溃疡性结肠炎大鼠肺损伤细胞凋亡机制及中药单体干预作用机制研究
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摘要
研究目的
溃疡性结肠炎(Ulcerative Colitis,UC),是一种以慢性炎症和溃疡形成为主要病理特点的结肠粘膜层的消化道疾病。该病病因复杂,发病环节多,治愈难度大,反复发作,并与结肠癌的发病关系密切,被世界卫生组织列为现代难治病之一。肺支气管病损作为炎症性肠病的肠外表现,发生率很高,文献报道可达50%,而关于溃疡性结肠炎肺损伤的发生机制研究至今甚少,治疗上多采用氨基水杨酸类药物和皮质类固醇药物治疗,效果不理想。本课题旨在通过中药单体鱼腥草素钠、甘草酸二铵及两者合用干预免疫复合物加三硝基苯磺酸-乙醇诱导的溃疡性结肠炎大鼠模型,探究溃疡性结肠炎肺损伤的细胞凋亡调控机制,揭示溃疡性结肠炎肺损伤生物学机制；阐明鱼腥草素钠、甘草酸二铵及两者合用治疗溃疡性结肠炎肺损伤的疗效、作用途径、靶点及对细胞凋亡调控的影响,为鱼腥草素钠、甘草酸二铵及两者合用治疗溃疡性结肠炎提供客观依据,并为临床治疗合并肺损伤的溃疡性结肠炎患者提供新的思路与方法,进而为中医治疗UC及其肺损伤从肺论治、从肠论治、肺肠同治的优势提供更为可靠的证据,升华肺与大肠表里脏腑气机升降相关理论。
研究内容与结果
本实验选用SPF级健康雄性Wistar大鼠,采用家兔结肠黏膜组织致敏加三硝基苯磺酸-50%乙醇灌肠的方法复制大鼠UC模型。造模0周(模型成立时)杀检取材后,将剩余模型大鼠随机分为五组,即模型组、西药组、鱼腥草组、甘草组、鱼腥草+甘草组。鱼腥草组给予鱼腥草素钠片水溶液灌胃治疗；甘草组给予甘草酸二铵胶囊水溶液灌胃治疗；鱼腥草+甘草组给予鱼腥草素钠片与甘草酸二铵胶囊的混合水溶液灌胃治疗；西药以柳氮磺吡啶肠溶片水溶液为阳性对照药灌胃治疗；正常组与模型组同步以纯净水灌胃作空白对照。于给药2、4周末,观察大鼠一般状态,麻醉后,腹主动脉取血,并取肺、结肠组织进行相关指标检测。
1溃疡性结肠炎大鼠一般状态及肺和结肠组织形态学变化特点
各时间点UC大鼠模型一般生活状态差,0周时模型组大鼠消瘦明显,并有呼吸气急、喘促等肺部症状。结肠组织HE染色图片显示0周时有明显的坏死灶侵及结肠粘膜的上皮层、黏膜层、黏膜下层,部分结肠组织出现凝固性坏死。肠壁少数坏死黏膜组织脱落后形成溃疡点,大量炎性细胞浸润粘膜下层；2周时病变与0周比较未见明显恢复；4周时结肠组织病变有一定程度的恢复,部分结肠组织粘膜下层仍有大量炎性细胞浸润及溃疡形成,粘膜上皮和固有膜内腺体减少、萎缩、破坏甚至消失。肺组织HE染色图片表现为0周时肺间质广泛的炎症,炎性细胞较多呈灶状分布、集中在支气管、血管的周围,部分肺泡变形；2周模型组大鼠肺组织病理与0周比较,未见明显改变,支气管、血管周围仍有大量的炎性细胞浸润肺,且呈灶状分布,可见到支气管壁有明显的增厚；4周时,UC模型组大鼠肺组织病变较0、2周时有一定程度的改善,但部分大鼠肺组织中仍有大量淋巴细胞呈灶状分布浸润,血管壁、支气管壁增厚,可见到部分支气管壁有一定程度的纤维组织增生形成。动物模型证实溃疡性结肠炎可以引起肺损伤,肺损伤是UC的肠外表现之一。
2溃疡性结肠炎大鼠外周血炎性细胞凋亡及中药单体的干预作用
2.1溃疡性结肠炎大鼠外周血淋巴细胞(lamina proprial lymphocyte, LPL)凋亡及中药的干预作用
为探讨外周血淋巴细胞凋亡异常与溃疡性结肠炎发生、发展的关系,以及中药治疗UC的可能作用机制,本研究采用流式细胞术动态检测、观察UC大鼠外周血中淋巴细胞凋亡率的变化以及中药对外周血淋巴细胞凋亡异常的干预作用。结果表明,2、4周时模型组大鼠外周血淋巴细胞凋亡率显著降低,提示淋巴细胞凋亡减慢可能是导致UC模型大鼠免疫功能紊乱的重要原因之一,从而与UC的发生密切相关。另外,外周血淋巴细胞凋亡率降低还可能与UC大鼠结肠组织病理学结果中的淋巴细胞浸润有关。
本实验应用的中西药物均能不同程度地促进淋巴细胞凋亡,从而调节UC大鼠的免疫功能,达到缓解UC症状的目的。甘草的短期应用(2周)对外周血淋巴细胞凋亡率的改善作用优于鱼腥草,而鱼腥草长期(4周)治疗较短期治疗对外周血淋巴细胞凋亡率的改善作用明显,鱼腥草与甘草合用对外周血淋巴细胞凋亡率的改善作用明显优于单用鱼腥草治疗,体现了两药合用所代表的肺肠同治的优越性。
2.2溃疡性结肠炎大鼠外周血中性粒细胞(polymorphonuclear neutrophils,PMN)凋亡及中药的干预作用
为探讨外周血中性粒细胞凋亡异常与溃疡性结肠炎发生、发展的关系,以及中药治疗UC的可能作用机制,本研究采用流式细胞术动态检测、观察UC大鼠外周血中性粒细胞凋亡率的变化以及中药对外周血中性粒细胞凋亡异常的干预作用。结果发现2周时UC模型组大鼠血中性粒细胞凋亡与正常组比较呈延迟现象;4周时,模型大鼠PMN凋亡率与正常组比较无明显差异,PMN凋亡延迟的现象得到恢复。结合造模后2周时UC大鼠的表现,UC大鼠粪便不成形,为黏液便或软变,结肠组织HE染色仍有溃疡、大量炎性细胞浸润,说明PMN凋亡抑制使PMN在血液循环和炎症部位的作用时间延长,提示模型大鼠处于UC活动期,结肠炎症反复、溃疡改变。同时,随着时间延长,模型大鼠PMN凋亡率明显恢复,而结肠组织病理损伤依然存在,提示4周时UC大鼠处于缓解期。表明PMN凋亡延迟可能是UC肠组织损伤的重要机制之一,并且PMN浸润是活动性UC的一个重要标志。PMN凋亡率的变化可能为临床UC病情观察及药物疗效评价提供一定的依据,进一步深入研究PMN凋亡,乃至适度控制其凋亡将为临床治疗UC开辟新的途径。
另外,中药单体甘草酸二铵单用、鱼腥草素钠与甘草酸二铵合用可能通过诱导中性粒细胞凋亡使UC大鼠提前进入缓解期,能在一定程度上减轻炎症反应,从而改善黏膜损伤,对UC炎症的发展发挥一定的治疗作用。LPL及PMN凋亡的减慢和溃疡性结肠炎的发生、发展存在一定关系,中药单体可能通过对外周血LPL与PMN凋亡率的调控,达到缓解UC目的。
3溃疡性结肠炎大鼠肺、结肠组织凋亡效应蛋白Caspase-3的含量及中药的干预作用
3.1溃疡性结肠炎大鼠结肠组织凋亡效应蛋白Caspase-3的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)动态观察溃疡性结肠炎大鼠结肠组织凋亡效应蛋白Caspase-3的含量,以探究UC大鼠结肠组织细胞凋亡是否存在异常,及中药对可能存在的凋亡异常的干预作用。结果表明,造模后2周及4周,UC模型组大鼠结肠组织中Caspase-3的含量与正常组比较均有明显的增加,提示UC模型大鼠结肠组织存在一定程度的细胞凋亡加速。Caspase-3是Caspase家族中的关键效应酶,是凋亡执行的重要效应分子,一般以23Ku的无活性前体存在于细胞质中,当细胞进入凋亡时才被激活,它参与多种因素如Fas系统、Bcl-2家族诱导的细胞凋亡。推测UC的发生可能与结肠组织凋亡效应蛋白Caspase-3的过量表达,诱导结肠组织细胞的凋亡有关,从而导致结肠组织损伤。
另外,西药柳氮磺胺吡啶能有效改善UC结肠组织细胞凋亡增加,但长期疗效不佳,而中药治疗UC在改善结肠组织Caspase-3过量表达方面优于西药,其中,中药甘草单用、甘草与鱼腥草合用短期应用优于单用鱼腥草。
3.2溃疡性结肠炎大鼠肺组织凋亡效应蛋白Caspase-3的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)观察溃疡性结肠炎大鼠肺组织凋亡效应蛋白Caspase-3的含量,以探究UC大鼠肺组织细胞凋亡是否存在异常,及中药对可能存在的凋亡异常的干预作用。结果表明,造模后2周及4周,UC模型组大鼠肺组织中Caspase-3的含量与正常组比较均有明显的增加,提示UC模型大鼠肺组织存在一定程度的细胞凋亡加速,UC大鼠肺组织细胞凋亡的发生与凋亡效应蛋白Caspase-3的过多表达存在一定关系。
另外,中药对UC肺损伤在细胞凋亡方面的干预作用优于西药柳氮磺胺吡啶。中药甘草在治疗2周时对肺组织Caspase-3干预作用尚不明显,治疗4周时能有效降低Caspase-3的含量。中药鱼腥草单用、鱼腥草与甘草合用均能有效改善UC大鼠肺损伤细胞凋亡的增加,且短期与长期治疗均有效。表明各中药及其所代表的治法可能通过干预凋亡效应蛋白Caspase-3的过量表达发挥治疗作用,可能为中医中药防治UC肺损伤提供了新的思路及治疗方法。
4溃疡性结肠炎大鼠肺、结肠组织凋亡调控蛋白Bcl-2、Bax的表达及中药的干预作用
4.1溃疡性结肠炎大鼠结肠组织凋亡调控蛋白Bcl-2、Bax的表达及中药的干预作用
