四神丸治疗DSS诱导的小鼠实验性结肠炎免疫机制的研究

【作者】 王旭丹；

【导师】 郝钰；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2015， 博士

【摘要】 炎症性肠病(inflammatory bowel disease, IBD)是一组世界范围内常见的、病因不明的慢性肠道炎症性疾病,主要包含了两个独立的疾病,即溃疡性结肠炎(Ulcerative colitis, UC)和克罗恩病(Crohn’s disease, CD)。IBD在西方国家发病率较高,近年来我国发病率呈上升趋势。本病也是大肠直肠癌的主要风险因子。IBD的发生是在易感基因的基础上,针对肠道共生微生物等抗原产生异常的免疫反应所致。在正常的生理状态下,肠黏膜固有层有含有大量的炎症效应T细胞和抑制炎症反应的调节性T细胞,二者处于高度的动态平衡状态。如果平衡被打破,如炎症效应过强或者调节效应减弱,均有可能导致肠黏膜炎症的发生。本病的治疗主要采用5-氨基水杨酸类制剂、糖皮质激素及免疫抑制和生物制剂如抗TNF-α单克隆抗体等。炎症性肠病在中医中多属于“泄泻”、“痢疾”等范畴,根据症状表现辨证论治。本病急性期多表现为大肠湿热症,可用葛根芩连片加减;慢性期多表现为脾肾阳虚症,可用四神丸加减。本研究利用DSS建立小鼠急性和慢性结肠炎模型,观察了四神丸和葛根芩连片对急性和慢性DSS结肠炎的治疗作用,结果发现,葛根芩连片仅对急性结肠炎有明显的治疗作用,而四神丸对急性和慢性结肠炎均有较好的疗效。四神丸具有温肾健脾、温阳化湿、涩肠止泻的功效,可作为治疗脾肾阳虚型慢性泄泻的基础方加减使用,因此也可作为治疗IBD的常用基础方药,其疗效的免疫机理目前尚不清楚。本研究利用小鼠DSS诱导结肠炎模型,对四神丸在结肠炎中所表现出来的治疗作用的免疫机制及其在急性和慢性结肠炎的作用差异进行了探讨。一、四神丸对DSS诱导小鼠急性和慢性结肠炎的治疗作用急性或慢性结肠炎试验中小鼠均随机分为4组,即正常对照组、DSS急性或慢性模型组、四神丸组(2.25 g/kg)、葛根芩连片组(6.5 g/kg)。4% DSS连续给小鼠自由饮用5天制备急性结肠炎模型；3%DSS给小鼠4次循环饮用(1-5天、8-12天、15-19天、22-26天)制备慢性结肠炎模型。急性模型于DSS饮水次日开始给药,连续给药8天；慢性模型于DSS饮水2次循环后开始给药,连续给药16天。测定小鼠疾病活动度指数(DAI)和结肠组织中髓过氧化物酶(MPO)活性,HE染色观察结肠组织学变化。结果显示：DSS诱导的急性结肠炎小鼠的DAI显著增加、结肠组织出现明显的炎症损伤且组织中MPO活力显著增加。经四神丸治疗后,可显著缓解DSS结肠炎小鼠的DAI、改善结肠的组织学损伤、降低结肠组织中MPO活性。中药葛根芩连片具有四神丸相似的治疗作用,但在缓解小鼠DAI上起效更早。慢性结肠炎中,模型小鼠DAI指数、组织学评分及MPO活力均显著高于正常组,但MPO活力增加的不如急性模型明显。四神丸的治疗可明显改善小鼠的DAI和组织损伤,但对MPO的降低作用并不明显,这与慢性结肠炎动物MPO活力增加并不显著有关。葛根芩连片对慢性DSS结肠炎的治疗效果并不明显。二、四神丸对DSS诱导的实验性结肠炎结肠组织中CX3CR1、CD103、IL-23表达的影响急性或慢性结肠炎试验中小鼠均随机分为3组,即正常对照组、DSS急性或慢性模型组、四神丸组(2.25 g/kg)。采用免疫组化法测定各组小鼠结肠组织中CX3CR1、 CD103、IL-23分子的表达。结果显示：急性DSS诱导的小鼠结肠炎中,黏膜组织中CX3CR1、CD103及IL-23表达均显著增加。四神丸治疗后能进一步增加CD103的表达,但对CX3CR1和IL-23的表达则没有明显的影响。慢性DSS诱导小鼠结肠炎中,CX3CR1、CD103及IL-23表达也显著增加,但增加的幅度有所下降。四神丸对慢性结肠炎中CX3CR1和CD103的表达无明显的影响,但可使IL-23的表达降低。三、四神丸对DSS诱导的结肠炎中效应T细胞的影响急性或慢性结肠炎试验中小鼠均随机分为3组,即正常对照组、DSS急性或慢性模型组、四神丸组(2.25 g/kg)。实验结束时取小鼠结肠进行器官培养,ELISA法测定培养上清中IFN-γ、IL-17A和IL-22的含量；分离小鼠结肠黏膜固有层单个核细胞(LPMC),采用胞内免疫荧光染色法测定表达IFN-γ和IL-17A的CD4+T细胞数；采用免疫组化法和Western blot法测定结肠组织中RORyt的表达。结果显示：急性DSS诱导的小鼠结肠炎组织培养上清中IFN-γ及LPMC中表达IFN-γ的CD4+T细胞均显著增加；IL-17A、LPMC中表达IL-17A的CD4+T细胞以及结肠组织中转录因子RORγt表达均显著增加。表明急性DSS结肠炎中存在较强的Thl和Th17反应。经四神丸治疗后,可显著降低模型小鼠结肠组织中IL-17A、抑制LPMC中IL-17A+T细胞数以及降低肠黏膜中RORγt的表达；但对IFN-γ及IFN-γ+T细胞无明显的影响。表明四神丸针对急性结肠炎疗效与其抑制Th17反应相关。慢性DSS诱导的结肠炎中,IFN-γ及表达IFN-γ的CD4+T细胞也显著升高,慢性结肠炎中IL-17A、LPMC1中表达IL-17A的CD4+T细胞以及结肠组织中转录因子RORγt表达虽然仍较对照组显著增加,但增加的幅度低于急性模型。四神丸给药后可显著降低IFN-γ+ CD4+T细胞数,但对IL-17A、IL-17A+CD4+T细胞及RORγt无明显的影响。表明四神丸在慢性结肠炎中对Th17无影响,对Th17没有显著的作用与慢性结肠炎中Th17反应明显减弱有关。但四神丸具有抑制慢性炎症中Thl的作用趋势,由于在急性炎症中并没有显示出这一作用,推测这一作用趋势可能为间接作用。IL-22作为Th17分泌另一种细胞因子,其变化趋势与IL-17A基本一致。急性DSS炎症中IL-22显著高于对照组,慢性DSS炎症增高并不明显。但四神丸在急性和慢性结肠炎中均有促进IL-22生成的趋势,这与四神丸对IL-17A的作用并不一致,可能与IL-22有更多的细胞来源有关。四、四神丸对DSS诱导的结肠炎Th17可塑性的影响急性或慢性结肠炎试验中小鼠均随机分为3组,即正常对照组、DSS急性或慢性模型组、四神丸组(2.25 g/kg)。实验结束分离小鼠结肠黏膜固有层单个核细胞(LPMC),采用胞内免疫荧光染色法测定共表达IFN-γ的Th17与共表达IL-10的Th17细胞数及二者之间的比例,结果显示：DSS急性模型中IFN-γ+Th17显著高于对照组；虽然DSS慢性模型中IFN-γ+Th17也显著高于对照组,但增加幅度较急性组明显下降,同时IL-10+Th17则较对照组显著上升。四神丸促进急性DSS模型中IL-10+Th17显著上升,但对IFN-γ+Th17无明显影响,从而使IFN-γ+Th17/IL-10+Th17较对照组显著降低。四神丸可使慢性结肠炎中IL-10+Th17和IFN-γ+Th17均显著降低,因此对IFN-γ+Th17/IL-10+Th17没有显著的影响。四神丸对急性DSS的治疗作用与其调整IFN-γ+Th17/IL-10+Th17比例密切相关。