灌肠方对溃疡性结肠炎大鼠结肠粘膜ITF、MUC2及TLR-4基因表达的影响
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摘要
溃疡性结肠炎(UC)是一种病因尚未完全阐明的慢性炎症性肠病。主要临床表现为持续或反复发作的腹痛、粘液脓血便、腹泻和里急后重。由于目前该病发病率逐年增加,发病机制尚未完全阐明,又缺乏有效的治疗药物,故寻找有效的防治药物也一直是研究的热点。中医药在UC的治疗上有较好的疗效,很多达到与西药相近的治疗效果,且可弥补西药较大毒副作用及停药后易复发的不足,有着广阔的应用和开发前景。灌肠方是中国中医科学院广安门医院在多年临床经验的基础上总结出治疗UC的中药复方,临床取得了较好的疗效,无明显毒副作用。本课题通过建立UC大鼠模型来研究灌肠方对UC的治疗作用及机制,特别是通过观察其对UC大鼠细胞因子(TNF-α、IL-1β、IL-6),肠三叶因子(ITF)、粘蛋白2(MUC2)、Toll样受体4(TLR-4)的基因表达的影响,深入揭示灌肠方治疗UC的作用机制,为中医药治疗UC提供一定的实验基础和理论依据。
1.灌肠方对UC大鼠治疗作用的病理观察
灌肠方是中国中医科学院广安门医院在多年临床经验的基础上总结出治疗UC的中药复方,临床取得了较好的疗效。为验证其临床疗效,并进一步探讨其作用机理,我们采用三硝基苯磺酸法制作溃疡性结肠炎大鼠模型,并在此模型上观察了灌肠方的治疗效果,灌肠方采用三个剂量给药治疗,阳性对照组用美沙拉嗪治疗,并设模型组及正常对照组。治疗结束进行病理观察,大体观察灌肠方对TNBS法UC大鼠模型结肠组织中一般情况的影响,光镜下观察肠粘膜组织炎症、水肿和充血程度。结果表明：模型组UC大鼠从症状上可见粘液稀便、甚至血便、消瘦、懒动、皮毛无光泽、食欲减退、体重减轻。模型组结肠组织损伤评分明显比正常对照组高(P<0.01),经过灌肠方及美沙拉嗪治疗后,灌肠方各组及美沙拉嗪组结肠组织损伤评分均有不同程度降低。
2.灌肠方对UC大鼠血清中促炎因子TNF-α、IL-1β、IL-6的影响
细胞因子是主要由免疫细胞所分泌的小分子多肽,在机体的免疫应答中具有重要作用。目前认为,肠道炎症的启动及其慢性化在很大程度上可能与促炎因子和抗炎因子的失衡有关：UC患者中促炎细胞因子水平升高,抗炎细胞因子水平下降,且升高的水平与疾病的严重程度呈正相关,持久的免疫调节失衡促使炎症趋于慢性化。因此,促炎细胞因子在UC发病发展中起重要作用。目前比较公认的与UC密切相关的促炎细胞因子有很多,其中对TNF-α、IL-1、IL-6的研究最具代表性。我们在灌肠方抗实验性UC作用的基础上,观察了该药对TNF-α、IL-1、IL-6的作用,不仅是对该药疗效的进一步验证,也是对其作用机制的探讨。采用三个剂量对TBNS法UC大鼠模型进行治疗,治疗结束采血并制备血清样本,采用双抗体夹心ABC-ELISA法检测血清样本中的含量。结果表明：模型组UC大鼠的组织中促炎细胞因子TNF-α、IL-1β、IL-6的含量高于正常对照组(P<0.01),灌肠方高、中、低剂量组、美沙拉嗪组TNF-α、IL-1β、IL-6含量均低于模型组(P<0.时或P<0.05),灌肠方大剂量组TNF-α、IL-1β、IL-6的含量分别与美沙拉嗪组比较,无显著性差异(P)0.05)。
3.灌肠方对溃疡性结肠炎大鼠结肠粘膜ITF和MUC2基因表达的影响
肠三叶因子(intestinal trefoilfactor, ITF/TFF3)属三叶因子家族(trefoil factor family, TFF)肽,是一类较新的肠黏膜保护因子,在肠黏膜的
保护和修复中起重要作用,且与UC的发病有着密切的关系。MUC2基因是一种粘蛋白核心肽基因,定位于11p15.3~15.5上,此基因的表达产物MUC2是一种分泌性粘蛋白,由于其广泛表达于肠粘膜中,因此被称为肠粘蛋白,对肠道具有保护和润滑作用。在炎症性肠病等病理情况下进行中药干预,以促进ITF基因和MUC2基因的上调表达,而使其更好的发挥粘膜保护及损伤修复作用,可能是炎症性肠病治疗的一条新的思路和方法。我们采用三个剂量对TBNS法UC大鼠模型进行治疗,治疗结束后采集结肠粘膜标本。用Trizol提取总RNA进行逆转录反应,以β-actin作为内参进行PCR反应,产物进行凝胶电泳分离,经染色后进行灰度分析,以ITFmRNA;和MUC2mRNA的灰度值与β-actin灰度值的比值作为相对表达量,进行组间比较分析。结果表明：模型组UC大鼠结肠粘膜ITFmRNA和MUC2mRNA的相对表达与正常组比较,略有增高,但没有显著差异(P>0.05)。与模型组比较,灌肠方和美沙拉嗪对UC大鼠结肠粘膜ITFmRNA和MUC2mRNA的表达都有明显的上调作用。
4.灌肠方对溃疡性结肠炎大鼠结肠粘膜TLR-4基因表达的影响