采用Western-bloting观察UC大鼠结肠组织凋亡调控蛋白Bcl-2、Bax的表达以及中药的干预作用。结果表明,2、4周正常组Bcl-2/Bax>1,细胞凋亡发生相对较少；模型组Bax蛋白表达持续增高,Bcl-2表达减少,Bcl-2/Bax 另外,中药与西药均能通过增加UC大鼠结肠组织Bcl-2,降低Bax的表达,有效恢复Bcl-2/Bax比值,对结肠组织细胞凋亡具有一定的抑制作用,从而促进结肠粘膜的修复。其中,鱼腥草长期干预UC,能有效增加Bcl-2/Bax比值,抑制结肠组织细胞凋亡,长期治疗作用优于柳氮磺胺吡啶；甘草单用、鱼腥草与甘草合用治疗UC,2周、4周时均能上调Bcl-2/Bax比值,减少结肠组织的细胞凋亡。提示中药不仅能有效改善UC急性期结肠组织的细胞凋亡加速,短期疗效显著,而且长期治疗亦有改善作用,并且体现了中医治疗UC从肺论治、肺肠同治的特点及有效性。
4.2溃疡性结肠炎大鼠肺组织凋亡调控蛋白Bcl-2、Bax的表达及中药的干预作用
采用Western-bloting观察UC大鼠肺组织凋亡调控蛋白Bcl-2、Bax的表达以及中药的干预作用。结果表明,造模后2周、4周时UC模型大鼠肺组织Bcl-2蛋白表达水平明显下降,Bax蛋白表达水平明显上升,Bcl-2/Bax比值显著降低,提示Bcl-2、Bax蛋白表达异常、Bcl-2/Bax比例失衡,并诱导肺组织细胞凋亡可能是UC肺损伤和“肠病及肺”的可能机制之一
另外,鱼腥草、甘草单用及鱼腥草与甘草合用通过上调Bcl-2、下调Bax,对肺组织的细胞凋亡加速均有一定的抑制作用,且优于西药柳氮磺胺吡啶；鱼腥草与甘草合用对UC大鼠肺组织凋亡的改善明显早于鱼腥草或甘草单用。说明中医肺病治肠以及肺肠同治改善UC肺损伤有其特殊意义,以干预Bcl-2/Bax细胞凋亡信号转导途径为中心,抑制肺组织细胞凋亡可能是治疗UC肺损伤肺病治肠以及肺肠同治的机制之一,为“肺与大肠相表里”相关研究的提供理论基础。
然而,UC肺损伤的发病机制十分复杂,且肺与大肠表里关系是多种物质和机制共同作用的结果,仅仅从Bcl-2/Bax调控细胞凋亡角度开展研究尚不能全面揭示UC及其肺损伤从肺论治、从肠论治、肺肠同治的可能生物学机制,更有待于今后从更广更深层面开展综合性相关研究。
5溃疡性结肠炎大鼠肺、结肠组织Fas/Fas-L系统对细胞凋亡的调控及中药的干预作用
5.1溃疡性结肠炎大鼠结肠组织Fas/Fas-L系统对细胞凋亡的调控及中药的干预作用
采用Western-bloting观察UC大鼠肺组织凋亡调控蛋白Fas/Fas-L的表达以及中药的干预作用。结果表明,UC模型组大鼠结肠组织中Fas、Fas-L蛋白表达量与正常组比较明显增加,提示Fas/Fas-L系统可能也参与溃疡性结肠炎大鼠肺组织细胞凋亡的调控,UC模型大鼠结肠组织细胞凋亡的加速可能与Fas、Fas-L蛋白的表达异常增多有关。
另外,柳氮磺胺吡啶、鱼腥草、甘草均能明显下调UC大鼠结肠组织的Fas或Fas-L蛋白表达,而鱼腥草与甘草合用对Fas、Fas-L的过多表达均有明显的抑制作用。说明中西药物对UC结肠组织的细胞凋亡均有一定程度的改善作用,但各有特点,且中药鱼腥草与甘草合用对UC大鼠结肠组织细胞凋亡的改善作用明显优于两药单用及西药柳氮磺胺吡啶。
UC大鼠结肠组织Fas或Fas-L蛋白表达的变化及药物对两者干预的变化,说明Fas/FasL系统可能直接或间接地参与了UC结肠组织的病理变化,以及各中药、各中药所代表的治法干预UC时发挥作用的可能作用途径及靶点。
5.2溃疡性结肠炎大鼠肺组织Fas/Fas-L系统对细胞凋亡的调控及中药的干预作用
采用Western-bloting观察UC大鼠肺组织凋亡调控蛋白Fas、Fas-L的表达以及中药的干预作用。结果表明,UC模型组大鼠肺组织中Fas、Fas-L蛋白的表达量明显高于正常组,给药4周后鱼腥草组及鱼腥草+甘草组肺组织Fas-L蛋白的表达量明显下降,西药组及甘草组对肺组织Fas-L蛋白表达无明显改善作用。结果提示Fas/Fas-L系统蛋白的过量表达参与UC肺组织细胞的凋亡加速,中药鱼腥草单用、鱼腥草与甘草合用对UC大鼠肺组织细胞凋亡异常的干预作用优于西药柳氮磺胺吡啶。
6溃疡性结肠炎肺、结肠组织核因子NF-κB的含量及中药的干预作用
6.1溃疡性结肠炎结肠组织核因子NF-κB的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)观察溃疡性结肠炎大鼠结肠组织凋亡效应蛋白NF-κB的含量,及中药的干预作用。结果表明,造模后2周、4周UC模型组大鼠结肠组织中NF-κB的含量均有明显的增加,结合模型组大鼠结肠组织Bcl-2、Bcl-2/Bax下调、Bax、Fas、Fas-L蛋白表达上调、Caspase-3含量的增加,提示NF-κB可能通过对Bcl-2/Bax、 Fas/Fas-L系统在基因转录水平的调控参与UC大鼠结肠组织的细胞凋亡
治疗2周时仅有鱼腥草+甘草组能明显降低大鼠结肠组织中NF-κB的含量,其他治疗组均无明显改善,治疗4周时,除鱼腥草组外,其他各治疗组大鼠结肠组织中NF-κB的含量均有明显降低。结果提示,中西药物在改善UC大鼠结肠组织NF-κB方面均有明显作用,其中西药柳氮磺胺吡啶短期干预效果不佳；鱼腥草与甘草合用对改善UC大鼠结肠组织NF-κB短期、长期均有效。短期给予鱼腥草或甘草干预UC大鼠结肠组织NF-κB与两中药合用比较,后者作用明显较好,体现了鱼腥草与甘草合用所代表的肺肠同治改善UC大鼠结肠组织NF-κB作用优于鱼腥草所代表的从肺论治及甘草所代表的从肠论治,同时推测中药干预治疗UC,复方作用优于单味药物的应用,且与临床中药应用于UC的实际情况有较高的一致性。
6.2溃疡性结肠炎肺组织核因子NF-κB的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)观察溃疡性结肠炎大鼠肺组织凋亡效应蛋白NF-κB的含量,及中药的干预作用。结果表明,造模2周时UC模型组大鼠肺组织中NF-κB的含量与正常组比较有明显的增加,结合2周时模型组大鼠肺组织Bcl-2、Bcl-2/Bax下调,Bax蛋白表达上调Caspase-3含量的增加,提示NF-κB可能通过对Bcl-2/Bax系统在基因转录水平的调控参与急性期UC大鼠肺组织的细胞凋亡。造模4周时UC模型组大鼠肺组织中NF-κB的含量与正常组比较未见有明显的改变,但肺组织仍存在Bcl-2/Bax比值下降,Fas、Fas-L蛋白表达、Caspase-3含量的增加,表明随着UC大鼠肺损伤一定程度的恢复,肺组织细胞凋亡依然存在。
治疗2周时,鱼腥草组、鱼腥草+甘草组与模型组比较大鼠肺组织中NF-κB的含量均有明显的下降,提示中药鱼腥草单用、鱼腥草与甘草合用对UC肺组织细胞凋亡在干预NF-κB方面明显优于西药,且中药鱼腥草与甘草合用优于甘草单用,说明鱼腥草与甘草所代表的肺肠同治对肺组织NF-κB的干预作用优于甘草所代表的从肠论治。
结论
通过中药单体鱼腥草素钠、甘草酸二铵及两者合用干预免疫复合物加三硝基苯磺酸-乙醇诱导的溃疡性结肠炎大鼠模型,探究溃疡性结肠炎肺损伤的细胞凋亡调控机制,揭示溃疡性结肠炎肺损伤的生物学机制；阐明鱼腥草、甘草及两者合用治疗溃疡性结肠炎肺损伤的疗效、作用途径、靶点及对细胞凋亡调控的影响,为鱼腥草、甘草及两者合用治疗溃疡性结肠炎提供客观依据,并为临床治疗合并肺损伤的溃疡性结肠炎患者提供新的思路与方法,进而为中医治疗UC及其肺损伤从肺论治、从肠论治、肺肠同治的优势提供更为可靠的证据,升华肺与大肠表里脏腑气机升降相关理论,为中医肺与大肠相表里的脏腑相关理论提供了依据。
1本实验采用TNBS-乙醇诱导复合结肠黏膜组织致敏UC大鼠模型,此模型可达到全身免疫异常与局部炎症病变的共存,产生与人类UC较为接近的临床症状及肠道病变,符合人UC病变由B细胞介导的体液免疫反应和T细胞介导的细胞免疫反应共同作用的病变机制,是比较理想的研究UC病理机制及中药干预作用的动物模型。
2炎性细胞凋亡延迟参与UC的发病,是造成溃疡性结肠炎炎症持续的重要原因。
2.1UC大鼠外周血淋巴细胞凋亡存在明显的延迟,提示淋巴细胞凋亡率的下降可能从一定程度上反映了UC大鼠免疫功能的障碍,淋巴细胞凋亡延迟可能是UC发生的重要病理机制之一。因此,通过对淋巴细胞凋亡延迟的干预,为改善UC患者免疫功能提供了新的思路。
2.2急性期UC大鼠外周血中性粒细胞(PMN)存在明显的凋亡延迟,PMN凋亡受到抑制使PMN在血液循环和炎症部位的作用时间延长,并释放活性介质,加重炎症反应,进一步引起组织损伤,是UC肠组织损伤的重要机制之一,其浸润还可作为活动性UC的一个重要标志,诱导中性粒细胞凋亡可能是药物干预使UC大鼠提前进入缓解期的原因之一,而缓解期UC大鼠外周血中性粒细胞凋亡延迟的现象不明显.
