羧胺三唑对小鼠实验性结肠炎的疗效评价与作用机制研究

【作者】 郝晓健；

【导师】 张德昌； 叶菜英；

【作者基本信息】 中国协和医科大学 ， 药理学， 2009， 博士

【摘要】 研究背景炎性肠病(inflammatory bowel diseases,IBD)尤其是溃疡性结肠炎(ulcerativecolitis,UC)在我国的发病率呈逐年上升趋势,但其病因、发病机制目前仍不清楚。糖皮质激素、柳氮磺胺吡啶(salazosulfapyridine,SASP)是治疗IBD的常规药物,但此两类药物长期应用毒副作用大,且对部分病人无效,因此开发毒副作用小而有效的治疗IBD的药物具有重要意义。羧胺三唑(carboxyamidotriazole,CAI)是一种能够抑制细胞外钙内流的化合物,其在体外和体内模型中均表现出一定的抑制肿瘤增殖和转移的作用,此外该化合物还具有抑制血管生成、花生四烯酸释放以及激活NF-κB通路的作用。本课题组通过动物实验已证明该药物对急性、亚急性和慢性炎症具有显著的抗炎作用。为考察该药物对UC的潜在治疗作用,我们通过建立葡聚糖硫酸钠(dextran sulphate sodium salt,DSS)诱导的小鼠结肠炎模型,并施予CAI,观察其疗效,并从组织水平、细胞水平和分子水平探讨CAI对这种结肠炎模型的保护作用及作用机制,为进一步的临床研究奠定基础。研究方法采用DSS诱导小鼠溃疡性结肠炎模型,观察小鼠的体重和粪便变化,用大便性状改变和有无便血这两项指标评价小鼠的症状。取静脉血并将小鼠处死,制作结肠标本,HE染色观察结肠组织学变化,进行粘膜损害程度评分,采用酶联免疫吸附法(ELISA)测定血清、结肠匀浆和腹腔巨噬细胞培养上清中肿瘤坏死因子(tumornecrosis factor,TNF)-α、白介素(interleukin,IL)-1β、IL-6水平,采用ELISA法测定血清和结肠匀浆中基质金属蛋白酶-2,9(matrix metalloproteinase,MMP-2,9)和转化生长因子β1(transforming growth factor,TGFβ1)的含量。采用免疫组化方法观察小鼠结肠中细胞间粘附分子(intercellular adhesion molecule,ICAM)-1、核转录因子(nuclear factor,NF)-κB和TNF-α的表达或活化情况。检测小鼠结肠匀浆中髓过氧化物酶(myeloperoxidase,MPO)的活性。检测小鼠结肠匀浆中丙二醛(malonaldehyde,MDA)的含量和超氧化物歧化酶(superoxide dismutase,SOD)的活性。采用硝酸还原法测定小鼠结肠匀浆中一氧化氮(NO)的含量。采用Western Blot法测定小鼠腹腔巨噬细胞培养上清中诱导型一氧化氮合酶(iNOS)、IκBα和磷酸化IκBα的表达情况。采用Masson’s染色法观察小鼠结肠组织中胶原沉淀的情况。研究结果1.3%DSS能够成功诱导小鼠溃疡性结肠炎模型,其肠道病理变化类似于人类溃疡性结肠炎的病理改变。溃疡性结肠炎小鼠肠粘膜充血、水肿、糜烂或溃疡形成,粘膜炎症细胞浸润(主要为中性粒细胞)及隐窝脓肿形成。2.CAI(20 mg/kg)对3%DSS诱导的小鼠溃疡性结肠炎具有保护作用。