The Study On The Mechanism Of Changrui Enema In The Treatment Of Radiation Proctitis By Regulating AQP1 And AQP3

Objective: Radiation proctitis(RP)is the most common complication of abdominal and pelvic malignant tumor radiotherapy,seriously affects the patients' prognosis and living standard.Here we assessed the therapeutic effect of Changrui retention enema on radiation proctitis,and elucidated its possible mechanism involved in the regulation of aquaporin 1(AQP1)and aquaporin 3(AQP3).Furthermore,the correlation among AQP1,AQP3,nuclear factor-kappa B(NF-?B),and vascular endothelial growth factor(VEGF)were also analized.Methods: 1.Expression of AQP1 and AQP3 in the treatment of radiation proctitis with Changrui enema(1)Model establishing,grouping and enema method: 60 wild healthy C57BL/6 mice were randomly divided into normal control group,model group,western medicine group,Changrui high-dose group,Changrui middle-dose group and Changrui low-dose group,10 in each group.In addition to the normal control group,other groups were given a single high dose of pelvic irradiation.During 1-14 days after irradiation,the normal control group and the model group were treated by distilled water enema.The mice of western medicine group were treated by dexamethasone combined with gentamicin enema.High,medium and low dose groups were treated with different concentrations of Changrui enema.(2)Observation and detection index: The general signs of the mice were observed.Mice raised for 2 weeks were killed by cervical dislocation method.The 1cm tissue from the upper rectum of anus was divided into two segments.The expression of AQP1 and AQP3 was detected by immunehistochemistry and western blot.2.Application of AQP1 knockout mice to study the mechanism of treatment of radiation proctitis with Changrui enema(1)Model establishing,grouping and enema method: 36 homozygous mice were averagely divided into normal control group,model group,western medicine group,Changrui high-dose group,Changrui middle-dose group and Changrui low-dose group,6 in each group.36 wild-type mice were grouped as above.Model establishment and enema method are the same as before.(2)Observation index: a.Pathological changes of rectal tissue in each group were observed by HE staining b.The liver index and spleen index were calculated to investigate whether radiation had an effect on the immunity of mice.(3)Detection index: Mice raised for 2 weeks were killed by cervical dislocation method.The 1cm tissue from the upper rectum of anus was divided into two segments.The expression of AQP1,AQP3,NF-?B and VEGF in rectum were examined by HE,immunohistochemistry,western blot and RT-PCR.Results: 1.Expression of AQP1 and AQP3 in the treatment of radiation proctitis with Changrui enema(1)Immunohistochemistry: The IHS of AQP1 in the model group was significantly higher compared with normal control group while the IHS of AQP3 in the model group was significantly lower compared with normal control group.It suggested that radiation proctitis could promote the expression of AQP1 and inhibit the expression of AQP3.The expression of AQP1 was significantly decreased while the expression of AQP3 was increased in the western medicine group and the high-dose group.Compared with the middle and low dose groups,the expression of AQP1 was the lowest and the AQP3 was the highest in the high-dose group.Therefore,the high-dose group of Changrui had the optimal curative effect.(2)The expression of protein: Compared with normal control group,the protein expression of AQP1 was significantly increased,while AQP3 was significantly decreased.The expression of AQP1 in each treatment group was lower than that in the model group,and the expression of AQP3 was higher than that in the model group.In the comparison between groups of Changrui enema,expression of AQP1 protein was the lowest and the AQP3 protein was the highest in the high dose group.(3)We also found a negative linear correlation between AQP1 and AQP3 in immunohistochemistry and western blot((r=-0.835,P<0.01;r=-0.879,P<0.01).2.Application of AQP1 knockout mice to study the mechanism of treatment of radiation proctitis with Changrui enema(1)Pathological changes: In the model group,the level of rectal inflammation in the AQP1 knockout mice was significantly lower than that in the wild-type mice.the signs of congestion and edema of rectal mucosa and submucosa of wild-type mice were more obvious than those of AQP1 knockout mice.In the western medicine group and the high-dose group,the pathological damage of rectal tissue was alleviated compared with the model group,and the degree of inflammation was basically the same.(2)Liver index and spleen index: The liver index and spleen index of the homozygous mice in the western medicine group were statistically significant compared with the other groups.but it could not be considered that the deletion of AQP1 gene had an effect on the immunity of mice.(3)Immunohistochemistry: Expression trends of AQP3,NF-?B and VEGF in homozygous group were basically the same as the wild-type group.The IHS of AQP3 in the model group was significantly lower than that in the normal control group,while NF-?B and VEGF were significantly higher compared with normal control group.NF-?B and VEGF in each drug group were lower than those in the model group,and the AQP3 was higher than the model group.In the wild-type group,the IHS of AQP1 in the model group was higher compared with normal control group.(4)Expression of protein: The trend of the homozygous group and the wild-type group was the same,results were as follows.The protein expression of NF-?B and VEGF in the model group was higher than that in the other groups,and the protein expression of NF-?B and VEGF in the high dose group was the lowest among the drug groups.Expression of AQP3 in the model group was lower than that in the other groups,excepting the low dose group.The highest expression of AQP3 was found in the high dose group.Expression of AQP1 in the model group was lower than that in the normal control group,and the expression was the lowest in the high dose group among the drug groups.We also found a negative linear correlation between AQP3 and VEGF(r=-0.817,P<0.01).(5)Gene expression: The expressions of AQP1,AQP3,NF-?B and VEGF gene was consistent with the results of western blot.Conclusion: 1.Radiation proctitis promoted the expression of AQP1 and inhibited the expression of AQP3.Changrui enema could down-regulate the expression of AQP1 and up-regulate the expression of AQP3 to treat radiation proctitis.2.The deletion of AQP1 gene significantly reduced the pathological damage of radiation proctitis and could be used as a new target for the treatment of radiation proctitis.3.There is a negative linear correlation between AQP1 and AQP3,and AQP1 cannot be considered to be associated with AQP3 and NF-?B.4.There may be a mutual regulation relationship between AQP1 and VEGF.AQP1 may participate in the formation of radiation proctitis by regulating osmotic pressure to promote the migration of endothelial cells to angiogenesis.5.AQP3 may participate in radiation proctitis by regulating water transport and metabolism,while promoting the repair of intestinal mucosal barrier to alleviate inflammation.