3UC大鼠结肠组织凋亡蛋白Bax表达持续增高,Bcl-2表达减少,Bcl-2/Bax<1,Fas. Fas-L蛋白的表达、Caspase-3、NF-κB含量显著增加,结肠组织细胞凋亡明显占优势,表明结肠组织细胞凋亡加速,并且UC大鼠结肠组织细胞凋亡叫能通过NF-κB-Bcl-2/Bax、 Fas/Fas-L系统-Caspase-3凋亡通路实现,从而初步明确了UC发生的可能细胞凋亡途径。因此,通过对该凋亡通路的阻断或干预,有效上调UC大鼠结肠组织Bcl-2,降低Bax、Fas. Fas-L、Caspase-3、NF-κB的表达,恢复Bcl-2/Bax比值,可能对结肠组织细胞凋亡加速具有一定的抑制作用,从而促进结肠粘膜的修复,为药物干预治疗UC提供分了生物学依据。
4急性期UC大鼠肺组织凋亡蛋白Bax表达增高,Bcl-2表达减少,Bcl-2/Bax<1, Caspase-3、NF-κB含量显著增加,存在凋亡加速；缓解期UC大鼠肺组织凋亡蛋白NF-κB的恢复,而其他凋亡蛋白的变化依然存在,提示UC肺损伤的发生可能与诸多凋亡调控蛋白相关。其中,NF-κB、Bcl-2/Bax、Fas/Fas-L系统、Caspase-3凋亡相关蛋白的异常表达并诱导肺组织细胞凋亡可能是UC肺损伤和“肠病及肺”的可能机制之一。通过对以上靶基因的调控,可能为UC肺损伤防治提供新的方向。
5综合本研究中肠与肺相关凋亡蛋白的检测结果,表明UC大鼠结肠与肺组织细胞凋亡增加均不同程度的通过NF-κB-Bcl-2/Bax、Fas/Fas-L系统-Caspase-3凋亡通路实现,然而在对此通路中发挥作用的凋亡调节蛋白的变化随疾病进展又有所不同。UC大鼠存在肺、结肠组织病理损伤及肺组织细胞凋亡加速,从模型上证实了肺与大肠相表里的肺肠相关理论。因此,以干预细胞凋亡信号转导途径为中心,抑制肺、结肠组织细胞凋亡的研究,可能是UC肺损伤及“肺与大肠相表里”相关研究的主要内容之一。然而,UC及其肺损伤的发病机制十分复杂,且肺与大肠表里关系是多种物质和机制共同作用的结果,仅仅从细胞凋亡角度开展研究尚不能全面揭示肺与大肠表里关系的生物学机制内涵,有待于今后从更广更深层面开展综合性相关研究。
6整体评价中药鱼腥草、甘草、鱼腥草与甘草合用,以及西药柳氮磺胺吡啶,在改善UC大鼠一般状态、肺、结肠组织病理、外周血炎性细胞、凋亡调控蛋白等方面均有一定的作用,但各有特点。中药作用总体优于西药柳氮磺胺吡啶,柳氮磺胺吡啶对UC大鼠结肠组织细胞凋亡的改善主要体现在急性期,而中药不仅能有效改善UC急性期肺、结肠组织的细胞凋亡加速,短期疗效显著,而且长期治疗亦有改善作用。鱼腥草与甘草合用对UC及其肺损伤细胞凋亡的干预作用较两味中药单用作用更为肯定,比如2周时鱼腥草+甘草组大鼠结肠组织NF-κB与模型组比较有明显的下降,而鱼腥草组、甘草组均无明显变化；2周时鱼腥草+甘草组大鼠肺组织Bcl-2/Bax与模型组比较有明显的上升,而鱼腥草组、甘草组均无明显变化,一定程度上体现了中药复方作用优于单味中药。另外,鱼腥草对UC大鼠结肠组织细胞凋亡亦存在一定程度的改善作用。通过对本研究中凋亡通路的研究,发现中药鱼腥草、甘草、鱼腥草与甘草合用,及西药柳氮磺胺吡啶可能通过对NF-κB-Bcl-2/Bax、Fas/Fas-L系统-Caspase-3途径中的一个或多个环节和／或靶点的干预发挥对UC大鼠肺、结肠组织细胞凋亡的改善作用,同时中药通过对以上靶基因的调控,可能为UC及其肺损伤防治提供新的方向。因此,根据各中药所代表的中医治法,鱼腥草与甘草合用所代表的肺肠同治对UC及其肺损伤的细胞凋亡加速的调控作用优于鱼腥草所代表的从肺论治法及甘草所代表的从肠论治法,体现了基于“肺与大肠相表里’的理论中医中药治疗UC及其肺损伤的独特优势。
Objective
Ulcerative Colitis (UC) is a kind of digestive tract disease characterized by chronic inflammation and ulcer formation in colon mucosa layer. UC is rated by the world health organization (WHO) as one of the modern diseases, which is difficult to be cured, with complex Etiology and many pathogcnesis links. Lung and bronchial lesions is one of parenteral manifestations in inflammatory bowel disease, with a high incidence, up to50%, according to literature reports. So far, there has been little research on the mechanism of lung injury in UC. UC is usually treated by amino salicylic acid treatment or cortex steroid, with insufficient effect. In this study, the UC animal model was induced by immune complex, TNBS and ethanol. Sodium houttuyfonate and diammonium glycynhizinate (DG) were used for animals, so as to explore apoptosis mechanism of lung injury in UC, and to reveal biological mechanism of lung injury in UC as well. Efficacy of sodium houttuyfonate and diammonium glycynhizinate was proved by their pathways, targets and the effects on apoptosis regulation,so as to provide an objective basis for the treatment of ulcerative colitis. And it may provide more reliable evidence for TCM treatment of UC and lung injury and further exploring theory of the lung and the large intestine being interioi-exteriorly related.
Methods&Results
I The characteristics of general status and morphological changes in rats with ulcerative colitis.
Healthy male Wistar rats were induced by immune complex, TNBS and ethanol, divided into five groups:model group, western medicine group, sodium houttuyfonate group, diammonium glycynhizinate group, and sodium houttuyfonate plus diammonium glyeyrrhizinate group. Otherwise, there were30rats for normal group.0,2and4weeks after model establishment, general status and morphological changes were observed in rats. General living status of UC rats was worse than the normal rats, with significantly lower weight, breathing, shortness of breath and other symptoms of lung on week O. Pathology of lung tissue, such as lung interstitial inflammatory infiltrates, interstitial fibrosis, etc. was observed on all the time points. Lung injury might be caused by UC which is confirmed by animal models in this study. Therefore, lung injury is one of the parenteral performances of UC.
2Apoptosis of inflammatory cell of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer
According to the principle of randomized, controlled experiment design, the rest of the model rats were randomly divided into five groups, namely model group, western medicine group, sodium houttuyfonate group, diammonium glycyrrhizinate group, and sodium houttuyfonate plus diammonium glycyrrhizinate group, after week0. Each treatment group was administrated by Chinese herb monomer or western medicine, while rats in normal group and model group were administrated by water as blank control. On the2nd and4th weekend, general condition of rats was observed. Otherwise, lung and colon tissue was taken for testing of indicators.
2.1Apoptosis of lymphocytes of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer
Flow cytometry was used for dynamic testing apoptosis of lymphocytes of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer, so as to discuss the relationship between abnormal lymphocytes apoptosis in peripheral blood and occurrence of UC, and to explore the mechanism of the TCM treatment of UC. Lymphocytes apoptosis rate in peripheral blood of rats with UC was significantly reduced. Combined the results of HE staining of colon tissue, it showed immune dysfunction of UC rats, structure damage of colon tissue, and infiltration of inflammatory cells. The results suggest that the lymphocyte apoptosis is involved in the pathogencsis of UC, and slow apoptosis of lymphocyte is closely related with occurrence of UC. Delay of lymphocytes apoptosis might be one of important pathological mechanisms, which may reflect immune function of UC rats.
The Chinese and western medicine applied in the experiment may improve immune function of UC rats and alleviate symptoms by promoting apoptosis of lymphocyte. The improvement of lymphocyte apoptosis in peripheral blood rate by short-term application of diammonium glycyrrhizinate was better than that of sodium houttuyfonate, whose long-term effeet was better than short ones. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate for improving lymphocyte apoptosis was significantly better than single use of sodium houttuyfonate, which suggests superiority of treatment of UC from both lung and intestine.
2.2Apoptosis of neutrophil of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer
Flow cytometry was used for dynamic testing apoptosis of neutrophil of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer, so as to discuss the relationship between abnormal neutrophil apoptosis in peripheral blood and occurrence of UC, and to explore the mechanism of the TCM treatment of UC. Ncutrophils apoptosis was delayed in peripheral blood in model group, compared with normal one on the2nd week, while there was no difference in rats of neutrophils apoptosis between normal and model groups on the4th week, which suggest restoration of the delay. Considering general status of UC rats on the2nd week, such as mucinous or soft feces, ulcer and a large number of inflammatory cells infiltrated in colon tissue from images of HE staining, showed prolonged inflammation effect in blood or local by apoptosis inhibition of neutrophil, which suggest UC rats was in active period, with colon inflammation and ulcers. Otherwise, apoptosis of neutrophil was recovered on the4th week, while pathological damage of colon tissue was still existed, which showed UC rats was in remission on the4th week. It reveals that delayed apoptosis of neutrophil was one of important mechanisms of colon tissue injury in UC, and infiltration of neutrophil might be an important symbol of active UC. Changes in the rate of neutrophil apoptosis may provide certain basis for obsewation of UC and efficacy evaluation of drug. Otherwise, it may be a new ways for further study of neutrophil apoptosis and moderately control of apoptosis for clinical treatment of UC.
In addition, neutrophil apoptosis was induced by use of sodium houttuyfonate and diammonium glycyrrhizinate, so UC rats got into remission in advance. Chinese herb monomer could reduce the inflammatory in a certain extent, so as to improve the mucosal injury, and to play a role in treatment of UC inflammation. Delayed apoptosis of lymphocyte and neutrophil was related with occurrence and development of ulcerative colitis. Chinese herb monomer may control apoptosis rats of lymphocyte and neutrophil in blood to achieve ease of UC.
3Observation of apoptosis related protein, Caspase-3in UC rats and effect of Chinese herb monomer
3.1Observation of apoptosis related protein, Caspase-3in colon tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of apoptosis related protein, Caspase-3in colon tissue of UC rats and effect of Chinese herb monomer, so as to confirm abnormal apoptosis in colon tissue, and to explore the effect of TCM treatment. Content of Caspase-3in colon tissue of UC rats was increased on the2nd and4th week, compared with normal rats, showing apoptosis acceleration of colon tissue existing in UC rats in a certain degree. Therefore, occurrence of UC may be related with excessive expression of Caspase-3in colon tissue of UC rats, which may induce apoptosis of colonic epithelial,and cause injury of colon tissue.
In addition, SASP could improve the increased apoptosis of the colon tissue, but with insufficient long-term effect, while Caspase-3was decreased by Chinese herb in the colon. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate and separate use of diammonium glycyrrhizinale was superior to separate use of sodium houttuyfonate.
3.2Observation of apoptosis related protein, Caspase-3in lung tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of apoptosis related protein, Caspase3in lung tissue of UC rats and effect of Chinese herb monomer, so as to confirm abnormal apoptosis in lung tissue, and to explore the effect of TCM treatment. Content of Caspase-3in lung tissue of UC rats was increased, compared with normal rats on the2nd and4th week, showing apoptosis acceleration of lung tissue existing in UC rats in a certain degree. Therefore, occurrence of lung injury in UC may be related with excessive expression of Caspase-3in lung tissue of UC rats, which may induce apoptosis and cause injury of lung tissue.
In addition, Chinese medicine was superior to SASP in improving the accelerated apoptosis in the lung. On the4th week Caspase-3of the lung was decreased significantly, with no evident changes on the2nd week by diammonium glycyrrhizinate. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate and separate use of sodium houttuyfonate could improve apoptosis of the lung in UC rats, with short and long-term effects. It suggests that the treatments represented by herbs could improve UC by decreasing Caspase-3level, and provide a new way to prevention and treatment of UC and its lung injury.
