| Purpose:Establishing a radiation proctitis in C57BL/6 mice model to study whether Chang-rui retention enema prevent and treat acute radiation proctitis via inhibit angiogenesis by adjust AQP1 and VEGF.Methods:1.Establish radiation proctitis model(1)Irradiation method:Establish radiation proctitis in C56BL/6 mice model by a single large dose 6MV-X ray local irradiation.The 36 C56BL/6 mice were randomly divided into 6 groups,10 mice in each group,irradiation doses were: 0Gy,21 Gy,23Gy,25 Gy,27Gy,30 Gy.(2)Observation index: a.mice were observed daily food intake,water intake,body weight,excrement,crissum skin,independent spirit activities.b.2 mice were killed in each group at 1W,2W,3W after irradiation,getted rectum tissue about 1cm above anus,HE staining,observed pathological changes of mice rectum tissue under light microscope.c.Evaluation model: Food and water intake decreased,abnormal stools and other symptoms.Histopathology of rectum was severe inflammatory changes,which similar to patients with clinical.2.Evaluation of curative effect of drug retention enema and the mechanism research(1)Grouping and medication: 70 mice were randomly divided into 7 groups:normal control group,positive control group,western medicine group,chinese medicines group,high dose group,middle dose group,low dose group,10 mice in each group,dealed with not irradiation and distilled water enema,irradiation and distilled water enema,irradiation and hexadecadrol?gentamicin enema,irradiation and baweixileisan enema,irradiation and high dose chang-rui enema,irradiation and middle dose chang-rui enema,irradiation and low dose chang-rui enema.Retention enema was performed at same time every day 1-14 d after irradiation.(2)Observation index: a.mice were observed daily food intake,water intake,excrement,crissum skin,independent spirit activities and signed scores.b.1W after irradiation 4 mice in each group were randomly selected to be killed,2W after irradiation the last mice in each group were killed and the rectal tissues were taked to observe the expression of protein,pathological changes.c.To study the effects of radiation on immunity in mice by weighing the mouse liver and spleen weigh,observing the liver index and spleen index.(3)Detection index: a.Immunohistochemical was used to test the level and position of rectal tissues in mice of Aquaporin 1(AQP1),Vascular endothelial growth fator(VEGF).b.Western blot was used to test the expression of protein in rectal tissues of Aquaporin 1(AQP1),Vascular endothelial growth fator(VEGF).Results: 1.Establish radiation proctitis model(1)Local radiation injuryed rectal tissues of mice,damage degree was bigger with the increase of irradiation dose,general features and pathological scores were higher.(2)27Gy group was consistent with the performance,pathology was a moderate to severe inflammation,so 27 Gy was selected as the irradiation dose of radiation proctitis model.2.Evaluation of curative effect of drug retention enema and the mechanism research(1)General signs score: The score of positive control group had statistical difference compared with the normal control group(P?0.01),the treatment groups had statistical difference compared with the positive control group(P?0.05),western medicine group,chinese medicine group had not statistical difference compared with the chang-rui enema groups(P?0.05),low dose group,middle dose group,high dose group had no statistical significance between each other,it is not mean chang-rui enema is better than other groups in terms of improving the general features.(2)The pathological score: The positive control group had statistical difference compared with the normal control group(P?0.01),the positive control group compared with western medicine group,chinese medicine group,chang-rui enema groups,there was statistically significant(P?0.05).Western medicine group compared with chang-rui enema groups,there was not statistically significant(P?0.05),chinese medicine group compared with middle dose group,high dose group,there was statistically significant(P?0.05),but the score of positive control group was higher than others,in line with pathological damage rank R considering the effect of chang-rui enema high dose group is best,better than baweixileisan,cannot think superior to dexamethasone combined gentamicin.(3)Liver index,spleen index: the spleen index of 2W western medicine group had statistical difference compared with the 2W positive normal group(P?0.01),the rest of the group were no statistical difference compared with each other(P?0.05),prompting the local radiation caused radiation proctitis,cannot have an effect on immunity in mice,also cannot determine the western medicine will affect immunity in mice.(4)Immunohistochemical of AQP1,VEGF: The average optical density of AQP1 and VEGF in positive control group was significantly higher than the normal control group,there was statistically significant(P?0.01).The average optical density of AQP1 and VEGF in treatment groups was lower than the positive control group,had statistical difference compared with positive control group(P?0.05 ? P?0.01),western medicine group,chinese medicine group had statistically difference compared with high dose group(P?0.05),high dose group had statistical difference compared with low dose group and middle dose group(P?0.05),suggested the effect of chang-rui enema was better than other groups.That AQP1 and VEGF level was positively linear correlation(r=0.820,P?0.01).(5)The expression of protein: the expression of AQP1,VEGF protein in 1W,2W positive control group was obviously higher than that of normal control group,there was statistically significant(P?0.05),the treatment groups had statistical difference compared with the positive control group(P?0.01),chang-rui enema groups compared with western medicine group,chinese medicine group had statistical difference(P?0.01),high dose group had statistical difference compared with low dose group and middle dose group(P?0.01),suggesting the best group of reducing AQP1 and VEGF protein expression was high dose group;Also found that AQP1 and VEGF level was positively linear correlation(r=0.962,P?0.01).Conclusion: 1.A single large dose 27 Gy 6MV-X ray local irradiation method could establish the model of radiation proctitis successfully.2.Drug retention enema was effective in the prevention and treatment of acute radiation proctitis,alleviated the degree of inflammation,damage,improved local microcirculation,and the effect of high dose group was better than other groups.3.Chang-rui enema cut the expression of AQP1 and VEGF,inhibited angiogenesis and achieved the goal of treatment of radiation proctitis. |
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