五、四神丸对DSS诱导的结肠炎Treg细胞的影响急性或慢性结肠炎试验中小鼠均随机分为3组,即正常对照组、DSS急性或慢性模型组、四神丸组(2.25g/kg)。实验结束时取小鼠结肠进行器官培养,ELISA法测定培养上清中IL-10.TGF-β和IL-35的含量；分离小鼠结肠黏膜固有层单个核细胞(LPMC),采用胞内免疫荧光染色法测定表达IL-10的CD4+T细胞数；采用Western blot法测定结肠组织中Foxp3的表达。结果显示：急性DSS结肠炎中虽然IL-35、LPMC中IL-10+Treg含量显著增加,但IL-10和Foxp3无明显变化,表明急性结肠炎中Treg反应增强特征并不明显。经四神丸给药后,对Treg分泌的细胞因子及特征性转录因子Foxp3也无明显的影响。慢性DSS结肠炎中,模型中IL-10、LPMC中IL-10+Treg及Foxp3表达均明显增加,表明慢性DSS炎症中确立了以IL-10为主的控制炎症的机制。经四神丸给药后,进一步促进IL-10和IL-35的升高,并能促进IL-10+Treg的生成及Foxp3的表达。因此,四神丸在慢性结肠炎中具有显著的放大Treg效应,从而有利于炎症的恢复。综上所述,本课题的研究发现：(1)四神丸对DSS诱导的小鼠急性和慢性结肠炎均具有显著的疗效。(2)四神丸治疗DSS急性结肠炎的免疫机制与其抑制Thl7反应及促进调节性Th17生成相关。(3)四神丸治疗DSS慢性结肠炎的免疫机制主要与促进表达IL-10的Treg生成相关,四神丸在慢性炎症中抑制Thl的反应可能是通过促进Treg实现的。(4)在急性和慢性结肠炎中,四神丸促进IL-22生成的趋势可能有利于上皮的修复,缓解炎症。更多还原

【Abstract】 The two major clinically defined forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic remittent or progressive inflammatory conditions that may affect the entire gastrointestinal tract and the colonic mucosa, respectively, and are associated with an increased risk for colon cancer. IBD is common in developed countries, the incidence rate rise in china in recent years. The pathogenesis of IBD is not fully defined, but is clearly multifactorial, resulting from multiple genes, developmental and environmental factors, which together result in a dysregulated innate and adaptive mucosal immune response.Under normal physiological conditions, lamina propria contains numerous inflammatory T cells and regulatory T cells which inhibit inflammation, the two T cells are in a state of dynamic equilibrium. If the balance is broken, such as inflammation effect is too strong or regulating effect too weak, may lead to intestinal inflammation. Therapy for IBD mainly is 5-amino salicylic acid, glucocorticoid and immunosuppressive and biological agents such as anti-TNF-a monoclonal.According its clinical symptoms, IBD belongs to "diarrhea" and "dysentery" in traditional Chinese medicine (TCM). Sishen pill has the function of warming kidney and invigorating spleen, warming yang to melt wet, inducing astringency and relieving diarrhea. Sishen pill often is used as a basis prescription for treating IBD caused by asdthenic splenonephro-yang, but immune mechanism of treating IBD is not clear. In this study, we observe the therapeutic effect of Sishen pill and its immune mechanism by the acute and chronic colitis induced by DSS in mice.The effect of Sishen pill treat acute and chronic colitis induced by DSSAcute colitis was induced by feeding mice with 4%(wt/vol) DSS dissolved in drinking water. After 1 day mice were administrated with Sishen pill (2.25 g/kg) or Gegenqinlian pellet (6.5 g/kg) for 8 days. Chronic experimental colitis was induced by multiple cycle administration of 3%(wt/vol) DSS drinking water on days 1-5,8-12,15-19, and 22-26. After 12 day mice were administrated with Sishen pill (2.25 g/kg) or Gegenqinlian pellet (6.5 g/kg) for 16 days. Changes in disease activity index (DAI) together with histological scores were evaluated, and myeloperoxidase (MPO) in colonic tissue was detected. Both Sishen pill and Gegenqinlian pellet therapy significantly