4Observation of apoptosis related protein, Bcl-2/Bax in UC rats and effect of Chinese herb monomer
4.1Observation of apoptosis related protein, Bcl-2/Bax in colon tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Bcl-2/Bax in colon tissue of UC rats and effect of Chinese herb monomer. In normal group Bcl-2/Bax>1, cell apoptosis is relatively little. While in model group, Bax was continuously increased, and Bcl-2was decreased with Bcl-2/Bax<1, which suggested that cell apoptosis was in the ascendant. Therefore, apoptosis acceleration and abnormal level of Bcl-2/Bax in colon tissue may be one of the molecular mechanisms of colon injury in UC
In addition, Bcl-2was increased with decreased Bax and increased ratio of Bcl-2/Bax by both Chinese medicine and western medicine, which induced inhibition of apoptosis of colon tissue, so as to facilitate repair of the colon mucosa. Sodium houttuyfonate could increase the ratio of Bcl-2/Bax, and inhibit apoptosis of colon tissue, which was superior to SASP with long-term effect. Separate use of diammonium glycyrrhizinate and combined use of sodium houttuyfonate and diammonium glycyrrhizinate could increase the ratio of Bcl-2/Bax, so as to decrease the increased apoptosis of colon tissue. It suggest that Chinese medicine could improve apoptosis of colon tissue at both short and long term stages, which reveals characteristics and effectiveness of TCM treatment of UC from the lung and both from the lung and the intestine.
4.2Observation of apoptosis related protein, Bel-2/Bax in lung tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Bcl-2/Bax in lung tissue of UC rats and effect of Chinese herb monomer. Bcl-2in lung tissue of UC rats was decreased on the2nd and4th week, while Bax was increased, with decreased Bcl-2/Bax, which suggested that increased apoptosis of lung tissue due to abnormal level of Bcl-2and Bax may be one of possible mechanisms of lung injury in UC. In addition, separate use of sodium houttuyfonate and diammonium glycyrrhizinate, and their combined use could increase Bcl-2and decrease Bax, with inhibited effect on apoptosis of lung tissue, which is better than SASP. Otherwise, combined use of sodium houttuyfonate and diammonium glycyrrhizinate was superior to their separate use. Lung disease was treated from the intestine and treated from both lung and intestine, which showed their special significances, providing theoretical basis for the theory of the lung and the large intestine being interior-exteriorly related.
However, the pathogenesis of lung injury is very complicated. The lung and the large intestine being interior-exteriorly related is the result of a variety of substances and mechanisms. Therefore, study of Bcl-2/Bax for UC and lung injury is insufficient, which needs future comprehensive research.
5Observation of apoptosis related protein, Fas/Fas-L in UC rats and effect of Chinese herb monomer
5.1Observation of apoptosis related protein,Fas/Fas-L in colon tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Fas/Fas-L in colon tissue of UC rats and effect of Chinese herb monomer. The content of Fas and Fas-L was increased in model group, compared with normal group, which suggest Fas/Fas-L may also participate in regulation of lung tissue apoptosis. Accelerated apoptosis of colon tissue may be related with abnormal increase of Fas and Fas-L.
In addition, SASP, sodium houttuyfonate and diammonium glycyrrhizinate could decrease Fas or Fas-L in the colon tissue, while combined use of sodium houttuyfonate and diammonium glycyrrhizinate could both decrease Fas and Fas-L. It showed that both herbs and SASP could improve apoptosis of colon tissue, with each characteristic, while the effect of combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than that of SASP.
The change of content of Fas and Fas-L and the intervention of drugs suggest that Fas and Fas-L may participate in pathological changes of colon tissue in UC rats, directly or indirectly. Otherwise, it showed pathways and targets by which herbs and therapies they represented may play a role.
5.2Observation of apoptosis related protein, Fas/Fas-L in lung tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Fas/Fas-L in lung tissue of UC rats and effect of Chinese herb monomer. The content of Fas and Fas-L was increased in lung tissue of UC rats, compared with normal group. Fas-L was decreased on the4th week in sodium houttuyfonate group and sodium houttuyfonate plus diammonium glycyrrhizinate group. There was no significant difference of Fas-L in lung tissue between western medicine group and model group, as well as diammonium glycyrrhizinate group. Tn addition, there was also no difference among other groups in Fas of the lung.
6Observation of NF-κB in UC rats and effect of Chinese herb monomer
6.1Observation of NF-κB in colon tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of NF-κB in colon tissue of UC rats and effect of Chinese herb monomer. The results showed that NF-κB, Bax, Fas, Fas-L and Caspase-3of colon were increased in UC rats on the2nd and4th week, with decreased Bcl-2and the ratio of Bcl-2and Bax. It suggested that NF-κB may regulat apoptosis of colon tissue on gene transcription levels through Bcl-2/Bax, and Fas/Fas-L system.
On the2nd week, NF-κB of colon was decreased in sodium houttuyfonate plus diammonium glycyrrhizinate group, without improvement in other groups. On the4th week, NF-κB of colon was decreased in treatment groups except sodium houttuyfonate group. The results showed that both Chinese and western medicine could improve NF-κB of colon, while the effect of short-term use of SASP (sulfasalazine) was not obvious, and the short-term and long-term combined use of sodium houttuyfonate and diammonium glycyrrhizinate were both fairly good. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than single use of which for intervention of NF-κB in colon tissue. It showed that treatment for both the lung and the large intestine was better than single one. In treatment of UC, compound of Chinese medicine was better than single application of Chinese herbs.6.2Observation of NF-κB in lung tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of NF-κB in lung tissue of UC rats and effect of Chinese herb monomer. The results showed that NF-κB, Bax and Caspase-3in lung tissue of UC rats was increased, while Bcl-2, the ratio of Bcl-2and Bax was decreased, compared with normal ones on the2nd week. It suggested that NF-κB may regulate apoptosis of lung tissue on gene transcription levels through Bcl-2/Bax system. No difference of NF-κB in lung tissue was shown between normal and model groups, while Fas, Fas-L and Caspase-3were still increased and the ratio of Bcl-2and Bax was decreased. It suggested that apoptosis of lung tissue still existed by recovery of lung injury.
NF-κB in lung tissue was obviously decreased in sodium houttuyfonate group and sodium houttuyfonate plus diammonium glycyrrhizinate group on the2nd week, which suggested that single use of sodium houttuyfonate and combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than SASP. Otherwise, combined use of Chinese herbs was better than single use of diammonium glycyrrhizinate, which showed that treatment for both the lung and the large intestine was better than single one.
Conclusion
UC rats were used to evaluate effect of Chinese herb monomers, in order to explore apoptosis regulation mechanism of lung injury and biological mechanism of lung injury in UC. Otherwise, single and combined use of sodium houttuyfonate and diammonium glycyrrhizinate was evaluated for their effect, pathways and targets, which provide an objective basis for the treatment of ulcerative colitis. New ways and methods might be provided for clinical treatment of lung injury in patients. And it may provide more reliable evidence for TCM treatment of UC and lung injury and further exploring theory of the lung and the large intestine being interior-exteriorly related.
1TNBS and ethanol with colonic mucosa tissue induced model rats were used in this experiment, which were relatively close to the clinical patients, with both systemic immune abnormalities and local inflammation pathological changes. The animal model is similar to pathological mechanism of UC in patients, with pathological mechanism of B cells and T cells induced immune response. Therefore, it is a suitable animal model for studying pathological mechanism and treatment effect of UC.
2Delayed apoptosis of inflammatory cells may be related with UC, and may be important cause of continuous inflammation of ulcerative colitis.
2.1Delayed lymphocytes apoptosis in peripheral blood was significant, which suggest that decreased rate of lymphocytes apoptosis may reflect immune function disorder in certain degree. Therefore, delayed lymphocytes apoptosis may be one of the important pathological mechanisms of UC. Thus, a new way to improve the immune function of patients with UC may be provided through intervention of lymphocyte apoptosis.
2.2Apoptosis of neutrophil (PMN) in peripheral blood was delayed in UC rats at acute stages. Therefore, apoptosis of PMN was inhibited by prolonged existence in blood and inflamed lesions, which induced inflammatory injury of tissue, and may be one of the important mechanisms of injury of colon tissue in UC Its seepage may also be used as an important symbol of active UC.
3Increased Bax, Fas, Fas-L, Caspase-3and NF-κB in colon tissue, with decreased Bcl-2, Bcl-2/Bax<1showed that accelerated apoptosis in the colon, which may be through apoptosis pathways, that is, NF-κB-Bcl-2/Bax and Fas/Fas-L systems Caspase-3, in order to preliminarily reveal possible apoptosis pathway in UC. Therefore, to increase Bcl-2. decrease Bax, Fas, Fas-L, Caspasc-3and NF-κB may inhibit accelerated apoptosis in colon tissue in certain degree, by interfering with the apoptosis pathways, so as to promote the repair of colonic mucosa and to provide evidence of molecular biology in treatment of UC.
4In acute phase, Bax, Caspase-3and NF-κB were increased in lung tissue, while Bcl-2was decreased. In remission, NF-κB was improved, while other apoptosis related proteins were still abnormal, which suggested that lung injury was related with number of apoptosis regulatory proteins. Abnormal level of NF-κB, Bcl-2/Bax and Fas/Fas-L systems, and Caspase-3may induce apoptosis in lung injury, which may be one of possible mechanisms of lung injury in UC and lung disease due to the bowel. It may provide a new way to prevent and cure lung injury in UC.
5The results showed thaf apoptosis in lung and colon was increased by possible pathways, that is, NF-κB-Bcl-2/Bax and Fas/Fas-L systems-Caspase-3. While apoptosis regulatory proteins may play different roles along with development of the disease. Theory of the lung and the large intestine being interior-exteriorly related was confirmed by UC animal model, in which tissue pathological injury of the lung and the colon with apoptosis acceleration of lung tissue existed. However, pathogenesis of UC and its lung injury is complicated, and relationship between the lung and the large intestine was correlated with a variety of substances. Thus, it needs future comprehensive research to reveal biological mechanisms of the lung and the large intestine being interior-exteriorly related, not only from the perspective of apoptosis mechanism.
6Sodium houttuyfonate and diammonium glycyrrhizinate with their combined use, and SASP were overall evaluated through the effects on general condition of rats UC, pathology of the lung and the colon, peripheral inflammatory cells, apoptosis regulatory proteins, etc. with their own characteristics. Chinese medicine was generally superior to SASP in this study, which could improve apoptosis of the colon tissue mainly at acute stages, while Chinese medicine could both improve accelerated apoptosis of the colon and the lung tissue at acute and later stages. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than their separate use on improving accelerated apoptosis of the colon and the lung tissue, which reveals compound Chinese medicine is superior to separate use of one herb. In addition, sodium houttuyfonate could also improve apoptosis of the colon tissue in UC rats. Sodium houttuyfonate and diammonium glycyrrhizinate with their combined use, and SASP could improve apoptosis of the colon and the lung through one or more targets of the pathway, that is, NF-κB-Bcl-2/Bax、Fas/Fas-L-Caspase-3. Otherwise, a new way for prevention and cure of UC with its lung injury was provided through regulating and controlling target genes by Chinese medicine. Therefore, treatment from both the lung and the colon was superior to treatment from separate viscera, which showed that unique advantages of the theory of the lung and the large intestine being interior-exteriorly related.