Toll样受体4(toll like receptor 4, TLR4)是近年来发现的天然免疫识别受体(pattern recognition receptors, PRR)之一,主要识别脂多糖(lipopolysaccharide, LPS)并介导其跨膜信号转导。目前有研究表明,TLR4在正常的结肠黏膜中呈现低表达,而在溃疡性结肠炎的结肠黏膜中则呈现异常的高表达,提示TLR4受体及其信号传导通路可能是溃疡性结肠炎发病机制发展过程的重要环节。因此我们观察了灌肠方对TBNS法UC大鼠模型结肠组织中TLR4mRNA表达的作用,以期对灌肠方治疗UC的作用机制进行较深入的探讨。采用三个剂量对TBNS法UC大鼠模型进行治疗,治疗结束后采集结肠粘膜标本。用Trizol提取总RNA进行逆转录反应,以β-actin作为内参进行PCR反应,产物进行凝胶电泳分离,经染色后进行灰度分析,以TLR4mRNA的灰度值与β-actin灰度值的比值作为相对表达量,进行组间比较分析。结果表明：模型组UC大鼠结肠粘膜TLR-4mRNA的相对表达与正常组比较,显著增高(P<0.05)。经灌肠方和美沙拉嗪治疗后,TLR-4mRNA的表达升高,灌肠方高、中、低剂量组及美沙拉嗪组与模型组比较均有非常显著性差异(P<0.01)。
5.结论动物模型复制成功。其中中药组存在量效关系,灌肠方大剂量组疗效于美沙拉嗪组比较,未见明显差异。促炎细胞因子与抗炎细胞因子之间的平衡失调所致的免疫异常是UC的重要发病机制。灌肠方能有效降低UC模型大鼠血清TNF-α、IL-1β、IL-6的含量,抑制促炎因子的释放。ITF和MUC2对结肠粘膜都有直接的保护作用,当二者结合后可以增加保护功能。灌肠方高、中剂量组和美沙拉嗪组结肠ITF和MUC2表达均明显上调。提示,灌肠方治疗UC的作用机理可能与加强ITF和MUC2的保护作用有关。Toll样受体(TLRs)是NF-K B激活的TLRs/NF-K B通路的重要环节。通过对Toll样受体表达的抑制,可以抑制TLRs/NF-K B通路的活化,从而对溃疡性结肠炎起到治疗作用。灌肠方各治疗组能有效的抑制TLR-4mRNA的表达,这可能是灌肠方能有效抑制UC的反应炎症,达到治疗效果的重要机理。
Ulcerative colitis (UC)is an idiopathic chronic inflammatory bowel disease(IBD). Its main manifestations are diarrhea, bloody mucopurulent stool, abdominal pain and tenesmus. Because the disease is difficult to cure, we have been lacking of effective remedies for treating it. In the past years, the scholars of home and abroad have been intent on the study of finding effective drug. Therapeutic effect of traditional Chinese medicine (TCM) on UC was similar to that of western medicine. But the defect of western medicine such as recur easily, toxical and side effects were seldom found in using TCM. The prospect of utilization and development of traditional medicine is cheerful. Guanchang Decoction was frequently used in Guang'anmen Hospital to treat UC patients. It showed affirmative therapeutic effect on patients without obvious toxical and side effects. This subject studied treatment of effect and mechanism of Guanchang Decoction by making rat model of UC. In particular, we use Guanchang Decoction to treat UC rats and then we observe thecontent is high or low about cytokines TNF-α、IL-1βand IL-6 which come from serum of the UC rats. We also observe other indexes such as:Gene Expression of ITF, MUC2 and TLR-4 in colon in mucosa of rats with ulcerative colitis.
