| Ulcerative colitis (UC) is a long term and wide range of disease that easily develops to cancer. The treatment of UC is a major clinical problem. Uyghur traditional Medicine has unique method in treating UC. Xipayi Kui Jie'an has been used for the treatment of UC effectively. However, the treatment mechanism of Xipayi Kui Jie'an is still unclear. In this study, UC animal model was established and which has been used for the treatment with Xipayi Kui Jie'an;the mRNA and the protein level of induced -nitricoxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-кB), interleukin-6 (IL-6) early oncogene c-jun, c-fos candidate gene were measured. Differentially expressed proteins in the tissue of the UC model group and the treatment group rats were tested by using the technology of high performance two-dimensional liquid chromatography. At the same time, the RNAi technology platform has been established, the strategy of RNAi gene therapy was used for the treatment of rat ulcerative colitis.1.Methods and Results1.1 Development of UC animal model and the intervention of Xipayi Kui Jie'an In this research, rat UC model was successfully developed by using 2, 4-dinitrochlorobenzene (DNCB) and acetic acid. The symptoms, physical signs and histopathology changes of UC rats were closely correspondent to the UC indexes. The methods of semi-quantity RT-PCR and Western blot were used to detect the different expression of mRNAs and proteins in normal control and model groups. The results showed that the expression levels of mRNA and protein of iNOS, COX-2, NF-кB, IL-6 gene were up-regulated, there was a statistical significance between the normal control group and the model group (P<0.05). Meanwhile, there was no statistical significance in the mRNA expression level of c-jun and c-fos between these two groups. The expression level of c-Jun protein was up-regulated in model group compared to the normal control group (P<0.05) ; no statistical significance has been found in the protein expression level for c-fos between these two groups. In order to study the therapeutic effect of Uyghur traditional medicine Xipayi Kui Jie'an, animals were divided into four groups, which include the model group, NS negative control group, the 5-ASA positive control group and the Xipayi Kui Jie'an treatment group (large-dosage group, middle-dosage group and small-dosage group). The results showed that, Xipayi Kui Jie'an, Uyghur traditional medicine has a therapeutic effect on UC rats, but there was no statistical significance in the therapeutic effect between the large-dosage group and the middle-dosage group (P>0.05). The statistical significance between the large-middle dosage groups and the small-dosage group (P<0.05). The methods of semi-quantity RT-PCR and Western blot were used to detect the different expression of mRNA and protein in each group. The result showed that expression levels of iNOS?COX-2 and c-Jun protein in the large-dosage group were down-regulated compared to the normal saline treated negative control group (NS) (P<0.05). The expressed mRNA of COX-2 and the protein of iNOS were significantly down-regulated in the middle-dosage group compared to the normal saline treated negative control group (NS) (P<0.05). There was no statistical significance between the Xipayi Kui Jie'an group and the ulcerative colitis (UC) model group in the expression level of mRNA and protein for each candidate gene (P>0.05). The COX-2 mRNA was significantly down-regulated in the amino salicylic acid positive control group (5-ASA) compared to the normal saline treated negative control group (NS) (P<0.05). There was no statistical significance between the amino salicylic acid positive control group (5-ASA) and the normal saline treated negative control group in the expression level of iNOS?c-jun mRNA and protein (P>0.05). Although the expression level of NF-кB?c-fos mRNA and protein were changed in each experimental groups, there was no statistical significance between the UC group and the normal saline treated negative control group (P>0.05).1.2 To analyse the differentially expressed proteins in UC model group and the Xipayi Kui Jie'an treated group by using the technology of high performance two-dimensional liquid chromatography.In this study, Proteomics technology was used to analysis the pathogenesis of Ulcerative colitis and the mechanism of Xipayi Kuijie'an. First of all, the lysis product of ulcerative colitis tissues were separated by using the technology of one-dimensional focusing chromatography and two-dimensional reverse liquid chromatography. Two-dimensional data was transformed into simulating pattern by ProteoVue software and the comparison and analysis of the expressed protein in each group were conducted by DeltaVue software. The data show 296 different expressed bands between the model group and the normal group, 132 bands between the model groups and NS negative control group, 286 bands between the model group and Uyghur traditional medicine Xipayi Kui Jie'an group, 186 bands between the normal group and NS negative control group, 162 bands between normal group and Uyghur traditional medicine Xipayi Kui Jie'an groups, 132 bands between normal saline treated negative control group, and Uyghur traditional medicine Xipayi Kui Jie'an group, which in the range of pH8.5～4.0 region. The expressed proteins in different groups was concentrated in the range of pH 7.0～8.5 of isoelectric point.1.3 The treatment of UC rat by using the gene gun jointed with RNAi technology1.3.1 Regulation of UC related gene by using RNAi technology In this study, pcDNA3. 1/CT-GFP-TOPO recombinant eukaryotic expression vectors of iNOS, COX-2, NF-κB, c-jun, c-fos genes and corresponding pSilencerTM 1.0-U6-siRNA relevant interference vectors were successfully constructed. The GFP fused target protein was expressed in the COS-7 cell. The pcDNA3. 1/CT-GFP-TOPO recombinant eukaryotic expression vectors and the pSilencerTM 1.0-U6-siRNA relevant interference vectors were co-transfected in the same cell line. The expression of GFP fused target protein was detected by inverted fluorescent microscope and the Western blot.1.3.2 Treatment of UC rat by using gene gun jointed with RNAi technology Interference vector of pSilencerTM1.0-U6-c-jun was successfully transformed into the colon tissue in vivo by gene gun technology and the expression level of c-jun in the colon tissue of the rats was regulated by this construct. The effective concentration of the target gene was confirmed to be 5μg and the time of activity was lasted for 9 days. The ulcerative colitis tissue of the rats was interfered by pSilencerTM1. 0-U6-c-jun interference vector, and the histopathological evaluation was determined. The result showed that the granulation tissue was facilitated effectively by the interference vector, and there was a statistical significance (P<0.05) in the healing ratio of ulcerative colitis between the groups, which has been used interference vector and blank vector.2.ConclusionUC model was developed by using DNCB and acetic acid, we found that early oncogene c-jun was expressed shortly in UC animal model. Histopothology results showed that Uyghur traditional medicine Xipayi Kui Jie'an is effective for treating UC;Medicine intervention experiment results showed that, the treatment effect of Xipayi Kui Jie'an might be related to the action of down regulation of iNOS, COX-2, c-jun expression. Uyghur traditional medicine Xipayi Kui Jie'an may regulate COX-2 in the transcription level, whereas the regulation to the gene of iNOS and c-jun may occur at the post-transcription level. Proteomics analysis results showed that the protein expression of different groups were concentrated in the range of pH7.0～8.5 region of isoelectric point. That result imply that the occurrence of ulcerative colitis in the rats and the therapeutic effect of Uyghur traditional medicine Xipayi Kui Jie'an are highly related to many different kinds of proteins. We also successfully constructed eukaryotic expression vector and the pSilencer interference carrier in COS-7 cells. After applied the gene gun-mediated RNAi technique on rat UC model, we found that the expression of c-jun is down regulated, and the technique of RNAi gene therapy is effective in treating UC.
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