溃疡性结肠炎(Ulcerative Colitis,UC),是一种以慢性炎症和溃疡形成为主要病理特点的结肠粘膜层的消化道疾病。该病病因复杂,发病环节多,治愈难度大,反复发作,并与结肠癌的发病关系密切,被世界卫生组织列为现代难治病之一。肺支气管病损作为炎症性肠病的肠外表现,发生率很高,文献报道可达50%,而关于溃疡性结肠炎肺损伤的发生机制研究至今甚少,治疗上多采用氨基水杨酸类药物和皮质类固醇药物治疗,效果不理想。本课题旨在通过中药单体鱼腥草素钠、甘草酸二铵及两者合用干预免疫复合物加三硝基苯磺酸-乙醇诱导的溃疡性结肠炎大鼠模型,探究溃疡性结肠炎肺损伤的细胞凋亡调控机制,揭示溃疡性结肠炎肺损伤生物学机制；阐明鱼腥草素钠、甘草酸二铵及两者合用治疗溃疡性结肠炎肺损伤的疗效、作用途径、靶点及对细胞凋亡调控的影响,为鱼腥草素钠、甘草酸二铵及两者合用治疗溃疡性结肠炎提供客观依据,并为临床治疗合并肺损伤的溃疡性结肠炎患者提供新的思路与方法,进而为中医治疗UC及其肺损伤从肺论治、从肠论治、肺肠同治的优势提供更为可靠的证据,升华肺与大肠表里脏腑气机升降相关理论。
研究内容与结果
本实验选用SPF级健康雄性Wistar大鼠,采用家兔结肠黏膜组织致敏加三硝基苯磺酸-50%乙醇灌肠的方法复制大鼠UC模型。造模0周(模型成立时)杀检取材后,将剩余模型大鼠随机分为五组,即模型组、西药组、鱼腥草组、甘草组、鱼腥草+甘草组。鱼腥草组给予鱼腥草素钠片水溶液灌胃治疗；甘草组给予甘草酸二铵胶囊水溶液灌胃治疗；鱼腥草+甘草组给予鱼腥草素钠片与甘草酸二铵胶囊的混合水溶液灌胃治疗；西药以柳氮磺吡啶肠溶片水溶液为阳性对照药灌胃治疗；正常组与模型组同步以纯净水灌胃作空白对照。于给药2、4周末,观察大鼠一般状态,麻醉后,腹主动脉取血,并取肺、结肠组织进行相关指标检测。
1溃疡性结肠炎大鼠一般状态及肺和结肠组织形态学变化特点
各时间点UC大鼠模型一般生活状态差,0周时模型组大鼠消瘦明显,并有呼吸气急、喘促等肺部症状。结肠组织HE染色图片显示0周时有明显的坏死灶侵及结肠粘膜的上皮层、黏膜层、黏膜下层,部分结肠组织出现凝固性坏死。肠壁少数坏死黏膜组织脱落后形成溃疡点,大量炎性细胞浸润粘膜下层；2周时病变与0周比较未见明显恢复；4周时结肠组织病变有一定程度的恢复,部分结肠组织粘膜下层仍有大量炎性细胞浸润及溃疡形成,粘膜上皮和固有膜内腺体减少、萎缩、破坏甚至消失。肺组织HE染色图片表现为0周时肺间质广泛的炎症,炎性细胞较多呈灶状分布、集中在支气管、血管的周围,部分肺泡变形；2周模型组大鼠肺组织病理与0周比较,未见明显改变,支气管、血管周围仍有大量的炎性细胞浸润肺,且呈灶状分布,可见到支气管壁有明显的增厚；4周时,UC模型组大鼠肺组织病变较0、2周时有一定程度的改善,但部分大鼠肺组织中仍有大量淋巴细胞呈灶状分布浸润,血管壁、支气管壁增厚,可见到部分支气管壁有一定程度的纤维组织增生形成。动物模型证实溃疡性结肠炎可以引起肺损伤,肺损伤是UC的肠外表现之一。
2溃疡性结肠炎大鼠外周血炎性细胞凋亡及中药单体的干预作用
2.1溃疡性结肠炎大鼠外周血淋巴细胞(lamina proprial lymphocyte, LPL)凋亡及中药的干预作用
为探讨外周血淋巴细胞凋亡异常与溃疡性结肠炎发生、发展的关系,以及中药治疗UC的可能作用机制,本研究采用流式细胞术动态检测、观察UC大鼠外周血中淋巴细胞凋亡率的变化以及中药对外周血淋巴细胞凋亡异常的干预作用。结果表明,2、4周时模型组大鼠外周血淋巴细胞凋亡率显著降低,提示淋巴细胞凋亡减慢可能是导致UC模型大鼠免疫功能紊乱的重要原因之一,从而与UC的发生密切相关。另外,外周血淋巴细胞凋亡率降低还可能与UC大鼠结肠组织病理学结果中的淋巴细胞浸润有关。
本实验应用的中西药物均能不同程度地促进淋巴细胞凋亡,从而调节UC大鼠的免疫功能,达到缓解UC症状的目的。甘草的短期应用(2周)对外周血淋巴细胞凋亡率的改善作用优于鱼腥草,而鱼腥草长期(4周)治疗较短期治疗对外周血淋巴细胞凋亡率的改善作用明显,鱼腥草与甘草合用对外周血淋巴细胞凋亡率的改善作用明显优于单用鱼腥草治疗,体现了两药合用所代表的肺肠同治的优越性。
2.2溃疡性结肠炎大鼠外周血中性粒细胞(polymorphonuclear neutrophils,PMN)凋亡及中药的干预作用
为探讨外周血中性粒细胞凋亡异常与溃疡性结肠炎发生、发展的关系,以及中药治疗UC的可能作用机制,本研究采用流式细胞术动态检测、观察UC大鼠外周血中性粒细胞凋亡率的变化以及中药对外周血中性粒细胞凋亡异常的干预作用。结果发现2周时UC模型组大鼠血中性粒细胞凋亡与正常组比较呈延迟现象;4周时,模型大鼠PMN凋亡率与正常组比较无明显差异,PMN凋亡延迟的现象得到恢复。结合造模后2周时UC大鼠的表现,UC大鼠粪便不成形,为黏液便或软变,结肠组织HE染色仍有溃疡、大量炎性细胞浸润,说明PMN凋亡抑制使PMN在血液循环和炎症部位的作用时间延长,提示模型大鼠处于UC活动期,结肠炎症反复、溃疡改变。同时,随着时间延长,模型大鼠PMN凋亡率明显恢复,而结肠组织病理损伤依然存在,提示4周时UC大鼠处于缓解期。表明PMN凋亡延迟可能是UC肠组织损伤的重要机制之一,并且PMN浸润是活动性UC的一个重要标志。PMN凋亡率的变化可能为临床UC病情观察及药物疗效评价提供一定的依据,进一步深入研究PMN凋亡,乃至适度控制其凋亡将为临床治疗UC开辟新的途径。
另外,中药单体甘草酸二铵单用、鱼腥草素钠与甘草酸二铵合用可能通过诱导中性粒细胞凋亡使UC大鼠提前进入缓解期,能在一定程度上减轻炎症反应,从而改善黏膜损伤,对UC炎症的发展发挥一定的治疗作用。LPL及PMN凋亡的减慢和溃疡性结肠炎的发生、发展存在一定关系,中药单体可能通过对外周血LPL与PMN凋亡率的调控,达到缓解UC目的。
3溃疡性结肠炎大鼠肺、结肠组织凋亡效应蛋白Caspase-3的含量及中药的干预作用
3.1溃疡性结肠炎大鼠结肠组织凋亡效应蛋白Caspase-3的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)动态观察溃疡性结肠炎大鼠结肠组织凋亡效应蛋白Caspase-3的含量,以探究UC大鼠结肠组织细胞凋亡是否存在异常,及中药对可能存在的凋亡异常的干预作用。结果表明,造模后2周及4周,UC模型组大鼠结肠组织中Caspase-3的含量与正常组比较均有明显的增加,提示UC模型大鼠结肠组织存在一定程度的细胞凋亡加速。Caspase-3是Caspase家族中的关键效应酶,是凋亡执行的重要效应分子,一般以23Ku的无活性前体存在于细胞质中,当细胞进入凋亡时才被激活,它参与多种因素如Fas系统、Bcl-2家族诱导的细胞凋亡。推测UC的发生可能与结肠组织凋亡效应蛋白Caspase-3的过量表达,诱导结肠组织细胞的凋亡有关,从而导致结肠组织损伤。
另外,西药柳氮磺胺吡啶能有效改善UC结肠组织细胞凋亡增加,但长期疗效不佳,而中药治疗UC在改善结肠组织Caspase-3过量表达方面优于西药,其中,中药甘草单用、甘草与鱼腥草合用短期应用优于单用鱼腥草。
3.2溃疡性结肠炎大鼠肺组织凋亡效应蛋白Caspase-3的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)观察溃疡性结肠炎大鼠肺组织凋亡效应蛋白Caspase-3的含量,以探究UC大鼠肺组织细胞凋亡是否存在异常,及中药对可能存在的凋亡异常的干预作用。结果表明,造模后2周及4周,UC模型组大鼠肺组织中Caspase-3的含量与正常组比较均有明显的增加,提示UC模型大鼠肺组织存在一定程度的细胞凋亡加速,UC大鼠肺组织细胞凋亡的发生与凋亡效应蛋白Caspase-3的过多表达存在一定关系。
另外,中药对UC肺损伤在细胞凋亡方面的干预作用优于西药柳氮磺胺吡啶。中药甘草在治疗2周时对肺组织Caspase-3干预作用尚不明显,治疗4周时能有效降低Caspase-3的含量。中药鱼腥草单用、鱼腥草与甘草合用均能有效改善UC大鼠肺损伤细胞凋亡的增加,且短期与长期治疗均有效。表明各中药及其所代表的治法可能通过干预凋亡效应蛋白Caspase-3的过量表达发挥治疗作用,可能为中医中药防治UC肺损伤提供了新的思路及治疗方法。
