| ObjectiveWhether NF-κB signal pathway activated or not related to the occurrence, development and prognosis of ulcerative colitis. The activation of NF-κB is the key link of UC, then form the pathogenesis that take the NF-κB as the center, the cell factors, adhesion molecules, chemotactic factors, abnormal immune responses, etc for network interactions and mutual constraints. In view of NF-κB plays an important role in development of disease, so regulating every links of NF-κB activation may be the target of drug intervention. Every pathways are all activated through the activation IKK and finally activate NF-κB, so the activative pathways of IKK/IκB/NF-κB has become the ideal target of drug intervention and been also a hot issue of recent drug research. Consequently the research on the changes of IκBα, IKKβ and UPP that are in the process of development and repair of UC and also intervention function of chinese traditional medicine, which has an important theoretical significance of treating UC by chinese traditional medicine.The previous researches have shown that KuiJieling has the therapeutic effect on three kinds of UC rat model-that is heterogeneous antigen immune model, DNDB model and TNBS model. KuiJieling can reduce colon ulcer points, reduce inflammatory cells infiltration. At the same time, it can reduce the content of promoting inflammation factors such as TNF-α IL-1β in colonic mucosal of TNBS method UC rat model, make NF-κB relative activity reduced significantly, and inhibit NF-κB p65protein expression. In order to discuss further more about the deep mechanism of the NF-κB activated inhibition, this study that based on the previous studies, starts with the activation and regulation of NF-κB-the source of cell factor, using the TNBS clyster to establish the UC rat model and preparation of caco-2inflammatory cells model induced by tumor necrosis factor-α(TNF-α), and then observe the effect of Kuijieling on the mRNA and protein expression of E1,E2and E3which are related molecules of IκBα, IKKβ and the ubiquitin-proteasome system from the overall and cell level. This research may reveal the deep mechanism of Kuijieling on treating UC through the mechanism of IκB phosphorylation and ubiquitination in NF-κB activation.Contents1,The effect of Kuijieling decoction(KD) on the mRNA and protein expression of IκBα and IKKP in colonic mucosa of Ulcerative Colitis Model Rats.Almost all known inducers of NF-κB can activate the NF-κB rapidly and momently by degrading IκBα. Phosphorylation that is the beginning of IκBα degradeation is catalyzed by activated IKK complex. The purpose of this research is to observe the effect of KD on the gene and protein expression of IκBα and IKKβ, as well as the protein expression of pIκBoc in colonic mucosa of UC model rats, then discuss the related mechanism.Methods:UC model rats were induced by TNBS, and the rats were randomly divided into model control group, SASP group, high-does Kuijieling, medium-does Kuijieling, low-does Kuijieling, at the same time, a blank control group. After the drug treatment, the rats were sacrificed and scraped their colonic mucosa. Real-time PCR was used to assay the expression of IκBαmRNA and IKKβ mRNA, Western blot was used to detect the expression of IκBα protein, pIκBα protein and IKKβ protein.Results:The expression of IκBαmRNA in UC model rats'mucous membrane tissue of colon in model group was significantly lower than that in normal group (P<0.01), the expression of IκBαmRNA in high-dosage groups of KD was obviously higher than that in model group(P<0.05); The expression of IKKβ mRNA in model group was higher than that in normal group, but no statistically significant (P>0.05), the expression of IKKβ mRNA in high-dosage group, medium-dosage group and low-dosage groups of KD were obviously lower than that in model group (P<0.05);The relative protein expression of IκBα in model group was obviously lower than that in normal group (P<0.05), the relative protein expression of IκBα in high-dosage group and medium-dosage group of KD were significantly higher than that in model group(P<0.01, P <0.05);The relative protein expression of pIκBα in model group was significantly higher than that in normal group(P<0.01), the relative protein expression of pIκBα in high-dosage groups and low-dosage group of KD were significantly lower than that in model group (P<0.01, P<0.05); The relative protein expression of IKKβ in model group was significantly higher than that in normal group (P<0.01), the relative protein expression of IKKβ in high-dosage group and medium-dosage group of KD were obviously lower than that in model group (P<0.05)2,The effect of Kuijieling decoction (KD) on the mRNA and protein expression of E1,UBC5,E3RSIκB in colonic mucosa of Ulcerative Colitis Model RatsThe phosphorylated IκB (pIκB) is recognized by the ubiquitin-proteasome system connection enzyme compound, which will add the ubiquitin molecules to IκB one by one. The poly-ubiquitinated IκB can make the IκB to gain a signal of "death label", which can be identified by special26S proteasome and then be degraded, therefore, NF-κB is freely and quickly to move into the nuclear and combine with the specific function gene, regulat expression of gene. In order to discuss the relation between IκBα degradation and enzyme expression in UPP, in this research the gene expression of E1,UBC5,E3RSIκB and protein expression of E1,UBC5,E3RSIκB in colonic mucosa of UC model rats were studied to evaluate the treatment mechanism of KD in restraining NF-κB.Methods:molding,grouping,dosing and drawing materials were the same as the first experiment. Reverse transcription PCR was used to assay the expression of ElmRNA, UBC5mRNA and E3RSIκB mRNA, Elisa was used to detect the expression of El protein, UBC5protein and E3RSIκB protein.Results:The relative gene expression of E1and UBC5in UC model rats' mucous membrane tissue of colon in model group had no significantly difference compaired to normal group(P>0.05), the expression of E1mRNA and UBC5mRNA in different doses of KD also had no significantly difference compaired to model group(P>0.05), the expression of E3RSIκBmRNA in model group was obviously higher than that in normal group (P<0.05), the expression of E3RSIκBmRNA in high-dosage group of KD was obviously lower than that in model group (P <0.05); The relative protein expression of E1and UBC5in model group had no significantly difference compaired to normal group(P>0.05),the protein expression of E1in different doses of KD also had no significantly difference compaired to model group(P>0.05),the protein expression of UBC5in high-dosage group of KD was obviously lower than that in model group (P <0.05), the protein expression quantity of E3RSIκB in model group was obviously higher than that in normal group (P<0.05), the protein expression of E3RSIκB in high-dosage group and small-dosage group of KD were significantly lower than that in model group(P<0.01, P<0.05).3,The effect of Kuijieling decoction (KD) on binding activity of NF-κB p65DNA in Caco-2cells induced by TNF-αOn the basis of previous research, medicated serum was used to culture Caco-2cells, TNF-a was used to induce cells and Bortezomib was used to as the ubiquitin-proteasome pathways blockers.The binding activity of NF-κB p65DNA was detected in Caco-2cells, which in order to discuss the effect of KD on NF-κB signal pathway in Caco-2cells from cell signal and then provide the theoretical and experimental basises for illustrating the pathogenesis and preventions of UC.Methods:Grouping Caco-2cells which were in the growth status of70%-80%. They were divided into seven groups as follows:normal group, model group, proteasome inhibitor bortezomib group, positive group (SASP), KD high, medium,low dose groups. Establishing inflammatory cell model stimulated by TNF-a. The Caco-2cells were collected and then extracted nucleoprotein. ELISA-based Trans AMTM NF-κB p65kit was used to detect the DNA binding activity of nuclear protein NF-κB p65.Results:The NF-κB p65relative activity of model group induced by TNF-a was significantly higher than the normal group (P<0.01),the NF-κB p65relative activity of KD high,medium dose group were significantly lower than the model group (P<0.01).4,The effect of Kuijieling decoction (KD) on the mRNA and protein expression of IκBα and IKKβ in Caco-2cells induced by TNF-aThe whole animal experimental results have show that KD has a raising effect on genes and proteins expression of IKBa and lowering effect on IKKβ as well as protein expression of pIκBα in colonic mucosa of TNBS ulcerative colitis model rats. As for further confirm the mechanism of restraining NF-κB activation, the expression of IKKβ,IκBa,and PIκBα were detected in Caco-2 cells which induced by TNF-α through the treatment of KD.Methods:the cell culturing, grouping and collecting samples were the same as the third experiment. Real-time PCR was used to assay the expression of IκBαamRNA and IKKβ mRNA, Western blot was used to detect protein levels of IκBα, pIκBα and IKK0.Results:The expression of IκBαmRNA in the Caco-2cell model group induced by TNF-α was obviously lower than that in normal group (P<0.05), the expression of IκBαmRNA in high-dosage groups of KD was obviously higher than that in model group(P<0.05); The expression of IKKβmRNA in the Caco-2cell model group induced by TNF-a was obviously higher than that in normal group(P<0.05), the expression of IKKβmRNA in high-dosage groups of KD was obviously lower than that in model group(P<0.05); The protein expression of IκBα in the Caco-2cell model group induced by TNF-a was obviously lower than that in normal group(P<0.05), the protein expression of IκBα in high-dosage and medium-dosage groups of KD were obviously higher than that in model group(P <0.05); The protein expression of pIκBα in the Caco-2cell model group induced by TNF-a was obviously higher than that in normal group(P<0.05), the protein expression of pIκBα in high-dosage and medium-dosage groups of KD were obviously lower than that in model