| Objective:To research the treatment effect of Kuijieankang decoction on rats with ulcerative colitis,and to explore the role of the possible mechanism and target point with a number of links and multi-level,With a view to provide theoretical and experimental basis for the treatment of ulcerative colitis in Chinese medicine.All above would get help from experimental study of traditional Chinese medicine Kuijieankang decoction on rats with ulcerative colitis.The experiment will study the general state of model rat with ulcerative colitis,visual observation of the rats' colon tissue,optical microscopy of the rats' colon tissue(HE staining),the rats' electrointestinogram(frequency and amplitude), Oxygen free radicals of the rats' colon(MDA contents and SOD activity),contents and protein expression of the rats' colon tissue inflammatory cytokines(IL-1β, IL-10 and TNF-α),as well as contents epidermal growth factor(EGF) and mRNA expression of the rats' of colon tissue.Materials and methods:1.Method of model facture60 healthy adult Wistar rats,weight 180-220g,were equally divided by male and female(male and female rear separately).After 1 week adapt feed in order to determine the health of rats.They were randomly divided into model group (48 rats),and the control group(12 rats).The UC model was established by TNBS/ethanol method and the control group was applied to normal saline enema. 3 days later,selected four model group rats and a control group rat randomly. Took the colon under anesthesia for morphological observation to confirm the success of model.2.GroupingThe 11 rats in control group are look as the nomal rats.Model rats were divided into 4 groups by random method:model group,Bupiyichang pill group, SASP group,and Kuijieankang decoction group,11 rats in each group.We fed male and female rats in different cages.3.Drugs givenFrom the fourth day after established model,Bupiyichang pill group's rats were given Bupiyichang pill 3ml intragastric administration daily,the concentration was 0.108g/ml,the amount was 1.62g/kg,converted as equivalent effect as health adult.SASP group's rats were given SASP 3ml intragastric administration daily,the concentration was 0.024g/ml,equivalent to 0.36g/kg dosage of health.And Kuijieankang decoction group's rats were given Kuijieankang decoction 3ml intragastric administration daily,the concentration was 1.2g/ml, equivalent to 18g/kg dosage of health,each rat of the normal group and the model group was given normal saline 3ml intragastric administration daily.All rats were given drugs at the same time every day for 21 days continuously.4.Perfusion and drew the materials4.1 PerfusionIn the night of the 21st day all rats were feed nothing but water.The rats were fixed after intraperitoneal anesthesia with 10%chloral hydrate,surgical incision of the chest cavity,perfus 30-50ml 37℃normal saline into the left ventricle quickly,and 30-50ml 4℃solution with 4%Paraformaldehyde(PBS confecte ph7.2-7.4) followed.4.2 Drew the materialsAfter the perfusion,surgical incision of the abdomen.Took the department of 6-8cm colon from 2cm above the anus,then cut along the mesenteric edge mesocaval vertically.Then rinsed with saline,tiled and fixed them with pin. We observed colon mucosa with the naked eye to find intestinal lesions.Then Sheared the most obvious part about 1cm in severe lesions,quick-frozen with dry ice,transferred to -80℃cryopreservation refrigerator.Collected several colon lesions of the various groups' rats where most notably,Placed them into 10%formaldehyde solution for more than 48 hours.Every organization would be cut into a small piece of 0.5cm×1cm,dehydration with gradient alcohol.These organizations would be cut into cross-chip after paraffin-embedded.Then they would be posted on the slides coated with Poly-L-lysine.Finally,they would be sent to the laboratory center of Liaoning university of Traitional Chinese Medicine.Results:1.The general state of rats10 days after intragastric administration,the rats of Kuijieankang decoction group,Bupiyichang pill group,and SASP group got marked improvement in mental state,more slightly shiny hair,activities increase,and an obvious increase in food intake.At the same time,traits of stool improved,pus and blood stool reduced gradually.21 days later the symptoms of all rats in treatment groups were significantly improved,mucus and pus and blood stool reduced obviously.However,the above symptoms were still slightly worse compared with normal group.The rats of Kuijieankang decoction group and SASP group had got the best restoration.Model group rats still existed matte fur,less activities,thin stool,ate less,and so forth.2.Visual observation Compared with normal group,the general concept score of model rats' colon mucosa injury was increased significantly.Handled the data with the statisticmethod,it was statistically significant(P＜0.05).Compared with model group,the general concept score of Bupiyichang pill group,SASP group, and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group,the general concept score had no significant difference.Handled the data with the statisticmethod,it wasn't statistical significance(P＞0.05).3.Observation under light microscopyCompared with normal