【作者】 宋宁；

【导师】 王汝俊；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2008， 博士

【摘要】 溃疡性结肠炎(UC)是一种主要累及直肠、结肠粘膜的慢性非特异性炎症,临床上至今没有特异性的根治措施,治疗多以抗炎、调节免疫为主。近年研究发现,肠道受到一系列损伤发生溃疡后,机体的修复机制开始启动来促进其快速愈合,其过程有许多生长因子参与。肠三叶因子(ITF)是一种由杯状细胞分泌的特异分布于肠道的新型细胞生长因子,具有很强的细胞保护作用,可明显减轻多种损伤因子介导的肠粘膜损害,它不仅具有一般生长因子所具有的促进细胞增殖与移行能力,还具有独特的生理功能并可同粘液糖蛋白结合,稳定肠粘液层。因此,ITF被认为是肠道的特异保护因子。中医药在UC的治疗上有着明显的优势,开展中药对ITF调节作用的深入研究,有望为UC的治疗提供新的契机。脾胃研究所在多年临床经验的基础上,以清热健脾活血为治则,总结出治疗UC的溃结灵复方(KD),临床取得了较好的疗效。前期研究采用三硝基苯磺酸法(TNBS)制作UC大鼠模型,发现KD能明显减少UC大鼠结肠溃疡个数和面积,改善结肠粘膜炎症和水肿等病理变化,同时又能降低模型大鼠血清中促炎因子IL-1β、TNF-α的含量和结肠组织中NF-κBp65蛋白的阳性细胞率,对模型大鼠结肠粘膜TLR2、TLR4基因表达也有抑制作用。本研究拟从促粘膜修复入手,观察KD对ITF的表达及其信号转导通路的关键蛋白MEK、ERK磷酸化的影响,探讨KD治疗UC的深层次作用机理。这一研究将使中医药治疗UC的机理研究深入到基因调控水平,并为开发高质量的抗UC中药新药打下坚实的基础,有着重要的理论意义和广阔的应用前景。1 KD对UC大鼠结肠粘膜超微结构的影响研究发现UC病变主要限于结肠粘膜和粘膜下层,且以溃疡和炎症变化为主。髓过氧化物酶(MPO)是中性粒细胞中含量较高的一种酶,与炎症变化密切相关。本实验在我们前期病理形态学研究的基础上,进一步观察了KD对UC大鼠结肠粘膜的超微结构影响及MPO含量的改变,以期对其作用机制进行初步的探讨。大鼠随机分为4N:正常组、模型对照组、KD组和阳性药SASP组。治疗十天后处死大鼠并采集结肠组织标本。透射电镜观察,发现UC模型组大鼠结肠粘膜上皮表面的微绒毛脱落,变短,扭曲,细胞器减少,细胞质液化溶解,腺上皮细胞间连接松散,提示炎症及溃疡引起了上皮细胞及细胞连接的损伤。杯状细胞分泌增强,有的细胞内颗粒呈排空状态,可能是在受到炎性介质刺激下,肠道杯状细胞分泌量代偿性增加(包括粘蛋白、三叶因子等),从而启动肠粘膜自身保护机制。经溃结灵治疗后,大鼠结肠粘膜上皮表面的微绒毛基本完整,腺上皮间相互连接紧密,胞浆内粘液颗粒较丰富,并向腺腔排出,表明溃结灵能够减轻结肠粘膜超微结构损伤,促进杯状细胞分泌保护因子,进而加快肠道粘膜重建和溃疡修复过程。MPO检测结果显示,模型组大鼠结肠粘膜MPO活性为0.3516±0.0738U/g,明显高于正常组0.1982±0.0719 U/g(P＜0.01);与模型组比较,溃结灵组和SASP组结肠MPO活性显著降低(分别为0.2876±0.0554 U/g,P＜0.05;0.2507±0.0303 U/g,P＜0.01),提示溃结灵能减轻TNBS大鼠的炎症程度,减少溃疡发生。2 KD对UC大鼠结肠粘膜ITF基因和蛋白表达的影响ITF被看作是粘膜损伤的快速反应肽,在胃肠道有两个重要功能,即上皮保护和促进粘膜愈合,与肠道炎症性疾病的发生和发展有着密切的关系。本研究观察KD对TNBS法大鼠模型结肠粘膜ITF基因及蛋白表达的作用,深入探讨其作用机制。大鼠随机分组同前,治疗十天后处死大鼠并快速采集结肠标本。选择结肠病变相同部位剪取小块组织,固定包埋切片,免疫组织化学染色,光镜下观察ITF蛋白表达情况,并进行半定量统计分析。余下粘膜用Trizol提取总RNA,RT-PCR法检测ITFmRNA的表达,以GAPDH作为内参,产物进行凝胶电泳分离,用凝胶成像仪自带分析软件进行灰度分析,以ITF与GAPDH的灰度值比值作为ITFmRNA的相对表达量,进行组间比较分析。结果显示,模型组结肠粘膜中ITFmRNA相对表达量为1.027±0.1263,略高于正常组(1.004±0.1046;P＞0.05);模型组结肠组织ITF蛋白表达与正常组比较无显著差异(P＞0.05)。溃结灵组和SASP组结肠粘膜中ITFmRNA相对表达分别为1.268±0.2113和1.299±0.3117,明显高于模型组(P＜0.05);溃结灵组和SASP组结肠组织中ITF蛋白表达也明显高于模型组(P＜0.01),提示KD对TNBS法UC大鼠模型结肠粘膜ITF基因及蛋白表达有明显上调作用,这可能是溃结灵治疗作用的一个关键靶位。3 KD对UC大鼠结肠粘膜MUC2基因表达的影响粘蛋白2(MUC2)也是杯状细胞合成的分泌型蛋白,是维持粘液层厚度及胶体形态的主要成分,在粘膜保护及肠道疾病修复机制中占有重要的地位。本研究观察了KD对TNBS法UC大鼠模型结肠粘膜MUC2mRNA表达的影响,并探讨其中的作用机理。分组和治疗同前述,治疗结束后,刮取结肠粘膜标本并提取总RNA,采用RT-PCR法检测MUC2mRNA的表达,以GAPDH作为内参,产物进行凝胶电泳分离,用凝胶成像仪自带分析软件进行灰度分析,以MUC2与GAPDH的灰度值比值作为MUC2mRNA的相对表达量,进行组间比较分析。结果显示,UC模型组大鼠结肠粘膜MUC2mRNA的相对表达与正常组比较,略有增高,但没有显著差异(分别为0.6423±0.1668和0.5924±0.1758,P＞0.05)。与模型组比较,溃结灵和SASP对UC大鼠结肠粘膜MUC2基因的表达都有明显的上调作用(分别为0.9811±0.1828和1.0491±0.2675,P＜0.01)。提示,KD能上调MUC2基因表达,从而加强肠道粘膜屏障的保护功能,促进受损区域上皮细胞重建,这可能是溃结灵治疗UC的作用机理之一。4 KD对UC大鼠结肠粘膜pERK1/2、pMEK1/2蛋白水平的影响Ras-Raf-MEK1/2-ERK1/2等MAPK信号转导通路是广泛存在于真核细胞内的一条重要信号转导通路,参与调节细胞的分化、增生、凋亡等过程。有研究发现生长因子等有丝分裂刺激因素是激活ERK途径的主要细胞外信号,被激活的ERK可诱导细胞产生增殖、分化等核反应。KD对TNBS法UC大鼠模型结肠粘膜ITF基因及蛋白的表达有明显的上调作用,在此基础上本研究对UC大鼠模型结肠粘膜ERK1/2、MEK1/2磷酸化水平进行检测,以期对KD治疗UC的较深层次的作用机理进行探讨。