| [Objective]:This study takes the formation of Aberrant crypt focus(ACF),a key node in the colitis oncologic transformation to colon cancer,as the entry point.Research object is colitis-cancer transformation mouse model induced by AOM/DSS.Verify the preventive effect of Tong-Xie-Yao-Fang on the colitis-cancer progression and ACF formation.Detect the expression of NF-?B,STAT3,IL-6,Wnt/?-catenin signaling pathway in the serum and colon tissues of mice and evaluate the effect of Tong-Xie-Yao-Fang and the effects of Wnt/?-catenin signaling pathway in different doses of Tong xie yao fang,which can help us to.To deeply explore the mechanism of Tong-Xie-Yao-Fang on ACF and colitis-cancer transformation.[Methods]:1.The establishment of the colitis-cancer transformation mouse model and evaluation of the outcome of ACF:On the first day,C57BL/6 mice were given intraperitoneal injection of AOM(12 mg/kg),then drunk DSS 3%(W/V)and sterile water circularly for 7 weeks(To complete 7-week DSS and sterile water circulation:drunk sterile water in the first week;drunk DSS in the second week;drunk sterile water in the third and fourth weeks;then drunk DSS for 5 days;finally drink sterile water for 16 days).They were draw the materials at the 4th,5th,6th and 7th weekends respectively.The general state,body weight,disease activity index score(DAI),colon length,tumor formation,etc.were monitored.HE staining was used to observe the pathological changes of colon tissue and the formation and quantity of ACF.Evaluate the formation of ACF and the pathological morphology in each time node.2.The preventive effect of Tong-Xie-Yao-Fang on the colitis-cancer progression and ACF formation:C57BL/6 mice were randomly divided into a blank group,a model group,a positive group,a high-dose group of Tong-Xie-Yao-Fang,a middle-dose group ofTong-Xie-Yao-Fang,and a low-dose group of Tong-Xie-Yao-Fang,with 40 rats in each group.In addition to the blank group,the other groups were induced by AOM/DSS to construct the model of colitis-cancer transformation.The corresponding drug intervention was given for 6 weeks that one week after the start of modeling.Draw the materials at the 4th,5th,6th and 7th weekends respectively.The general state,body weight,DAI score,colon length,tumor formation,etc.were monitored.Evaluate the preventive effect of Tong-Xie-Yao-Fang by the pathological morphology and formation of ACF in each time node,screening for the optimal dose3.The mechanism of Tong-Xie-Yao-Fang prevent of ACFTake the serum and colon tissues of mice for test.Detect the expression of inflammation-related factor IL-6 in serum by ELISA,detect NF-?B and STAT3 of colon tissue by immunohistochemical staining,detect the expression of Wnt and ?-catenin in colon tissue by Western blot[Result]:1.Successfully build a stable animal model of colitis-cancer transformation Histopathological sections showed that the model group at 4 weeks:colonic epithelium,lamina propria,extensive inflammatory cell infiltration,and crypt inflammation.Part of the specimen section showed intestinal mucosal shedding.The more serious inflammation present the closer to the anus.Model group at 5 weeks showed:the structure of the crypt is disordered,the gland is deformed,the arrangement is disordered,and the ACF is partially visible.Model group at 6 weeks showed:Most of the colon showed dysplasia,the nucleus was heavily stained,the proportion of nucleoplasm was large,the cells arranged disorderly,and the polar disappeared.Model group at 7 weeks showed:All mice developed carcinogenesis and formed colon tumors2.Tong-Xie-Yao-Fang prevent the ACF and colitis-cancer transformation in mouse model.The general state,body weight,DAI score,colon length,tumor formation:the positive group,high-dose and middle-dose group of Tong-Xie-Yao-Fang showed better at general state,mental state,activity,and the stool was gradually formed,the blood in the stool was seen occasionally,the weight loss and colon shortening was smaller,the tumor formation rate is lower.Except the blank group,the DAI scores were increased The positive group,the high-dose and middle-dose groups had lower DAI scores Histopathological section observation:except the blank group,no adenocarcinoma was formed on the 4th,5th,6th,and 7th weekends.The preventive effect of Tong-Xie-Yao-Fang on ACF at the end of the 5th week:compared with the model group,positive group and middle-dose group of Tong-Xie-Yao-Fang showed less ACF formation(P<0.01).3.Effect of Tong-Xie-Yao-Fang on IL-6 from serum and NF-?B?STAT3?Wnt/?-catenin from colon tissue:?IL-6 content in mouse serum:Compared with the blank group,the serum IL-6 levels in the model group were significantly higher(P<0.01)Compared with the model group,the IL-6 content in the positive group was decreased,and the difference was statistically significant(P<0.01);Compared with the model group,the IL-6 content in the middle dose group of Tong-Xie-Yao-Fang decreased,the difference was statistically significant(P<0.05).It indicated that Tong-Xie-Yao-Fang can down-regulate serum IL-6 in mice.?Expression of NF-?B and STAT3 in mouse colon tissue:Compared with the blank group,the expression of NF-?B and STAT3 in the colon tissue of the model group was up-regulated.Compared with the model group,the expressions of NF-?B and STAT3 in the colon tissue of the positive drug group and the Tong-Xie-Yao-Fang group were significantly down-regulated.Compared with the positive drug group,the expression of NF-?B and STAT3 in the colon tissue of the mice in the middle dose group of Tong-Xie-Yao-Fang was higher.?Expression of Wnt/?-cateninin in mouse colon tissue:Compared with the blank group,the expression of Wnt and ?-catenin in the colon tissue of the model group,the positive group and the Tong-Xie-Yao-Fang group significantly increased,the difference was extremely significant(P<0.01);compared with the model group,the expressions of Wnt and ?-catenin in colon tissue of mice in the Tong-Xie-Yao-Fang middle-dose group and the positive group were significantly decreased(P<0.01).[Conclusion]:Tong-Xie-Yao-Fang can reduce the formation of ACF and delay the progression of colitis carcinogenesis.Its mechanism may related to that reducting the inflammatory factors IL-6,NF-?B and STAT3 in serum and colon tissues of mouse model and regulating Wnt/?-catenin signaling pathway. |
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