Effect Of Guanchang Decoction On Gene Expression Of ITF, MUC2 And TLR4 In Colonic Mucosa Of Rats With Ulcerative Colitis

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 RatsGuanchang 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 RatsIt 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. ConclusionThis 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.