蛇葡萄素、白芍总苷减轻大鼠实验性结肠炎的作用及机制研究
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摘要
炎症性肠病(inflammatory bowel disease, IBD),是指一组病因不明的慢性非特异性的肠道炎症性疾病,包括克罗恩病(Crohn's disease)和溃疡性结肠炎(ulcerative colitis)两个独立的疾病。炎症性肠病病因目前尚未完全阐明,可能与多种因素有关,目前认为该病的发病与宿主遗传易感性、胃肠道益生菌和致病菌失衡、肠上皮黏膜完整性受损及机体免疫功能失调等综合因素的相互作用有关。炎症性肠病是世界范围的疾病,其分布具有显著的地理特点,在欧美等西方国家如斯堪的那维亚、西欧和北美国家多见,而亚洲国家的发病率与患病率相对较低,但近年来我国的患病率呈明显上升趋势。正因炎症性肠病病因尚不完全明确,故而其治疗也颇为困难,目前治疗炎症性肠病的主要药物包括水杨酸类、糖皮质激素、免疫抑制剂及生物制剂如抗肿瘤坏死因子(抗-TNF)单克隆抗体等,用于调节异常的免疫反应。然而,这些药物对炎症性肠病的疗效有限且副作用明显,对部分炎症性肠病患者联合使用上述药物仍不能有效控制疾病的进展。因此,开发新的有效的、副作用少的药物用于炎症性肠病的治疗显得非常迫切和必要。
第一部分蛇葡萄素减轻大鼠实验性结肠炎的作用及机制研究
蛇葡萄素(Ampelopsin)是一种多酚羟基双氢黄酮醇,化学名称：(2R,3R)-3,5,7-三羟基-2-(3,4,5-三羟基苯基)苯并二氢吡喃-4-酮,分子式：C15H12O8,分子量：320.25,又名二氢杨梅素。近年来国内外学者对蛇葡萄素进行了大量的研究,证实了蛇葡萄素除有下调TNF-α、IL-6和IL-1p促炎性细胞因子调节免疫、抗炎、抑菌、抗病毒、护肝的生物活性外,在降糖、降脂、抗肿瘤、抗自由基等多方面也有显著的效果,而且毒性低微。本文假设蛇葡萄素对炎症性肠病也有治疗作用,我们初步以蛇葡萄素进行的预试验表明：蛇葡萄素明显改善实验性结肠炎大鼠的症状和结肠病理改变。本研究以三硝基苯磺酸(2,4,6-trinitrobenzene sulfonic acid, TNBS)／乙醇保留灌肠诱导大鼠实验性结肠炎,观察蛇葡萄素灌胃给药对大鼠实验性结肠炎的干预作用,并对其分子机制进行初步探讨。
目的：观察蛇葡萄素灌胃给药对TNBS/乙醇诱导的大鼠实验性结肠炎的干预作用及机制探讨。
方法：乙醚轻度麻醉下,给予雄性Sprague-Dawley (SD)大鼠0.25ml TNBS的50%乙醇溶液(含TNBS30mg)灌肠,诱导大鼠实验性结肠炎的发生。60只大鼠随机分为6组,每组10只,分别为正常对照组(A组)、模型对照组(B组)、柳氮磺吡啶(SASP)对照组(C组)、蛇葡萄素(Ampelopsin)低剂量组(D组)、蛇葡萄素中剂量组(E组)、蛇葡萄素高剂量组(F组)。TNBS/乙醇灌肠造模24小时后,蛇葡萄素低、中、高剂量组大鼠分别给予125、250、500mg/kg/day的蛇葡萄素进行灌胃给药：柳氮磺吡啶对照组大鼠给予柳氮磺吡啶100mg/kg/day灌胃给药；正常对照组和模型对照组大鼠以生理盐水(2.0ml/只)灌胃,每天3次,持续给药7天。试验期间每天观察大鼠的饮食、饮水、体重、大便、毛色、活动、精神状态等情况,进行疾病活动指数(disease activity index,DAI)评分；灌胃给药7天后处死全部大鼠,打开腹腔,分离结肠,对大鼠结肠组织炎症作大体评分(colon macroscopic damage index, CMDI);取距肛门端8cm处结肠用福尔马林固定,制作病理切片,对大鼠结肠炎症进行病理组织学评分(histopathological score,HPS)；以免疫组化S-P法检测大鼠结肠组织NF-κB p65蛋白的表达；另取大鼠结肠组织以分光光度法进行髓过氧化物酶(myeloperoxidase, MPO)活性测定;以酶联免疫法(ELISA)检测大鼠血清和结肠组织TNF-α、IL-1β和IL-10的蛋白含量,逆转录-聚合酶链式反应(RT-PCR)检测大鼠结肠组织TNF-α、IL-1β和IL-10mRNA的表达。
结果：①给予含30mg TNBS原液的乙醇溶液灌肠后第2天,模型对照组大鼠开始出现大便次数增多、血便、体重逐渐下降,造模后第8天,模型对照组大鼠表现为拱背、竖毛、毛发失去光泽甚至明显脱毛、蜷卧懒动、饮食明显减少、体重明显减轻,可见明显脓血便,大鼠DAI明显高于正常对照组(3.17±0.31vs0.10±0.16,P<0.01)。蛇葡萄素中、高剂量组、柳氮磺吡啶对照组大鼠也出现腹泻、血便和体重减轻,但程度较轻,灌胃给药后期逐渐改善。造模后第8天蛇葡萄素中、高剂量组、柳氮磺吡啶对照组DAI明显低于模型对照组(1.07±0.26、1.00±0.22、1.13±0.17vs3.17±0.31,P<0.01,respectively),蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间DAI比较无显著差异(1.07±0.26,1.00±0.22vs1.13±0.17, P>0.05, respectively)。蛇葡萄素低剂量组DAI明显低于模型对照组(2.04±0.39vs3.17±0.31,P<0.01),但明显高于柳氮磺吡啶对照组(1.74±0.32vs1.13±0.17, P<0.01).②TNBS溶液灌肠后8天处死所有大鼠,打开大鼠腹腔,分离结肠,沿肠系膜纵轴剖开,在冰上观察结肠充血水肿、炎症、溃疡等变化。模型对照组大鼠结肠可见与周围组织如肝脏、脾脏、输尿管及小肠等周围组织脏器严重粘连,局部肠管明显扩张,甚至可见巨结肠,大鼠结肠肠壁增厚、颜色灰暗,肠黏膜明显充血、水肿,糜烂、坏死明显,可见多发溃疡及较大溃疡病灶,溃疡呈线状或灶状,溃疡面可见灰黑色假膜样物覆盖,周围可见黏膜增厚,肠腔狭窄,CMDI评分明显高于正常对照组(4.38±0.74vs0.20±0.42,P<0.01)。柳氮磺吡啶对照组、蛇葡萄素中、高剂量组CMDI明显低于模型对照组(2.10±0.57、2.20±0.42、2.00±0.47vs4.38±0.74,P<0.01, respectively),蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(2.20±0.42,2.00±0.47vs2.10±0.57, P>0.05, respectively).蛇葡萄素低剂量组CMDI低于模型对照组(2.89±0.60vs4.38±0.74,P<0.01),但明显高于柳氮磺吡啶对照组(2.89±0.60vs2.10±0.57,P<0.01)。③显微镜下,正常对照组大鼠结肠组织未见或仅见极少中性粒细胞浸润,结肠黏膜完整,固有层内肠腺丰富,排列紧密。模型对照组大鼠结肠组织病理学表现为黏膜上皮大面积坏死脱落,腺体破坏且结构紊乱,杯状细胞减少,隐窝炎及隐窝脓肿形成,黏膜下层不同程度充血、水肿、毛细血管扩张,大量中性粒细胞、淋巴细胞浸润肠壁全层,肠壁水肿明显,黏膜、黏膜下层、甚至肌层可见大面积溃疡灶,溃疡底部伴有炎性肉芽组织增生,HPS明显高于正常对照组(4.13±0.64vs0.40±0.52,P<0.01)。与模型对照组相比,柳氮磺吡啶对照组、蛇葡萄素中、高剂量组大鼠结肠HPS明显低于模型对照组(1.90±0.57、2.00±0.47、1.80±0.63vs4.13±0.64,P<0.01, respectively),陀葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(2.00Q0.47、1.80±0.63vs1.90±0.57,P>0.05,respectively)。蛇葡萄素低剂量组大鼠结肠HPS低于模型对照组(3.00±0.50vs4.13±0.64,P<0.01),但明显高于柳氮磺吡啶对照组(3.00±0.50vs1.90±0.57,P<0.01)。④正常对照组大鼠结肠组织仅见微弱的NF-κB p65表达,模型对照组大鼠NF-KB p65表达明显增高,阳性着色细胞主要为结肠黏膜上皮细胞、隐窝上皮细胞、结肠黏膜下层和固有层中性粒细胞和单核细胞,阳性细胞胞质和胞核都含有棕褐色颗粒,但以胞核表达为主,其阳性细胞吸光值明显高于正常对照组(0.64±0.06vs0.21±0.03,P<0.01)。柳氮磺吡啶对照组、蛇葡萄素中、高剂量组NF-KBp65表达明显低于模型对照组(0.41±0.06、0.39±0.06、0.38±0.06vs0.64±0.06,P<0.01,respectively);蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(0.39±0.06、0.38±0.06vs0.41±0.06,P>0.05,respectively)。蛇葡萄素低剂量组大鼠结肠组织NF-KB p65表达明显低于模型对照组(0.55±0.04vs0.64±0.06,P<0.01)；但明显高于柳氮磺吡啶对照组(0.55±0.04vs0.41±0.06,P<0.01)。⑤与正常对照组比较,模型对照组大鼠结肠的髓过氧化物酶(MPO)活性增加约6倍,柳氮磺吡啶对照组、蛇葡萄素中、高剂量组大鼠结肠组织MPO活性较模型对照组降低50%,与模型对照组比较有显著性差异(17.90±2.51、18.50±2.80、16.70±2.45vs36.00±4.47,P<0.01,respectively);蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(18.50±2.80、16.70±2.45vs17.90±2.51,P>0.05respectively)。蛇葡萄素低剂量组大鼠结肠组织MPO活性低于模型对照组(24.67±3.12vs36.00±4.47,P<0.01)；但明显高于柳氮磺吡啶对照组(24.67±3.12vs17.90±2.51,P<0.01)。⑥给予TNBS溶液灌肠后第八天,模型对照组血清TNF-α、IL-1p的蛋白含量明显高于正常对照组(80.29±5.95vs25.15±2.88、61.95±5.53vs23.85±2.95,P<0.01,1rspectively), IL-10的蛋白含量明显低于正常对照组(28.31±3.21vs39.60±4.42,P<0.01)。