| Ulcerative colitis(ulcerative colitis,UC)was known as chronic nonspecific ulcerative colitis,which belongs to inflammatory bowel disease(inflammatory bowel disease with Crohn's disease(Crohn,disease,CD).Chronic ulcerative colitis is a chronic non-specific chronic intestinal disease,and its etiology and pathogenesis is not very clear,which maybe seriously affecting the quality of life and lead to serious colon cancer because of its recurrent disease and delayed healing.At present,the clinical treatment of UC is relatively difficulty.There is no complete and effective treatment of the disease.At the same time,the incidence of UC is increasing in china.Therefore,it is of great significance to explore the ideal experimental animal model,which is valuable for the study of its etiology,pathogenesis,diagnosis and treatment in UC.The model were induced by oxazolone(oxazolone,OXZ)and2,4,6-trinitrobenzene sulfonic acid(2,4,6-trinitrobenzene sulfonic acid TNBS).In this essay,we investigate the effect of time,drug concentration,ethanol concentration and respectively on the two UC models in mice induced by OXZ and TNBS respectively.Male mice were selected in the experiment.The higher doses of OXZ and the mild dose of TNBS in the ethanol of 40% were used to established the chronic UC,and in accordance with a new certain cycle induction method.In order to study the therapeutic effect of rhubarb on chronic ulcerative colitis(UC)model in mice,Sulfasalazine(Sulfasalazine,SASP)was used as a positive control drug.1 Single factor study on two ulcerative colitis models in mice1.1.The effect of Ethanol concentration on ulcerative colitis model21 healthy adult female Kunming mice were randomly divided into 7groups,three mice in each group were the normal group,OXZ 30% group(0 30),OXZ group(0 40),40%(0 50),TNBS 30% group(T 30),TNBS40% group(T 30)and TNBS 50%(T 50),the concentration of OXZ is0.8%,reaction time was 2 min,the concentration of TNBS is 2.1mg in each,reaction time was 2 min.7 groups of mice were killed at the end of the second days after modeling,and the methods of modeling and model evaluation were as follows.The results showed that the DAI value of two groups of mice increased with the increase of ethanol concentration at the time of 2 min,the concentration of OXZ was 0.8%,and the dose of TNBS was 2.1mg.When the concentration of ethanol was 50%,the mice died.The DAI value in the 40% ethanol group was higher than that in 30% ethanol,but the difference was not statistically significant.Therefore,the 40% ethanol induced group was suitable for UC in mice and the new chronic model method.1.2 The effect of gender on ulcerative colitis model30 healthy adult KM mice,half male and half female,were randomly divided into 6 groups,female control group(n=5),male control group(n=5),OXZ model group(n=5),female OXZ male model group(n=5),TNBS model group(n=5)and female TNBS male model group(n=5).The control group was normal control group.The OXZ model group was established OXZ UC model according to the OXZ model,the TNBS model group was the same as above..The state of mental state,hair color,eating and defecation of mice were recorded every day,and the disease activity index(disease activity index,DAI)was detected.Second days after enema,separation of colon,colon tissue of mice was assessed(CMDI)value,local ulcer and congestion area and colon weight index,and the lesion detection index of HPS and interleukin-4(interleukin 4,IL-4),tumor necrosis factor alpha(tumor alpha TNF-alpha necrosis factor-.Level).The results showed that there was no significant difference between the twogroups in each index.The DAI value of mice in OXZ-F model group was significantly lower than that of OXZ-M model group,the DAI value of mice in TNBS-F model group was significantly lower than that of TNBS-M model group.There was no significant difference between the OXZ model group and the normal group,the other three groups were significantly different from the normal group.The CMDI scores of the four groups were significantly higher than those of the normal mice.Moreover,the CMDI value of mice in OXZ-F model group was significantly lower than that of OXZ-M model group,but the CMDI value of TNBS-F model group was significantly lower than that of TNBS-M model group mice.The expression level of OXZ in female IL-4 group was lower than that in two groups,the difference was statistically significant.The expression level of TNF-? in OXZ female group was lower than that of male model group,and the difference was statistically significant(P<0.01).There was no significant difference between the other groups in gendar(P>0.05).1.3 The effect of dose on ulcerative colitis model21 healthy adult female Kunming mice were randomly divided into 7groups.3 mice in each group.Normal group(N group),OXZ 0.6 group(group A),OXZ 0.8 group(B group),OXZ 1 group(group C),TNBS 0.1group(group D),TNBS 0.3 group(group E)and TNBS 0.5 group(group F).Reaction time is 2 minutes.The concentration of ethanol is 40%.The induced method and model evaluation method as above.The colonic morpha of ulcerative colitis model in mice induced by a single dose of OXZ showed that any damage were not found in the normal group,the colon of 0.60% OXZ dose group had mild ulcer and hyperemia,0.80% OXZ group has obvious ulcer,and C mice colon damage is stable and consistent.The colon of OXZ 1% dose group(Fig.1-4)appeared obvious necrosis phenomenon,which