一类新药羧胺三唑在多种模型中的抗炎作用及部分机制研究

【作者】 郑茹；

【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， 2010， 博士

【摘要】 研究背景类风湿性关节炎、非特异性炎性肠病等自身免疫性疾病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、非甾体抗炎药物是治疗此类疾病的常用药物,不过这些药物长期应用毒副作用大,且对部分患者无效,因此开发毒性小且能够有效治疗此类疾病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在某些情况下抑制NF-κB通路激活的作用。本课题组通过体内实验已证明该药物对急性、亚急性和慢性炎症均具有显著的抗炎作用。为考察该药物对某些特定炎性疾病的潜在治疗作用,我们通过建立佐剂性关节炎模型和TNBS诱导的大鼠结肠炎模型,观察CAI的疗效,并从组织水平、细胞水平和分子水平探讨CAI的抗炎作用机制,为进一步的临床研究奠定基础。研究方法建立佐剂性关节炎模型,以足爪体积、体重、食物消耗量和关节炎评分为评价指标考察CAI对大鼠原发性病变和继发性病变的疗效。取静脉血并将大鼠处死,HE染色观察滑膜组织学变化情况,采用ELISA法测定血清和足爪匀浆中TNF-α和IL-1β水平。测定大鼠足爪匀浆中PGE2和NO的水平。建立TNBS诱导的结肠炎模型,以死亡率、体重、食物消耗量、临床评分、宏观评分和组织学评分为评价指标考察CAI对TNBS诱导大鼠结肠炎的疗效。取静脉血并将大鼠处死,HE染色观察结肠组织学变化情况,进行粘膜损害程度评分。测定结肠匀浆中MPO的活性。采用ELISA法测定血清和结肠匀浆中TNF-α和IL-6的水平。采用免疫组化方法观察大鼠结肠中NF-κB和ICAM-1的活化或表达情况。检测大鼠结肠匀浆中MDA的含量和SOD的活性。采用硝酸还原法测定大鼠结肠匀浆中NO的含量。采用Masson’s染色法观察大鼠结肠组织中胶原沉淀的情况,采用ELISA法测定结肠匀浆中TGF-β1的水平。以腹腔巨噬细胞为对象考察CAI的作用机制,MTT法检测CAI对正常大鼠和佐剂性关节炎大鼠腹腔巨噬细胞活力的影响。采用ELISA法测定正常大鼠、佐剂性关节炎大鼠和结肠炎大鼠腹腔巨噬细胞中多种细胞因子的水平。测定CAI对巨噬细胞中PEG2和NO等炎性介质的影响。采用Western Blot法测定腹腔巨噬细胞中多种炎症相关信号蛋白的表达和活化情况。采用免疫细胞化学法测定NF-κB的活化情况。利用中性红法测定大鼠腹腔巨噬细胞的吞噬作用。通过碳粒廓清实验、鸡红细胞致小鼠溶血素生成实验和DNFB诱导的迟发型超敏反应考察CAI对正常小鼠免疫系统的影响。研究结果1.CAI(10、20和40 mg／kg)能够剂量依赖性的改善佐剂性关节炎大鼠的原发性病变和继发性病变,CAI(20和40 mg/kg)能够明显改善实验动物的足爪肿胀程度、体重、食物消耗量和关节评分。CAI (20 mg/kg)对已出现关节症状但又未发生完全不可逆性病变的动物具有一定疗效。CAI (20 mg/kg)与地塞米松合用后,药物的疗效优于两种药物单独使用时的情况,且动物的体重减轻及其他不良反应与单独使用地塞米松时相当。2. CAI (20 mg/kg)对大鼠足跖组织中的PGE2含量没有明显影响。而地塞米松则能够显著降低佐剂性关节炎大鼠足跖组织中PGE2的含量(p＜0.001)。CAI (20 mg/kg)和地塞米松均能够明显降低佐剂性关节炎大鼠足跖中的NO含量(p＜0.01和p＜0.001)。3. CAI (20 mg/kg)分别可以使足跖组织中TNF-α和IL-1β水平下降54.1%和26.3%(均为p＜0.05),抑制作用与地塞米松相当,血清中TNF-α和IL-1β水平同样受到CAI(均为p＜0.05)和地塞米松的显著抑制。4.造模剂剂量摸索实验的结果显示,75 mg/kg TNBS/乙醇能够成功诱导大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。溃疡性结肠炎大鼠肠粘膜充血、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。CAI(20、30和40 mg/kg)能够剂量依赖性的改善实验性结肠炎大鼠的急性结肠炎症症状和结肠纤维化情况,CAI(30和40 mg/kg)能够明显改善实验动物的死亡率、体重、食物消耗量、临床评分、宏观评分和组织学评分等情况。大体见CAI(30和40 mg/kg)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。5.与空白对照组相比,TNBS诱导结肠炎大鼠结肠匀浆中MPO的活性升高,给予实验动物CAI (40 mg/kg)后,MPO的活性明显降低(p＜0.05)。6. CAI (40 mg/kg)分别可以使结肠组织中TNF-α和IL-6水平下降52.68%(p＜0.01)和57.15%(p＜0.01),其对TNF-α的抑制作用强于SASP,但略低于Infliximab,但CAI对IL-6的抑制作用强于SASP和Infliximab。血清中TNF-α和IL-6水平同样受到CAI(抑制率分别为75.35%[p＜0.001]和32.57%[p＜0.01])的显著抑制。7.给予实验动物CAI (40 mg/kg)后,结肠炎大鼠结肠组织中MDA和NO的含量显著降低,而SOD的活性则显著升高。与PEG400组相比,MDA和NO的含量分别降低25.97%和41.58%(均为p＜0.05),SOD的活性升高49.23%(p＜0.01)。8.免疫组化的检测结果显示,溃疡性结肠炎大鼠结肠组织中NF-κB p65核转位和ICAM-1表达明显升高,经CAI治疗后,大鼠结肠组织中NF-κB p65核转位和ICAM-1表达明显下降。9.溃疡性结肠炎大鼠经CAI (40mg/kg)治疗后,结肠匀浆中TGF-β1含量显著下降(p＜0.01)。Masson’s三色染色的结果显示,CAI能够显著降低大鼠结肠组织中胶原纤维的沉积。10.CAI对正常大鼠和佐剂性关节炎大鼠腹腔巨噬细胞的活力均没有影响,CAI对正常大鼠和佐剂性关节炎大鼠腹腔巨噬细胞的吞噬作用也没有影响。11.对于正常大鼠腹腔巨噬细胞而言,CAI作用18 h后,CAI(20和40μmol／L)能够明显抑制TNF-α的生成量(p＜0.05和p＜0.01)。CAI(10、20和40μmol／L)能够明显抑制IL-6的生成量(p＜0.05、p＜0.01和p＜0.001)。CAI(10、20和40μmol／L)能够明显抑制INF-γ的生成量(p＜0.05、p＜0.01和p＜0.01)。