| Above all the immune factors, cytokines played an important role in the morbility and development of inflammatory bowel disease (IBD). The study on morbility and therapy of ulcerative colitis (UC) had been made considerable progress by investigating these cytokines. However, the key component element was unknown. The mechanism of IBD has been revealed step by step with the investigation of cytokines, adherence factor, especially the Nuclear Factor-κB (NF-kB) and Toll-like receptors (TLRs) for the past few years. Furthermore, It was helpful to the treatment of IBD to study the TLRs/NF-κB signal transduction pathway. Now more and more experts has paid close attention to inhibiting the key elements of TLRs/NF-kB pathway to obstruct the irritable inflammation of IBD. 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. The key point of investigation on treating IBD with TCM was empirical study on mechanism of action. The exact effect target could be revealed with such investigation. In this experiment, the action mechanism of Kuijieling decoction, which showed more affirmative effects on treating UC patients, would be researched deeply. The on . The key elements of TLRs/NF-kB pathway Such as the activation of NF-kB and expression of TLR2, 4 were observed in the experiment go evaluate the interventional effect of Kuijieling decoction. The research on mechanism of TCM on treating UC would be penetrate deeply to gene level ,which would be helpful to develop new TCM. 1 Observation of Kuijieling Decoction in Pathological Changes on UC Model RatsKuijieling decoction was frequently used in PiWei institute to treat UC patients. The therapeutic principle of the decoction was clearing away heat, invigorating the spleen and activating blood .It showed affirmative therapeutic effect on patients without obvious toxical and side effects. To validate the clinical curative effect and investigate the mechanism of action, effect of Kuijieling Decoction on UC model rats induced by TNBSwas observed. All the rats were divided into six groups as follows: normal control (NC) group, model control (MC) group ,Kuijieling low dose (KLD) group, Kuijieling medium dose (KMD) group, Kuijieling high dose (KHD) group and SASP group. The rats were killed to observe pathological changes after 10-days treatment. The number of ulcer were calculated, the area of ulcer were masured and the index of injury were culated with macroscopic observation. The degrees of inflammation, hyperemia and edema in colonic mucosa were graded with microscopic observation. The results showed: There were several ulcers in the colonic mucosa of MC group and the degrees of inflammation and edema were serious. The number of ulcer on the colonic mucosa of KMD group and KHD group were significantly decreased compared to the MC group (P<0.05, P<0.01). The index of injury of KMD group and KHD group were significantly decreased compared to the MC group (P <0.01) . The degrees of inflammation of KMD group and KHD group were significantly relieved compared to the model control group (P<0.05, P<0.01) . The degrees of hyperemia and edema of KHD group were significantly relieved compared to the model control group (P<0.05). The conclusion was: the number and area of ulcer of model rats were significantly reduced with the administration of Kuijieling Decoction. Pathological changes such as inflammatory cell infiltration and edema were improved. It showed affirmative curative effect of kuijieling on treating UC model rats induced by TNBS.2 Effect of Kuijieling Decoction on Proinflammatory Cytokines IL-lp, TNF-a in Serum of UC Model RatsIt was proposed that cytokines especially the proinflammatory cytokines such as the interleukin-ip( IL-ip) and the tumor necrosis factor- a (TNF- a ) played an important role in the morbility and development of UC .To confirm the curative effect and study the treatment mechanism of Kuijieling Decoction, the effect of IL-ip and TNF-a in serum of UC model rats was studied. The grouping and administration were same as before. The rats were killed after 10-days administration to get their serum. The contents of IL-10 and TNF-a in serum were measured with double antibody sandwich ABC-ELISA. The results showed that the contents of TNF-a and IL-ip in serum of the MC group were significantly increased compared to the normal control group (P<0.01) .The contents of TNF-a and IL-ip in serum of KMD group and KHD group were significantly decreased compared to the MC group (P<0.01). The conclusion was: By using Kuijieling Decoction, the contents of TNF-a and IL-ip in