柳氮磺吡啶对照组、蛇葡萄素中、高剂量组TNF-α、IL-1p的蛋白含量明显低于模型对照组(44.09±4.71、46.15±4.78、43.18±4.16vs80.29±5.95,38.14±4.04、40.55±4.09、37.64±3.65vs61.95±5.53,P<0.01,respectively),IL-10的蛋白含量明显高于模型对照组(67.10±4.23、68.03±5.00、65.38±4.89vs28.31±3.21, P<0.01, respectively);蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(P>0.05)。蛇葡萄素低剂量组血清TNF-α、IL-1β的蛋白含量低于模型对照组(64.56±5.88vs80.29±5.95、49.00±4.66vs61.95±5.53,P<0.01,respectively),IL-10的蛋白含量高于模型对照组(33.61±3.77vs28.31±3.21,P<0.01)：蛇葡萄素低剂量组血清TNF-α、IL-1β的蛋白含量明显高于柳氮磺吡啶对照组(64.56±5.88vs44.09±4.71,49.00±4.66vs38.14±4.04,P<0.01,respectively),IL-10的蛋白含量明显低于柳氮磺吡啶对照组(33.61±3.77vs67.10±4.23,P<0.01)。⑦模型对照组大鼠结肠TNF-α、IL-1βmRNA(1.35±0.27vs0.34±0.11、1.43±0.21vs O.38±0.10,P0.05)。蛇葡萄素低剂量组TNF-α、IL-1β mRNA(0.91±0.22vs1.35±0.27.1.07±0.20vs1.43±0.21,P<0.01, respectively)的表达和TNF-α、IL-1β蛋白的含量低于模型对照组(73.11±7.19vs88.16±8.19、69.36±9.75vs85.88±11.06,P<0.01,respectively),IL-10mRNA(0.50±0.15vs0.27±0.13,P<0.01)的表达和IL-10蛋白的含量高于模型对照组(36.08±3.83vs28.10±3.26,P<0.01)；蛇葡萄素低剂量组TNF-α、IL-1β mRNA(0.91±0.22vs O.65±0.15.1.07±0.20vs0.68±0.17,P<0.01,respectively)的表达和TNF-α、IL-1β蛋白的含量明显高于柳氮磺吡啶对照组(73.11±7.19vs53.80±5.65、69.36±9.75vs52.35±7.34,P<0.01,respectively),IL-10mRNA(0.50±0.15vs1.42±0.19,P<0.01)的表达和IL-10蛋白的含量明显低于柳氮磺吡啶对照组(36.08±3.83vs83.21±7.43,P<0.01)。
结论：早期以蛇葡萄素灌胃给药干预,能减轻TNBS诱导大鼠实验性结肠炎,其作用机制可能与蛇葡萄素下调结肠NF-κB p65表达、Th1促炎性细胞因子TNF-α、IL-1β和上调Th2抑炎性细胞因子IL-10有关。
第二部分白芍总苷减轻大鼠实验性结肠炎的作用及机制研究
白芍是中国传统中药植物,为毛莨科植物芍药(paeonia lactiflora)的干燥根,是中医学用于治疗风湿类疾病、自身免疫性疾病、肝炎及肝硬化等疾病的重要组方之一,引起了医药学界的广泛重视与开发研究。国内外学者通过多年的研究,已成功提取出白芍的药效成分单体,主要为一组糖苷类物质,包括芍药甙(paeoniflorin).芍药花甙(paeonin)、芍药内酯甙(albiflorin)、羟基芍药甙(hydroxy-paeoniflorin)、苯甲酰芍药甙(benzoylpaeoniflorin),统称为白芍总苷(total glucosides of paeony, TGP),其中芍药苷占总苷量的90%以上,是白芍的主要有效成分。对白芍总苷的药理及临床进行研究发现,白芍总苷可有效抑制炎症局部前列腺素E的合成,抑制淋巴细胞增殖及巨噬细胞产生促炎性细胞因子,如TNF-α、IL-1β、IL-8及IL-6等,纠正CD4+/CD8+T淋巴细胞比例失衡,从而多途径调节自身免疫紊乱,以及具有抗炎、止痛、保肝的作用。白芍总苷对类风湿性关节炎(RA)有确切疗效,已在临床上广泛应用,并显示出对系统性红斑狼疮、强直性脊柱炎等多种自身免疫性疾病治疗的良好应用前景。
本文假设白芍总苷对炎症性肠病也有治疗作用,本研究以TNBS/乙醇灌肠诱导复制大鼠实验性结肠炎模型,观察白芍总苷灌胃给药对大鼠实验性结肠炎的干预作用,并对其分子机制进行初步探讨。
目的：观察白芍总苷灌胃给药对TNBS/乙醇诱导的大鼠实验性结肠炎的干预作用及机制探讨。
方法：乙醚轻度麻醉下,给予雄性Sprague-Dawley (SD)大鼠0.25ml TNBS的50%乙醇溶液(含TNBS30mg)灌肠,诱导大鼠实验性结肠炎的发生。60只大鼠随机分为6组,每组10只,分别为正常对照组(A组)、模型对照组(B组)、柳氮磺吡啶(SASP)对照组(C组)、白芍总苷(TGP)低剂量组(D组)、白芍总苷中剂量组(E组)、白芍总苷高剂量组(F组)。TNBS/乙醇灌肠造模24小时后,白芍总苷低、中、高剂量组大鼠分别给予25、50、100mg/kg/day的白芍总苷进行灌胃给药：柳氮磺吡啶对照组大鼠给予柳氮磺吡啶100mg/kg/day灌胃给药；正常对照组和模型对照组以生理盐水(2.0ml/只)灌胃,每天3次,持续给药7天。试验期间每天观察大鼠的饮食、饮水、体重、大便、毛色、活动、精神状态等情况,进行疾病活动指数(disease activity index, DAI)评分；灌胃给药7天后处死全部大鼠,打开腹腔,分离结肠,对大鼠结肠组织炎症作大体评分(colon macroscopic damage index, CMDI);取距肛门端8cm处结肠用福尔马林固定,制作病理切片,对大鼠结肠炎症进行病理组织学评分(histopathological score, HPS);另取大鼠结肠组织以分光光度法进行髓过氧化物酶(myeloperoxidase, MPO)活性测定,同时以酶联免疫法(ELISA)检测大鼠血清TNF-α、IL-1β和IL-10的蛋白含量,逆转录-聚合酶链式反应(RT-PCR)检测大鼠结肠组织TNF-α、IL-1β和IL-10mRNA的表达,Western blot检测大鼠结肠组织TNF-α、IL-1β和IL-10蛋白的表达。
结果：①给予含30mg TNBS原液的乙醇溶液灌肠后第2天,模型对照组大鼠开始出现大便次数增多、血便,体重逐渐下降,造模后第8天,可见明显脓血便、饮食明显减少、体重减轻、毛发失去光泽甚至明显脱毛、喜扎堆,反应迟缓、懒动,DAI明显高于正常对照组(2.74±0.49vs0.07±0.14,P<0.01)。柳氮磺吡啶对照组、白芍总苷中、高剂量组大鼠给予TNBS溶液灌肠后出现腹泻、血便和体重减轻,但程度较轻,灌胃给药后期逐渐改善。造模后第8天白芍总苷低剂量组DAI低于模型对照组(2.22±0.29vs2.74±0.49,P<0.05),柳氮磺吡啶对照组、白芍总苷中、高剂量组DAI明显低于模型对照组(1.10±0.22、1.00±0.16、0.97±0.11vs2.74±0.49,P<0.01,respectively).白芍总苷低剂量组DAI明显高于柳氮磺吡啶对照组(2.22±0.29vs1.10±0.22,P<0.01)；白芍总苷中、高剂量组、柳氮磺吡啶对照组组间DAI比较无显著差异(1.00±0.16、0.97±0.1l vs1.10±0.22,P>0.05,respectively).②TNBS溶液灌肠后8天处死所有大鼠,打开大鼠腹腔,分离结肠,沿肠系膜纵轴剖开,在冰上观察结肠充血水肿、炎症、溃疡等变化。模型对照组大鼠结肠与周围组织如输尿管、小肠及脾脏等脏器严重粘连,局部结肠肠管扩张,肠壁增厚、颜色灰暗,肠黏膜明显充血、水肿,可见较大溃疡病灶,病灶处有灰黑色假膜样物覆盖,CMDI明显高于正常对照组(3.67±0.71vs0.10±0.32,P<0.01)。TNBS溶液灌肠后给予柳氮磺吡啶、白芍总苷低、中、高剂量干预后,大鼠结肠病变有不同程度减轻,白芍总苷低剂量组CMDI低于模型对照组(2.89±0.60vs3.67±0.71,P<0.05);柳氮磺吡啶对照组、白芍总苷中、高剂量组CMD)I明显低于模型对照组(1.60±0.52.1.80±0.42.1.70±0.67vs3.67±0.71,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量组CMDI明显高于柳氮磺吡啶对照组(2.89±0.60vs1.60±0.52,P<0.01),白芍总苷中、高剂组、柳氮磺吡啶对照组组间比较无显著差异(1.80±0.42,1.70±0.67vs1.60±0.52,P>0.05,respectively)。③显微镜下,正常对照组大鼠结肠组织未见或仅见极少中性粒细胞浸润,结肠黏膜完整,固有层内肠腺丰富,排列紧密。模型对照组大鼠结肠组织病理学表现为固有层充血水肿,大量中性粒细胞、淋巴细胞浸润,广泛黏膜上皮坏死脱落,隐窝炎及脓肿形成,腺体破坏且结构紊乱,溃疡形成,肉芽肿、纤维组织增生,结肠HPS明显高于正常对照组(3.33±0.71vs0.30±0.48,P<0.01)。与模型对照组相比,白芍总苷低剂量组大鼠结肠HPS低于模型对照组(2.67±0.50vs3.33±0.71,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组大鼠结肠HPS明显低于模型对照组(1.80±0.63、1.90±0.57、1.60±0.52vs3.33±0.71,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量结肠HPS明显高于柳氮磺吡啶对照组(2.67±0.50vs1.80±0.63,P<0.01),白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(1.90±0.57、1.60±0.52vs1.80±0.63,P>0.05,respectively).