| 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.
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