4溃疡性结肠炎大鼠肺、结肠组织凋亡调控蛋白Bcl-2、Bax的表达及中药的干预作用
4.1溃疡性结肠炎大鼠结肠组织凋亡调控蛋白Bcl-2、Bax的表达及中药的干预作用
采用Western-bloting观察UC大鼠结肠组织凋亡调控蛋白Bcl-2、Bax的表达以及中药的干预作用。结果表明,2、4周正常组Bcl-2/Bax>1,细胞凋亡发生相对较少；模型组Bax蛋白表达持续增高,Bcl-2表达减少,Bcl-2/Bax
4.2溃疡性结肠炎大鼠肺组织凋亡调控蛋白Bcl-2、Bax的表达及中药的干预作用
采用Western-bloting观察UC大鼠肺组织凋亡调控蛋白Bcl-2、Bax的表达以及中药的干预作用。结果表明,造模后2周、4周时UC模型大鼠肺组织Bcl-2蛋白表达水平明显下降,Bax蛋白表达水平明显上升,Bcl-2/Bax比值显著降低,提示Bcl-2、Bax蛋白表达异常、Bcl-2/Bax比例失衡,并诱导肺组织细胞凋亡可能是UC肺损伤和“肠病及肺”的可能机制之一
另外,鱼腥草、甘草单用及鱼腥草与甘草合用通过上调Bcl-2、下调Bax,对肺组织的细胞凋亡加速均有一定的抑制作用,且优于西药柳氮磺胺吡啶；鱼腥草与甘草合用对UC大鼠肺组织凋亡的改善明显早于鱼腥草或甘草单用。说明中医肺病治肠以及肺肠同治改善UC肺损伤有其特殊意义,以干预Bcl-2/Bax细胞凋亡信号转导途径为中心,抑制肺组织细胞凋亡可能是治疗UC肺损伤肺病治肠以及肺肠同治的机制之一,为“肺与大肠相表里”相关研究的提供理论基础。
然而,UC肺损伤的发病机制十分复杂,且肺与大肠表里关系是多种物质和机制共同作用的结果,仅仅从Bcl-2/Bax调控细胞凋亡角度开展研究尚不能全面揭示UC及其肺损伤从肺论治、从肠论治、肺肠同治的可能生物学机制,更有待于今后从更广更深层面开展综合性相关研究。
5溃疡性结肠炎大鼠肺、结肠组织Fas/Fas-L系统对细胞凋亡的调控及中药的干预作用
5.1溃疡性结肠炎大鼠结肠组织Fas/Fas-L系统对细胞凋亡的调控及中药的干预作用
采用Western-bloting观察UC大鼠肺组织凋亡调控蛋白Fas/Fas-L的表达以及中药的干预作用。结果表明,UC模型组大鼠结肠组织中Fas、Fas-L蛋白表达量与正常组比较明显增加,提示Fas/Fas-L系统可能也参与溃疡性结肠炎大鼠肺组织细胞凋亡的调控,UC模型大鼠结肠组织细胞凋亡的加速可能与Fas、Fas-L蛋白的表达异常增多有关。
另外,柳氮磺胺吡啶、鱼腥草、甘草均能明显下调UC大鼠结肠组织的Fas或Fas-L蛋白表达,而鱼腥草与甘草合用对Fas、Fas-L的过多表达均有明显的抑制作用。说明中西药物对UC结肠组织的细胞凋亡均有一定程度的改善作用,但各有特点,且中药鱼腥草与甘草合用对UC大鼠结肠组织细胞凋亡的改善作用明显优于两药单用及西药柳氮磺胺吡啶。
UC大鼠结肠组织Fas或Fas-L蛋白表达的变化及药物对两者干预的变化,说明Fas/FasL系统可能直接或间接地参与了UC结肠组织的病理变化,以及各中药、各中药所代表的治法干预UC时发挥作用的可能作用途径及靶点。
5.2溃疡性结肠炎大鼠肺组织Fas/Fas-L系统对细胞凋亡的调控及中药的干预作用
采用Western-bloting观察UC大鼠肺组织凋亡调控蛋白Fas、Fas-L的表达以及中药的干预作用。结果表明,UC模型组大鼠肺组织中Fas、Fas-L蛋白的表达量明显高于正常组,给药4周后鱼腥草组及鱼腥草+甘草组肺组织Fas-L蛋白的表达量明显下降,西药组及甘草组对肺组织Fas-L蛋白表达无明显改善作用。结果提示Fas/Fas-L系统蛋白的过量表达参与UC肺组织细胞的凋亡加速,中药鱼腥草单用、鱼腥草与甘草合用对UC大鼠肺组织细胞凋亡异常的干预作用优于西药柳氮磺胺吡啶。
6溃疡性结肠炎肺、结肠组织核因子NF-κB的含量及中药的干预作用
6.1溃疡性结肠炎结肠组织核因子NF-κB的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)观察溃疡性结肠炎大鼠结肠组织凋亡效应蛋白NF-κB的含量,及中药的干预作用。结果表明,造模后2周、4周UC模型组大鼠结肠组织中NF-κB的含量均有明显的增加,结合模型组大鼠结肠组织Bcl-2、Bcl-2/Bax下调、Bax、Fas、Fas-L蛋白表达上调、Caspase-3含量的增加,提示NF-κB可能通过对Bcl-2/Bax、 Fas/Fas-L系统在基因转录水平的调控参与UC大鼠结肠组织的细胞凋亡
治疗2周时仅有鱼腥草+甘草组能明显降低大鼠结肠组织中NF-κB的含量,其他治疗组均无明显改善,治疗4周时,除鱼腥草组外,其他各治疗组大鼠结肠组织中NF-κB的含量均有明显降低。结果提示,中西药物在改善UC大鼠结肠组织NF-κB方面均有明显作用,其中西药柳氮磺胺吡啶短期干预效果不佳；鱼腥草与甘草合用对改善UC大鼠结肠组织NF-κB短期、长期均有效。短期给予鱼腥草或甘草干预UC大鼠结肠组织NF-κB与两中药合用比较,后者作用明显较好,体现了鱼腥草与甘草合用所代表的肺肠同治改善UC大鼠结肠组织NF-κB作用优于鱼腥草所代表的从肺论治及甘草所代表的从肠论治,同时推测中药干预治疗UC,复方作用优于单味药物的应用,且与临床中药应用于UC的实际情况有较高的一致性。
6.2溃疡性结肠炎肺组织核因子NF-κB的含量及中药的干预作用
采用酶联免疫吸附法(ELISA)观察溃疡性结肠炎大鼠肺组织凋亡效应蛋白NF-κB的含量,及中药的干预作用。结果表明,造模2周时UC模型组大鼠肺组织中NF-κB的含量与正常组比较有明显的增加,结合2周时模型组大鼠肺组织Bcl-2、Bcl-2/Bax下调,Bax蛋白表达上调Caspase-3含量的增加,提示NF-κB可能通过对Bcl-2/Bax系统在基因转录水平的调控参与急性期UC大鼠肺组织的细胞凋亡。造模4周时UC模型组大鼠肺组织中NF-κB的含量与正常组比较未见有明显的改变,但肺组织仍存在Bcl-2/Bax比值下降,Fas、Fas-L蛋白表达、Caspase-3含量的增加,表明随着UC大鼠肺损伤一定程度的恢复,肺组织细胞凋亡依然存在。
治疗2周时,鱼腥草组、鱼腥草+甘草组与模型组比较大鼠肺组织中NF-κB的含量均有明显的下降,提示中药鱼腥草单用、鱼腥草与甘草合用对UC肺组织细胞凋亡在干预NF-κB方面明显优于西药,且中药鱼腥草与甘草合用优于甘草单用,说明鱼腥草与甘草所代表的肺肠同治对肺组织NF-κB的干预作用优于甘草所代表的从肠论治。
结论
通过中药单体鱼腥草素钠、甘草酸二铵及两者合用干预免疫复合物加三硝基苯磺酸-乙醇诱导的溃疡性结肠炎大鼠模型,探究溃疡性结肠炎肺损伤的细胞凋亡调控机制,揭示溃疡性结肠炎肺损伤的生物学机制；阐明鱼腥草、甘草及两者合用治疗溃疡性结肠炎肺损伤的疗效、作用途径、靶点及对细胞凋亡调控的影响,为鱼腥草、甘草及两者合用治疗溃疡性结肠炎提供客观依据,并为临床治疗合并肺损伤的溃疡性结肠炎患者提供新的思路与方法,进而为中医治疗UC及其肺损伤从肺论治、从肠论治、肺肠同治的优势提供更为可靠的证据,升华肺与大肠表里脏腑气机升降相关理论,为中医肺与大肠相表里的脏腑相关理论提供了依据。
1本实验采用TNBS-乙醇诱导复合结肠黏膜组织致敏UC大鼠模型,此模型可达到全身免疫异常与局部炎症病变的共存,产生与人类UC较为接近的临床症状及肠道病变,符合人UC病变由B细胞介导的体液免疫反应和T细胞介导的细胞免疫反应共同作用的病变机制,是比较理想的研究UC病理机制及中药干预作用的动物模型。
2炎性细胞凋亡延迟参与UC的发病,是造成溃疡性结肠炎炎症持续的重要原因。
2.1UC大鼠外周血淋巴细胞凋亡存在明显的延迟,提示淋巴细胞凋亡率的下降可能从一定程度上反映了UC大鼠免疫功能的障碍,淋巴细胞凋亡延迟可能是UC发生的重要病理机制之一。因此,通过对淋巴细胞凋亡延迟的干预,为改善UC患者免疫功能提供了新的思路。
2.2急性期UC大鼠外周血中性粒细胞(PMN)存在明显的凋亡延迟,PMN凋亡受到抑制使PMN在血液循环和炎症部位的作用时间延长,并释放活性介质,加重炎症反应,进一步引起组织损伤,是UC肠组织损伤的重要机制之一,其浸润还可作为活动性UC的一个重要标志,诱导中性粒细胞凋亡可能是药物干预使UC大鼠提前进入缓解期的原因之一,而缓解期UC大鼠外周血中性粒细胞凋亡延迟的现象不明显.