④与正常对照组比较,模型对照组大鼠结肠的MPO活性增加约5倍,MPO活性明显高于正常对照组(31.11±3.55vs6.10±0.73,P<0.01)。白芍总苷低剂量组大鼠结肠MPO活性低于模型对照组(27.56±2.70vs31.11±3.55,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组大鼠结肠MPO活性较模型对照组降低50%,明显低于模型对照组(15.80±1.69、16.10±1.37、14.90±1.66vs31.11±3.55,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量组结肠MPO活性明显高于柳氮磺吡啶对照组(27.56±2.70vs15.80±1.69,P<0.01),白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(16.10±1.37、14.90±1.66vs15.80±1.69,P>0.05,respectively).⑤给予TNBS溶液灌肠八天后,模型对照组血清TNF-α、IL-1β的蛋白含量明显高于正常对照组(84.08±14.32vs21.98±3.66、66.13±9.99vs27.71±2.45,P<0.01,respectively),IL-10的蛋白含量明显低于正常对照组(26.87±4.00vs40.83±3.73,P<0.01)。白芍总苷低剂量组血清TNF-α、IL-1β的蛋白含量低于模型对照组(68.71±10.65vs84.08±14.32、54.51±7.82VS66.13±9.99,P<0.05,respectively),IL-10的蛋白含量高于模型对照组(31.61±3.22vs26.87±4.00,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组血清TNF-α、IL-1β的蛋白含量明显低于模型对照组(47.69±7.45、49.26±6.34、45.99±7.15vs84.08±14.32,40.39±6.45、40.86±5.14、38.99±6.54vs66.13±9.99,P<0.01,respectively),IL-10的蛋白含量明显高于模型对照组(73.78±8.15、71.39±8.35、75.75±8.28vs26.87±4.00,P<0.01,respectively)。与柳氮磺吡啶对照组比较,白芍总苷低剂量组血清TNF-α、IL-1β的蛋白含量明显高于柳氮磺吡啶对照组(68.71±10.65vs47.69±7.45,54.51±7.82vs40.39±6.45,P<0.01,respectively),IL-10的蛋白含量明显低于柳氮磺吡啶对照组(31.61±3.22vs73.78±8.15,P<0.01)；白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(49.26±6.34、45.99±7.15vs47.69±7.45,40.86±5.14、38.99±6.54vs40.39±6.45,71.39±8.35、75.75±8.28vs73.78±8.15,P>0.05,respectively).⑥模型对照组大鼠结肠TNF-α、IL-1β mRNA(1.35±0.44vs0.36±0.12.1.57±0.40vs0.42±0.21,P<0.01,respectively)和TNF-α、IL-1β蛋白的表达明显高于正常对照组(1.18±0.36vs0.23±0.08、1.27±0.31vs0.32±0.07,P<0.01,respectively), IL.10mRNA(0.25±0.12vs0.9l±0.11,P<0.01)和IL-10蛋白的表达明显低于正常对照组((0.37±0.14vs1.09±0.08,P<0.01))。白芍总苷低剂量组TNF-α、IL-1β mRNA(0.97±0.21vs1.35±0.44、1.19±0.28vs1.57±0.40,P<0.05,Fespectively)和TNF-α、IL-1β蛋白的表达低于模型对照组((0.81±0.19vs1.18±0.36、0.97±0.24vs1.27±0.31,P<0.05,respectively),IL-10mRNA(0.41±0.13vs0.25±0.12,P<0.05)和IL-10蛋白的表达高于模型对照组((0.53±0.12vs0.37±0.14,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组TNF-α、IL-1β mRNA(0.60±0.19.0.61±0.18.0.58±0.20vs1.35±0.44,0.74±O.23.0.72±0.24.O.70±0.25vs1.57±0.40,P<0.01,respectively)和TNF-α、IL-1β蛋白的表达明显低于模型对照组(0.51±0.14、0.50±0.13、0.48±0.15vs1.18±0.36；0.63±0.15.0.62±O.16.0.60±0.15vs1.27±0.31,P<0.01,respectively),IL-10mRNA(1.23±0.23.1.19±0.21.1.27±0.24vs0.25±0.12,P<0.01,respectively)和IL-10蛋白的表达明显高于模型对照组(1.37±0.16、1.35±0.17、1.39±0.18vs0.37±0.14,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量组TNF-α、IL-1β mRNA(0.97±0.21vs0.60±0.19.1.19±0.28vs0.74±0.23,P<0.01,repectively)和TNF-α、IL-1β蛋白的表达明显高于柳氮磺吡啶对照组(0.81±0.19vs0.51±0.14、0.97±0.24vs0.63±0.15, P<0.01, respectively), IL-10mRNA(0.41±0.13vs1.23±0.23, P<0.01)和IL-10蛋白的表达明显低于柳氮磺吡啶对照组(0.53±0.12vs1.37±0.16,P<0.01)；白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(P>0.05)。
结论：早期以白芍总苷灌胃给药干预,能减轻TNBS诱导大鼠实验性结肠炎,其作用机制可能与白芍总苷下调Thl促炎性细胞因子TNF-α、IL-1β和上调Th2抑炎性细胞因子IL-10有关。
Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is an idiopathic, non-specific inflammatory disorder of the intestinal tract with unknown causes. It is currently believed that genetic susceptibility, dysfunction of immune regulation in intestinal mucosa and intestinal bacteria are involved in the pathogenesis of IBD. Immune regulation dysfunction might be the direct cause for onset of IBD. It is now well accepted that abnormal immune response mediated by proinflammatory cytokines such as TNF-α,IL-1β, IL-6and IL-8, and antiinflammatory cytokines IL-10plays an important role in balance of Thl and Th2as well as inflammatory severity and activity in IBD. Currently, there is a lack of an effective therapy to cure the diseases since causes and mechanisms of IBD are not totally understood. Aminosalicylates, corticosteroids, immunomodulating and immunosuppressive drugs are used for treatment of IBD by controlling active inflammation and regulating abnormal immune response. However, such treatments have multiple adverse effects, particularly for long-term administration, and relapse is high upon drug discontinuance. Biological agents such as anti-TNF-a monoclonal antibody can induce the alleviation of IBD, but at a high cost and put the patients at risk to develop treatment-related cancers, and surgical resection of the colon and ileostomy is the ultimate alternative in many cases. Therefore, there is an urgent for the development of new and specific therapies for IBD with few side effects.
