Pharmacological Mechanisms Of Runchang Tongbian Effects Of Angelicae Sinensis Radix Based On Expressions Of AQPs In Mice Colon

Objectives: The effect of Runchang Tongbian is one of traditional effects of Angelicae sinensis radix(Danggui),which was recorded in the books of traditional Chinese medicine(TCM),and there is exact effect in treatment of blood-deficiency constipation(Xuexu Bianmi)in Chinese Medicine clinical practice,but its pharmacological mechanism is unknown.In this study,we established the Xuexu Bianmi model of mice,researched the relationship between expression of aquaporins(AQPs)in colon and formation of constipation,researched and explored the pharmacological mechanism of Runchang Tongbian of Danggui from the level of macro effectiveness and micro molecular biology,so as to explain the scientific connotation of its traditional effects.Methods: 1.The visualization research of literatures on TCM treatment for constipation The literatures on TCM treatment for constipation were searched in China Academic Journals Full-text Database(1994.1-2016.7),VIP Chinese Science and Technology Periodicals Full-text Database(1989.1-2016.7),Wanfang Digital Periodicals Full-text Database(1997.1-2016.7)and Chinese Biomedical Literature Database(1978.1-2016.7)with the search words of "Chinese materia medica","Chinese herbal medicine"," Chinese patent medicine","herbal extracts",and "constipation".Three kinds of software of BICOMS,g CLUTO and Ucinet were used to study social network visualization research of literatures on TCM treatment for constipation,so as to comprehensively grasp the current research state of TCM treatment for constipation and provide theoretical reference for the smooth implementation of the study.2.Establishment and evaluation of Xuexu Bianmi model of mice(1)The mice of blood-deficiency model were subcutaneously injected with Acetylphenyhydrazine(APH)and intraperitoneally injected with Cyclophosphamide(CPA);the mice of constipation model were orally administrated with diphenoxylate(DPN);the mice of Xuexu Bianmi model were subcutaneously injected with Acetylphenyhydrazine(APH)and intraperitoneally injected with Cyclophosphamide(CPA)and orally administrated with diphenoxylate(DPN).Then to evaluate the three models of mice with the Xuexu and Bianmi syndromes as the main criterion,in combination with the levels of RBC,WBC,HGB,HCT in peripheral blood and the character of feces.Finally to give Danggui decoction respectively tothe mice of three models to intervene and provide counterevidence.(2)To analyze the differentiation expression of such macro and micro indicators as the character of feces,water content of the colon,the tissue structure and mucus secretion function of colon,the expression of AQPs in colon,so as to establish mice model for pharmacological mechanism study of Runchang Tongbian effect of Danggui.3.The impact of Danggui on macro efficacy indicators of Xuexu Bianmi mice model 96 Kunming mice,male and female in half,were randomly divided into six groups according to gender and weight: normal control group,Xuexu Bianmi group,positive control group,and high-dose,middle-dose and low-dose Danggui groups.Except the normal control group,the rest mice groups were subcutaneously injected with Acetylphenyhydrazine(APH)and intraperitoneally injected with Cyclophosphamide(CPA)and orally administrated with diphenoxylate(DPN)to copy Xuexu Bianmi model.At the same time,the Danggui groups were administered with different doses of Danggui decoction.The general status,the character of feces,the levels of RBC,WBC,HGB and HCT in peripheral blood,the defecation time,the water content in stool and in colon in mice of all groups were tested and the colon tissue was stained with hematoxylin-eosin(HE)and AB-PAS to observe the changes of it and the mucus secretion,so as to analyze the impacts on macro efficacy indicators,changes of colon tissue and mucus secretion of Danggui treatment for Xuexu Bianmi mice model.4.The impact of Danggui on the expression of AQP4 and AQP8 and its regulatory mechanism To establish Xuexu Bianmi mice model and give Danggui treatment and then the expression of AQP4 and AQP8 in colon were detected by the immunohistochemical technique(IHC);the levels of AQP4 m RNA and AQP8 m RNA in colon were tested by real-time fluorescence quantification polymerase chain reaction(RT-PCR);the levels of AQP4 and AQP8 in colon were tested by western blotting(WB);the levels of phospholipase C(PLC),phosphoinositide(IP3),calmodulin(Ca M),adenylate cyclase(AC),cyclic adenosine monophosphate(c AMP)and protein kinase A(PKA)in colon were detected by Enzyme-linked immune technology(ELISA).5.The preliminary study on the efficacy material base of Danggui's Runchang Tongbian effect The Xuexu Bianmi mice were administered with Danggui decoction,angelica sinensis polysaccharide(ASP)and angelica volatile oil.Then the general status,the characters of feces,the levels of RBC,WBC,HGB and HCT in peripheral blood,the defecation time,the water content in stool and in colon in mice of all groups were tested.Results: 1.The investigation fields of Chinese herbal medicine treatment for constipation include ?The basic and clinical study of Chinese herbal medicine treatment for slow transit constipation(STC)?,?The clinical symptoms and efficacy study of integrated TCM and Western medicine treatment for chronic functional constipation