group,the pathology score of model rats' colon was increased significantly.Handled the data with the statisticmethod,it was statistically significant(P＜0.05).Compared with model group,the pathology score of Bupiyichang pill group,SASP group,and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group, the pathology score had no significant difference.Handled the data with the statisticmethod,it wasn't statistical significance(P＞0.05).4.ElectrointestinogramComparing the electrointestinogram of model rats with normal group,a two-way change emerged.Both the frequency and the amplitude increased obviously. Handled the data with the statisticmethod,it was statistically significant (P＜0.05).Compared with model group,both the frequency and the amplitude of Bupiyichang pill group,SASP group,and Kuijieankang decoction group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And compared with Bupiyichang pill group,both indicators of Kuijieankang decoction group and SASP group dropped markedly.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with SASP group,the frequency dropped.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).While the amplitude had no statistical significance(P＞0.05).5.Oxygen free radicalsComparing Oxygen free radicals of the rats' colon in model group with normal group,the MDA contents of the former were much accentuated,and the SOD activity was much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group,and Kuijieankang decoction group with model group,the MDA contents were much reduced, and the SOD activity was much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with Bupiyichang pill group,the MDA contents were reduced,and the SOD activity was accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with SASP group,the MDA content were reduced. Handled the data with the statisticmethod,it was statistical significance(P＜0.05).While the SOD activity was't statistical significance(P＞0.05).6.Inflammatory cytokinesComparing the rats' colon tissue inflammatory cytokines of model group with normal group,the contents and protein expression of IL-1βand INF-αwere much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group,and Kuijieankang decoction group with model group,the contents and protein expression of IL-1βand TNF-αwere much reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were much accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Kuijieankang decoction group with Bupiyichang pill group,the contents and protein expression of IL-1βand TNF-αwere reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with SASP group,the contents and protein expression of IL-1βwasn't statistical significance(P＞0.05).The contents and protein expression of TNF-αwere reduced.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).The contents and protein expression of IL-10 were accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).7.Epidermal growth factorComparing model group with normal group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all reduced obviously.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Comparing Bupiyichang pill group,SASP group and Kuijieankang decoction group with model group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated obviously.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).And comparing Kuijieankang decoction group with Bupiyichang pill group,the contents of EGF, the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Compare Kuijieankang decoction group with SASP group,the contents of EGF,the optical density and the rate of EGF mRNA between the strength and internal reference were all accentuated.Handled the data with the statisticmethod,it was statistical significance(P＜0.05).Conclusion:1.The rat model of ulcerative colitis was successfully established through observation to the rats' general state,visual observation and light microscopy observation.2.Kuijieankang decoction played a significant role of ulcer repair in the morphology to rats with ulcerative colitis.3.Kuijieankang decoction could obviously reduce the frequency and the amplitude of ulcerative colitis rats' electrointestinogram.4.To rats with ulcerative colitis,Kuijieankang decoction could increase the SOD activity of colon,clear Oxygen free radicals,inhibit lipid per oxidation, reduce MDA contents,and reduce colon mucosa injury.5.Kuijieankang decoction could control inflammatory response,by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. Therefore it could make a recovery of ulcerative colitis rats' immune function, improve immunity,and reduce colon mucosa injury.6.To rats with ulcerative colitis,Kuijieankang decoction could increase cellular proliferation capacity,tissue growth and repair capacity,protect colon mucosa,by increasing EGF contents and expression of EGF mRNA of colon. Then promote the healing of ulcer.From the following five different levels including histomorphology, electromyography physiology,biochemistry,immunohistochemistry and molecular biology,the experimental study showed that Kuijieankang decoction used cooling and heat prescription together,and it could invigorate spleen and Qi as well as activate blood and resolve stasis. |
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