Part I:Ampelopsin attenuates2,4,6-trinitrobenzene sulfonic acid/ethanol-induced colitis in rats
Ampelopsin is a flavonoids extracted from Rattan tea which is a traditional Chinese herbal remedy prepared from the stems and leaves of the plant ampelopsis grossedentata. Ampelopsin was reported as a major bioactive component in ampelopsis grossedentata and first isolated from ampelopsis meliaefolia by Kotake and Kubota in1940.Recent studies have shown that ampelopsin has a broad range of biological functions including inhibition of apoptosis, hypoglycemic, anti-hypertension, anti-bacterial, anti-inflammatory, antioxidant, anti-tumor, hepatoprotective, and neuroprotective effects. These findings support the hypothesis that ampelopsin is also effective against IBD. Therefore, ampelopsin may be a bright potential candidate Chinese drug for treatment of IBD.
Objective The present study is to investigate therapeutic effects of ampelopsin on2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of ampelopsin for treatment of inflammatory bowel disease (IBD).
Methods Sixty Sprague-Dawley (SD) rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100mg/kg/day), and low, medium and high-dose ampelopsin groups (125,250and500mg/kg/day, respectively).24hours following colonic instillation of TNBS, ampelopsin and SASP were given by gastric gavage three times a day for7days.Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS) and myeloperoxidase (MPO) activity were evaluated. Levels of serum and colon TNF-α,IL-1β and IL-10were measured by enzyme-linked immunosorbent assay (ELISA), expression of TNF-α,IL-1β and IL-10mRNA in colonic tissues was detected by reverse transcription-polymerase chain reaction (RT-PCR), and expression of NF-κB p65protein in colonic tissues was detected by immunohistochemical methods, respectively.
Results①Normal control rats treated with ethanol enema gained body weight and defecated normally. In the model control group, pasty to liquid gross bloody stool and weight loss were observed in all rats. When ampelopsin (250or500mg/kg/day) or SASP (100mg//kg/day) was administered on day7, bloody stools were not evident, stools were better formed, and weight loss was lessened. The colons from rats in the model control group were seriously adherent to small intestines and spleen, and showed marked hyperemia, inflammation, necrosis, ulcer, whereas the colons from rats in the normal control group showed no or a slight inflammation. Treatment with ampelopsin (250or500mg/kg/day) or SASP markedly decreased both hyperemia and inflammation in the colons. Administration of ampelopsin (250or500mg/kg/day) significantly improved the DAI and CMDI8days after TNBS instillation. And there was no significant difference compared with administration of SASP (P>0.05).②Ulcerations, massive transmural infiltration of inflammatory cells, thickening of the colon wall, goblet cell depletion, and extensive fibrosis found throughout colons in the model control group. Administration of ampelopsin (250or500mg/kg/day) improved these signs and the HPS significantly. And there was no significant difference compared with administration of SASP (P>0.05).③Rectal instillation of TNBS led to the marked enhancement of NF-κB p65protein expression in colonic tissues. Compared with the normal group, administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of NF-κB p65protein expression in colonic tissues. The difference was not significant when compared with administration of SASP (P>0.05).④MPO activity was very low in the normal control group, and increased significantly nearly6-fold in rats after TNBS enema in the model control group. In contrast, this increase was significantly blunted by about50%by administration of ampelopsin (250or500mg/kg/day). And there was no significant difference compared with administration of SASP (P>0.05).⑤ectal instillation of TNBS led to the marked enhancement of levels of serum TNF-α and IL-1β, and the marked decrease of levels of serum IL-10. Administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of levels of serum TNF-α and IL-1β and at the same time increased the decrease of levels of serum IL-10. And there was no significant difference when compared with administration of SASP(P>0.05).⑥Rectal instillation of TNBS led to the marked enhancement of mRNA expression of TNF-α and IL-1β, and the marked decrease of that of IL-10in colonic tissues. Administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of mRNA expression of TNF-α and IL-1β and at the same time significantly increased the decrease of that of IL-10. And there was no significant difference when compared with administration of SASP (P>0.05).⑦Protein expression of TNF-α and IL-1β was markedly elevated and that of IL-10was markedly decreased in the colons after rectal TNBS instillation, which was consistent with results of the mRNA expression.250or500mg/kg/day ampelopsin treatment significantly abrogated the elevation in protein expression of TNF-α,IL-1β and at the same time significantly increased the decrease of protein expression of IL-1O.The difference was not significant when compared with administration of SASP (P>0.05). On the other hand, administration of ampelopsin at the dose of125mg/kg/day showed modest improvement on colonic inflammation and regulatory effects on expression of TNF-α,IL-1β and IL-10, but the values were lower than administration of SASP (P<0.01).
Conclusion Ampelopsin attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the down-regulation the enhancement of NF-κB p65protein expression in colonic tissues and adjustment of Thl/Th2cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10.
Part II:Total glucosides of peony attenuates2,4,6-trinitrobenzene sulfonic acid/ethanol-induced colitis in rats
Total glucosides of peony (TGP) is a group of glucosides extracted from peony, including paeoniflorin, hydroxy-paeoniflorin, paeonin, albiflorin and benzoylpaeoniflorin. Paeoniflorin is a monoterpene glucoside, the major active ingredient of TGP, accounting for over90%of total glucosides. Now TGP has been a common prescription drug used for the treatment of autoimmune diseases including rheumatoid arthritis, ankylosing spondylitis and systemic lupus erythematosus and shown significant efficacy in the clinic. Recent studies have shown that TGP has anti-inflammatory and immunoregulatory functions, by inhibiting Th1cytokines and enhancing Th2cytokines. These findings support the hypothesis that TGP is also effective against IBD. Therefore, TGP may be a bright potential candidate Chinese drug for treatment of IBD.
Objective The present study is to investigate therapeutic effects of total glucosides of peony (TGP) on2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of TGP for treatment of inflammatory bowel disease (IBD).
Methods Sixty Sprague-Dawley (SD) rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100mg/kg/day), and low, medium and high-dose TGP groups (25,50and100mg/kg/day, respectively).24hours following colonic instillation of TNBS, TGP and SASP were given by gastric gavage three times a day for7days.Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS) and myeloperoxidase (MPO) activity were evaluated. Levels of serum TNF-α,IL-1β and IL-10were measured by ELISA, and expression of TNF-α,IL-1β and IL-10mRNA and protein in colonic tissues was detected by RT-PCR and Western blot, respectively.
Results①Normal control rats treated with ethanol enema gained body weight and defecated normally. In the model control group, pasty to liquid gross bloody stool and weight loss were observed in all rats. When TGP (50or100mg/kg/day) or SASP was administered on day7, bloody stools were not evident, stools were better formed, and weight loss was lessened. The colons from rats in the model control group were seriously adherent to small intestines and spleen, and showed marked hyperemia, inflammation, necrosis, ulcer, whereas the colons from rats in the normal control group showed no or a slight inflammation. Treatment with TGP (50or100mg/kg/day) or SASP markedly decreased both hyperemia and inflammation in the colons. Administration of TGP (50or100mg/kg/day) significantly improved the DAI and CMDI8days after TNBS instillation. And there was no significant difference compared with administration of SASP (P>0.05).②Ulcerations, massive transmural infiltration of inflammatory cells, thickening of the colon wall, goblet cell depletion, and extensive fibrosis found throughout colons in the model control group. Administration of TGP (50or100mg/kg/day) improved these signs and the HPS significantly.③MPO activity was very low in the normal control group, and increased significantly nearly5-fold in rats after TNBS enema in the model control group. In contrast, this increase was significantly blunted by about50%by administration of TGP (50or100mg/kg/day).④Rectal instillation of TNBS led to the marked enhancement of levels of serum TNF-α and IL-1β and the marked decrease of levels of serum IL-10. Administration of TGP (50or100mg/kg/day) significantly reduced the enhancement of levels of serum TNF-α and IL-1β and at the same time increased the decrease of levels of serum IL-10. And there was no significant difference when compared with administration of SASP (P>0.05).⑤Rectal instillation of TNBS led to the marked enhancement of mRNA expression of TNF-α and IL-1β, and the marked decrease of that of IL-10in colonic tissues. Administration of TGP (50or100mg/kg/day) significantly reduced the enhancement of mRNA expression of TNF-α and IL-1β and at the same time significantly increased the decrease of that of IL-10.⑥Protein expression of TNF-α and IL-1β was markedly elevated and that of IL-10was markedly decreased in the colons after rectal TNBS instillation, which was consistent with results of the mRNA expression.50or100mg/kg/day TGP treatment significantly abrogated the elevation in protein expression of TNF-α,IL-1β and at the same time significantly increased the decrease of protein expression of IL-10. The difference was not significant when compared with administration of SASP (P>0.05). On the other hand, administration of TGP at the dose of25mg/kg/day showed modest improvement on colonic inflammation and regulatory effects on expression of TNF-α,IL-1β and IL-10, but the values were lower than administration of SASP (P<0.01).