分组和治疗同前,治疗结束后采集结肠粘膜标本并提取全细胞蛋白,运用蛋白免疫印迹(Western blot)方法对pERK1/2、pMEK1/2的蛋白表达水平进行检测,以β-action作为内参,以目的蛋白与β-action的密度比值作为目的蛋白的相对含量,进行组间比较分析。结果显示,模型组大鼠结肠粘膜中pERK1/2、pMEK1/2蛋白相对表达量分别为0.3974±0.1017和0.6994±0.1372,均高于正常组(分别为0.2037±0.1234和0.4092±0.1177,P＞0.05,P＜0.01)。与模型对照组比较,KD组pERK1/2、pMEK1/2蛋白相对表达量明显增高,分别为0.7060±0.1607和0.8928±0.1801(P＜0.01,P＜0.05)。阳性药SASP组pERK1/2、pMEK1/2蛋白相对表达量为0.7299±0.2710和0.9053±0.1591,也显著高于模型组(P＜0.01,P＜0.05)。提示KD对TNBS法UC大鼠模型结肠粘膜pERK1/2、pMEK1/2蛋白表达有显著上调作用,这可能是其治疗UC的作用环节之一。5结论ITF具有很好的胃肠粘膜保护作用,在炎症性肠病时上调表达,可促进损伤粘膜的修复。其作用机制涉及ITF与粘糖蛋白相互作用,增强粘液凝胶层对粘膜表面有害物质的抵抗,以及促进细胞迁移增殖等。对ITF的深入研究,特别是对其在炎性肠病粘膜修复中作用的研究,颇具应用价值。本研究选用清热健脾活血中药复方KD治疗大鼠实验性UC,取得了较好的疗效,结果表明,①KD能明显降低UC模型大鼠结肠中MPO的活性,改善结肠粘膜的超微结构;②显著上调UC模型大鼠结肠粘膜中MUC2基因、ITF基因及蛋白的表达水平;③明显提高UC模型大鼠结肠粘膜中ERK1/2和MEK1/2磷酸化水平。综合研究结果分析,KD治疗UC的作用机制,一方面可能是通过上调ITF和MUC2的表达,加强二者相互作用,促进了粘液凝胶层的形成,进而增强肠道粘膜防御屏障的保护能力;另一方面,也可能是通过上调ITF表达从而激活ERK信号转导通路,刺激UC大鼠肠道上皮细胞移行增殖,加速损伤修复的。进一步,我们还需对ITF及MAPK信号传导通路在肠损伤中的作用作更为深入和全面的动态研究,明确其作用的分子机制及具体的靶基因和蛋白,以便为临床UC治疗提供新的思路与方法。更多还原

【Abstract】 Ulcerative colitis(UC)is a chronic and non-specific inflammatory disease of the rectal and colonic mucosa.Up to now,there have no specific radical measures to cure UC.Anti-inflammatory and regulating immunity are the main treatments of UC.Recently,lots of researches found that after intestinal tract is destroyed by a series of impairments and intestinal ulcers,repair mechanisms of body start to run to enhance the healing of ulcers,which include a number of growth factors.As a new cell growth factor,intestinal trefoil factor(ITF)expressed in intestinal tract is a major secretory product of mucous epithelia.ITF has a strong cells protective effect to alleviate intestinal impairments,such as promoting proliferation and migration of intestinal epithelial cell,increasing the intestinal mucosa defence by associated with mucins.Therefore,ITF is regarded as a specific protective factor.The therapeutic effect of Chinese medicine is superiority than western medicine in the process of UC,so the study of ITF regulated by Chinese medicine is a new way to develop the treatment of UC.Kuijieling decoction(KD)was frequently used to treat UC patients in Pi Wei institute.The therapeutic principle of KD is clearing away heat, invigorating the spleen and activating blood.It showed affirmative therapeutic effect on patients.Some experiments about effect of KD on UC model rats induced by TNBS were observed before.The observation showed that the number and area of ulcer in model rats were significantly reduced with the administration of KD.Pathological changes such as inflammatory cell infiltration and edema were improved.It showed affirmative curative effect of KD on treating UC model rats induced by TNBS.At the same time KD could reduce contents of TNF-αand IL-1βin serum of UC model rats and positive rate of NF-κB