1. Observation of Guanchang Decoction Decoction in Pathological Changes on UC Model Rats
Guanchang Decoction was frequently used in Guang'anmen Hospital to treat UC patients. It showed affirmative therapeutic effect on patients To validate the clinical curative effect and investigate the mechanism of action, effect of Guanchang Decoction on UC model rats induced by TNBS was observed.All the rats were randomly divided into six groups, namely the normal control group, the pathologic control group, the Mesalazine group, the high dose group of Guanchang Decoction, medium dose group of Guanchang Decoction, low dose group of Guanchang Decoction. The rats were killed to observe the general condition and pathological changes after treatment. The results showed:The rats in the model group can been observed the changes of the symptoms such as intermittent diarrhea with mucus and blood at stool,. emaciation, laziness, no luster in hair, poor appetite, loss of weight and increase of colonic damage scores. The colon tissue damaged score in pathologic group is higher than that in normal group(P<0.01);After treatment, their symptoms and sighs obviously improved. The colon tissue damaged score in pathologic group was higher than that in the high, medium, low dose group of Guanchang Decoction and Mesalazine group.
2. Effect of Guanchang Decoction on Proinflammatory Cytokines TNF-α、IL-1β、IL-6 in Serum of UC Model Rats
It was proposed that cytokines especially the proinflammatory cytokines such as the tumor necrosis factor-α(TNF-α)、interleukin-1β(IL-1β)and interleukin-6 (IL-6)the played an important role in the morbility and development of UC. To confirm the curative effect and study the treatment mechanism of Guanchang Decoction, the effect of TNF-α、IL-1β、IL-6 in serum of UC model rats was studied. The grouping and administration were same as before. The rats were killed after administration to get their serum. The contents of TNF-α、IL-1β、IL-6 in serum were measured with double antibody sandwich ABC-ELISA. The results showed:The level of TNF-α、IL-1βand IL-6 in pathologic group was significantly higher than that in normal group(P<0.01); The level of TNF-α、IL-1βand IL-6 in Guanchang Decoction high, medium, low dose group, Mesalazine group was lower than in pathologic group (P<0.01 or P<0.05); The level of TNF-α、IL-1βand IL-6 in Guanchang Decoction high dose group was lower than that in SASP group two groups have not significant difference(P>0.05).
3. Effect of Guanchang Decoction on Gene Expression of ITF and MUC2 in Colonic Mucosa of UC Model Rats.
ITF was regarded as a fast reaction peptide in the course of mucosa impairments. ITF has two important functions in the intestinal tract:protecting the epithelial cell and promoting the repair of mucosa. So ITF plays an important role in the morbility and development of UC. Mucin2 (MUC2) is also secretory product of mucous epithelia, which is main ingredients of maintaining thickness of slime layer and colloidal shape. So it plays a key role in mucosal protection and repair mechanism of UC. To investigate the deepgoing treatment mechanism of Guanchang Decoction on UC, the genes expression of and were detected in colonic mucosa of UC model rats induced by TNBS. The grouping and administration were same as before. After treated period, the rats were killed to get their colonic mucosa. Total RNA was isolated from these rats'colonic mucosa. RT PCR was used to detect ITF and MUC2 expressions andβ-actin was used as internal index. The products of PCR were separated with gel electrophoresis and stained. To compare the relative expression which was calculated from the gray scale ratio of ITF、MUC2 andβ-acting. The results showed:Relative gene expression of ITF, MUC2 in pathologic group were slightly higher than that in normal group, two groups have not significant difference(P>0.05). Relative gene expression of ITF、MUC2 in Guanchang Decoction high, medium dose group was significantly higher than that in pathologic group (P<0.05)