Conclusion TGP attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the adjustment of Thl/Th2cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10.
第一部分蛇葡萄素减轻大鼠实验性结肠炎的作用及机制研究
蛇葡萄素(Ampelopsin)是一种多酚羟基双氢黄酮醇,化学名称：(2R,3R)-3,5,7-三羟基-2-(3,4,5-三羟基苯基)苯并二氢吡喃-4-酮,分子式：C15H12O8,分子量：320.25,又名二氢杨梅素。近年来国内外学者对蛇葡萄素进行了大量的研究,证实了蛇葡萄素除有下调TNF-α、IL-6和IL-1p促炎性细胞因子调节免疫、抗炎、抑菌、抗病毒、护肝的生物活性外,在降糖、降脂、抗肿瘤、抗自由基等多方面也有显著的效果,而且毒性低微。本文假设蛇葡萄素对炎症性肠病也有治疗作用,我们初步以蛇葡萄素进行的预试验表明：蛇葡萄素明显改善实验性结肠炎大鼠的症状和结肠病理改变。本研究以三硝基苯磺酸(2,4,6-trinitrobenzene sulfonic acid, TNBS)／乙醇保留灌肠诱导大鼠实验性结肠炎,观察蛇葡萄素灌胃给药对大鼠实验性结肠炎的干预作用,并对其分子机制进行初步探讨。
目的：观察蛇葡萄素灌胃给药对TNBS/乙醇诱导的大鼠实验性结肠炎的干预作用及机制探讨。
方法：乙醚轻度麻醉下,给予雄性Sprague-Dawley (SD)大鼠0.25ml TNBS的50%乙醇溶液(含TNBS30mg)灌肠,诱导大鼠实验性结肠炎的发生。60只大鼠随机分为6组,每组10只,分别为正常对照组(A组)、模型对照组(B组)、柳氮磺吡啶(SASP)对照组(C组)、蛇葡萄素(Ampelopsin)低剂量组(D组)、蛇葡萄素中剂量组(E组)、蛇葡萄素高剂量组(F组)。TNBS/乙醇灌肠造模24小时后,蛇葡萄素低、中、高剂量组大鼠分别给予125、250、500mg/kg/day的蛇葡萄素进行灌胃给药：柳氮磺吡啶对照组大鼠给予柳氮磺吡啶100mg/kg/day灌胃给药；正常对照组和模型对照组大鼠以生理盐水(2.0ml/只)灌胃,每天3次,持续给药7天。试验期间每天观察大鼠的饮食、饮水、体重、大便、毛色、活动、精神状态等情况,进行疾病活动指数(disease activity index,DAI)评分；灌胃给药7天后处死全部大鼠,打开腹腔,分离结肠,对大鼠结肠组织炎症作大体评分(colon macroscopic damage index, CMDI);取距肛门端8cm处结肠用福尔马林固定,制作病理切片,对大鼠结肠炎症进行病理组织学评分(histopathological score,HPS)；以免疫组化S-P法检测大鼠结肠组织NF-κB p65蛋白的表达；另取大鼠结肠组织以分光光度法进行髓过氧化物酶(myeloperoxidase, MPO)活性测定;以酶联免疫法(ELISA)检测大鼠血清和结肠组织TNF-α、IL-1β和IL-10的蛋白含量,逆转录-聚合酶链式反应(RT-PCR)检测大鼠结肠组织TNF-α、IL-1β和IL-10mRNA的表达。
结果：①给予含30mg TNBS原液的乙醇溶液灌肠后第2天,模型对照组大鼠开始出现大便次数增多、血便、体重逐渐下降,造模后第8天,模型对照组大鼠表现为拱背、竖毛、毛发失去光泽甚至明显脱毛、蜷卧懒动、饮食明显减少、体重明显减轻,可见明显脓血便,大鼠DAI明显高于正常对照组(3.17±0.31vs0.10±0.16,P<0.01)。蛇葡萄素中、高剂量组、柳氮磺吡啶对照组大鼠也出现腹泻、血便和体重减轻,但程度较轻,灌胃给药后期逐渐改善。造模后第8天蛇葡萄素中、高剂量组、柳氮磺吡啶对照组DAI明显低于模型对照组(1.07±0.26、1.00±0.22、1.13±0.17vs3.17±0.31,P<0.01,respectively),蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间DAI比较无显著差异(1.07±0.26,1.00±0.22vs1.13±0.17, P>0.05, respectively)。蛇葡萄素低剂量组DAI明显低于模型对照组(2.04±0.39vs3.17±0.31,P<0.01),但明显高于柳氮磺吡啶对照组(1.74±0.32vs1.13±0.17, P<0.01).②TNBS溶液灌肠后8天处死所有大鼠,打开大鼠腹腔,分离结肠,沿肠系膜纵轴剖开,在冰上观察结肠充血水肿、炎症、溃疡等变化。模型对照组大鼠结肠可见与周围组织如肝脏、脾脏、输尿管及小肠等周围组织脏器严重粘连,局部肠管明显扩张,甚至可见巨结肠,大鼠结肠肠壁增厚、颜色灰暗,肠黏膜明显充血、水肿,糜烂、坏死明显,可见多发溃疡及较大溃疡病灶,溃疡呈线状或灶状,溃疡面可见灰黑色假膜样物覆盖,周围可见黏膜增厚,肠腔狭窄,CMDI评分明显高于正常对照组(4.38±0.74vs0.20±0.42,P<0.01)。柳氮磺吡啶对照组、蛇葡萄素中、高剂量组CMDI明显低于模型对照组(2.10±0.57、2.20±0.42、2.00±0.47vs4.38±0.74,P<0.01, respectively),蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(2.20±0.42,2.00±0.47vs2.10±0.57, P>0.05, respectively).蛇葡萄素低剂量组CMDI低于模型对照组(2.89±0.60vs4.38±0.74,P<0.01),但明显高于柳氮磺吡啶对照组(2.89±0.60vs2.10±0.57,P<0.01)。③显微镜下,正常对照组大鼠结肠组织未见或仅见极少中性粒细胞浸润,结肠黏膜完整,固有层内肠腺丰富,排列紧密。模型对照组大鼠结肠组织病理学表现为黏膜上皮大面积坏死脱落,腺体破坏且结构紊乱,杯状细胞减少,隐窝炎及隐窝脓肿形成,黏膜下层不同程度充血、水肿、毛细血管扩张,大量中性粒细胞、淋巴细胞浸润肠壁全层,肠壁水肿明显,黏膜、黏膜下层、甚至肌层可见大面积溃疡灶,溃疡底部伴有炎性肉芽组织增生,HPS明显高于正常对照组(4.13±0.64vs0.40±0.52,P<0.01)。与模型对照组相比,柳氮磺吡啶对照组、蛇葡萄素中、高剂量组大鼠结肠HPS明显低于模型对照组(1.90±0.57、2.00±0.47、1.80±0.63vs4.13±0.64,P<0.01, respectively),陀葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(2.00Q0.47、1.80±0.63vs1.90±0.57,P>0.05,respectively)。蛇葡萄素低剂量组大鼠结肠HPS低于模型对照组(3.00±0.50vs4.13±0.64,P<0.01),但明显高于柳氮磺吡啶对照组(3.00±0.50vs1.90±0.57,P<0.01)。④正常对照组大鼠结肠组织仅见微弱的NF-κB p65表达,模型对照组大鼠NF-KB p65表达明显增高,阳性着色细胞主要为结肠黏膜上皮细胞、隐窝上皮细胞、结肠黏膜下层和固有层中性粒细胞和单核细胞,阳性细胞胞质和胞核都含有棕褐色颗粒,但以胞核表达为主,其阳性细胞吸光值明显高于正常对照组(0.64±0.06vs0.21±0.03,P<0.01)。柳氮磺吡啶对照组、蛇葡萄素中、高剂量组NF-KBp65表达明显低于模型对照组(0.41±0.06、0.39±0.06、0.38±0.06vs0.64±0.06,P<0.01,respectively);蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(0.39±0.06、0.38±0.06vs0.41±0.06,P>0.05,respectively)。蛇葡萄素低剂量组大鼠结肠组织NF-KB p65表达明显低于模型对照组(0.55±0.04vs0.64±0.06,P<0.01)；但明显高于柳氮磺吡啶对照组(0.55±0.04vs0.41±0.06,P<0.01)。⑤与正常对照组比较,模型对照组大鼠结肠的髓过氧化物酶(MPO)活性增加约6倍,柳氮磺吡啶对照组、蛇葡萄素中、高剂量组大鼠结肠组织MPO活性较模型对照组降低50%,与模型对照组比较有显著性差异(17.90±2.51、18.50±2.80、16.70±2.45vs36.00±4.47,P<0.01,respectively);蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(18.50±2.80、16.70±2.45vs17.90±2.51,P>0.05respectively)。蛇葡萄素低剂量组大鼠结肠组织MPO活性低于模型对照组(24.67±3.12vs36.00±4.47,P<0.01)；但明显高于柳氮磺吡啶对照组(24.67±3.12vs17.90±2.51,P<0.01)。⑥给予TNBS溶液灌肠后第八天,模型对照组血清TNF-α、IL-1p的蛋白含量明显高于正常对照组(80.29±5.95vs25.15±2.88、61.95±5.53vs23.85±2.95,P<0.01,1rspectively), IL-10的蛋白含量明显低于正常对照组(28.31±3.21vs39.60±4.42,P<0.01)。