p65 in colonic mucosa of UC rats.The gene expressions of TLR2, TLR4 in colonic mucosa of UC model rats induced by TNBS were obviously inhibited after using KD.In this article,the gene and protein expressions of ITF and the key elements of MEK/ERK pathway such as the activation of MEK and ERK were observed to evaluate the interventional effect of KD.The mechanism of KD on treating UC would be detected deeply to gene level,which would be helpful to develop theory of TCM.1 Effect of KD on ultrastructure in Colonic Mucosa of UC Model RatsThe researches showed that the features of UC were inflammation and ulcer in the submucosa and mucosa of the colon.Myeloperoxidase is an enzyme association with the changes of inflammation,which is higher existed in the neutrophilic granulocyte.So the changes of MPO contents and epithelial cell ultrastructures in colonic mucosa in UC model rats were studied.UC model rats were induced by TNBS.The rats were divided into four groups randomly as follows: normal control(NC)group,model control(MC)group,Kuijieling dose(KD) group and salicylazosulfapyridine(SASP)group.After 10-days treatment the rats sacrificed to get their colonic tissue.The ultrastructural changes were obsered by transmission electron microscope.In model group,the ultrastructures of colonic mucosa were subversion,the microvilli of epithelium were sparse,shorter,irregularly curled or fall off;the organelles became fewer;the cytoplasm liquefied and dissolved.The links between epithelium were incompact,and some goblet cells showed the stronger function of secretion and evacuation.Above changes in model group hinted that the epithelial cell were impaired by inflammation and ulcer,and the goblet cell might be stimulated by inflammation mediator to secrete more substances including mucin and ITF.So the repair mechanism in colonic mucosa would be stated.In KD groups,the ultrastructural pathological changes were improved, such as the microvilli on the surface of the mucosal epithelium were basically regular and intact,epithelia linked tightly,the goblet cells had affluent mucus particle and evacuation.It showed that KD could improve the morph and function of goblet cells and alleviate the ultrastructure damage in colonic mucosa of UC Model Rats.The levels of MPO in colonic mucosa of model group were 0.3516±0.0738U/g,obviously higher than that of normal control group (0.1982±0.0719 U/g,P＜0.01).Compared with model group,the MPO levels of KD and SASP group were decreased significantly(respectively0.2876±0.0554U/g, P＜0.05,0.2507±0.0303 U/g,P＜0.01).The results showed the anti-inflammatory effect of KD on treating UC model rats induced by TNBS.2 Effect of KD on Gene and Protein Expression of ITF in Colonic Mucosa of UC Model RatsITF 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.In this