连续10天给予小鼠20 mg/kg的CAI后,小鼠的体重、粪便和血便情况均有不同程度的改善。大体见CAI治疗组动物结肠充血、肠壁增厚明显减轻,粘膜缺损区浅小;组织学观察见粘膜损害累及的结肠范围缩小,毛细血管扩张、淋巴滤泡增生、粘膜下层水肿和隐窝结构破坏程度明显减轻。系列浓度的CAI(5、10、20和30 mg/kg)对3%DSS诱导的小鼠溃疡性结肠炎具有剂量依赖性的保护作用。同时CAI(20 mg/kg)对2%DSS诱导的小鼠溃疡性结肠炎也具有治疗作用。3.与正常对照组相比,饮用DSS的小鼠血清、结肠匀浆和腹腔巨噬细胞培养上清中TNF-α、IL-1β和IL-6水平升高,经CAI治疗后均降低。小鼠血清中TNF-α、IL-1β和IL-6的含量分别降低了66.75%、20.19%和26.49%,小鼠结肠匀浆中TNF-α、IL-1β和IL-6的含量分别降低了55.00%、74.95%和64.33%,小鼠腹腔巨噬细胞培养上清中TNF-α、IL-1β和IL-6的含量分别降低了80.41%、13.03%和58.58%。4.免疫组化的检测结果显示,溃疡性结肠炎小鼠结肠组织中TNF-α、NF-κB p65和ICAM-1表达明显升高,经CAI治疗后,小鼠结肠组织中TNF-α、NF-κBp65和ICAM-1的表达明显下降。5.与正常对照组相比,饮用DSS的小鼠结肠匀浆中MPO的活性升高,给予实验动物CAI后,MPO的活性明显降低,下降了13.58%。6.给予实验动物CAI后,溃疡性结肠炎小鼠结肠组织中MDA的含量显著降低,而SOD的活性则显著升高。与PEG400组相比,MDA的含量降低19.57%,SOD的活性升高29.14%。7.饮用DSS的小鼠腹腔巨噬细胞培养上清中NO水平升高,但经CAI治疗后显著降低,小鼠腹腔巨噬细胞培养上清中NO含量降低了24.53%。经CAI治疗后,小鼠腹腔巨噬细胞中iNOS的表达量下降。8.CAI能够显著抑制DSS引起的小鼠腹腔巨噬细胞中IκBα的降解以及磷酸化IκBα含量的升高。9.与正常对照组相比,饮用DSS的小鼠血清和结肠匀浆中MMP-2和MMP-9的含量升高,但经CAI治疗后,血清和结肠匀浆中MMP-2的含量分别下降了27.75%和47.14%;然而MMP-9的含量却未发生明显改变。10.溃疡性结肠炎小鼠经CAI治疗后,结肠匀浆中TGF-β1含量下降了52.43%。Masson’s三色染色的结果显示,CAI能够显著降低小鼠结肠组织中胶原纤维的沉积。结论CAI对DSS诱导的小鼠实验性结肠炎具有预防和治疗作用,且上述作用呈现出剂量依赖关系。CAI可显著改善结肠炎小鼠的腹泻和便血,减轻肠道充血和水肿及粘膜损害的范围和程度。CAI能够抑制NF-κB的活化,并通过清除粘膜活性氧,抑制TNF-α、IL-1β和NF-κB的激活,阻断相互激活的正反馈通路,阻断IL-6、IL-1β和ICAM-1的产生,最终表现为TNF-α、IL-1β、IL-6和ICAM-1低水平表达,从而减轻肠道炎性损害,改善结肠炎症状。CAI能够降低模型小鼠结肠中TGF-β1的含量,进而减少病变结肠中胶原纤维的含量,最终起到延缓病变结肠纤维化的作用。综上所述,根据我们的研究结果可以推测,CAI作为一种口服有效且副作用较少的药物,对溃疡性结肠炎的临床治疗具有较高的潜在应用价值。更多还原