and elderly functional constipation?,?The observation of the curative effects of Chinese drugs preparation in treatment of habitual constipation and elderly functional constipation on?,?The treatment of abdominal distension constipation with Chinese herbal medicine of purgative effect?,?The clinical treatment study on intractable constipation and Changzao constipation?.There are 1044 types of Chinese herbal medicine for treatment of constipation concerned in the research literatures,of which Danggui was of the highest frequency of 1446,followed by RHEI RADIX ET RHIZOMA(945),AURANTZZ FRUCTUS IMMATURUS(943),CISTANCHES HERBA(880),ASTRAGALI RADIX(846)etc.The 95 kinds of Chinese herbal medicine with the frequency more than 49 were analyzed by social network analysis to find that Danggui was in the center of the network and RHEI RADIX ET RHIZOMA,CISTANCHES HERBA,MAGNOLIAE OFFICINALIS CORTEX,FRUCTUS IMMATURUS,CANNABIS SEMEN,ATRACTYLODIS MACROCEPHALAE RHIZOMA,ASTRAGALI RADIX were all around Danggui.2.The mice models of subcutaneous injection of Acetylphenyhydrazine(APH)in combination with intraperitoneal injection of Cyclophosphamide(CPA)and oral administration of diphenoxylate(DPN)were not conformed to the standards of the Xuexu Bianmi mice model.For the mice model copied by APH + CPA + DPN,there appeared the Xuexu Bianmi syndromes as follows,crouching and listless,thin,dry and lusterless in hair,pale in the color of ears,nose,lip and tail,dry and hard in the stool.Compared with the normal control group,the levels of RBC,WBC,HGB,HCT in peripheral blood were significantly decreased,the defecation time was significantly prolonged,the defecation was decreased,the colonic mucosa and gland were atrophied,mucous membrane layer was thinned,mucus secretion was decreased,and the expression of AQP4 and AQP8 was increased significantly.3.The Xuexu and Bianmi syndromes were improved,the levels of RBC,WBC,HGB and HCT in peripheral blood were all increased,the defecation time was significantly shortened,the water content of colon were increased with three doses of Danggui decoction(16.67,8.33,4.17g/kg.d)for the Xuexu Bianmi mice model.The water content of the stool was increased in 16.67 and 8.33 kg.d groups.The atrophy of colon mucosa and gland was improved,the mucus secretion was increased,and the colon lubrication function was improved with three doses of Danggui decoction(16.67,8.33,4.17g/kg.d)for the Xuexu Bianmi mice model.4.The results of IHC show that the expression of AQP4 and AQP8 could be reduced significantly by three doses of Danggui decoction.The results of RT-PCR show that the expression of AQP4 m RNA and AQP8 m RNA was significantly decreased in three doses of Danggui decoction.The results of WB show that the expression of AQP4 in three dose of Danggui decoction and AQP8 in 16.67 and 8.33g/kg.d doses of Danggui decoction were significantly decreased.The results of ELISA show that the levels of PLC,IP3 and Ca M were significantly increased for the Xuexu Bianmi mice model,but the level of PLC was significantly reduced with 16.67g/kg.d dose of Danggui decoction;the level of IP3 was reduced,but there was no statistical significance between the groups;the level of Ca M was reduced significantly by three doses of Danggui decoction.The results of ELISA show that the levels of AC,c AMP and PKA were significantly increased for the Xuexu Bianmi mice model,but they could be reduced significantly by three doses of Danggui decoction.5.The Xuexu and Bianmi syndromes were improved,the levels of RBC,WBC,HGB and HCT in peripheral blood were significantly increased,the defecation time was significantly shortened,the water content of colon and feces was increased with Danggui decoction for the Xuexu Bianmi mice model.The Xuexu syndromes were improved,the levels of RBC,WBC,HGB and HCT were significantly increased,the water content of colon was increased,but the defecation time was not changed in ASP group.Whereas the Bianmi syndromes were improved,the defecation time was significantly shortened,the levels of RBC,WBC,HGB and HCT were not affected in angelica volatile oil group.Conclusions: 1.Chinese herbal medicine treatment for constipation mainly focused on clinical research and the basic research was relatively ignored.The treatment of constipation was mainly compounds of Chinese herbal medicine,and Danggui was one of the most commonly used ones.2.The mice model of APH+CPA+DPN was in accordance with the Xuexu Bianmi syndromes.The levels of RBC,WBC,HGB and HCT,the defecation time,the defecation quantity,the colon mucosa and gland,the mucus secretion,the expression of AQP4 and AQP8 were all changed,which can be used as evaluating indicators.3.The symptoms of Xuexu Bianmi were obviously improved,the water content of the stooland colon was increased,the stool was softened and promoted to discharge,with administration of Danggui decoction to realize its effect of Runchangtongbian.4.To adjust the PLC-IP3-Ca M and AC-c AMP-PKA signaling pathways and influence the expression of AQPs in colon,restrain water absorption of the proximal colon,improve mucus secretion and lubrication function of the distal colon may be one of the mechanisms of Runchang Tongbian effects of Danggui.5.Angelica volatile oil and APS may be important ingredients of Runchangtongbian effects of Danggui,but still they can not represent the overall efficacy of Danggui.