柳氮磺吡啶对照组、蛇葡萄素中、高剂量组TNF-α、IL-1p的蛋白含量明显低于模型对照组(44.09±4.71、46.15±4.78、43.18±4.16vs80.29±5.95,38.14±4.04、40.55±4.09、37.64±3.65vs61.95±5.53,P<0.01,respectively),IL-10的蛋白含量明显高于模型对照组(67.10±4.23、68.03±5.00、65.38±4.89vs28.31±3.21, P<0.01, respectively);蛇葡萄素中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(P>0.05)。蛇葡萄素低剂量组血清TNF-α、IL-1β的蛋白含量低于模型对照组(64.56±5.88vs80.29±5.95、49.00±4.66vs61.95±5.53,P<0.01,respectively),IL-10的蛋白含量高于模型对照组(33.61±3.77vs28.31±3.21,P<0.01)：蛇葡萄素低剂量组血清TNF-α、IL-1β的蛋白含量明显高于柳氮磺吡啶对照组(64.56±5.88vs44.09±4.71,49.00±4.66vs38.14±4.04,P<0.01,respectively),IL-10的蛋白含量明显低于柳氮磺吡啶对照组(33.61±3.77vs67.10±4.23,P<0.01)。⑦模型对照组大鼠结肠TNF-α、IL-1βmRNA(1.35±0.27vs0.34±0.11、1.43±0.21vs O.38±0.10,P
结论：早期以蛇葡萄素灌胃给药干预,能减轻TNBS诱导大鼠实验性结肠炎,其作用机制可能与蛇葡萄素下调结肠NF-κB p65表达、Th1促炎性细胞因子TNF-α、IL-1β和上调Th2抑炎性细胞因子IL-10有关。
第二部分白芍总苷减轻大鼠实验性结肠炎的作用及机制研究
白芍是中国传统中药植物,为毛莨科植物芍药(paeonia lactiflora)的干燥根,是中医学用于治疗风湿类疾病、自身免疫性疾病、肝炎及肝硬化等疾病的重要组方之一,引起了医药学界的广泛重视与开发研究。国内外学者通过多年的研究,已成功提取出白芍的药效成分单体,主要为一组糖苷类物质,包括芍药甙(paeoniflorin).芍药花甙(paeonin)、芍药内酯甙(albiflorin)、羟基芍药甙(hydroxy-paeoniflorin)、苯甲酰芍药甙(benzoylpaeoniflorin),统称为白芍总苷(total glucosides of paeony, TGP),其中芍药苷占总苷量的90%以上,是白芍的主要有效成分。对白芍总苷的药理及临床进行研究发现,白芍总苷可有效抑制炎症局部前列腺素E的合成,抑制淋巴细胞增殖及巨噬细胞产生促炎性细胞因子,如TNF-α、IL-1β、IL-8及IL-6等,纠正CD4+/CD8+T淋巴细胞比例失衡,从而多途径调节自身免疫紊乱,以及具有抗炎、止痛、保肝的作用。白芍总苷对类风湿性关节炎(RA)有确切疗效,已在临床上广泛应用,并显示出对系统性红斑狼疮、强直性脊柱炎等多种自身免疫性疾病治疗的良好应用前景。
本文假设白芍总苷对炎症性肠病也有治疗作用,本研究以TNBS/乙醇灌肠诱导复制大鼠实验性结肠炎模型,观察白芍总苷灌胃给药对大鼠实验性结肠炎的干预作用,并对其分子机制进行初步探讨。
目的：观察白芍总苷灌胃给药对TNBS/乙醇诱导的大鼠实验性结肠炎的干预作用及机制探讨。
方法：乙醚轻度麻醉下,给予雄性Sprague-Dawley (SD)大鼠0.25ml TNBS的50%乙醇溶液(含TNBS30mg)灌肠,诱导大鼠实验性结肠炎的发生。60只大鼠随机分为6组,每组10只,分别为正常对照组(A组)、模型对照组(B组)、柳氮磺吡啶(SASP)对照组(C组)、白芍总苷(TGP)低剂量组(D组)、白芍总苷中剂量组(E组)、白芍总苷高剂量组(F组)。TNBS/乙醇灌肠造模24小时后,白芍总苷低、中、高剂量组大鼠分别给予25、50、100mg/kg/day的白芍总苷进行灌胃给药：柳氮磺吡啶对照组大鼠给予柳氮磺吡啶100mg/kg/day灌胃给药；正常对照组和模型对照组以生理盐水(2.0ml/只)灌胃,每天3次,持续给药7天。试验期间每天观察大鼠的饮食、饮水、体重、大便、毛色、活动、精神状态等情况,进行疾病活动指数(disease activity index, DAI)评分；灌胃给药7天后处死全部大鼠,打开腹腔,分离结肠,对大鼠结肠组织炎症作大体评分(colon macroscopic damage index, CMDI);取距肛门端8cm处结肠用福尔马林固定,制作病理切片,对大鼠结肠炎症进行病理组织学评分(histopathological score, HPS);另取大鼠结肠组织以分光光度法进行髓过氧化物酶(myeloperoxidase, MPO)活性测定,同时以酶联免疫法(ELISA)检测大鼠血清TNF-α、IL-1β和IL-10的蛋白含量,逆转录-聚合酶链式反应(RT-PCR)检测大鼠结肠组织TNF-α、IL-1β和IL-10mRNA的表达,Western blot检测大鼠结肠组织TNF-α、IL-1β和IL-10蛋白的表达。
结果：①给予含30mg TNBS原液的乙醇溶液灌肠后第2天,模型对照组大鼠开始出现大便次数增多、血便,体重逐渐下降,造模后第8天,可见明显脓血便、饮食明显减少、体重减轻、毛发失去光泽甚至明显脱毛、喜扎堆,反应迟缓、懒动,DAI明显高于正常对照组(2.74±0.49vs0.07±0.14,P<0.01)。柳氮磺吡啶对照组、白芍总苷中、高剂量组大鼠给予TNBS溶液灌肠后出现腹泻、血便和体重减轻,但程度较轻,灌胃给药后期逐渐改善。造模后第8天白芍总苷低剂量组DAI低于模型对照组(2.22±0.29vs2.74±0.49,P<0.05),柳氮磺吡啶对照组、白芍总苷中、高剂量组DAI明显低于模型对照组(1.10±0.22、1.00±0.16、0.97±0.11vs2.74±0.49,P<0.01,respectively).白芍总苷低剂量组DAI明显高于柳氮磺吡啶对照组(2.22±0.29vs1.10±0.22,P<0.01)；白芍总苷中、高剂量组、柳氮磺吡啶对照组组间DAI比较无显著差异(1.00±0.16、0.97±0.1l vs1.10±0.22,P>0.05,respectively).②TNBS溶液灌肠后8天处死所有大鼠,打开大鼠腹腔,分离结肠,沿肠系膜纵轴剖开,在冰上观察结肠充血水肿、炎症、溃疡等变化。模型对照组大鼠结肠与周围组织如输尿管、小肠及脾脏等脏器严重粘连,局部结肠肠管扩张,肠壁增厚、颜色灰暗,肠黏膜明显充血、水肿,可见较大溃疡病灶,病灶处有灰黑色假膜样物覆盖,CMDI明显高于正常对照组(3.67±0.71vs0.10±0.32,P<0.01)。TNBS溶液灌肠后给予柳氮磺吡啶、白芍总苷低、中、高剂量干预后,大鼠结肠病变有不同程度减轻,白芍总苷低剂量组CMDI低于模型对照组(2.89±0.60vs3.67±0.71,P<0.05);柳氮磺吡啶对照组、白芍总苷中、高剂量组CMD)I明显低于模型对照组(1.60±0.52.1.80±0.42.1.70±0.67vs3.67±0.71,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量组CMDI明显高于柳氮磺吡啶对照组(2.89±0.60vs1.60±0.52,P<0.01),白芍总苷中、高剂组、柳氮磺吡啶对照组组间比较无显著差异(1.80±0.42,1.70±0.67vs1.60±0.52,P>0.05,respectively)。③显微镜下,正常对照组大鼠结肠组织未见或仅见极少中性粒细胞浸润,结肠黏膜完整,固有层内肠腺丰富,排列紧密。模型对照组大鼠结肠组织病理学表现为固有层充血水肿,大量中性粒细胞、淋巴细胞浸润,广泛黏膜上皮坏死脱落,隐窝炎及脓肿形成,腺体破坏且结构紊乱,溃疡形成,肉芽肿、纤维组织增生,结肠HPS明显高于正常对照组(3.33±0.71vs0.30±0.48,P<0.01)。与模型对照组相比,白芍总苷低剂量组大鼠结肠HPS低于模型对照组(2.67±0.50vs3.33±0.71,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组大鼠结肠HPS明显低于模型对照组(1.80±0.63、1.90±0.57、1.60±0.52vs3.33±0.71,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量结肠HPS明显高于柳氮磺吡啶对照组(2.67±0.50vs1.80±0.63,P<0.01),白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(1.90±0.57、1.60±0.52vs1.80±0.63,P>0.05,respectively).④与正常对照组比较,模型对照组大鼠结肠的MPO活性增加约5倍,MPO活性明显高于正常对照组(31.11±3.55vs6.10±0.73,P<0.01)。