对于佐剂性关节炎大鼠腹腔巨噬细胞而言,CAI(20 mg／kg)可以使佐剂性关节炎大鼠腹腔巨噬细胞中TNF-α、IL-6和INF-γ的水平分别下降63.56%(p＜0.01)、14.52%(p＜0.05)和80.81%(p＜0.01),CAI虽然也能够使IL-10升高,但该结果与PEG400组相比无显著性差异。对于结肠炎大鼠腹腔巨噬细胞而言,CAI (40mg/kg)能显著降低TNBS诱导结肠炎大鼠腹腔巨噬细胞中TNF-α和IL-6的生成量(p＜0.001),抑制率分别为77.05%和56.66%。12.对于正常大鼠腹腔巨噬细胞而言,分别将浓度为5、10、20和40μmol／L的CAI与大鼠腹腔巨噬细胞共同作用18 h(同时加LPS刺激)后,可剂量依赖性地抑制NO的产生。CAI能够对iNOS的表达产生明显的抑制作用,且与CAI对NO生成量抑制作用的变化趋势一致。对于佐剂性关节炎大鼠腹腔巨噬细胞而言,与PEG400组相比,CAI (20mg/kg)对大鼠腹腔巨噬细胞上清中PGE2的生成量没有影响。CAI (20 mg/kg)能够明显降低佐剂性关节炎大鼠腹腔巨噬细胞上清中NO的含量(p＜0.01)。CAI (20 mg/kg)对COX-2和iNOS蛋白表达的影响与上述结果一致。13.对于正常大鼠腹腔巨噬细胞而言,DMSO组巨噬细胞的胞核和胞浆中NF-κB p65的染色均较深,而CAI(20μmol／L)组巨噬细胞的胞核中几乎没有染色,胞浆中的染色也较浅,提示CAI能够抑制p65入核。分别将浓度为5、10、20和40μmol／L的CAI与大鼠腹腔巨噬细胞共同作用18 h(同时加LPS刺激)后,将对LPS诱导的IκBα降解产生明显的阻断作用,CAI能够减少LPS诱导的IκBα磷酸化,上述作用均呈现出剂量依赖性。对于佐剂性关节炎大鼠腹腔巨噬细胞而言,CAI (20 mg/kg)能够对p65核转位以及IκB-α的降解和磷酸化产生明显的阻断作用。CAI (20mg/kg)能够抑制佐剂性关节炎大鼠腹腔巨噬细胞中JNK和p38 MAPK的磷酸化。但是,该药物对非磷酸化JNK和p38的表达没有影响。14. CAI (20 mg/kg)对正常小鼠的碳粒廓清能力没有影响,CAI (20 mg/kg)对鸡红细胞致小鼠溶血素的生成量无抑制作用,CAI (20 mg/kg)对DNFB诱导的小鼠迟发型超敏反应没有抑制作用。结论CAI对大鼠佐剂性关节炎和TNBS诱导的大鼠结肠炎均有较好的治疗作用。多种动物模型和细胞水平的研究结果均提示,CAI能够抑制NF-κB相关通路的活化,并通过清除活性氧,抑制TNF-α、IL-6和NF-κB的激活,阻断相互激活的正反馈通路,阻断IL-6、IL-1β等细胞因子的产生,最终表现为多种促炎性细胞因子表达水平降低,从而改善多种炎症症状。此外,CAI对巨噬细胞的作用主要表现在抑制细胞因子分泌,而对巨噬细胞的吞噬功能没有影响,CAI不是通过抑制免疫系统发挥抗炎作用。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、副作用较少且价格低廉的药物,对自身免疫性疾病的临床治疗具有较高的潜在应用价值。更多还原

【Abstract】 BackgroundThe incidence of the autoimmune diseases, such as rheumatoid arthritis, non-specific inflammatory bowel disease, is increasing constantly in china cross past decades. However, we know little about its etiology and pathogenesis until now. Glucocorticoids, non-steroidal anti-inflammatory drugs are commonly used agents for the autoimmune diseases, but their long-term application is limited by adverse effects and resistance. There is no doubt that a potent agent with favorable safety profile will fulfill this unmet clinical need. Carboxyamidotriazole (CAI) is a compound that inhibits calcium influx and proliferation of tumor cell. CAI has been proven active against proliferation and metastasis in both in vitro and in vivo models. Its other activities, as reported by literatures, include anti-angiogenesis, inhibition of arachidonic acid release and 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. This study is a step forward on our way to explore CAI’s anti-inflammatory effect in inflammatory diseases such as ulcerative colitis and adjuvant arthritis. Animal models and in vitro testing systems were established for mechanism investigation, and CAI was canvassed in multiple levels from in vivo through molecular.MethodsRat adjuvant arthritis model was established and validated. The effect of CAI on primarily and secondary lesion was evaluated using criteria of paw volume, body weight, food consumption and disease activity score. After collecting blood samples, rats were sacrificed and synovial membrane was