| Ulcerative colitis(UC)is a chronic nonspecific inflammatory bowel disease of the rectum and colon which is not clear.It is the main symptom of abdominal pain,diarrhea,mucous pus and stool,and the heavy weight in the post.It belongs to the category of "long diarrhea","long diarrhea","intestinal wind" and so on.Because of its unknown etiology,many links,hard to heal,easy to become cancerous,it has been listed as one of the most difficult modern diseases by WHO.Inflammatory bowel disease is common in European and American countries,but it is prevalent in many countries all over the world,and the incidence rate is increasing.The incidence rate in China is also increasing year by year.Therefore,searching for effective drugs for ulcerative colitis is an important task for Chinese medicine practitioners.The root of Rose odorata Sweet var.gigantea of Rosa family.It has the effect of astringent and astringent acid,which can kill pathogenic bacteria,protect the digestive tract mucosa,accelerate the circulation of the intestinal wall and accelerate the healing of the intestinal mucosa.It is commonly used in the treatment of diarrhea and bacteria.Dysentery is a kind of Yi nationality folk medicine with long history.At present,the research work of the Yi Nationality Medicine on the ROE is mainly completed by our research group.In the previous work,we first determined the effective parts of the treatment of diarrhoea by the crude extract,and on the basis of this,it was the first time that the extract of the ROE can effectively treat the ulcerative colitis induced by dextran sodium sulfate.The early exploratory study also found that the treatment of ulcerative colitis is mainly through reducing the content of TNF-a and IL-6 in the inflammatory site,inhibiting the activity of NF-kB,activating the Nrf2 pathway,resisting inflammation,reaching the anti-inflammatory and treating ulcerative colitis.Therefore,this project is based on the NF-kB/Nrf2 pathway to study the anti-inflammatory mechanism and material action basis of the treatment of ulcerative colitis with ROE.1.Using animal models to evaluate the efficacy and mechanism of extract from the ROE on the regulation of experimental ulcerative colitis based on the Nrf2 pathway.A model of ulcerative colitis in mice was established with 3% DSS solution.After eighth days of administration,the head was sacrificed(water weight,weight,blood sample,spleen and intestinal segment).The oxidant stress index(SOD,MDA,MPO)was used to evaluate the changes of oxidative stress index in each group.Compared with the model group,it was found that the oxidative stress index of the ROE extract group had a dose dependent change,the antioxidant capacity was enhanced,and the ELISA test kit was used to determine the various cytokines(IL-1b,IL-6,TNF-?)level,compared with the model group,the level of cytokine in the ROE extract group was significantly lower and the inflammatory condition was improved.The NOS activity of each group was measured and the NOS level of the ROE extract group decreased with the increase of dose,and the inflammation was alleviated;the length of the colon and the weight of the spleen were measured.The colon length of the ROE extract group was dose-dependent,and the blood sample in the colon was reduced,the weight of the spleen was reduced,and the inflammation was improved.The colon tissue was embedded in the paraffin embedded HE staining section.The intestinal wall of the model group was severely damaged,and a large number of inflammatory cells were immersed and congested.The destruction of the intestinal wall was reduced,the infiltration of inflammatory cells decreased,and the inflammatory condition was improved.The serum and colon homogenate supernatant was detected by Western-Blot method.The results showed that the group of ROE extract could play an antioxidant role in alleviating inflammation,and the expression of inflammatory protein NF-kB was significantly reduced..To sum up,the extract from the ROE can alleviate the experimental ulcerative colitis and reduce the inflammatory state of ulcerative colitis.2.In vitro evaluation of anti-inflammatory effect and mechanism of extract from solid fruit by using inflammatory cells in vitro based on Nrf2 pathwayThe MTT method was used to determine the toxicity range of the ROE extract to RAW246.7 cells,There was no obvious toxic effect on RAW246.7 cells in the range of 1~400 ?g/mL.After the experiment,three concentrations of 100 ?g/mL,200 ?g/mL and 400 ?g/mL were selected for further verification.The inflammatory model was established by LPS with the change of TNF-? content as the standard.It was determined that 5?g/mL LPS stimulated RAW246.7 cells,and ELISA kit was used to determine the level of cytokines(IL-1b,IL-6,TNF-a),indicating that the extract of the ROE extract significantly inhibited the level of cytokine and decreased inflammation with the increase of dose,and NOS activity was inhibited with the increase of the amount of extract of ROE,reducing NO promotion.The release of inflammatory factors,the cell protein of each group was detected by Western-Blot method,and the expression of Nrf2 protein increased with the increase of dose.On the contrary,the protein expression of NF-kB decreased significantly.The result indicated that the extract of the ROE can increase the expression of antioxidant protein and lower the expression of inflammatory protein,and introduced the NF-kB.Protein inhibitor(BAY 11-7082)detected the expression of all related proteins.It was found that BAY 11-7082 could not only inhibit the expression of NF-kB protein,but also reduce the expression of Nrf2 protein.In conclusion,the extract of the ROE can inhibit the inflammatory reaction induced by LPS,and is related to the antioxidant and inflammatory pathways.3.In vitro evaluation of the effect of extract from solid fruit on apoptosis of Nrf2 cells and its mechanismMTT method was used to detect the effect of the extract of ROE on the activity of two human colon cancer cells.The extract of the ROE can inhibit the increment of two kinds of human colon cancer cells,and select the extract of 0.1mg/mL,0.2 mg/mL and 0.4 mg/mL for the follow-up experiment.The DAPI dye is a kind of fluorescent dye capable of combining with DNA strongly,we used the DAPI dye to observe the damage of the nucleus,and the ROE extract can damage the nucleus of colon cancer in a dosedependent manner.Flow cytometry is one of the most advanced quantitative analysis techniques of the cell.We use flow cytometry to detect the apoptosis rate of colon cancer cells.The results showed that the extract of ROE can increase the apoptosis of colon cancer cells,and the cell protein was collected by Western-Blot method,and the expression of apoptotic protein Bax increased significantly with the increase of additive amount,while the expression of the apoptosis protein Bcl-2 decreased,and the apoptosis of colon cancer cells and the antioxidant protein were found.Nrf2 related,apoptosis increased and Nrf2 protein expression decreased significantly,the results of two kinds of human colon cancer cells were consistent.To sum up,the extract of the ROE can increase the apoptosis rate of human colon cancer cells. |
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