白芍总苷低剂量组大鼠结肠MPO活性低于模型对照组(27.56±2.70vs31.11±3.55,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组大鼠结肠MPO活性较模型对照组降低50%,明显低于模型对照组(15.80±1.69、16.10±1.37、14.90±1.66vs31.11±3.55,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量组结肠MPO活性明显高于柳氮磺吡啶对照组(27.56±2.70vs15.80±1.69,P<0.01),白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(16.10±1.37、14.90±1.66vs15.80±1.69,P>0.05,respectively).⑤给予TNBS溶液灌肠八天后,模型对照组血清TNF-α、IL-1β的蛋白含量明显高于正常对照组(84.08±14.32vs21.98±3.66、66.13±9.99vs27.71±2.45,P<0.01,respectively),IL-10的蛋白含量明显低于正常对照组(26.87±4.00vs40.83±3.73,P<0.01)。白芍总苷低剂量组血清TNF-α、IL-1β的蛋白含量低于模型对照组(68.71±10.65vs84.08±14.32、54.51±7.82VS66.13±9.99,P<0.05,respectively),IL-10的蛋白含量高于模型对照组(31.61±3.22vs26.87±4.00,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组血清TNF-α、IL-1β的蛋白含量明显低于模型对照组(47.69±7.45、49.26±6.34、45.99±7.15vs84.08±14.32,40.39±6.45、40.86±5.14、38.99±6.54vs66.13±9.99,P<0.01,respectively),IL-10的蛋白含量明显高于模型对照组(73.78±8.15、71.39±8.35、75.75±8.28vs26.87±4.00,P<0.01,respectively)。与柳氮磺吡啶对照组比较,白芍总苷低剂量组血清TNF-α、IL-1β的蛋白含量明显高于柳氮磺吡啶对照组(68.71±10.65vs47.69±7.45,54.51±7.82vs40.39±6.45,P<0.01,respectively),IL-10的蛋白含量明显低于柳氮磺吡啶对照组(31.61±3.22vs73.78±8.15,P<0.01)；白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(49.26±6.34、45.99±7.15vs47.69±7.45,40.86±5.14、38.99±6.54vs40.39±6.45,71.39±8.35、75.75±8.28vs73.78±8.15,P>0.05,respectively).⑥模型对照组大鼠结肠TNF-α、IL-1β mRNA(1.35±0.44vs0.36±0.12.1.57±0.40vs0.42±0.21,P<0.01,respectively)和TNF-α、IL-1β蛋白的表达明显高于正常对照组(1.18±0.36vs0.23±0.08、1.27±0.31vs0.32±0.07,P<0.01,respectively), IL.10mRNA(0.25±0.12vs0.9l±0.11,P<0.01)和IL-10蛋白的表达明显低于正常对照组((0.37±0.14vs1.09±0.08,P<0.01))。白芍总苷低剂量组TNF-α、IL-1β mRNA(0.97±0.21vs1.35±0.44、1.19±0.28vs1.57±0.40,P<0.05,Fespectively)和TNF-α、IL-1β蛋白的表达低于模型对照组((0.81±0.19vs1.18±0.36、0.97±0.24vs1.27±0.31,P<0.05,respectively),IL-10mRNA(0.41±0.13vs0.25±0.12,P<0.05)和IL-10蛋白的表达高于模型对照组((0.53±0.12vs0.37±0.14,P<0.05)；柳氮磺吡啶对照组、白芍总苷中、高剂量组TNF-α、IL-1β mRNA(0.60±0.19.0.61±0.18.0.58±0.20vs1.35±0.44,0.74±O.23.0.72±0.24.O.70±0.25vs1.57±0.40,P<0.01,respectively)和TNF-α、IL-1β蛋白的表达明显低于模型对照组(0.51±0.14、0.50±0.13、0.48±0.15vs1.18±0.36；0.63±0.15.0.62±O.16.0.60±0.15vs1.27±0.31,P<0.01,respectively),IL-10mRNA(1.23±0.23.1.19±0.21.1.27±0.24vs0.25±0.12,P<0.01,respectively)和IL-10蛋白的表达明显高于模型对照组(1.37±0.16、1.35±0.17、1.39±0.18vs0.37±0.14,P<0.01,respectively).与柳氮磺吡啶对照组比较,白芍总苷低剂量组TNF-α、IL-1β mRNA(0.97±0.21vs0.60±0.19.1.19±0.28vs0.74±0.23,P<0.01,repectively)和TNF-α、IL-1β蛋白的表达明显高于柳氮磺吡啶对照组(0.81±0.19vs0.51±0.14、0.97±0.24vs0.63±0.15, P<0.01, respectively), IL-10mRNA(0.41±0.13vs1.23±0.23, P<0.01)和IL-10蛋白的表达明显低于柳氮磺吡啶对照组(0.53±0.12vs1.37±0.16,P<0.01)；白芍总苷中、高剂量组、柳氮磺吡啶对照组组间比较无显著差异(P>0.05)。
结论：早期以白芍总苷灌胃给药干预,能减轻TNBS诱导大鼠实验性结肠炎,其作用机制可能与白芍总苷下调Thl促炎性细胞因子TNF-α、IL-1β和上调Th2抑炎性细胞因子IL-10有关。
Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is an idiopathic, non-specific inflammatory disorder of the intestinal tract with unknown causes. It is currently believed that genetic susceptibility, dysfunction of immune regulation in intestinal mucosa and intestinal bacteria are involved in the pathogenesis of IBD. Immune regulation dysfunction might be the direct cause for onset of IBD. It is now well accepted that abnormal immune response mediated by proinflammatory cytokines such as TNF-α,IL-1β, IL-6and IL-8, and antiinflammatory cytokines IL-10plays an important role in balance of Thl and Th2as well as inflammatory severity and activity in IBD. Currently, there is a lack of an effective therapy to cure the diseases since causes and mechanisms of IBD are not totally understood. Aminosalicylates, corticosteroids, immunomodulating and immunosuppressive drugs are used for treatment of IBD by controlling active inflammation and regulating abnormal immune response. However, such treatments have multiple adverse effects, particularly for long-term administration, and relapse is high upon drug discontinuance. Biological agents such as anti-TNF-a monoclonal antibody can induce the alleviation of IBD, but at a high cost and put the patients at risk to develop treatment-related cancers, and surgical resection of the colon and ileostomy is the ultimate alternative in many cases. Therefore, there is an urgent for the development of new and specific therapies for IBD with few side effects.