reduced DAI and histological scores of mice in acute DSS colitis. They also alleviate inflammatory cell infiltration of colonic tissue in the model. Although Gegenqinlian pellet has fast effect on DAI in actue DSS model, Sishen has better effectiveness. Sishen pill therapy significantly reduced DAI and histological scores of mice in chronic DSS colitis, but Gegenqinlian pellet show no obvious effect in the model. Amelioration efficacy of Sishen pill on mice acute colitis and chronic colitis induced by DSS were obtained. Gegenqinlian pellet only ameliorate acute colits induced by DSS. The effectiveness of Sishen pill is slightly better than Gegenqinlian pellet, but there is no significant difference between two prescriptions.Effect of Sishen Pill on CX3CR1, CD103, IL-23 expression in experimental colitis induced by DSS in miceAcute colitis induced by DSS in mice significantly increased the expression of CX3CR1, CD 103 and IL-23 in tissues. Sishen pill after treatment can increase the expression of CD103, but no obvious effect of the expression of CX3CR1 and IL-23. The expression of CX3CR1, CD103 and IL-23 also significantly increased in chronic colitis induced by DSS in mice, but the extent of increase of CX3CR1 and IL-23 decrease. There is no significant effect of Sishen Pill on CX3CR1 and CD103 expression in chronic colitis, but the expression of IL-23 was decreased.Effect of Sishen Pill on inflammatory T cells in experimental colitis induced by DSS in miceTh1 and Th17 are mainly inflammatory effector T cells in the intestinal mucosa. IFN-y is characteristic cytokine of Th1, and IL-17A is characteristic cytokine of Th17. IFN-y production level from colon organ culture supernatant and CD4+ T cells of expressing IFN-y in LPMC increased significantly in acute DSS model mice compared with control normal mice. IL-17A level from colon organ culture supernatant, CD4+T cells of expressing IL-17A and characteristic transcription factor RORyt of Th17 increased significantly as well. These data indicate the enhanced Th1 and Th17 reaction in acute colitis induced by DSS. After Sishen pill treatment, IL-17A production level, CD4+T cells of expressing IL-17A in LPMC, and RORyt in intestinal mucosa decreased significantly compared with acute colitis model group; but there is no obvious influence on IFN-y and CD4+T cells of expressing IFN-y in LPMC. The data show the Sishen pill treatment effect is associated with its inhibition of Th17 response.IFN-y production level from colon organ culture supernatant and CD4+T cells of expressing IFN-y in LPMC increased significantly in chronic DSS colitis model. Although IL-17A production level, CD4+T cells of expressing IL-17A in LPMC, and RORyt still increased significantly compared with the control group in chronic DSS colitis, the rate of increase is lower than acute model. After administration of Sishen pill can significantly reduce the number of CD4+T cells of expressing IL-17A in LPMC, but no obvious effect on level of IL-17A, IL-17A+T cells and RORyt. The results show that the Sishen pill had no effect on