3UC大鼠结肠组织凋亡蛋白Bax表达持续增高,Bcl-2表达减少,Bcl-2/Bax<1,Fas. Fas-L蛋白的表达、Caspase-3、NF-κB含量显著增加,结肠组织细胞凋亡明显占优势,表明结肠组织细胞凋亡加速,并且UC大鼠结肠组织细胞凋亡叫能通过NF-κB-Bcl-2/Bax、 Fas/Fas-L系统-Caspase-3凋亡通路实现,从而初步明确了UC发生的可能细胞凋亡途径。因此,通过对该凋亡通路的阻断或干预,有效上调UC大鼠结肠组织Bcl-2,降低Bax、Fas. Fas-L、Caspase-3、NF-κB的表达,恢复Bcl-2/Bax比值,可能对结肠组织细胞凋亡加速具有一定的抑制作用,从而促进结肠粘膜的修复,为药物干预治疗UC提供分了生物学依据。
4急性期UC大鼠肺组织凋亡蛋白Bax表达增高,Bcl-2表达减少,Bcl-2/Bax<1, Caspase-3、NF-κB含量显著增加,存在凋亡加速；缓解期UC大鼠肺组织凋亡蛋白NF-κB的恢复,而其他凋亡蛋白的变化依然存在,提示UC肺损伤的发生可能与诸多凋亡调控蛋白相关。其中,NF-κB、Bcl-2/Bax、Fas/Fas-L系统、Caspase-3凋亡相关蛋白的异常表达并诱导肺组织细胞凋亡可能是UC肺损伤和“肠病及肺”的可能机制之一。通过对以上靶基因的调控,可能为UC肺损伤防治提供新的方向。
5综合本研究中肠与肺相关凋亡蛋白的检测结果,表明UC大鼠结肠与肺组织细胞凋亡增加均不同程度的通过NF-κB-Bcl-2/Bax、Fas/Fas-L系统-Caspase-3凋亡通路实现,然而在对此通路中发挥作用的凋亡调节蛋白的变化随疾病进展又有所不同。UC大鼠存在肺、结肠组织病理损伤及肺组织细胞凋亡加速,从模型上证实了肺与大肠相表里的肺肠相关理论。因此,以干预细胞凋亡信号转导途径为中心,抑制肺、结肠组织细胞凋亡的研究,可能是UC肺损伤及“肺与大肠相表里”相关研究的主要内容之一。然而,UC及其肺损伤的发病机制十分复杂,且肺与大肠表里关系是多种物质和机制共同作用的结果,仅仅从细胞凋亡角度开展研究尚不能全面揭示肺与大肠表里关系的生物学机制内涵,有待于今后从更广更深层面开展综合性相关研究。
6整体评价中药鱼腥草、甘草、鱼腥草与甘草合用,以及西药柳氮磺胺吡啶,在改善UC大鼠一般状态、肺、结肠组织病理、外周血炎性细胞、凋亡调控蛋白等方面均有一定的作用,但各有特点。中药作用总体优于西药柳氮磺胺吡啶,柳氮磺胺吡啶对UC大鼠结肠组织细胞凋亡的改善主要体现在急性期,而中药不仅能有效改善UC急性期肺、结肠组织的细胞凋亡加速,短期疗效显著,而且长期治疗亦有改善作用。鱼腥草与甘草合用对UC及其肺损伤细胞凋亡的干预作用较两味中药单用作用更为肯定,比如2周时鱼腥草+甘草组大鼠结肠组织NF-κB与模型组比较有明显的下降,而鱼腥草组、甘草组均无明显变化；2周时鱼腥草+甘草组大鼠肺组织Bcl-2/Bax与模型组比较有明显的上升,而鱼腥草组、甘草组均无明显变化,一定程度上体现了中药复方作用优于单味中药。另外,鱼腥草对UC大鼠结肠组织细胞凋亡亦存在一定程度的改善作用。通过对本研究中凋亡通路的研究,发现中药鱼腥草、甘草、鱼腥草与甘草合用,及西药柳氮磺胺吡啶可能通过对NF-κB-Bcl-2/Bax、Fas/Fas-L系统-Caspase-3途径中的一个或多个环节和／或靶点的干预发挥对UC大鼠肺、结肠组织细胞凋亡的改善作用,同时中药通过对以上靶基因的调控,可能为UC及其肺损伤防治提供新的方向。因此,根据各中药所代表的中医治法,鱼腥草与甘草合用所代表的肺肠同治对UC及其肺损伤的细胞凋亡加速的调控作用优于鱼腥草所代表的从肺论治法及甘草所代表的从肠论治法,体现了基于“肺与大肠相表里’的理论中医中药治疗UC及其肺损伤的独特优势。
Objective
Ulcerative Colitis (UC) is a kind of digestive tract disease characterized by chronic inflammation and ulcer formation in colon mucosa layer. UC is rated by the world health organization (WHO) as one of the modern diseases, which is difficult to be cured, with complex Etiology and many pathogcnesis links. Lung and bronchial lesions is one of parenteral manifestations in inflammatory bowel disease, with a high incidence, up to50%, according to literature reports. So far, there has been little research on the mechanism of lung injury in UC. UC is usually treated by amino salicylic acid treatment or cortex steroid, with insufficient effect. In this study, the UC animal model was induced by immune complex, TNBS and ethanol. Sodium houttuyfonate and diammonium glycynhizinate (DG) were used for animals, so as to explore apoptosis mechanism of lung injury in UC, and to reveal biological mechanism of lung injury in UC as well. Efficacy of sodium houttuyfonate and diammonium glycynhizinate was proved by their pathways, targets and the effects on apoptosis regulation,so as to provide an objective basis for the treatment of ulcerative colitis. And it may provide more reliable evidence for TCM treatment of UC and lung injury and further exploring theory of the lung and the large intestine being interioi-exteriorly related.
Methods&Results
I The characteristics of general status and morphological changes in rats with ulcerative colitis.
Healthy male Wistar rats were induced by immune complex, TNBS and ethanol, divided into five groups:model group, western medicine group, sodium houttuyfonate group, diammonium glycynhizinate group, and sodium houttuyfonate plus diammonium glyeyrrhizinate group. Otherwise, there were30rats for normal group.0,2and4weeks after model establishment, general status and morphological changes were observed in rats. General living status of UC rats was worse than the normal rats, with significantly lower weight, breathing, shortness of breath and other symptoms of lung on week O. Pathology of lung tissue, such as lung interstitial inflammatory infiltrates, interstitial fibrosis, etc. was observed on all the time points. Lung injury might be caused by UC which is confirmed by animal models in this study. Therefore, lung injury is one of the parenteral performances of UC.
2Apoptosis of inflammatory cell of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer
According to the principle of randomized, controlled experiment design, the rest of the model rats were randomly divided into five groups, namely model group, western medicine group, sodium houttuyfonate group, diammonium glycyrrhizinate group, and sodium houttuyfonate plus diammonium glycyrrhizinate group, after week0. Each treatment group was administrated by Chinese herb monomer or western medicine, while rats in normal group and model group were administrated by water as blank control. On the2nd and4th weekend, general condition of rats was observed. Otherwise, lung and colon tissue was taken for testing of indicators.
2.1Apoptosis of lymphocytes of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer
Flow cytometry was used for dynamic testing apoptosis of lymphocytes of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer, so as to discuss the relationship between abnormal lymphocytes apoptosis in peripheral blood and occurrence of UC, and to explore the mechanism of the TCM treatment of UC. Lymphocytes apoptosis rate in peripheral blood of rats with UC was significantly reduced. Combined the results of HE staining of colon tissue, it showed immune dysfunction of UC rats, structure damage of colon tissue, and infiltration of inflammatory cells. The results suggest that the lymphocyte apoptosis is involved in the pathogencsis of UC, and slow apoptosis of lymphocyte is closely related with occurrence of UC. Delay of lymphocytes apoptosis might be one of important pathological mechanisms, which may reflect immune function of UC rats.
The Chinese and western medicine applied in the experiment may improve immune function of UC rats and alleviate symptoms by promoting apoptosis of lymphocyte. The improvement of lymphocyte apoptosis in peripheral blood rate by short-term application of diammonium glycyrrhizinate was better than that of sodium houttuyfonate, whose long-term effeet was better than short ones. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate for improving lymphocyte apoptosis was significantly better than single use of sodium houttuyfonate, which suggests superiority of treatment of UC from both lung and intestine.
2.2Apoptosis of neutrophil of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer
Flow cytometry was used for dynamic testing apoptosis of neutrophil of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer, so as to discuss the relationship between abnormal neutrophil apoptosis in peripheral blood and occurrence of UC, and to explore the mechanism of the TCM treatment of UC. Ncutrophils apoptosis was delayed in peripheral blood in model group, compared with normal one on the2nd week, while there was no difference in rats of neutrophils apoptosis between normal and model groups on the4th week, which suggest restoration of the delay. Considering general status of UC rats on the2nd week, such as mucinous or soft feces, ulcer and a large number of inflammatory cells infiltrated in colon tissue from images of HE staining, showed prolonged inflammation effect in blood or local by apoptosis inhibition of neutrophil, which suggest UC rats was in active period, with colon inflammation and ulcers. Otherwise, apoptosis of neutrophil was recovered on the4th week, while pathological damage of colon tissue was still existed, which showed UC rats was in remission on the4th week. It reveals that delayed apoptosis of neutrophil was one of important mechanisms of colon tissue injury in UC, and infiltration of neutrophil might be an important symbol of active UC. Changes in the rate of neutrophil apoptosis may provide certain basis for obsewation of UC and efficacy evaluation of drug. Otherwise, it may be a new ways for further study of neutrophil apoptosis and moderately control of apoptosis for clinical treatment of UC.
In addition, neutrophil apoptosis was induced by use of sodium houttuyfonate and diammonium glycyrrhizinate, so UC rats got into remission in advance. Chinese herb monomer could reduce the inflammatory in a certain extent, so as to improve the mucosal injury, and to play a role in treatment of UC inflammation. Delayed apoptosis of lymphocyte and neutrophil was related with occurrence and development of ulcerative colitis. Chinese herb monomer may control apoptosis rats of lymphocyte and neutrophil in blood to achieve ease of UC.
3Observation of apoptosis related protein, Caspase-3in UC rats and effect of Chinese herb monomer
3.1Observation of apoptosis related protein, Caspase-3in colon tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of apoptosis related protein, Caspase-3in colon tissue of UC rats and effect of Chinese herb monomer, so as to confirm abnormal apoptosis in colon tissue, and to explore the effect of TCM treatment. Content of Caspase-3in colon tissue of UC rats was increased on the2nd and4th week, compared with normal rats, showing apoptosis acceleration of colon tissue existing in UC rats in a certain degree. Therefore, occurrence of UC may be related with excessive expression of Caspase-3in colon tissue of UC rats, which may induce apoptosis of colonic epithelial,and cause injury of colon tissue.
In addition, SASP could improve the increased apoptosis of the colon tissue, but with insufficient long-term effect, while Caspase-3was decreased by Chinese herb in the colon. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate and separate use of diammonium glycyrrhizinale was superior to separate use of sodium houttuyfonate.
3.2Observation of apoptosis related protein, Caspase-3in lung tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of apoptosis related protein, Caspase3in lung tissue of UC rats and effect of Chinese herb monomer, so as to confirm abnormal apoptosis in lung tissue, and to explore the effect of TCM treatment. Content of Caspase-3in lung tissue of UC rats was increased, compared with normal rats on the2nd and4th week, showing apoptosis acceleration of lung tissue existing in UC rats in a certain degree. Therefore, occurrence of lung injury in UC may be related with excessive expression of Caspase-3in lung tissue of UC rats, which may induce apoptosis and cause injury of lung tissue.
In addition, Chinese medicine was superior to SASP in improving the accelerated apoptosis in the lung. On the4th week Caspase-3of the lung was decreased significantly, with no evident changes on the2nd week by diammonium glycyrrhizinate. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate and separate use of sodium houttuyfonate could improve apoptosis of the lung in UC rats, with short and long-term effects. It suggests that the treatments represented by herbs could improve UC by decreasing Caspase-3level, and provide a new way to prevention and treatment of UC and its lung injury.