serum of UC model rats were reduced.3 Effect of Kuijieling Decoction on Immunosuppressive Cytokines IL-4 in Serum of UC Model RatsOf all the immunologic mechanisms of UC, the unbalance between proinflammatorycytokines and immunosuppressive cytokines was important. To investigate the therapy mechanism of Kuijieling Decoction, the effect of immunosuppressive cytokines—IL-4 in Serum of UC model rats was observed. The grouping and administration were same as before. The content of IL-4 in serum was measured with double antibody sandwich ABC-ELISA. The results showed that the content of IL-4 in serum of the MC group was significantly increased compared to the NC group (P<0.01) .The content of IL-4 in serum of KHD group was markedly decreased compared to the model control group (P< 0.01) .The changes of IL-4 in UC patients or UC rats were under dispute. Even some results were contradictory. By using Kuijieling Decoction, the content of IL-4 in serum was reduced. What kind of role it played in UC should be investigated further.4 Effect of Kuijieling Decoction on Expression of NF-kB P65 in Colonic Mucosa of UC Model RatsAt present the investigation of NF-kB, TLRs/ NF-kB pathway was becoming a focus point of IBD research, which could reveal the nosogenesis of IBD. To investigate the deepgoing therapy mechanism of Kuijieling Decoction on UC, the expression of NFKB/p65 in colonic mucosa was detected with immunohistochemical method. The grouping and administration were same as before. The tissue sections of colon were stained with immunohistochemical staining. The positive rate of p65 expression was analysis with statistics. The results showed that the positive rate of p65 was significantly higher in the MC group than that of NC group (P<0.01). The positive rate of p65 was significantly lower in the KMD and KHD group than that of MC group (P<0.01). The positive rate of NF-kB p65 in colonic mucosa of UC model rats was reduced after the administration of Kuijieling Decoction.5 Effect of Kuijieling Decoction on Gene Expression of TLR2,4 in Colonic Mucosa of UC Model RatsTLRs 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 Kuijieling Decoction on UC, the genes expression of TLR2 and TLR4 were detected in colonic mucosa of UC model rats induced by TNBS. The grouping and administration were same as before. After 10-days 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 TLR2 and TLR4 expressions and P -actin was used as internal index. The products of PCR were separated with gel electrophoresis and stained .To compare the relativeexpression which was calculated from the gray scale ratio of TLR2, 4 and 3 - acting. The results showed that relative gene expression of TLR2, 4 in MC group were significantly higher than that in NC group (P<0.01). Relative gene expression of TLR2 in KMD and KHD group was significantly lower than that in MC group (P<0.05). Relative gene expression of TLR4 in KHD group was significantly lower than that in MC group (P< 0.05). The gene expression of TLR2, 4.in colonic mucosa of UC model rats induced by TNBS were inhibitted after using Kuijieling Decoction.TLRs/ NF-kB pathway played crucial roles in the occurrence and development of IBD. Since NF-kB could regulate "many key cytokines in the morbility of UC, it maybe participate in inflammation and immunity reaction of UC. Combined with upstream stimulating factors, TLRs transferred activated signals to the intracellular regions and activated transcription factors such as NF-kB, then the productions of inflammatory mediators that could result in injury in mucosa of IBD were regulated. Once provoked by activated NF-kB, the inflammatory reaction was enlarged and couldn't terminate at once even if the initial etiopathogenisis were removed. NF-kB, TLRs and upstream (downstream) factors such as IL-ip, TNF-a were key components of TLRs/ NF-kB pathway. Proceeding of UC could be prevented efficiently by inhibiting these components. In short, Kuijieling Decoction could be effectually on treating UC model rats with clearing away heat, invigorating the spleen and activating blood therapy. By using Kuijieling Decoction, the contents of TNF-a, IL-ip and IL-4 in serum of model rats were reduced, the positive rate of NF-kB p65 in colonic mucosa of UC model rats was reduced, and the genes expression for TLR2 and TLR4 in colonic mucosa were inhibitted. The results showed many component elements of TLRs/ NF-kB pathway were regulated after using Kuijieling Decoction , which could be its partial mechanism of treating UC.
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