Th17, but had inhibitory effect on Th1 which may be due to indirect actions.IL-22, another cytokine of Th17, show analogous change trend compare with IL-17A in actue and chronic colitis induced by DSS. Interestingly, Sishen pill promote IL-22 production level from colon organ both acute and chronic colitis, which is inconsistent with Sishen pill on IL-17A. This phenomenon may be related to the more cells to produce IL-22.Effect of Sishen Pill on Th17 plasticity in experimental colitis induced by DSS in miceTh17 plasticity is that Th17 co-express characteristic cytokine such as IFN-y from Thl or IL-10 from Treg while the expression IL-17A. There is significant increase in IFN-γ+Thl7 compare with the control group in acute DSS colitis. Although IFN-y+Thl7 also increased significantly in chronic DSS colitis, the rate of increase is lower than acute model, and IL-10+Thl7 increased significantly compared with control group. Sishen Pill could increase significantly IL-10+Thl7 in acute DSS colitis, but had no significant effect on IFN-γ+Th17, so that IFN-γ+Th17/IL-10+Th17 decreased significantly compared with the control group. Sishen pill decreased IFN-y+Th17 and IL-10+Th17 at the same time, and so therefore has no significant effect on IFN-γ+Th17/IL-10+Th17 compare with model group in chronic DSS colitis. Therefore, therapeutic effect of Sishen pill on actue DSS colitis is closely related to IFN-y+Th17/IL-10+Th17 ratio adjustment.Effect of Sishen Pill on DSS Treg cells in experimental colitis induced by DSS in miceTreg is an important regulatory T cells to control ntestinal mucosal inflammation, IL-10 is a characteristic cytokine secretion of Treg. Treg also can secrete IL-35 and TGF-β. Although IL-35 production level from colon organ culture supernatant and IL-10+T cells in LPMC increased significantly, the feature of Treg increase is not obvious in acute DSS colitis. Administration of the Sishen pill had no significant effect on cytokines and characteristic transcription factor Foxp3 of Treg.In chronic DSS colitis, IL-10 production, IL-10+Treg, and Foxp3 expression increased significantly in model group, which show that the mechanism of controlling inflammation depending IL-10 had been established. The Sishen pill could further promote IL-10 and IL-35 production, increase IL-10+Treg and Foxp3. Therefore, Treg reaction could be amplified after Sishen pill administration in chronic colitis, thus contributing to the recovery of inflammation.In summary, Sishen pill has significant curative effect on acute and chronic colitis induced by DSS in mice. The immune mechanisms of Sishen pill in acute colitis is related to the inhibition of Thl7 reaction and promote regulatory Th17 generation. The immune mechanisms of Sishen pill in chronic acute colitis mainly promotes the expression of IL-10 in Treg generation, inhibition of the Thl response in chronic colitis may be through the promotion of Treg. In acute and chronic colitis, Sishen pill shows increasing the production of IL-22 which may contribute to repair the epithelium and alleviate inflammation.更多还原