌注不同剂量的云母可以剂量相关地改善TNBS诱导性结肠炎大鼠的体重减少现象,同时减少大鼠结肠的大体和病理积分。该部分研究提示云母治疗结肠炎有量效关系。为了进一步探讨云母起效的可能机制,我们接着对云母作用的体内和体外机制进行深入的研究。过度的免疫反应是UC炎症和组织损伤的内在重要因素,而细胞因子在调节肠道免疫反应中扮演重要角色,与UC的发生及转归密切相关。与UC发病有关的细胞因子主要有TNF-a(Tumor Necrosis Factor-α,TNF-α)、IL-1β(Interleukin-1β,IL-1β)、IL-8等。我们采用大鼠TNBS诱导性结肠炎模型在体内水平深入研究了云母灌肠治疗后大鼠结肠局部TNF-α、IL-1β的基因和蛋白的改变情况。TNBS大鼠结肠炎模型制模的当天随机分组,分别给予低剂量云母、中剂量云母、高剂量云母和5-ASA灌肠。用药14天后,PT-PCR检测结肠组织中TNF-α、IL-1β、粘蛋白2(Mucin 2,MUC2)和肠三叶因子(Intestinal Trefoil Factor,ITF)的mRNA含量。ELISA方法检测结肠组织中TNF-α、IL-1β蛋白含量,免疫组化和AB-PAS(Alcian blue/periodic acid-Schiff reaction)染色检测粘蛋白分布情况。结果显示TNBS诱导性结肠炎大鼠的结肠组织中TNF-α、IL-1β的mRNA和蛋白水平均明显增加,云母和5-ASA灌肠可以减少结肠炎大鼠结肠中TNF-α、IL-1β的mRNA和蛋白量。同时还发现TNBS诱导性结肠炎大鼠结肠中MUC2 mRNA明显下降,云母和5-ASA灌肠治疗后有所上升。但是,各组结肠中ITF mRNA无明显改变。该部分研究提示云母灌肠治疗可以明显降低TNBS诱导性结肠炎局部的TNF-α、IL-1β的mRNA和蛋白水平。最后采用THP-1和HT-29两种细胞株TNF-α、IL-1β和IL-8三种细胞因子的标准品在体外研究了云母对这三种细胞因子的作用情况。ELISA方法检测云母对脂多糖(Lipopolysaccharide,LPS)刺激后的THP-1和HT-29两种细胞株分泌的细胞因子TNF-α、IL-1β及IL-8的影响情况。ELISA方法检测不同剂量的云母和TNF-α、IL-1β及IL-8三种标准品共同孵育1小时后这三种细胞因子的变化情况。结果显示云母可以剂量依赖性地减少THP-1细胞株分泌的TNF-α、IL-1β和HT-29细胞株分泌的IL-8蛋白含量。TNF-α、IL-1β及IL-8这三种细胞因子的标准品在和云母共同孵育1小时后都有明显的剂量依赖性的下降。通过上述实验,我们成功建立一种新的符合人类UC的大鼠化学诱导性结肠炎模型;明确了云母灌肠对两种化学诱导性结肠炎的治疗作用并摸索出量效关系。云母治疗作用机制与降低肠道局部的TNF-α、IL-1β的mRNA和蛋白有关,云母能吸附肠道各种细胞所释放的细胞因子可能是其起效的机制之一。更多还原

【Abstract】 Ulcerative colitis(UC) is an inflammatory disease of unknown cause that exhibits a clinical course with remission and exacerbations,characterized by abdominal pain, rectal bleeding and diarrhea.Inflammation usually occurs in the rectum and lower part of the colon.The number of cases increased rapidly in China.With long term of bowel symptoms and systemic symptoms,the quality of life and social function of patients are also affected severely.The research of UC is a hot topic nowadays.One study showed 70.2%were proctosigmoiditis or proctitis.For the treatment of UC confined to the rectum and sigmoid,topical administration of medicines,such as glucocorticoids and 5-aminosalicylic acid(5-ASA),to the injured colonic mucosa has been shown to be more effective and less hazardous than systemic administration. However,current topical treatments for UC have their limitations.For example,the high price of 5-ASA prevents it to be wildly used in our country.When applying glucocorticoids topically,there is still a significant absorption of glucocorticoids through the inflamed mucosa that causes systemic effects.It is necessary to develop alternative therapeutic approaches that are effective,affordable and have less adverse effects. Muscovite is one kind of natural clay consisting of an insoluble double silicate of aluminium and magnesium.In traditional Chinese medicine it has been documented that muscovite has antidiarrheal activity and can protect mucosa.It’s possible that rectal administration of muscovite can alleviate inflammation of UC.Adequate animal model of UC is a useful tool of research on the therapeutic agents.Several models of experimental colitis resembling ulcerative colitis have been reported previously.The most widely used models are induced by administering toxic chemical such as dextran sulfate sodium(DSS),trinitrobenzene sulfonic acid(TNBS). However,DSS-induced colitis results in inflammation mainly in the distal colonic mucosa.The histopathology of this model showed some characteristics resemblance to UC,enabling research into the pathogenesis and therapy of this disease.But an obvious obstacle with regard to the DSS model is that DSS is very expensive.Thus it is difficulty to apply this model widely in some regions.TNBS-induced colitis is a traditional model of inflammatory bowel disease and is widely used.In order to investigate whether rectal administration of muscovite can alleviate inflammation of UC,we established two animal models of UC to doublely check its effect.Besides TNBS-induced colitis,we developed an inexpensive DSS/ethanol model of UC rats.Female rats were randomized into four groups.In group A and B rats received 2%dextran sulfate sodium in the drinking water for 3 days.On day 4,rats in group A and B received 0.5ml 30%ethanol or saline intracolonically respectively.As a control,rats in group C and D only received intracolonic 30%ethanol or saline respectively.Before enema,the rats were lightly anesthetized with ether.30%ethanol or saline was instilled into the lumen of the colon through the rubber catheter.Disease Activity Index(DAI) was used to quantify the clinical evolution of every group.At various times(24h,3 th day,7th day,14 th day and 21 th day) after intracolonic administration of 30%ethanol or control saline,5 rats from each treatment group were randomly selected and killed to assess for colonic inflammatory and ulcerative responses with respect to gross morphologic damage and histopathologic scoring system.The results showed the DSS/ethanol induced significant weight loss,diarrhea and hematochezia in rats.Ulcerations and inflammation of the distal part of rat colon were developed rapidly.Histological examination showed an increased infiltration of polymorphonuclear leukocytes,lymphocytes and existence of cryptic abscesses and dysplasia.The weight of rat with colitis was