| Ulcerative colitis(UC) is an inflammatory disease of the rectal and colonic mucosa. The aetiology of UC remains unknown. UC seems to result from a complex series of interactions between susceptibility genes, the environment and the immune system. An involvement of the immune system plays an important role in the pathogenesis of UC. With the fast development of immunology, molecular biology and investigation of cytokines, adherence factor, especially nuclear factor-κB (NF-κB) and toll-like receptors(TLRs) for the past few years, the mechanism of UC has been revealed step by step. It was helpful to the treatment of UC by studying TLRs/NF-κB signal transduction pathway. Now more and more experts have paid close attentions to inhibiting key elements of TLRs/NF-κB pathway to obstruct the irritable inflammation of UC. Therapeutic effect of traditional Chinese medicine(TCM) on UC was similar to that of Western medicine. But the defect of Western medicine such as easy recurrence, toxicity and side effects were seldom found in using TCM. The prospect of utilization and development of TCM is cheerful. The key point of investigation on treating UC with TCM was empirical studied on mechanism of action. The exact effect target could be revealed with such investigation.Kuijieling decoction(KD) was frequently used to treat UC patients in PiWei institute. 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 toxicity and side effects. In the past investigation, effect of KD on UC model rats induced by TNBS was observed. It was observed that the number and area of ulcer of 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 model rats. The gene expression of TLR2, TLR4 in colonic mucosa of UC model rats induced by TNBS were inhibitted after using KD. The key elements of TLRs/NF-κB pathway such as the activation of NF-κB and expression of TLR2, TLR4 were observed in the experiment go evaluate the interventional effect of KD. The research on mechanism of TCM on treating UC would be penetrated deeply to gene level, which would be helpful to develop theory of TCM.1 Effect of KD on IL-1β, TNF-αin Colonic Mucosa of UC Model RatsIt was proposed that cytokines, especially the proinflammatory cytokines such as the interleukin-1β(IL-1β) and the tumor necrosis factor-α(TNF-α) played important roles in the morbility and development of UC. To study the treatment mechanism of KD, the effect of IL-1βand TNF-αin colonic mucosa of UC model rats was studied. UC model rats were induced by TNBS. All the rats were randomly divided into four groups as follows: normal control(NC) group, model control(MC) group, Kuijieling medium dose(KMD) group and Kuijieling high dose(KHD) group. After 10-days treatment the rats were killed to get their colonic mucosa. Contents of IL-1βand TNF-αin colonic mucosa were measured with double antibody sandwich ABC-ELISA. The results showed that the contents of TNF-αand IL-1βin colonic mucosa of MC group were respectively 99.35±36.67 pg/mL and 160.90±51.78 pg/mL, significantly increased compared to those in the NC group(respectively 33.06±10.97 pg/mL and 35.64±11.17pg/mL, P＜0.01). The contents of TNF-αin colonic mucosa of KMD group was 40.66±6.58pg/mL, significantly decreased compared to MC group(P＜0.01). The contents of IL-1βin colonic mucosa of KMD group and KHD group were respectively 67.30±32.57pg/mL and 69.32±31.24pg/mL, significantly decreased compared to MC group (P＜0.01). The conclusion was: by using KD, the contents of TNF-αand IL-1βin colonic mucosa of UC model rats were reduced.2 Effect of KD on Gene Expression of ICAM-1 in Colonic Mucosa of UC Model RatsRecently people have payed more and more attentions on relationship between UC and cell adhesion molecules(CAM), and intercellular adhesion molecules(ICAM-1) plays an important role in the morbility and development of UC. To study the treatment mechanism of Kuijieling Decoction, gene expression of ICAM-1 in colonic mucosa of UC model rats was studied. UC model rats were induced by TNBS. The rats were randomly divided into six groups as follows: NC group, MC group, Kuijieling low dose(KLD) group, KMD group, KHD group and SASP group. After 10-days treatment the rats were killed to get their colonic mucosa. Total RNA was isolated from colonic mucosa by Trizol. RT-PCR was used to detect ICAM-1 expressions andβ-actin was used as internal index. The products of PCR were separated with gel electrophoresis. The relative expression of ICAM-1 which was calculated from the gray scale ratio of ICAM-1 andβ-actin was compared. The results showed that relative gene expression of ICAM-1 in MC group was 1.044±0.180, significantly higher than that in NC group (0.359±0.119, P＜0.01). Relative gene expression of ICAM-1 in KMD was 0.797±0.210, significantly lower than that in MC group(P＜0.05). Relative gene expression of ICAM-1 in KHD and SASP group were respectively 0.555±0.184 and 0.635±0.242, significantly lower than that in MC group(P＜0.01). The gene expression of ICAM-1 in colonic mucosa of UC model rats induced by TNBS were inhibited after using KD.3 Effect of KD on DNA Binding