article the gene and protein expression of ITF in colonic mucosa of UC model rats was studied to evaluate the treatment mechanism of KD.The grouping and administration were same as before.After 10-days treatment the rats sacrificed to get their colonic tissue.Tissue sections of colon were stained with immunohistochemical staining.Positive expression of ITF was analysised with statistics.Total RNA was isolated from colonic mucosa by Trizol.RT-PCR was used to detect ITF expressions and GAPDH was used as internal index.The products of PCR were separated with agarose gel electrophoresis.The relative expression of ITF was calculated from the gray scale ratio of ITF and GAPDH. The results showed that relative gene expression of ITF in MC group was 1.027±0.1263,slightly higher than that in NC group(1.004±0.1046,P＞0.05). Relative protein expression of ITF in MC group was no significantly difference comparing to NC group(P＞0.05).Relative gene expression of ITF in KD and SASP were respectively 1.268±0.2113 and 1.299±0.3117,significantly higher than that in MC group(P＜0.05).Relative protein expression of ITF in KD and SASP group were significantly higher than that in MC group(P＜0.01).The gene and protein expression of ITF in colonic mucosa of UC model rats induced by TNBS were increased after using KD.3 Effect of KD on Gene Expression of MUC2 in Colonic Mucosa of UC Model RatsMucin2(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.The grouping and administration were same as before.The rats sacrificed after 10-days administration to get their fresh colonic mucosa.Total RNA was isolated from colonic mucosa by Trizol.RT-PCR was used to detect MUC2 expressions and GAPDH was used as internal index.The products of PCR were separated with gel electrophoresis.The relative expression of MUC2 was calculated from the gray scale ratio of MUC2 and GAPDH.The results showed that relative gene expression of MUC2 in MC group was slightly higher than that in NC group(respectively 0.6423±0.1668 and 0.5924±0.1758,P＞0.05). Relative gene expression of MUC2 in KD and SASP were 0.9811±0.1828 and 1.0491±0.2675,significantly higher than that in MC group(P＜0.01).It showed the gene expression of MUC2 in colonic mucosa of UC model rats induced by TNBS were increased after using KD,which reinforced the protection function of mucosal barrier and could be partial mechanism of treating UC.4 Effect of KD on Proteins Level of pMEK1/2 and pERK1/2 in Colonic Mucosa of UC Model RatsMAPK pathway is an important message pathway,which generally resides in eukaryotic cells.It participates in regulating the process of cell differentiation、proliferation、cleavage and apoptosis,and so on.At present there are at least four kinds of MAPK pathways in mammalian intestinal cells,the first identificated way is Ras-Raf-MEK1/2-ERK1/2 