suggested the dose is too large.In the groups of ulcerative colitis model induced by TNBS,the general morphology of the colon in TNBS 0.1mg dose group did not show obvious hyperemia and ulcers,which was similar to the normal group.The colon inTNBS 0.3mg dose group showed congestion and extremely mild ulcer,which indicated that this dose is lighter,but can cause tissue damage as the lower dose.TNBS 0.5mg dose group can cause obvious ulcer phenomenon.In addition,the DAI value of the mice increased significantly with the increase of the dose of the drug.2 explore the method of a new chronic ulcerative colitis model induced by OXZ and TNBS together in mice and Study on the pharmacodynamics of Rhubarb in the treatment of the mice with new induced method2.1 The preparation of rhubarb suppositoryTake the 200 g 36 type semi synthetic fatty acid ester(Semi-synthetic fatty,acidglycerides,SSFAG)in a beaker as blank suppository matrix of rhubarb,which was melted in the 45? constant temperature water bath.Taking 2.3584 g rhubarb(through six sieve)precisely to the blank matrix suppository,and fully mixed.Cleaning the bolt die and make it dry,and the amount of lubricant [soap: glycerol: 90% ethanol(1:1:5)] was wiped in the model die.Then placing the model in the temperature of 4? in refrigerator30 min.Utill completely solidification,the suppository were removed and the the overflow were scraped,then the rhubarb suppository were accomplished.The round shape with smooth hollow suppository was selected,excepted the suppository or bubble suppository.Make the quality difference maintained within 0.1g which was used to study the effect of the late new mouse UC.2.2 explore the method of a new chronic ulcerative colitis model induced by OXZ and TNBS together in mice and Study on the pharmacodynamics of Rhubarb in the treatment of the mice with new induced method60 healthy adult male Kunming mice were randomly divided into five groups: normal control group(group N,n=12),model group(group M,n=12),blank suppository group(group I,n=12),rhubarb suppository group(Group II,n=12)and SASP group(Group III,n=12).The new chronic UC model in mice were induced by OXZ and TNBS together in enema according to a certain cycle induction method,and daily mental state,haircolor,eating stool were recorded,and the disease activity index(disease activity index,DAI)of mice was detected every day.After the induction were accomplished the three therapeutic groups were given corresponding suppository once a day,and the model group and normal group without any treatment.At the same time,three mice were sacrificed in each group every weeks in a month respectively,which were N1,N2,N3,N4,M1,M2,M3 and M4 group,and the same as I,II and III group.The colons were separated,and the colonic tissue of mice CMDI were determined as long as the CI,immunohistochemical staining technique was used to detect the expression level of IL-4 and TNF-?.The mice's colon morphology can be seen after the molding cycle,each group for 4 weeks showed different degrees of congestive ulcer,except the normal group.The model group in two months after the ulcer still showed serious damage.The DAI values of the four M groups were higher than that of the normal group,and the difference was statistically significant(P<0.01).In the first week,there was no statistically significant difference between the treatment groups and the M group in the same week.Second weeks,group III was significantly lower than the M group,the difference was statistically significant(P<0.05).The DAI value of group II and group III was significantly lower than that of group M at third weeks,and the difference was statistically significant(P<0.01).In the fourth week,the treatment group was significantly lower than the M group,the difference was statistically significant(P<0.01).The CI values in each M group were higher than those in group N,the difference was statistically significant.The other treatment groups were higher than the N group in different degree.There was no significant difference between the 4 groups of CI values of group I3,I4,II2,II3,III2,III3 and III4(P>0.05)as compared with the N group.The CMDI values of the M group and each treatment group in the four weeks were significantly higher than those in the N group,and thedifference was statistically significant(P<0.01).The expression levels of immunohistochemistry IL-4 and TNF-? in each treatment group were significantly different from those of the normal group(P<0.01).The expression levels of IL-4 in 3 groups,2,4,3,II and III were significantly different from those in group M.Except for I1,I3,II1,II2,III1 and III2,there was no significant difference in the level of TNF-?expression compared with the M group,and there were significant differences between the 3 groups as compared with the M group.The staining of colon tissue sections showed that the morphology and structure of colon tissue in N group were clear and complete,and both TNF-? and IL-4 were expressed in the model.The morphology and structure of colon tissues were irregular,the glands increased or lost,the crypt structures were broken,and there were a large number of IL-4 and TNF-? positive cells in the surrounding M group and the treatment group.At the 10*40magnification of the microscope,5 images were taken at random,and the positive cell rate was calculated respectively. |
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