separated for HE staining. The specimens of serum or paw homogenate were subjected to ELISA for cytokines and mediators including:tumor necrosis factor alpha (TNF-a), interleukin 1β(IL-1β), interleukin 6 (IL-6), prostaglandin E2 (PGE2) and nitric oxide (NO). TNBS-induced colitis model of rat was established and validated. The effect of CAI was evaluated using criteria of mortality, body weight, food consumption, clinical score, macroscopic and microscopic finding. After collecting blood samples, rats were sacrificed and colon section was removed for HE staining and evaluation. The following parameters of serum or colon homogenate were determined:MPO activity, TNF-a level (ELISA), IL-6 level (ELISA), TGFβ1 level (ELISA), NO content, MDA concentration and SOD activity. IHC was employed to evaluate the status of NF-κB and ICAM-1 in colon tissues. Collagen accumulation in rat colon was evaluated by Masson’s staining. For mechanism studies in peritoneal macrophages, the cytotoxicity of CAI was evaluated by MTT assay. ELISA was performed to quantify cytokines and mediators. Western Blot was conducted to investigate the expression and activation of inflammation-related proteins in peritoneal macrophages. The status of NF-κB was profiled by Immunocytochemistry. An in vitro neutral red assay was employed to evaluate the influence of CAI on phagocytosis. To address the concern about potential obstruction to normal immune system, CAI was tested in carbon clearance assay, CRBCs-induced hemolysin formation assay and DNFB-induced contact hypersensitivity assay.Results1. CAI (10,20 and 40 mg/kg) improved both primarily and secondary lesion in rat adjuvant arthritis model in a dose-dependent manner. In doses> 10 mg/kg (20 and 40 mg/kg), the effect of CAI on paw edema, body weight, food consumption and joint score was statistically significant when compared with vehicle. CAI (20 mg/kg) was of therapeutic value to those affected but not irreversible joint damage caused by arthritis. The combination of CAI (20 mg/kg) and dexamethasone has a superior efficacy over each alone, and a comparable safety profile with dexamethasone.2.20 mg/kg CAI had little effect on the PGE2 level of rat paw tissue, which was significantly decreased by dexamethasone (p<0.001). Both CAI (20 mg/kg) and dexamethasone suppressed NO level in rat paw tissue (p＜0.01 and p＜0.001, respectively).3. After treated with 20 mg/kg CAI, paw tissue TNF-a and IL-1βlevels decreased by 54.1% and 26.3% (both p<0.05), respectively. A similar repression effect was seen in dexamethasone group. These observations are consistent with what was found in serum samples.4. In the model validation study,75 mg/kg TNBS/EtOH was found to be the optimal dose to induce rat ulcerative colitis. The intestinal pathological changes in model animals were representative of human ulcerative colitis, including intestinal