group(P<0.05); The protein expression of IKKβ in the Caco-2cell model group induced by TNF-a was obviously higher than that in normal group (P<0.05), the protein expression of IKKβ in high-dosage and medium-dosage groups of KD were obviously lower than that in model group(P<0.05).5,The effect of Kuijieling decoction(KD) on the mRNA and protein expression of E1,UBC5,E3RSIκB in Caco-2cells induced by TNF-αFrom the former cell experiments, the mRNA and protein expression of IκBα were reduced significantly, while the binding activity of NF-κB p65DNA was elevated in TNF-a irritative Caco-2cells. In the protease inhibitors group, the expression of IκBα was increased significantly, and NF-κB activity was contained, all of which showed that ubiquitin-proteasome system plays an important part in IκBα degradation and NF-κB activation. This study was to perspective the effect of UPP on NF-κB signal pathway in TNF-a irritative Caco-2cells that will provide the theoretical and experimental basis for illustrating the pathogenesis and prevention of UC, and then discuss the mechanisms of restraining NF-κB activation. Methods:The cell culturing,grouping and col lecting samples were the same as the third experiment. Reverse transcription PCR was used to assay the expression of E1mRNA, UBC5mRNA and E3RSIκB mRNA, Elisa was used to detect protein contents of El, UBC5and E3RSIκB.Results:The expression of E1mRNA and UBC5mRNA in the Caco-2cell model group induced by TNF-a had no significantly difference compaired to normal group(P>0.05), and the mRNA expression of E1and UBC5in all KD groups also had no significantly difference compaired to model group(P>0.05); The expression of E3RSIκBmRNA in the Caco-2cell model group induced by TNF-α was obviously higher that in the normal group(P<0.05), the expression of E3RSIκBimRNA in high-dosage group and medium-dosage group of KD were significantly lower than that in the model group(P<0.01, P<0.05); The protein expression of El and UBC5in the Caco-2cell model group induced by TNF-α had no significantly difference compaired to normal group(P>0.05), the protein expression of El in all KD groups had no significantly difference compaired to model group (P>0.05), the protein expression of UBC5in medium-dosage group of KD was obviously lower than that in the model group(P<0.05); The protein expression of E3RSIκB in the Caco-2cell model group induced by TNF-a was obviously higher than that in the normal group(P<0.05), the protein content of high-dosage group and medium-dosage group in KD were obviously lower than that in the model group(P<0.05).ConclusionsComprehensive analysis results had indicated that the mRNA and protein expression of IκBα in UC model rats'mucous membrane tissue of colon were obviously decreased, while the expression of E3RSIκBmRNA was obviously increased, the protein expression of IKKβ, pIκBa and E3RSIκB were also obviously increased; Under the Caco-2cell inflammation model induced by TNF-a, the NF-κB p65DNA binding activity, the expression of IKKβ mRNA, E3RSIκBmRNA and their protein, and the pIκBα protein were obviously increased, while the mRNA and protein expression of IκBα were obviously decreased, which were in agreement with the in vivo experiment. When UC illness does occurred, the activity of IKKβ and E3RSIκB were increased, which catalyzed IκBα to phosphorylation and ubiquitination degradation, then with the degradation of IκBα, the NF-κB pathway was activated; The activated NF-κB was combined with the κB sites of gene promoter and enhancer specificity in many cells, and then promoted transcription and expression, which caused the imbalance of inflammatory factor and the occurrence of inflammatory cells infiltration. The results through the treatment of KD in the whole animal and cell experiment had showed that:the expression of IκBα was obviously risen, however, the expression of IKKβ^E3RSIκB,pIκBα were obviously down-regulated, and the NF-κB p65DNA binding activity was obviously repressed, which was similar with the group of Bortezomib.Accroding to the study of the influence of KD on the expressions of IκBα, pIκBα, IKKβ, E3RSIκB in TNBS UC model method rat colonic mucosa and TNF-α induced Caco-2cell inflammation model, this thesis presents some conclusions as follows:1. TNF-a may degrade the IκBα protein through the pathway of ubiquitin proteasome,and then lead to increase of the NF-κB p65DNA binding activity;2. E3protein ligase E3RSIκB may take part in the ubiquitination degradation of IκBα in the inflammatory cell model which constructed by TNF-α;3. KD may affect the process of UC to occur, develop and transform through suppressing the activity of IKKβ,E3RSIκB to prevent IκBα phosphorylation and ubiquitination so as to indirectly suppress the activation of NF-κB signal transduction pathway, which may be the corresponding target that NF-κB excessive activation was inhibited by KD. In some degree, our study reflected the feature of TCM-multitarget and multistrata.
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