【Abstract】 BackgroundThe worldwide incidence and mortality of inflammatory bowel diseases(IBD) are increasing,especially for ulcerative colitis(UC),while its etiology and mechanism have not been fully understood yet.The routine treatment of IBD involves glucocorticoids and salazosulfapyridine(SASP),which are effective in most case but unsustainable for their toxicities.It is an unmet clinical need that calls for novel anti-IBD agents with favorable efficacy and acceptable safety.Carboxyamidotriazole(CAI) is a compound that inhibits calcium influx and proliferation of tumor cell and endothelial cell.CAI has been proven active against proliferation and metastasis in both in vitro and in vivo models,and being developed as an anti-tumor agent.Its other activities,as reported by literatures,include anti-angiogenesis,inhibition of arachidonic acid release and activation of NF-κB pathway. In our early studies,the potential anti-inflammatory activity of CAI was established in a battery of models that cover acute and chronic inflammation.We hereby investigated the effect of CAI,together with its underlying mechanism,on mouse UC model prepared with dextran sulphate sodium salt(DSS).MethodsThe mouse UC model was prepared with DSS on C57/BL strain.The model was evaluated using following criteria:body weight,stool change,and presence of hemafecia. After collection of blood sample,the mice were sacrificed to allow fixing the colon sections.The damage of colon mucous membrane was scored following HE stain of colon sections.ELISA was employed to test a series of cytokines in serum,colon homogenate and conditioned medium of peritoneal macrophage,including:tumor necrosis factor alpha(TNF-α),interleukin 1β(IL-1β) and interleukin 6(IL-6).For measuring matrix metalloproteinase 2 and 9(MMP-2,9) as well as transforming growth factorβ1(TGF-β1),the specimens of serum and colon homogenate were subjected to ELISA.Immunohistochemistry was utilized in determining the expression and/or activation of intercellular adhesion molecule-1(ICAM-1),nuclear factorκB(NF-κB) and TNF-α.The study also quantified the activities of myeloperoxidase(MPO) and superoxide dismutase(SOD) in colon homogenate,the activity of SOD in serum and the content of malonaldehyde(MDA) in both colon homogenate and serum.The nitrogen monoxidum(NO) level in colon homogenate was determined by reduction method.The expression patterns of Inducible nitric oxide synthase,IκBαand phosphorylated IκBα were examined by western blot.The purpose of Masson’s stain was to evaluate the extent of fibrosis in colon.Results1.In C57 mice,DSS in strength of 3%was sufficient to produce pathological changes similar to that seen in human UC,making it an ideal agent for preparation the model through this study.The phenotype of the model was characterized by mucous hyperemia,edema,erosion and ulceration in colon,as well as inflammatory cell (mainly neutrophils) infiltration and crypt abscess.2.20 mg/kg CAI(p.o.) was proven to be protective against UC induced by 3%DSS in mice.After 10 days consecutive administration of CAI(20 mg/kg),the symptom of the disease(as indicated by body weight,stool change,and presence of hemafecia) was significantly improved.The mice treated with CAI showed alleviated enteremia and edema in colon and diminished mucous membrane lesions in macroscopic examination.Microscopy results revealed less severe telangiectasis,lymph foilicie hyperplasia,and submucosa edema and crypt disorganization compared with control group.Based on the data from a similar experiment involving an ascending doses of CAI(5,10,20 and 30 mg/kg),the dose dependent protective effect of CAI against UC induced by 3%DSS in mice was established.Therapeutic action of 20 mg/kg CAI(p.o.) was also evident in mouse UC model prepared with 2.5%DSS.3.Compared with normal mice,model mice had significant high levels of TNF-α, IL-1βand IL-6 in their serum,colon homogenate and conditioned medium of peritoneal macrophage.CAI treatment can palliate these elevations,with inhibition rates of 66.75%,20.19%and 26.49%in serum,55.00%,74.95%and 4.33%in colon homogenate,80.41%,13.03%and 58.58%in conditioned medium for NF-α,IL-1βand IL-6 respectively.4.Immunohistochemistry results indicated a downward trend in expressions of TNF-α, NF-κB p65 and ICAM-1 after treatment with CAI,which were upregulated in UC model mice.5.The activity of MPO in colon homogenate prepared from DSS-treated mice was higher than normal control group.CAI was tested to be active in mitigating this disparation by 13.58%.6.MDA depression and SOD activation were other 2 activities that can be contributed to CAI,as concluded in this study.In colon homogenate,when compared with PEG400 control group,the MDA level decreased by 19.57%,while SOD activity increased by 29.14%for CAI group.7.The conditioned medium of peritoneal macrophage collected from DSS-treated mice was high in NO level.This change was milder in CAI group with a decrease of 24.53%.In consist with its effect on NO level;CAI treatment exerted a negative influence on iNOS expression in peritoneal macrophages.8.The degradation of IκBαand elevation of pIκBαinduced by DSS in peritoneal macrophages can be significantly inhibited by CAl.9.The levels of MMP-2 and MMP-9 in model mice were relatively higher than normal control.For CAI treatment group,the results shown here suggested a down regulation of MMP-2 by 27.75%and 47.14%in serum and colon homogenate respectively,when compared with PEG400 group.However,no effect of CAI on MMP-9 content was observed so far.10.In CAI group,the content of TGF-β1 in colon homogenate was 52.43%lower than PEG400 control group.Results gained from Masson’s stain suggested CAI is potent in blocking collagen fiber accumulation.ConclusionCAI is proven to possess protective action and therapeutic action against UC induced by DSS in mice,and these actions are delivered in a dose dependent manner.CAI can significantly improve the symptoms of diarrhea and hemafecia caused by UC. Necroscopy fidings shows alleviated hyperemia,edema and mucous membrane damages in CAI group.CAI inhibits NF-κB activation,while scavenging active oxygen at colon mucous membrane,thus blocking downstream signal molecules such as TNF-α,IL-1β, IL-6 and ICAM-1 by suppressing their expressions.All these mechanisms collectively protect the colon from inflammation.In addition,CAI is a down regulator of TGF-β1, which is a mediator of collagen fiber accumulation in colon and plays a crucial role in ulcerative colitis fibrosis.In summary,the results we provided herein highlight CAI a promising oral available anti-ulcerative colitis agent with favorable efficacy and safety profile,which is well worth further development.更多还原