pathway.Some researches showed that caryocinetic stimulating factors(such as growth factors)are main extracellular signal to activate ERK pathways.The activated ERK could induct cells and then some nuclear reactions take place, such as differentiation and proliferation.The gene and protein expression of ITF in colonic mucosa of UC model rats induced by TNBS were increased after using KD.On base of these,protein levels of pMEK1/2 and pERK1/2 were detected in colonic mucosa of UC model rats for further study.The grouping and administration were same as before.Whole-cell protein was extracted from colonic mucosa.Western blot technique was used to detect protein levels of pMEK1/2and pERK1/2.β-actin was used as internal index.Relative expression of target protein was calculated from the gray scale ratio of target protein andβ-actin.The results showed that relative protein expression of pERK1/2 and pMEK1/2 in MC group were respectively 0.3974±0.1017 and 0.6994±0.1372, higher than that in NC group(respectively 0.2037±0.1234 and 0.4092±0.1177, P＞0.05,P＜0.01).Relative protein expression of pERK1/2 and pMEK1/2 in KD group were respectively 0.7060±0.1607 and 0.8928±0.1801,significantly higher than that in MC group(P＜0.01,P＜0.05).Relative protein expression of pERK1/2 and pMEK1/2 in SASP group was 0.7299±0.2710 and 0.9053±0.1591,significantly higher than that in MC group(P＜0.01,P＜0.05).The relative protein expression of pMEK1/2 and pERK1/2 in colonic mucosa of UC model rats induced by TNBS were enhanced after using KD,which could be its partial mechanism of KD to treat UC.5 conclusionITF has better protective function to gastrointestinal mucosa.In IBD, up-regulation expression of ITF could recover the impaired mucosa.The mechanism involves in interaction of ITF and MUC2,such as reinforcing the gel layer to resist noxious substance,promoting cell immigration and proliferation.Therefore,ITF is worth to be studied in IBD.KD could be effectual on treating UC model rats with clearing away heat, invigorating the spleen and activating blood therapy.KD could reduce contents of MPO,improve ultrastructural changes;increase gene and protein expression of ITF,raise gene expression of MUC2 and protein expression of pMEK1/2and pERK1/2 in colonic mucosa of UC model rats.In short,KD 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.On the other hand,the increasing ITF expression could activate ERK pathways,which induct some nuclear reactions of cells,such as differentiation、proliferation.These results would offer a new target for Chinese medicine and recipe.The deeper dynamic researches on the effect of ITF and MAPK pathway in IBD should be done for next step, which could identify the molecular mechanism and target protein or gene.The study could provide a new idea for clinical to treat UC.更多还原