4. Effect of" Guanchang Decoction on Gene Expression of TLR-4 in Colonic Mucosa of UC Model Rats.
TLRs were important component elements of TLRs/NF-kB pathway, which could be activated by NF-kB. If the gene expression of TLRs was inhibited, the activation of TLRs/NF-kB could be inhibited. It was important to inhibite the activation of TLRs/NF-kB in the period of the treatment for UC patients. To investigate the deepgoing treatment mechanism of Guanchang Decoction on UC, the genes expression of and were detected in colonic mucosa of UC model rats induced by TNBS. The grouping and administration were same as before. After treated period, the rats were killed to get their colonic mucosa. Total RNA was isolated from these rats'colonic mucosa. RT-PCR was used to detect TLR-4 expressions andβ-actin was used as internal index. The products of PCR were separated with gel electrophoresis and stained.To compare the relative expression which was calculated from the gray scale ratio of TLR-4 andβ-acting. The results showed:Relative gene expression of TLR-4 in pathologic group were significantly higher than that in normal group (P<0.05). Relative gene expression of TLR-4 in Guanchang Decoction high, medium dose group was significantly lower than that in pathologic group (P<0.05).
5. Conclusion
This model was successful. The treatment of Guanchang Decoction was effective on the UC rats. Guanchang Decoction treating the rats with UC has dose-effect relationship. Blance between pro-inflammatory cytokines and anti-inflammatory cytokine is extremely important in the formation of the UC. Guanchang Decoction could reduce the content of TNF- p、IL-1βand IL-6, inhibit the release of pro-inflammatory cytokines. ITF and MUC2 have better protective function to gastrointestinal mucosa. The mechanism involves in interaction of ITF and MUC2, such as gel layer to resist noxious substance, promoting cell reinforcing the immigration and proliferation. Guanchang Decoction could up-regulate ITF and MUC2 expressions, strengthen the interaction of ITF and MUC2, promote the formation of protective layer to defend the intestinal mucosa of UC. TLRs were important component elements of TLRs/ NF-kB pathway, which could beactivated by NF-κB. If the gene expression of TLRs was inhibited, the activation of TLRs/NF-kB could be inhibited. It was important to inhibite the activation of TLRs/NF-kB in the period of the treatment for UC patients. The gene expression of TLR-4, in colonic mucosa of UC model rats induced byTNBS were inhibitted after using Guangchang Decoction.
1.灌肠方对UC大鼠治疗作用的病理观察
灌肠方是中国中医科学院广安门医院在多年临床经验的基础上总结出治疗UC的中药复方,临床取得了较好的疗效。为验证其临床疗效,并进一步探讨其作用机理,我们采用三硝基苯磺酸法制作溃疡性结肠炎大鼠模型,并在此模型上观察了灌肠方的治疗效果,灌肠方采用三个剂量给药治疗,阳性对照组用美沙拉嗪治疗,并设模型组及正常对照组。治疗结束进行病理观察,大体观察灌肠方对TNBS法UC大鼠模型结肠组织中一般情况的影响,光镜下观察肠粘膜组织炎症、水肿和充血程度。结果表明：模型组UC大鼠从症状上可见粘液稀便、甚至血便、消瘦、懒动、皮毛无光泽、食欲减退、体重减轻。模型组结肠组织损伤评分明显比正常对照组高(P<0.01),经过灌肠方及美沙拉嗪治疗后,灌肠方各组及美沙拉嗪组结肠组织损伤评分均有不同程度降低。
