| Objective：To study the intervention and influence of Si-Shen bolus on colitis associatedcancer in mice;to observe mice’ general status and organs of colitis associated cancer;To clearSi-Shen bolus’s chemical prevention for colitis associated cancer; to study Si-Shen bolusaffectting on colon cancer’s COX-2protein expression induced by colitis; to analysis Si-Shenbolus’ possible mechanisms of chemical prevention effect of colitis associated cancer;toobserve the effect of colon tissue’s Nrf2ARE signal pathway in mice;to discuss Si-Shenbolus’ possible mechanisms of chemical prevention effect of colitis associated cancer;toanalysis test data and clear the best intake way of Si-Shen bolus.Materials and Methods:Part Ⅰ:Si-Shen bolus’ influence to mice’ organs、cancer rate and pathology with colitisassociated cancer.We gave mice strong inflammatory agent dextran sodium sulfate(DSS)drinking water for two weeks, and gave strong carcinogenic agent1,2-dimethyl hydrazine(DMH) by intraperitoneal injection at the same time.We manufactured mouse model of colitisassociated cancer finally. Si-Shen bolus was accordanced in strict with the original formula,proportion and dosage, powder, grinding, decocting, extraction, circumfluence, concentration,drying and powder and set aside.50mice were randomly divided into5groups: normalcontrol group, model group, oral group, enema group, western medicine control group, eachgroup10mice. Control group does not make any processing. Model group mice was givendrink water containing20g/L dextran sodium sulfate (DSS).A week later, mice was givenabdominal cavity injection containing40mg/kg,1,2-dimethyl hydrazine (DMH) in salinesolution,the next day at a time, a total of three times. After two weeks stop drinking DSS,drinking water to drink water, until death. Other groups’early treatment was the same ofmodel group. Oral group was given0.2ml(calculated by surface area, with traditionalChinese medicine2g/ml) Si-Shen bolus extract. Enema group was given0.2ml (calculatedby surface area, with traditional Chinese medicine2g/ml)Si-Shen bolus extract by enemaevery day. Western medicine control group was given willow nitrogen sulphanilamidepyridine distilled water solution for a0.2ml(calculated by surface area) every day. Duringthis period closely observe general status in mice, the symptom such as prolapse and hemafecia. After20weeks,we gathered the spleen, thymus, colon, and weighed with filterpaper. We observe mice colon tissue and calculated tumor rate.We observe each group micecolon tissue’s section of HE staining under microscopic. PartⅡ:prevention mechanismresearch based on the Nrf2/ARE pathways induced colitis associated cancer. Animal feeding,Chinese medicine treatment, animal group, building disposal,processing factors, buildingtime,based method were same of the first part. By Western blot method,we detected theNrf2ARE signal pathways’ Nrf2protein expression.By extraction of total protein in thecolonic tissue,Polypropylene phthalic amide gel electrophoresis,protein from gel tomembrane,we analyse Nrf2protein’s optical density value. PartⅢ:prevention mechanismresearch based on COX-2protein induced colitis associated cancer. Animal feeding, Chinesemedicine treatment, animal group, building disposal,processing factors, building time,basedmethod were same of the first part. By reverse transcription polymerase chain reaction (rt-pcr)we detected of cox-2protein related genes mRNA, Calculated cox-2related genes mRNAexpression. By immunohistochemical we detected cox-2protein expression,and analysedoptical density.Result:1. Part Ⅰ:Si-Shen bolus’ influence to mice’ organs、cancer rate and pathology with colitisassociated cancer.1.1Changes in body weightModel group, oral group, enema group, western medicine control group was significantlylower than the normal control group (P <0.01), weight loss in model group was most. Inaddition to the normal control group, other is no statistical significance between groups.1.2Bloody and rectoceleMost bloody appeared in the model group, total4only, at40%. Followed by oral groupand western medicine group,1only, at10%. Normal control group and enema group was atleast,0only.Most rectocele appeared in model group,total3,up to33%.1.3The thymus, spleen and colon tissue weightCompared to control group, the spleen and colon quality of model group increased significantly (P <0.01), thymus quality is significantly lower (P <0.01).Oral group, enema group, western medicine control group compared with the modelgroup, only the data of enema group’s thymus was better than the model group, and havestatistical significance (P<0.05). The data of the three groups’s spleen was significantly betterthan the model group (P<0.01). The data of three groups’s colon tissue was significantlybetter than the model group (P<0.01) too.Oral group,enema group, western medicine control group compare between eachother,from the data, enema group effect is better than oral group, oral group effect is betterthan western medicine control group. The data of enema