hyperemia, edema, mucosal erosion and ulceration and inflammatory cell infiltration (mainly neutrophils) and the crypt abscess formation. CAI (20,30 and 40 mg/kg) dose-dependently protected model animal from acute symptoms and fibrosis of colon. In CAI 30 mg/kg group and 40 mg/kg group, the improvement achieved was reflected in criteria of mortality, body weight, food consumption, clinical score, macroscopic finding and microscopic finding. The rat treated with CAI (dose≥30 mg/kg) 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.5. The activity of MPO in colon homogenate prepared from TNBS-treated rat was higher than normal control. The difference in MPO activity between CAI (40 mg/kg) group and control group was statistically significant (p<0.05), with a lower activity in CAI group.6. In 40 mg/kg CAI group, the TNF-a and IL-6 levels in colon homogenate were decreased by 52.68% (p＜0.01) and 57.15% (p<0.01), respectively. The inhibition rate of CAI on TNF-a was greater than that of SASP, but inferior to Infliximab. For IL-6, CAI was shown to be a more potent inhibitor than both SASP and Infliximab. The TNF-a and IL-6 levels in serum samples were also decreased after CAI treatment, with calculated inhibition rate of 75.35% (p＜0.001) and 32.57% (p＜0.01), respectively.7. After treatment with CAI (40 mg/kg), the MDA and NO contents in colon tissue collected from colitis rats were decreased by 25.97% and 41.58% (both p＜0.01, compared with PEG400), respectively. Meanwhile, SOD as measured by its activity, was 49.23% (p＜0.01) higher compared with PEG400.8. The results from IHC indicated that, the altered NF-κB p65 translocation and ICAM-1 expression in model rat colon tissue were significantly reversed in after CAI administration.9. In 40 mg/kg CAI group, the content of TGF-β1 in colon homogenate was significant lower than control group (p<0.01). Results from Masson’s stain suggested CAI is active in blocking collagen fiber accumulation in colon.10. Irrespective of the origin of the macrophages (source from normal rats or adjuvant arthritis), CAI did not affect the vitality nor the phagocytosis of the macrophages.11. In in vitro studies focusing on macrophages, the TNF-a concentration in the conditioned medium of normal rat macrophages was significantly decreased after 20 and 40μmol/L CAI treatment (for 18 h; p<0.05 and p＜0.01, respectively). Similar effect was observed for IL-6 and INF-γ. For 10,20 and 40μmol/L CAI, the significance of inhibition to IL-6 was p<0.05, p＜0.01 and p＜0.001, respectively. The significance of inhibition to INF-y was p<0.05, p＜0.01 and p<0.01, respectively. In studies of same design but with macrophages from adjuvant arthritis rats, the effect of CAI (20 mg/kg) on TNF-a, IL-6 and INF-y levels in conditioned medium of macrophages was consistent with what was established with normal rat macrophages. The inhibition