decreased while the gross morphologic and histopathologic scores were increased.The successfully established DSS/ethanol model is characterized by a clinical course,localization of the lesions and histopathological features similar to human UC while it is much cheaper than DSS model.First we examined the effect of rectal administration of muscovite on DSS/ethanol induced colitis.The experimental animals were randomly divided into 4 groups to take the rectal administration of muscovite,5-ASA and the equal amount of control respectively from beginning of the induction of the colitis rats to the end of study while another group was administrated rectally with only saline served as normal control. After 10 days of treatment,the animals were assessed for colonic inflammatory and ulcerative responses with respect to change of body weight,gross morphologic damage and histopathologic scoring system.The results showed the loss of weight in the colitis groups was significantly higher than that of normal group(P＜0.01).Rectal administration of muscovite improved the loss of body weight of DSS/ethanol induced colitis.Treatment of rats with muscovite or 5-ASA decreased both morphologic and histopathologic scores of DSS/ethanol induced rats.This part showed that rectal administration of muscovite could ameliorate colonic inflammation of DSS/ethanol induced colitis.Then we examined the effect of administration of different dosage of muscovite on TNBS-induced colitis in rats.Female SD rats with TNBS-induced colitis were treated with rectal administration of different dosage of muscovite daily for 14 days.The changes in body weight,macroscopic damage and histologic scores were subsequently evaluated.The effect of muscovite was compared with that of 5-ASA.We found rectal administration of muscovite improved the loss of body weight,macroscopic and histologic scores of TNBS-induced colitis in a dose-dependent manner.This part showed that rectal administration of different dosage of muscovite could ameliorate colonic inflammation of TNBS-induced colitis.Based on these observations we proved that muscovite is useful for the treatment of these two kinds of colitis.Further experiments were also done to ascertain the possible mechanisms involved in the anti-inflammtory effect of muscovite.An exaggerated intestinal immune response in colon plays a key role in the pathophysiology of UC.Pathological processes accompanying UC are associated with aberrant expression of many proinflammatory cytokines,including tumor necrosis factor-α(TNF-α),Interleukin-1β(IL-1β) and IL-8.To ascertain the possible mechanisms involoed in the anti-inflammtory effect of muscovite,we examined the effect of muscovite on the mucosal inflammatory changes by studying the expression of IL-1βand TNF-αin the colonic tissues in rats with TNBS-induced colitis.Female SD rats with TNBS-induced colitis were treated with rectal administration of different dosage of muscovite daily for 14 days.Gene expression of TNF-α,IL-1β,mucin2 (MUC2) and intestinal trefoil factor(ITF) in the colonic tissues was assessed by semiquantitative reverse-transcription polymerase chain reaction(RT-PCR) while protein levels of TNF-αand IL-1βwere detected by enzyme-linked immunosorbent assay(ELISA).MUC2 expression in colonic mucosa was detected by immunohistochemistry and Alcian blue-periodic acid Schiff(AB-PAS).The results showed TNBS-induced expression of TNF-α,IL-1βwas reduced by administration of muscovite and 5-ASA.Reduction of MUC2 expression in colitis rats was reversed by muscovite and 5-ASA treatment.However,the expression of ITF mRNA in colonic mucosa was not affected.This part showed rectal administration of different dosage of muscovite can decrease the expression of TNF-α,IL-1β. In the end,the capacity of muscovite to adsorb cytokines in vitro was determine by measuring the residual amount of standard TNF-α,IL-1βand IL-8 after these cytokines were incubated with muscovite for 1 hours as well as the changes in the amount of TNF-α,IL-1βsecreted by lipopolysaccharide(LPS)-stimulated THP-1 cells and IL-8 secreted by LPS- stimulated HT-29 cells.Muscovite inhibited the expression of TNF-α, IL-1βsecreted by THP-1 and IL-8 secreted by HT-29 cells in a dose-dependent manner. The levels of standard TNF-α,IL-1βand IL-8 were also decreased dramatically after incubating with muscovite for 1 hour.In conclusion,we successfully established a model of chemincal-induced experimental colitis which is characterized by a clinical course,localization of the lesions and histopathological features similar to human UC.We confirmed that rectal administration of muscovite can ameliorate colonic inflammation of two different kinds of chemical-induced experimental colitis and can ameliorate colonic inflammation of TNBS-induced colitis in dose-depend manner.The mechanism of therapeutic effect of muscovite is related with the fact that rectal administration of different dosage of muscovite can decrease the mRNA and protein expression of TNF-α,IL-1β.The possible mechanisms of muscovite include a direct adsorptive action of cytokines production by mucosal cells.更多还原