【作者】 周国胜；

【导师】 吴正祥；

【作者基本信息】 安徽医科大学 ， 内科学， 2009， 硕士

【摘要】 研究背景炎症性肠病(inflammatory bowel disease,IBD)在发达国家和发展中国家中是一个非常突出的健康问题,尤其在发达国家更为突出。IBD为胃肠道慢性反复发作与缓解的炎症性疾病,是一种非特异性胃肠道疾病,主要包括溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn’s disease,CD)。其病因和发病机制迄今尚未阐明,目前认为IBD受遗传、感染、环境、免疫和精神心理等因素影响,属于难治性疾病。随着IBD发病率和患病率的增高,预防和治疗的难题引起同行的高度重视。目前,炎症性肠病的治疗主要依赖5-氨基水杨酸、糖皮质激素、免疫抑制剂等,包括嘌呤类抗代谢物和环孢霉素,然而激素抵抗和依赖已成重要的临床问题,其它免疫抑制剂如硫唑嘌呤和环孢霉素A也表现出严重的副作用。近年来,生物制剂如抗肿瘤坏死因子抗体和抗白细胞介素抗体应用于治疗炎症性肠病,虽取得一些成功,但也有其不足之处,而且其治疗方案复杂,价格又昂贵。因此,寻找新型有效的药物一直是该领域的研究热点。青藤碱(sinomenine)是从青风藤中提取出来的选择性生物碱单体,研究表明青藤碱具有显著的抗炎和免疫抑制作用,清除自由基等作用,发挥免疫调节功能,并且毒副作用小,肠道易吸收。临床广泛应用于关节炎的治疗,并取得良好疗效。在IBD的研究中,报道较少。目的观察青藤碱(sinomenine)对2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)/乙醇诱导结肠炎大鼠的抗炎和保护作用,并初步探讨其作用机制,为临床应用青藤碱治疗炎症性肠病提供一定的实验理论依据。方法60只成年雄性健康清洁级Sprague-Dawley(SD)大鼠随机均分为正常对照组(Normal group,N group,N组)、结肠炎模型组(Model group,M group,M组)、5-氨基水杨酸治疗组(5-aminosalicylic acid group,5-ASA group,C组)、青藤碱高剂量治疗组(High dose sinomenine group,HD group,HD组)、青藤碱中剂量治疗组(Middle dose sinomenine group,MD group,MD组)以及青藤碱低剂量治疗组(Low dose sinomenine group,LD group,LD组)。除正常对照组外,余5组采用三硝基苯磺酸/乙醇灌肠法制备结肠炎模型,造模24 h后,开始灌胃,正常对照组和结肠炎模型组每只大鼠给予2 mL生理盐水灌胃,5-ASA治疗组每只大鼠给予100mg/kg·d 5-ASA灌胃,青藤碱高、中、低剂量治疗组每只大鼠分别给予120、80、40 mg/kg·d青藤碱灌胃。评价各组大鼠疾病活动指数(disease activity index,DAI)、结肠大体形态损伤指数(colon macroscopic damage index,CMDI)及组织学损伤指数(tissues damage index,TDI)。ELISA法检测大鼠血清IL-1β、TNF-α、MMP-2以及MMP-9的含量,免疫组织化学SP法检测大鼠结肠组织MMP-2、MMP-9以及pp38MAPK的表达。结果正常对照组、结肠炎模型组、5-ASA治疗组以及青藤碱高、中、低剂量治疗组的DAI评分分别为0.00±0.00、3.47±0.48、1.00±0.55、0.93±0.54、1.60±0.81和2.43±0.75;CMDI评分分别为0.30±0.48、6.50±1.27、1.30±1.16、1.00±0.82、2.30±1.70和4.40±2.01;TDI评分分别为0.20±0.42、5.10±0.74、1.10±1.20、0.90±0.99、2.80±1.40和4.10±1.10;大鼠血清IL-1β含量分别为24.39±5.08 pg/mL、88.48±5.12 pg/mL、35.44±4.31 pg/mL、35.80±3.05 pg/mL、51.77±2.73 pg/mL和62.20±5.07 pg/mL;大鼠血清TNF-α含量分别为23.54±4.20 pg/mL、74.72±5.30pg/mL、35.37±4.44 pg/mL、32.50±4.91 pg/mL、49.76±4.38 pg/mL和61.63±7.16 pg/mL;大鼠血清MMP-2的含量分别为0.76±0.44 ng/mL、10.40±2.10 ng/mL、2.44±0.83ng/mL、2.50±0.82 ng/mL、3.76±1.44 ng/mL和5.34±1.07 ng/mL;大鼠血清MMP-9的含量分别为2.83±1.15 ng/mL、15.85±2.39 ng/mL、5.88±1.84 ng/mL、5.32±1.81ng/mL、7.69±2.16 ng/mL和10.14±2.61 ng/mL;大鼠结肠组织MMP-2的表达分别为0.60±0.52、5.00±1.41、1.30±1.25、1.00±0.94、2.20±1.03和3.50±1.58;大鼠结肠组织MMP-9的表达分别为0.50±0.53、6.30±0.67、1.50±1.35、1.40±0.97、2.90±1.20和4.50±1.43;大鼠结肠组织pp38MAPK的表达分别为1.30±0.48、5.80±1.03、2.10±0.74、2.00±0.82、3.10±1.37和4.30±0.95。与正常对照组大鼠进行比较,上述指标在结肠炎模型组中显著升高,差异具有统计学意义(P值均＜0.01)。与结肠炎模型组比较,5-ASA治疗组和青藤碱高、中剂量组的大鼠DAI、CMDI和TDI显著降低;大鼠血清中的IL-1β、TNF-α、MMP-2和MMP-9的含量显著降低;大鼠结肠组织MMP-2、MMP-9和pp38MAPK的表达也显著降低,经单因素方差分析及t检验,差异有统计学意义(P值均＜0.01);而青藤碱高剂量治疗组大鼠与5-ASA治疗组大鼠进行比较,二者作用相当(P＞0.05)。青藤碱高剂量治疗组大鼠与青藤碱低剂量治疗组大鼠进行比较,上述指标均显著降低,差异有统计学意义(P值均＜0.01),提示青藤碱对TNBS诱导的结肠炎大鼠干预存在剂量反应关系。结论青藤碱可改善大鼠结肠炎症情况,显著降低血清和结肠组织IL-1β、TNF-α、MMP-2和MMP-9,其作用机制可能与抑制细胞因子的表达,同时抑制基质金属蛋白酶和pp38MAPK的表达,减轻上皮细胞破坏,保护肠黏膜屏障有关。更多还原