2.灌肠方对UC大鼠血清中促炎因子TNF-α、IL-1β、IL-6的影响
细胞因子是主要由免疫细胞所分泌的小分子多肽,在机体的免疫应答中具有重要作用。目前认为,肠道炎症的启动及其慢性化在很大程度上可能与促炎因子和抗炎因子的失衡有关：UC患者中促炎细胞因子水平升高,抗炎细胞因子水平下降,且升高的水平与疾病的严重程度呈正相关,持久的免疫调节失衡促使炎症趋于慢性化。因此,促炎细胞因子在UC发病发展中起重要作用。目前比较公认的与UC密切相关的促炎细胞因子有很多,其中对TNF-α、IL-1、IL-6的研究最具代表性。我们在灌肠方抗实验性UC作用的基础上,观察了该药对TNF-α、IL-1、IL-6的作用,不仅是对该药疗效的进一步验证,也是对其作用机制的探讨。采用三个剂量对TBNS法UC大鼠模型进行治疗,治疗结束采血并制备血清样本,采用双抗体夹心ABC-ELISA法检测血清样本中的含量。结果表明：模型组UC大鼠的组织中促炎细胞因子TNF-α、IL-1β、IL-6的含量高于正常对照组(P<0.01),灌肠方高、中、低剂量组、美沙拉嗪组TNF-α、IL-1β、IL-6含量均低于模型组(P<0.时或P<0.05),灌肠方大剂量组TNF-α、IL-1β、IL-6的含量分别与美沙拉嗪组比较,无显著性差异(P)0.05)。
3.灌肠方对溃疡性结肠炎大鼠结肠粘膜ITF和MUC2基因表达的影响
肠三叶因子(intestinal trefoilfactor, ITF/TFF3)属三叶因子家族(trefoil factor family, TFF)肽,是一类较新的肠黏膜保护因子,在肠黏膜的
保护和修复中起重要作用,且与UC的发病有着密切的关系。MUC2基因是一种粘蛋白核心肽基因,定位于11p15.3~15.5上,此基因的表达产物MUC2是一种分泌性粘蛋白,由于其广泛表达于肠粘膜中,因此被称为肠粘蛋白,对肠道具有保护和润滑作用。在炎症性肠病等病理情况下进行中药干预,以促进ITF基因和MUC2基因的上调表达,而使其更好的发挥粘膜保护及损伤修复作用,可能是炎症性肠病治疗的一条新的思路和方法。我们采用三个剂量对TBNS法UC大鼠模型进行治疗,治疗结束后采集结肠粘膜标本。用Trizol提取总RNA进行逆转录反应,以β-actin作为内参进行PCR反应,产物进行凝胶电泳分离,经染色后进行灰度分析,以ITFmRNA;和MUC2mRNA的灰度值与β-actin灰度值的比值作为相对表达量,进行组间比较分析。结果表明：模型组UC大鼠结肠粘膜ITFmRNA和MUC2mRNA的相对表达与正常组比较,略有增高,但没有显著差异(P>0.05)。与模型组比较,灌肠方和美沙拉嗪对UC大鼠结肠粘膜ITFmRNA和MUC2mRNA的表达都有明显的上调作用。
4.灌肠方对溃疡性结肠炎大鼠结肠粘膜TLR-4基因表达的影响
Toll样受体4(toll like receptor 4, TLR4)是近年来发现的天然免疫识别受体(pattern recognition receptors, PRR)之一,主要识别脂多糖(lipopolysaccharide, LPS)并介导其跨膜信号转导。目前有研究表明,TLR4在正常的结肠黏膜中呈现低表达,而在溃疡性结肠炎的结肠黏膜中则呈现异常的高表达,提示TLR4受体及其信号传导通路可能是溃疡性结肠炎发病机制发展过程的重要环节。因此我们观察了灌肠方对TBNS法UC大鼠模型结肠组织中TLR4mRNA表达的作用,以期对灌肠方治疗UC的作用机制进行较深入的探讨。采用三个剂量对TBNS法UC大鼠模型进行治疗,治疗结束后采集结肠粘膜标本。用Trizol提取总RNA进行逆转录反应,以β-actin作为内参进行PCR反应,产物进行凝胶电泳分离,经染色后进行灰度分析,以TLR4mRNA的灰度值与β-actin灰度值的比值作为相对表达量,进行组间比较分析。结果表明：模型组UC大鼠结肠粘膜TLR-4mRNA的相对表达与正常组比较,显著增高(P<0.05)。经灌肠方和美沙拉嗪治疗后,TLR-4mRNA的表达升高,灌肠方高、中、低剂量组及美沙拉嗪组与模型组比较均有非常显著性差异(P<0.01)。
5.结论动物模型复制成功。其中中药组存在量效关系,灌肠方大剂量组疗效于美沙拉嗪组比较,未见明显差异。促炎细胞因子与抗炎细胞因子之间的平衡失调所致的免疫异常是UC的重要发病机制。灌肠方能有效降低UC模型大鼠血清TNF-α、IL-1β、IL-6的含量,抑制促炎因子的释放。ITF和MUC2对结肠粘膜都有直接的保护作用,当二者结合后可以增加保护功能。灌肠方高、中剂量组和美沙拉嗪组结肠ITF和MUC2表达均明显上调。提示,灌肠方治疗UC的作用机理可能与加强ITF和MUC2的保护作用有关。Toll样受体(TLRs)是NF-K B激活的TLRs/NF-K B通路的重要环节。通过对Toll样受体表达的抑制,可以抑制TLRs/NF-K B通路的活化,从而对溃疡性结肠炎起到治疗作用。灌肠方各治疗组能有效的抑制TLR-4mRNA的表达,这可能是灌肠方能有效抑制UC的反应炎症,达到治疗效果的重要机理。
Ulcerative colitis (UC)is an idiopathic chronic inflammatory bowel disease(IBD). Its main manifestations are diarrhea, bloody mucopurulent stool, abdominal pain and tenesmus. Because the disease is difficult to cure, we have been lacking of effective remedies for treating it. In the past years, the scholars of home and abroad have been intent on the study of finding effective drug. Therapeutic effect of traditional Chinese medicine (TCM) on UC was similar to that of western medicine. But the defect of western medicine such as recur easily, toxical and side effects were seldom found in using TCM. The prospect of utilization and development of traditional medicine is cheerful. Guanchang Decoction was frequently used in Guang'anmen Hospital to treat UC patients. It showed affirmative therapeutic effect