4Observation of apoptosis related protein, Bcl-2/Bax in UC rats and effect of Chinese herb monomer
4.1Observation of apoptosis related protein, Bcl-2/Bax in colon tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Bcl-2/Bax in colon tissue of UC rats and effect of Chinese herb monomer. In normal group Bcl-2/Bax>1, cell apoptosis is relatively little. While in model group, Bax was continuously increased, and Bcl-2was decreased with Bcl-2/Bax<1, which suggested that cell apoptosis was in the ascendant. Therefore, apoptosis acceleration and abnormal level of Bcl-2/Bax in colon tissue may be one of the molecular mechanisms of colon injury in UC
In addition, Bcl-2was increased with decreased Bax and increased ratio of Bcl-2/Bax by both Chinese medicine and western medicine, which induced inhibition of apoptosis of colon tissue, so as to facilitate repair of the colon mucosa. Sodium houttuyfonate could increase the ratio of Bcl-2/Bax, and inhibit apoptosis of colon tissue, which was superior to SASP with long-term effect. Separate use of diammonium glycyrrhizinate and combined use of sodium houttuyfonate and diammonium glycyrrhizinate could increase the ratio of Bcl-2/Bax, so as to decrease the increased apoptosis of colon tissue. It suggest that Chinese medicine could improve apoptosis of colon tissue at both short and long term stages, which reveals characteristics and effectiveness of TCM treatment of UC from the lung and both from the lung and the intestine.
4.2Observation of apoptosis related protein, Bel-2/Bax in lung tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Bcl-2/Bax in lung tissue of UC rats and effect of Chinese herb monomer. Bcl-2in lung tissue of UC rats was decreased on the2nd and4th week, while Bax was increased, with decreased Bcl-2/Bax, which suggested that increased apoptosis of lung tissue due to abnormal level of Bcl-2and Bax may be one of possible mechanisms of lung injury in UC. In addition, separate use of sodium houttuyfonate and diammonium glycyrrhizinate, and their combined use could increase Bcl-2and decrease Bax, with inhibited effect on apoptosis of lung tissue, which is better than SASP. Otherwise, combined use of sodium houttuyfonate and diammonium glycyrrhizinate was superior to their separate use. Lung disease was treated from the intestine and treated from both lung and intestine, which showed their special significances, providing theoretical basis for the theory of the lung and the large intestine being interior-exteriorly related.
However, the pathogenesis of lung injury is very complicated. The lung and the large intestine being interior-exteriorly related is the result of a variety of substances and mechanisms. Therefore, study of Bcl-2/Bax for UC and lung injury is insufficient, which needs future comprehensive research.
5Observation of apoptosis related protein, Fas/Fas-L in UC rats and effect of Chinese herb monomer
5.1Observation of apoptosis related protein,Fas/Fas-L in colon tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Fas/Fas-L in colon tissue of UC rats and effect of Chinese herb monomer. The content of Fas and Fas-L was increased in model group, compared with normal group, which suggest Fas/Fas-L may also participate in regulation of lung tissue apoptosis. Accelerated apoptosis of colon tissue may be related with abnormal increase of Fas and Fas-L.
In addition, SASP, sodium houttuyfonate and diammonium glycyrrhizinate could decrease Fas or Fas-L in the colon tissue, while combined use of sodium houttuyfonate and diammonium glycyrrhizinate could both decrease Fas and Fas-L. It showed that both herbs and SASP could improve apoptosis of colon tissue, with each characteristic, while the effect of combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than that of SASP.
The change of content of Fas and Fas-L and the intervention of drugs suggest that Fas and Fas-L may participate in pathological changes of colon tissue in UC rats, directly or indirectly. Otherwise, it showed pathways and targets by which herbs and therapies they represented may play a role.
5.2Observation of apoptosis related protein, Fas/Fas-L in lung tissue of UC rats and effect of Chinese herb monomer
Western-bloting was used for dynamic observation of apoptosis related protein, Fas/Fas-L in lung tissue of UC rats and effect of Chinese herb monomer. The content of Fas and Fas-L was increased in lung tissue of UC rats, compared with normal group. Fas-L was decreased on the4th week in sodium houttuyfonate group and sodium houttuyfonate plus diammonium glycyrrhizinate group. There was no significant difference of Fas-L in lung tissue between western medicine group and model group, as well as diammonium glycyrrhizinate group. Tn addition, there was also no difference among other groups in Fas of the lung.
6Observation of NF-κB in UC rats and effect of Chinese herb monomer
6.1Observation of NF-κB in colon tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of NF-κB in colon tissue of UC rats and effect of Chinese herb monomer. The results showed that NF-κB, Bax, Fas, Fas-L and Caspase-3of colon were increased in UC rats on the2nd and4th week, with decreased Bcl-2and the ratio of Bcl-2and Bax. It suggested that NF-κB may regulat apoptosis of colon tissue on gene transcription levels through Bcl-2/Bax, and Fas/Fas-L system.
On the2nd week, NF-κB of colon was decreased in sodium houttuyfonate plus diammonium glycyrrhizinate group, without improvement in other groups. On the4th week, NF-κB of colon was decreased in treatment groups except sodium houttuyfonate group. The results showed that both Chinese and western medicine could improve NF-κB of colon, while the effect of short-term use of SASP (sulfasalazine) was not obvious, and the short-term and long-term combined use of sodium houttuyfonate and diammonium glycyrrhizinate were both fairly good. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than single use of which for intervention of NF-κB in colon tissue. It showed that treatment for both the lung and the large intestine was better than single one. In treatment of UC, compound of Chinese medicine was better than single application of Chinese herbs.6.2Observation of NF-κB in lung tissue of UC rats and effect of Chinese herb monomer
Enzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of NF-κB in lung tissue of UC rats and effect of Chinese herb monomer. The results showed that NF-κB, Bax and Caspase-3in lung tissue of UC rats was increased, while Bcl-2, the ratio of Bcl-2and Bax was decreased, compared with normal ones on the2nd week. It suggested that NF-κB may regulate apoptosis of lung tissue on gene transcription levels through Bcl-2/Bax system. No difference of NF-κB in lung tissue was shown between normal and model groups, while Fas, Fas-L and Caspase-3were still increased and the ratio of Bcl-2and Bax was decreased. It suggested that apoptosis of lung tissue still existed by recovery of lung injury.
NF-κB in lung tissue was obviously decreased in sodium houttuyfonate group and sodium houttuyfonate plus diammonium glycyrrhizinate group on the2nd week, which suggested that single use of sodium houttuyfonate and combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than SASP. Otherwise, combined use of Chinese herbs was better than single use of diammonium glycyrrhizinate, which showed that treatment for both the lung and the large intestine was better than single one.
Conclusion
UC rats were used to evaluate effect of Chinese herb monomers, in order to explore apoptosis regulation mechanism of lung injury and biological mechanism of lung injury in UC. Otherwise, single and combined use of sodium houttuyfonate and diammonium glycyrrhizinate was evaluated for their effect, pathways and targets, which provide an objective basis for the treatment of ulcerative colitis. New ways and methods might be provided for clinical treatment of lung injury in patients. And it may provide more reliable evidence for TCM treatment of UC and lung injury and further exploring theory of the lung and the large intestine being interior-exteriorly related.
1TNBS and ethanol with colonic mucosa tissue induced model rats were used in this experiment, which were relatively close to the clinical patients, with both systemic immune abnormalities and local inflammation pathological changes. The animal model is similar to pathological mechanism of UC in patients, with pathological mechanism of B cells and T cells induced immune response. Therefore, it is a suitable animal model for studying pathological mechanism and treatment effect of UC.
2Delayed apoptosis of inflammatory cells may be related with UC, and may be important cause of continuous inflammation of ulcerative colitis.
2.1Delayed lymphocytes apoptosis in peripheral blood was significant, which suggest that decreased rate of lymphocytes apoptosis may reflect immune function disorder in certain degree. Therefore, delayed lymphocytes apoptosis may be one of the important pathological mechanisms of UC. Thus, a new way to improve the immune function of patients with UC may be provided through intervention of lymphocyte apoptosis.
2.2Apoptosis of neutrophil (PMN) in peripheral blood was delayed in UC rats at acute stages. Therefore, apoptosis of PMN was inhibited by prolonged existence in blood and inflamed lesions, which induced inflammatory injury of tissue, and may be one of the important mechanisms of injury of colon tissue in UC Its seepage may also be used as an important symbol of active UC.
3Increased Bax, Fas, Fas-L, Caspase-3and NF-κB in colon tissue, with decreased Bcl-2, Bcl-2/Bax<1showed that accelerated apoptosis in the colon, which may be through apoptosis pathways, that is, NF-κB-Bcl-2/Bax and Fas/Fas-L systems Caspase-3, in order to preliminarily reveal possible apoptosis pathway in UC. Therefore, to increase Bcl-2. decrease Bax, Fas, Fas-L, Caspasc-3and NF-κB may inhibit accelerated apoptosis in colon tissue in certain degree, by interfering with the apoptosis pathways, so as to promote the repair of colonic mucosa and to provide evidence of molecular biology in treatment of UC.
4In acute phase, Bax, Caspase-3and NF-κB were increased in lung tissue, while Bcl-2was decreased. In remission, NF-κB was improved, while other apoptosis related proteins were still abnormal, which suggested that lung injury was related with number of apoptosis regulatory proteins. Abnormal level of NF-κB, Bcl-2/Bax and Fas/Fas-L systems, and Caspase-3may induce apoptosis in lung injury, which may be one of possible mechanisms of lung injury in UC and lung disease due to the bowel. It may provide a new way to prevent and cure lung injury in UC.
5The results showed thaf apoptosis in lung and colon was increased by possible pathways, that is, NF-κB-Bcl-2/Bax and Fas/Fas-L systems-Caspase-3. While apoptosis regulatory proteins may play different roles along with development of the disease. Theory of the lung and the large intestine being interior-exteriorly related was confirmed by UC animal model, in which tissue pathological injury of the lung and the colon with apoptosis acceleration of lung tissue existed. However, pathogenesis of UC and its lung injury is complicated, and relationship between the lung and the large intestine was correlated with a variety of substances. Thus, it needs future comprehensive research to reveal biological mechanisms of the lung and the large intestine being interior-exteriorly related, not only from the perspective of apoptosis mechanism.
6Sodium houttuyfonate and diammonium glycyrrhizinate with their combined use, and SASP were overall evaluated through the effects on general condition of rats UC, pathology of the lung and the colon, peripheral inflammatory cells, apoptosis regulatory proteins, etc. with their own characteristics. Chinese medicine was generally superior to SASP in this study, which could improve apoptosis of the colon tissue mainly at acute stages, while Chinese medicine could both improve accelerated apoptosis of the colon and the lung tissue at acute and later stages. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than their separate use on improving accelerated apoptosis of the colon and the lung tissue, which reveals compound Chinese medicine is superior to separate use of one herb. In addition, sodium houttuyfonate could also improve apoptosis of the colon tissue in UC rats. Sodium houttuyfonate and diammonium glycyrrhizinate with their combined use, and SASP could improve apoptosis of the colon and the lung through one or more targets of the pathway, that is, NF-κB-Bcl-2/Bax、Fas/Fas-L-Caspase-3. Otherwise, a new way for prevention and cure of UC with its lung injury was provided through regulating and controlling target genes by Chinese medicine. Therefore, treatment from both the lung and the colon was superior to treatment from separate viscera, which showed that unique advantages of the theory of the lung and the large intestine being interior-exteriorly related.
引文
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