group’s thymus was better than oralgroup and western medicine control group, and have statistical significance (P<0.05). Thedata of enema group and oral group’s spleen and colon was better than the western medicinecontrol control group, and there was statistical significance (P<0.01).1.4The effect on tumor formation rateModel group mice using only DMH+DSS，after21weeks,most mice formated to coloncancer from colitis.It’s cancer rate is90%, and the number of tumor was more than one.Thesmall intestine and other organs showed no tumor. Tumor diameter was2-6mm, found in themucosal surface,convex to the intestinal lumen, color dark red, crisp, surface ulcers.Thetumor formation rate of Oral group and enema group was20%.The number of tumor was1,and the smaller diameter. The mice without colon tumor slightly thickened, showedinflammation sex embellish, stayed in the colitis state, not to the development of colon cancer.Control group ‘s tumor formation rate was40%, higher than the two group of Si-Shen bolustreatment, but lower than that in the model group. It’s colon tumor number was more than thetwo groups of Si-Shen bolus treatment.1.5The colon tissue PH dyeing pathological comparisonNormal control group had normal gland structure, glandular structures arranged orderly,no inflammatory cells invasion, Mucous membrane was complete. Model group glandsstructure was disorder and Invasive growth.Model building was successful. There was acertain amount of inflammatory cells invasion in oral group,enema group and westernmedicine control group. There was thickening of the gland structure,but glands keep normalstructure.It stayed in colitis,not to colon cancer.There was no signs of surrounding tissue invasion. enema group and western medicine control group’s Inflammation degree was lessthan oral group’s.2.PartⅡ:prevention mechanism research based on the Nrf2/ARE pathways induced colitisassociated cancer.Nrf2protein expression quantity of model group was obviously decreased than thenormal control group (P <0.01). Oral and enema group Nrf2protein expression issignificantly higher than model group (P <0.01). Oral and enema group Nrf2proteinexpression is significantly higher than western medicine control group (P <0.01) too.Therewas no difference and statistical significance compared with model group and westernmedicine control group（P＞0.05）.3.PartⅢ:prevention mechanism research based on COX-2protein induced colitis associatedcancer.3.1Cox-2related gene mRNA RT-PCR detectionCompared with the control group, model group expression of cox-2related gene mRNAwas significantly higher (P <0.01). Cox-2related gene mRNA of oral group, enema groupand western medicine control group expression was significantly inhibited (P <0.01)compared with model group. But the difference between the three has no statisticalsignificance (P>0.05).3.2Mice colon tissue COX2protein immunohistochemicalYellow particles are the positive expression. Look from the naked eye，the yellowparticles in model group was obviously more than other groups. There was almost no positiveexpression in normal control group.But Oral group, enema group and western medicinecontrol group have different level of yellow granular distribution in their pictures.Model group’s cox-2protein immunohistochemical staining optical density valueincreased significantly compared with normal control group (P <0.01). Oral group,enemagroup and western medicine control group optical density value were all less than modelgroup optical density value, and there was statistical significance (P <0.01).But comparisonbetween oral group,enema group and western medicine control group, enema group’s opticaldensity value was less than less than other two groups.It has statistically significant (P <0.05). Conclusion:1. Si-Shen bolus can reduce colitis associated cancer symptoms such as bloody and rectocelein mice.2. Si-Shen bolus can inhibit changes of spleen,thymus,colon in colitis associated cancer mice.3. Si-Shen bolus can reduce colon cancer rate in colitis associated cancer mice.4. Si-Shen bolus can prevent colitis to colon cancer in mice after lesions, and Si-Shen bolushave chemoprevention effect for colitis associated cancer in mice.5. Si-Shen bolus can inhibit the Nrf2protein decrease in colitis associated cancer.6. Si-Shen bolus’s chemoprevention effect for colitis associated cancer may be related toinhibition of Nrf2protein decrease.7. Si-Shen bolus can inhibit colon tissue’s Cox-2related gene mRNA expression in colitisassociated cancer mice at genetic level.8. Si-Shen bolus can reduce colon tissue’s Cox-2protein expression in colitis associatedcancer miceat protein level.9. Si-Shen bolus’s chemoprevention effect for colitis associated cancer may be related toinhibition of Cox-2related gene mRNA expression and reducing Cox-2protein expression incolon tissue.10.From the point of delivery way, oral and enema was no significant difference inSi-Shen bolus’s chemoprevention effect for colitis associated cancer。... |
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