on patients without obvious toxical and side effects. This subject studied treatment of effect and mechanism of Guanchang Decoction by making rat model of UC. In particular, we use Guanchang Decoction to treat UC rats and then we observe thecontent is high or low about cytokines TNF-α、IL-1βand IL-6 which come from serum of the UC rats. We also observe other indexes such as:Gene Expression of ITF, MUC2 and TLR-4 in colon in mucosa of rats with ulcerative colitis.
1. Observation of Guanchang Decoction Decoction in Pathological Changes on UC Model Rats
Guanchang Decoction was frequently used in Guang'anmen Hospital to treat UC patients. It showed affirmative therapeutic effect on patients To validate the clinical curative effect and investigate the mechanism of action, effect of Guanchang Decoction on UC model rats induced by TNBS was observed.All the rats were randomly divided into six groups, namely the normal control group, the pathologic control group, the Mesalazine group, the high dose group of Guanchang Decoction, medium dose group of Guanchang Decoction, low dose group of Guanchang Decoction. The rats were killed to observe the general condition and pathological changes after treatment. The results showed:The rats in the model group can been observed the changes of the symptoms such as intermittent diarrhea with mucus and blood at stool,. emaciation, laziness, no luster in hair, poor appetite, loss of weight and increase of colonic damage scores. The colon tissue damaged score in pathologic group is higher than that in normal group(P<0.01);After treatment, their symptoms and sighs obviously improved. The colon tissue damaged score in pathologic group was higher than that in the high, medium, low dose group of Guanchang Decoction and Mesalazine group.
2. Effect of Guanchang Decoction on Proinflammatory Cytokines TNF-α、IL-1β、IL-6 in Serum of UC Model Rats
It was proposed that cytokines especially the proinflammatory cytokines such as the tumor necrosis factor-α(TNF-α)、interleukin-1β(IL-1β)and interleukin-6 (IL-6)the played an important role in the morbility and development of UC. To confirm the curative effect and study the treatment mechanism of Guanchang Decoction, the effect of TNF-α、IL-1β、IL-6 in serum of UC model rats was studied. The grouping and administration were same as before. The rats were killed after administration to get their serum. The contents of TNF-α、IL-1β、IL-6 in serum were measured with double antibody sandwich ABC-ELISA. The results showed:The level of TNF-α、IL-1βand IL-6 in pathologic group was significantly higher than that in normal group(P<0.01); The level of TNF-α、IL-1βand IL-6 in Guanchang Decoction high, medium, low dose group, Mesalazine group was lower than in pathologic group (P<0.01 or P<0.05); The level of TNF-α、IL-1βand IL-6 in Guanchang Decoction high dose group was lower than that in SASP group two groups have not significant difference(P>0.05).