Part I:Ampelopsin attenuates2,4,6-trinitrobenzene sulfonic acid/ethanol-induced colitis in rats
Ampelopsin is a flavonoids extracted from Rattan tea which is a traditional Chinese herbal remedy prepared from the stems and leaves of the plant ampelopsis grossedentata. Ampelopsin was reported as a major bioactive component in ampelopsis grossedentata and first isolated from ampelopsis meliaefolia by Kotake and Kubota in1940.Recent studies have shown that ampelopsin has a broad range of biological functions including inhibition of apoptosis, hypoglycemic, anti-hypertension, anti-bacterial, anti-inflammatory, antioxidant, anti-tumor, hepatoprotective, and neuroprotective effects. These findings support the hypothesis that ampelopsin is also effective against IBD. Therefore, ampelopsin may be a bright potential candidate Chinese drug for treatment of IBD.
Objective The present study is to investigate therapeutic effects of ampelopsin on2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of ampelopsin for treatment of inflammatory bowel disease (IBD).
Methods Sixty Sprague-Dawley (SD) rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100mg/kg/day), and low, medium and high-dose ampelopsin groups (125,250and500mg/kg/day, respectively).24hours following colonic instillation of TNBS, ampelopsin and SASP were given by gastric gavage three times a day for7days.Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS) and myeloperoxidase (MPO) activity were evaluated. Levels of serum and colon TNF-α,IL-1β and IL-10were measured by enzyme-linked immunosorbent assay (ELISA), expression of TNF-α,IL-1β and IL-10mRNA in colonic tissues was detected by reverse transcription-polymerase chain reaction (RT-PCR), and expression of NF-κB p65protein in colonic tissues was detected by immunohistochemical methods, respectively.
Results①Normal control rats treated with ethanol enema gained body weight and defecated normally. In the model control group, pasty to liquid gross bloody stool and weight loss were observed in all rats. When ampelopsin (250or500mg/kg/day) or SASP (100mg//kg/day) was administered on day7, bloody stools were not evident, stools were better formed, and weight loss was lessened. The colons from rats in the model control group were seriously adherent to small intestines and spleen, and showed marked hyperemia, inflammation, necrosis, ulcer, whereas the colons from rats in the normal control group showed no or a slight inflammation. Treatment with ampelopsin (250or500mg/kg/day) or SASP markedly decreased both hyperemia and inflammation in the colons. Administration of ampelopsin (250or500mg/kg/day) significantly improved the DAI and CMDI8days after TNBS instillation. And there was no significant difference compared with administration of SASP (P>0.05).②Ulcerations, massive transmural infiltration of inflammatory cells, thickening of the colon wall, goblet cell depletion, and extensive fibrosis found throughout colons in the model control group. Administration of ampelopsin (250or500mg/kg/day) improved these signs and the HPS significantly. And there was no significant difference compared with administration of SASP (P>0.05).③Rectal instillation of TNBS led to the marked enhancement of NF-κB p65protein expression in colonic tissues. Compared with the normal group, administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of NF-κB p65protein expression in colonic tissues. The difference was not significant when compared with administration of SASP (P>0.05).④MPO activity was very low in the normal control group, and increased significantly nearly6-fold in rats after TNBS enema in the model control group. In contrast, this increase was significantly blunted by about50%by administration of ampelopsin (250or500mg/kg/day). And there was no significant difference compared with administration of SASP (P>0.05).⑤ectal instillation of TNBS led to the marked enhancement of levels of serum TNF-α and IL-1β, and the marked decrease of levels of serum IL-10. Administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of levels of serum TNF-α and IL-1β and at the same time increased the decrease of levels of serum IL-10. And there was no significant difference when compared with administration of SASP(P>0.05).⑥Rectal instillation of TNBS led to the marked enhancement of mRNA expression of TNF-α and IL-1β, and the marked decrease of that of IL-10in colonic tissues. Administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of mRNA expression of TNF-α and IL-1β and at the same time significantly increased the decrease of that of IL-10. And there was no significant difference when compared with administration of SASP (P>0.05).⑦Protein expression of TNF-α and IL-1β was markedly elevated and that of IL-10was markedly decreased in the colons after rectal TNBS instillation, which was consistent with results of the mRNA expression.250or500mg/kg/day ampelopsin treatment significantly abrogated the elevation in protein expression of TNF-α,IL-1β and at the same time significantly increased the decrease of protein expression of IL-1O.The difference was not significant when compared with administration of SASP (P>0.05). On the other hand, administration of ampelopsin at the dose of125mg/kg/day showed modest improvement on colonic inflammation and regulatory effects on expression of TNF-α,IL-1β and IL-10, but the values were lower than administration of SASP (P<0.01).
Conclusion Ampelopsin attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the down-regulation the enhancement of NF-κB p65protein expression in colonic tissues and adjustment of Thl/Th2cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10.
Part II:Total glucosides of peony attenuates2,4,6-trinitrobenzene sulfonic acid/ethanol-induced colitis in rats
Total glucosides of peony (TGP) is a group of glucosides extracted from peony, including paeoniflorin, hydroxy-paeoniflorin, paeonin, albiflorin and benzoylpaeoniflorin. Paeoniflorin is a monoterpene glucoside, the major active ingredient of TGP, accounting for over90%of total glucosides. Now TGP has been a common prescription drug used for the treatment of autoimmune diseases including rheumatoid arthritis, ankylosing spondylitis and systemic lupus erythematosus and shown significant efficacy in the clinic. Recent studies have shown that TGP has anti-inflammatory and immunoregulatory functions, by inhibiting Th1cytokines and enhancing Th2cytokines. These findings support the hypothesis that TGP is also effective against IBD. Therefore, TGP may be a bright potential candidate Chinese drug for treatment of IBD.
Objective The present study is to investigate therapeutic effects of total glucosides of peony (TGP) on2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of TGP for treatment of inflammatory bowel disease (IBD).
Methods Sixty Sprague-Dawley (SD) rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100mg/kg/day), and low, medium and high-dose TGP groups (25,50and100mg/kg/day, respectively).24hours following colonic instillation of TNBS, TGP and SASP were given by gastric gavage three times a day for7days.Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS) and myeloperoxidase (MPO) activity were evaluated. Levels of serum TNF-α,IL-1β and IL-10were measured by ELISA, and expression of TNF-α,IL-1β and IL-10mRNA and protein in colonic tissues was detected by RT-PCR and Western blot, respectively.
Results①Normal control rats treated with ethanol enema gained body weight and defecated normally. In the model control group, pasty to liquid gross bloody stool and weight loss were observed in all rats. When TGP (50or100mg/kg/day) or SASP was administered on day7, bloody stools were not evident, stools were better formed, and weight loss was lessened. The colons from rats in the model control group were seriously adherent to small intestines and spleen, and showed marked hyperemia, inflammation, necrosis, ulcer, whereas the colons from rats in the normal control group showed no or a slight inflammation. Treatment with TGP (50or100mg/kg/day) or SASP markedly decreased both hyperemia and inflammation in the colons. Administration of TGP (50or100mg/kg/day) significantly improved the DAI and CMDI8days after TNBS instillation. And there was no significant difference compared with administration of SASP (P>0.05).②Ulcerations, massive transmural infiltration of inflammatory cells, thickening of the colon wall, goblet cell depletion, and extensive fibrosis found throughout colons in the model control group. Administration of TGP (50or100mg/kg/day) improved these signs and the HPS significantly.③MPO activity was very low in the normal control group, and increased significantly nearly5-fold in rats after TNBS enema in the model control group. In contrast, this increase was significantly blunted by about50%by administration of TGP (50or100mg/kg/day).④Rectal instillation of TNBS led to the marked enhancement of levels of serum TNF-α and IL-1β and the marked decrease of levels of serum IL-10. Administration of TGP (50or100mg/kg/day) significantly reduced the enhancement of levels of serum TNF-α and IL-1β and at the same time increased the decrease of levels of serum IL-10. And there was no significant difference when compared with administration of SASP (P>0.05).⑤Rectal instillation of TNBS led to the marked enhancement of mRNA expression of TNF-α and IL-1β, and the marked decrease of that of IL-10in colonic tissues. Administration of TGP (50or100mg/kg/day) significantly reduced the enhancement of mRNA expression of TNF-α and IL-1β and at the same time significantly increased the decrease of that of IL-10.⑥Protein expression of TNF-α and IL-1β was markedly elevated and that of IL-10was markedly decreased in the colons after rectal TNBS instillation, which was consistent with results of the mRNA expression.50or100mg/kg/day TGP treatment significantly abrogated the elevation in protein expression of TNF-α,IL-1β and at the same time significantly increased the decrease of protein expression of IL-10. The difference was not significant when compared with administration of SASP (P>0.05). On the other hand, administration of TGP at the dose of25mg/kg/day showed modest improvement on colonic inflammation and regulatory effects on expression of TNF-α,IL-1β and IL-10, but the values were lower than administration of SASP (P<0.01).
Conclusion TGP attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the adjustment of Thl/Th2cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10.
引文
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