Activity of NF-κB P65 in Colonic Mucosa of UC Model RatsExpressions of IL-1β, TNF-αand ICAM-1 are associated with activation of NF-κB. DNA binding activity of NF-κB P65 was evaluated to investigate therapy mechanism of KD on UC. The grouping and administration were same as before. The rats were killed after 10-days administration to get their fresh colonic mucosa to extract nuclear proteins. DNA binding activity of NF-κB P65 were detected by Trans AM TM NF-κB p65 kit based on ELISA. The results showed that relative activation of NF-κB in MC group was 0.440±0.119, significantly higher than that in NC group(0.261±0.042, P＜0.01). Relative activation of NF-κB in KHD and SASP group were respectively 0.261±0.056 and 0.269±0.106, significantly lower than that in MC group (P＜0.05). Relative activation of NF-κB in colonic mucosa of UC model rats induced by TNBS were inhibited after using KD.4 Effect of KD on Proteins Level of TLR2, TLR4 in Colonic Mucosa of UC Model RatsTLRs are important component elements of TLRs/NF-κB pathway, which are pattern recognition receptors(PRRS) and recognize pathogen-associated molecular patterns(PAMPs) that leads to activation of NF-κB by a series of signal transduction molecules. Protein level of TLR2 and TLR4 were detected in colonic mucosa of UC model rats induced by TNBS for further study of treatment mechanism of KD on UC. The grouping and administration were same as before. Whole-cell protein were extracted from colonic mucosa. Western blot technique was used to detect protein level of TLR2 and TLR4.β-actin was used as internal index. Relative expression of target protein was calculated from the gray scale ratio of target protein andβ-acting. The results showed that relative protein expression of TLR2 and TLR4 in MC group were respectively 0.663±0.137 and 0.843±0.201, significantly higher than that in NC group(respectively 0.254±0.052 and 0.472±0.072, P＜0.01). Relative protein expression of TLR2 in KMD and KHD group were respectively 0.472±0.160 and 0.376±0.104, significantly lower than that in MC group (P＜0.05, P＜0.01). Relative protein expression of TLR4 in KHD group was 0.620±0.178, significantly lower than that in MC group(P＜0.05). The relative protein expression of TLR2 and TLR4 in colonic mucosa of UC model rats induced by TNBS were inhibited after using KD.5 Effect of KD on Expression of IκB-α,IKK-αin Colonic Tissue of UC Model RatsIκB-αand IKK-αare middle links of TLRs/ NF-κB pathway. Phosphorylation of IκB is the key step of activation of NF-κB. Activation of IKK is a step that restricts the speed of activation of NF-κB. UC rats induced by TNBS is associated with activation of NF-κB and excessive expression of TLR2 and TLR4 protein. KD can down-mediate excessive activation of NF-κB and excessive expression of TLR2 and TLR4 protein. At the base of these, expression of IκB-αand IKK-αin colonic tissue were detected. The grouping and administration were same as before. Tissue sections of colon were stained with immunohistochemical staining. Positive rate of IκB-αor IKK-αexpression was analysised with statistics. The results showed that the positive rate of IκB-αor IKK-αin MC group were respectively 36.3±11.9％and 35.3±12.0％, significantly higher than those in NC group(respectively 14.8±5.1％and 11.0±4.4％, P＜0.01). The positive rate of IκB-αin KHD group was 26.2±8.6％, significantly lower than that in MC group(P＜0.05). The positive rate of IKK-αin KMD group was 25.3±7.0％, significantly lower than that in MC group(P＜0.05). The positive rate of IKK-αin KHD and SASP group were respectively 20.2±6.0％and 22.3±6.2％, significantly lower than that in MC group(P＜0.01). The positive rate of IκB-αor IKK-αin colonic tissue. of UC model rats was reduced after the administration of KD.In general, TLRs/NF-κB pathway played crucial roles in the occurrence and development of UC. Since NF-κB could regulate many key cytokines such as TNF-α, IL-1βand ICAM-1 in the morbility of UC, it maybe participate in inflammation and immunity reaction of UC. Combined with upstream stimulating factors such as TNF-α, IL-1βand so on, TLRs transferred activated signals to the intracellular regions and activated transcription factors such as NF-κB, then production of inflammatory mediators that could result in injury in mucosa of UC was regulated. Once provoked by activated NF-κB, the inflammatory reaction were enlarged and couldn't terminate at once even if the initial etiopathogenisis were removed. NF-κB, TLRs and upstream or downstream factors such as IL-1β, TNF-α, ICAM-1 were key components of TLRs/NF-κB pathway. Proceeding of UC could be prevented efficiently by inhibiting these components. In short, 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 TNF-αand IL-1β, gene expression of ICAM-1, relative activation of NF-κB and protein expression of TLR2 and TLR4 in colonic tissue of UC model rats. Positive rate of IκB-αor IKK-αin colonic mucosa of UC model rats were inhibited. The results showed that KD could regulate many component elements of TLRs/NF-κB pathway, which could be its partial mechanism of treating UC. |