【Abstract】 Research background Inflammatory bowel disease(IBD),a significant health problem both in the developed and developing countries,especially in the developed countries,which comprised two chronic relapsing and remitting inflammatory disorders of the gastrointestinal tract,which was a non-specificity inflammatory disease in gastrointestinal tract,mainly included ulcerative colitis(UC) and Crohn’s disease(CD). Up to now,little was known about the underlying pathogenesis of IBD,however,it was considered a disease of multifactorial origin reflecting an interplay of genetic, immunological,infection,psychology and environmental factors,which might underlie the initiation and perpetuation of gastrointestinal inflammation.With the heightening of incidence and prevalence in IBD,the difficulty of prevention and therapeutics evoked extensive attention.At present,pharmacologic management of IBD had relied mainly on 5-aminosalicylates,corticosteroids,and immunosuppressants,including purine antimetabolites and cyclosporine.However,corticosteroid dependence was a clinically important problem,and other immunosuppressive agents such as azathioprine and cyclosporine A also exhibited serious side effects.Recently,biological products such as anti-TNF-αand anti-IL-12 antibody had been used with some success in IBD.This approach,however,was also not without side effects,and treatment protocols were complicated and expensive.Therefore,additional studies were warranted to identify new anti-inflammatory agents to treat IBD.Sinomenine was a selective alkaloidal monomer which extracted from sinomenium acutum.Sinomenine possessed anti-inflammatory effects,depressed humoral immunity and cellular immunity,removed nitric oxide free radicals,produced anti-inflammatory effection,and almost without toxic and side-effect,was easily absorbed in intestinal tract.Extensively clinically applying sinomenine in therapeutics of arthritis,and acquired good therapeutic effects. But in the study of IBD,there was almost no report which be concerned with sinomenine.Objective To observe the anti-inflammatory and protective effects of sinomenine on experimental colitis-induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ alcohol in rats,and investigate it’s mechanism of action on colitis.In order to supply determinate experimental theory for clinical application of sinomenine in therapeutics of IBDMethods Sixty Sprague-Dawley(SD) rats were randomly divided into six groups (n=10):normal control group(N group),model colitis group(M group),5-aminosalicylic acid treatment group(5-ASA treatment group,C group),High dose sinomenine treatment group(HD group),Middle dose sinomenine treatment group(MD group) and Low dose treatment sinomenine group(LD group).Excepted normal control group,other group were colitis models of rats which were established by intracolon enema with trinitrobenzene sulfonic acid(TNBS)/ ethanol.In the normal control group,no colitis induction was performed,but colitis was induced in all other groups.Followed the induction of colitis,5-ASA and sinomenine were applied to homologous treatment groups of rats.Began to proceeding intragastric administration, after twenty-four hours by establishing colitis models.The each of rats in normal control group and model colitis group with physiological saline(2 mL);the each of rats in 5-ASA treatment group with 5-ASA(100 mg/kg·d);the each of rats in High、Middle and Low sinomenine treatment group with sinomenine in dose of 120、80 and 40 mg/kg·d respectively.Disease activity index(DAI) and colon macroscopic damage index(CMDI) as well as tissues damage index(TDI) of rats in