rate was 63.56% (p＜0.01),14.52% (p<0.05) and 80.81% (p＜0.01), for TNF-a, IL-6 and INF-y respectively. Although elevated IL-10 level in the CAI group was evident, this result was not significantly different from the PEG400 group. The production of TNF-a and IL-6 in macrophages from TNBS-induced colitis rats was significantly diminished by CAI (40 mg/kg) with inhibition rate of 77.05% and 56.66%, respectively (both p<0.001).12. NO production (elicited by LPS stimulation) of normal rat macrophages was down-regulated after 18-hr incubation with 5,10,20 and 40μmol/L CAI in a dose dependent manner, as compared with control group. A similar inhibition effect of CAI was observed on iNOS expression and NO production with correlation between each other. For peritoneal macrophages collected from adjuvant arthritis rats, the difference between CAI group (20 mg/kg) and PEG400 group on the PGE2 or COX-2 content was negligible. The data on NO production and iNOS expression was consistent with previous findings made on normal rat macrophages.13. In normal rat peritoneal macrophages, NF-κB p65 staining was found to be present in both nuclei and cytoplasm in DMSO-treated (control) group, while barely distinguishable in nuclei and very weak in cytoplasm in 20μmol/L CAI-treated group; indicating a blockade of p65 translocation by CAI.5,10,20 and 40μmol/L CAI was added into rat peritoneal macrophages and incubated for 18 hrs followed by LPS stimulation. The degradation of IκBa and formation of pIκBa induced by LPS in peritoneal macrophages can be significantly inhibited by CAI in a dose-dependent manner. The p65 translocation, IκBa degradation and pIκBa formation were also repressed by CAI (20 mg/kg) in adjuvant arthritis rat’s peritoneal macrophages. CAI (20 mg/kg) also inhibited the phosphorylation of JNK and p38 MAPK in adjuvant arthritis rat’s peritoneal macrophages, rather than the expression of JNK and p38.14. CAI at a dose of 20 mg/kg was proven to be neutral in following in vitro tests: carbon clearance assay, CRBCs-induced hemolysin formation assay and DNFB-induced contact hypersensitivity assay.Conclusions CAI was proven to be effective to adjuvant arthritis and TNBS-induced ulcerative colitis. As evidenced by data collected in in vivo and in vitro studies, the effect of CAI includes:NF-κB inhibition, active oxygen clearance and inflammatory cytokine (such as TNF-a and IL-6) suppression. For the macrophages, the phagocytosis will not be disturbed when CAI inhibits macrophage’s secretion of cytokines, which is the fuel of inflammation. The anti-inflammatory activity of CAI is free from immusupperession and quite different from classic NSAIDs in terms of mechanism. In summary, the data presented herein highlight CAI a promising oral available and economical compound that is likely to become a rising star in therapy of autoimmune diseases.更多还原