3. Effect of Guanchang Decoction on Gene Expression of ITF and MUC2 in Colonic Mucosa of UC Model Rats.
ITF was regarded as a fast reaction peptide in the course of mucosa impairments. ITF has two important functions in the intestinal tract:protecting the epithelial cell and promoting the repair of mucosa. So ITF plays an important role in the morbility and development of UC. Mucin2 (MUC2) is also secretory product of mucous epithelia, which is main ingredients of maintaining thickness of slime layer and colloidal shape. So it plays a key role in mucosal protection and repair mechanism of UC. To investigate the deepgoing treatment mechanism of Guanchang Decoction on UC, the genes expression of and were detected in colonic mucosa of UC model rats induced by TNBS. The grouping and administration were same as before. After treated period, the rats were killed to get their colonic mucosa. Total RNA was isolated from these rats'colonic mucosa. RT PCR was used to detect ITF and MUC2 expressions andβ-actin was used as internal index. The products of PCR were separated with gel electrophoresis and stained. To compare the relative expression which was calculated from the gray scale ratio of ITF、MUC2 andβ-acting. The results showed:Relative gene expression of ITF, MUC2 in pathologic group were slightly higher than that in normal group, two groups have not significant difference(P>0.05). Relative gene expression of ITF、MUC2 in Guanchang Decoction high, medium dose group was significantly higher than that in pathologic group (P<0.05)
4. Effect of" Guanchang Decoction on Gene Expression of TLR-4 in Colonic Mucosa of UC Model Rats.
TLRs were important component elements of TLRs/NF-kB pathway, which could be activated by NF-kB. If the gene expression of TLRs was inhibited, the activation of TLRs/NF-kB could be inhibited. It was important to inhibite the activation of TLRs/NF-kB in the period of the treatment for UC patients. To investigate the deepgoing treatment mechanism of Guanchang Decoction on UC, the genes expression of and were detected in colonic mucosa of UC model rats induced by TNBS. The grouping and administration were same as before. After treated period, the rats were killed to get their colonic mucosa. Total RNA was isolated from these rats'colonic mucosa. RT-PCR was used to detect TLR-4 expressions andβ-actin was used as internal index. The products of PCR were separated with gel electrophoresis and stained.To compare the relative expression which was calculated from the gray scale ratio of TLR-4 andβ-acting. The results showed:Relative gene expression of TLR-4 in pathologic group were significantly higher than that in normal group (P<0.05). Relative gene expression of TLR-4 in Guanchang Decoction high, medium dose group was significantly lower than that in pathologic group (P<0.05).
5. Conclusion
This model was successful. The treatment of Guanchang Decoction was effective on the UC rats. Guanchang Decoction treating the rats with UC has dose-effect relationship. Blance between pro-inflammatory cytokines and anti-inflammatory cytokine is extremely important in the formation of the UC. Guanchang Decoction could reduce the content of TNF- p、IL-1βand IL-6, inhibit the release of pro-inflammatory cytokines. ITF and MUC2 have better protective function to gastrointestinal mucosa. The mechanism involves in interaction of ITF and MUC2, such as gel layer to resist noxious substance, promoting cell reinforcing the immigration and proliferation. Guanchang Decoction could up-regulate ITF and MUC2 expressions, strengthen the interaction of ITF and MUC2, promote the formation of protective layer to defend the intestinal mucosa of UC. TLRs were important component elements of TLRs/ NF-kB pathway, which could beactivated by NF-κB. If the gene expression of TLRs was inhibited, the activation of TLRs/NF-kB could be inhibited. It was important to inhibite the activation of TLRs/NF-kB in the period of the treatment for UC patients. The gene expression of TLR-4, in colonic mucosa of UC model rats induced byTNBS were inhibitted after using Guangchang Decoction.
引文
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