each group were evaluated respectively.The serum levels of IL-1β,TNF-α,MMP-2 as well as MMP-9 were detected by enzyme-linked immunosorbent assay(ELISA).The expressions of MMP-2,MMP-9 as well as pp38MAPK in colonic tissues were detected using immunohistochemical method.Results In normal control group,model colitis group,5-ASA treatment group, High、Middle and Low dose of sinomenine treatment group,the DAI were 0.00±0.00, 3.47±0.48,1.00±0.55,0.93±0.54,1.60±0.81 and 2.43±0.75,respectively;the CMDI were 0.30±0.48,6.50±1.27,1.30±1.16,1.00±0.82,2.30±1.70 and 4.40±2.01 respectively;the TDI were 0.20±0.42,5.10±0.74,1.10±1.20,0.90±0.99,2.80±1.40 and 4.10±1.10 respectively;the serum levels of IL-1βwere 24.39±5.08 pg/mL, 88.48±5.12 pg/mL,35.44±4.31 pg/mL,35.80±3.05 pg/mL,51.77±2.73 pg/mL and 62.20±5.07 pg/mL respectively;the serum levels of TNF-αwere 23.54±4.20 pg/mL, 74.72±5.30 pg/mL,35.37±4.44 pg/mL,32.50±4.91 pg/mL,49.76±4.38 pg/mL and 61.63±7.16pg/ mL respectively;the serum levels of MMP-2 were 0.76±0.44 ng/mL, 10.40±2.10 ng/mL,2.44±0.83 ng/mL,2.50±0.82 ng/mL,3.76±1.44 ng/mL and 5.34±1.07 ng/mL respectively;the serum levels of MMP-9 were 2.83±1.15 ng/mL, 15.85±2.39 ng/mL,5.88±1.84 ng/mL,5.32±1.81 ng/mL,7.69±2.16 ng/mL and 10.14±2.61ng/mL respectively;the expressions of MMP-2 in colonic tissues of rats were 0.60±0.52,5.00±1.41,1.30±1.25,1.00±0.94,2.20±1.03 and 3.50±1.58 respectively;the expressions of MMP-9 in colonic tissues of rats were 0.50±0.53,6.30±0.67,1.50±1.35,1.40±0.97,2.90±1.20 and 4.50±1.43 respectively;the expressions of PP38MAPK in colonic tissues of rats were 1.30±0.48,5.80±1.03,2.10±0.74,2.00±0.82,3.10±1.37 and 4.30±0.95 respectively.Compared with normal control group,all index above-mentioned significantly increased in model colitis group (all P＜0.01).Compared with model colitis group,the DAI,CMDI as well as TDI in 5-ASA treatment group,High and Middle dose of sinomenine treatment group were significantly ameliorated(all P＜0.01);The serum levels of IL-1β,TNF-α,MMP-2 as well as MMP-9 were significantly reduced(all P＜0.01).The expressions of MMP-2, MMP-9 as well as pp38MAPK in colonic tissues of rats in 5-ASA treatment group, High and Middle dose of sinomenine treatment group significantly decreased(all P＜0.01),however,equalitily effects between 5-ASA treatment group and High dose sinomenine treatment group(P＞0.05).Compared with High dose sinomenine treatment group,all index above-mentioned in Low dose sinomenine treatment group significantly increased(all P＜0.01),suggested that the therapeutics of sinomenine on experimental colitis-induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ alcohol in rats presented dose-response relationship.All above results were determined by using one-way analysis of variance(ANOVA) and Student’s t test.Conclusions The present results of study indicates that sinomenine can ameliorate the colonic inflammation,reduce the serum levels of IL-1β,TNF-α,MMP-2 and MMP-9, and decrease the expression of MMP-2,MMP-9,pp38MAPK.It’s mechanism of anti-inflammatory effects is possiblely related with reducing the expression of cytokine, MMP-2,MMP-9,as well as pp38MAPK,alleviating epithelial cells cytoclasis,and protecting intestines mucous membrane barrier.更多还原