Therapeutic Effect And Mechanism Of Chang-an-jun-tai On The Treatment Of Rats With Diarrhea-predominant Irritable Bowel Syndrome Based On SCF/C-KIT/TRPV1

ObjectivesAs a functional intestinal disorder,the mechanism of irritable bowel syndrome(IBS)remains unclear.Modern medicine also has no ideal treatment.Traditional Chinese medicine has unique advantages in treating functional diseases.Chang-An-Jun-Tai Granules(CAJT)is a self-made prescription of Professor Fengbin Liu,which consists of Tongxie Yaofang,Sijunzitang and Xianglianwan.It has the functions of softening liver and relieving pain,strengthening spleen and stopping diarrhea.In the clinical treatment of diarrhea-predominant irritable bowel syndrome(IBS-D),it has good curative effect,no obvious side effects and adverse events were found.However,the mechanism for its therapeutic effect is unclear,so our team raised the following research purposes:1.To predict the effective active ingredients,target and related pathways of CAJT Granules in the treatment of IBS by network pharmacology of traditional Chinese medicine.2.To explore the anti-diarrhea,analgesic and anti-intestinal hyperfunction effects of CAJT granules through animal experiments and to evaluate its efficacy in the treatment of IBS-D.3.To research the relevant target action mechanism of CAJT granules based on the predicted results of network pharmacology.MethodsExperiment 1.A network pharmacology study to uncover the potential mechanism of CAJT granules1.Through the Chinese Medicine System Pharmacology Analysis Platform(TCMSP),we searched 9 kinds of traditional Chinese medicines of CAJT Granules,and set the oral utilization(OB?30.0)and drug-likeness(DL?0.18)to screen the potentially active active ingredient of CAJT granules;2.Obtain the chemical structure of the potential active ingredient through TCMSP and Pub Chem database and import them into the Swiss Target Prediction database,define the research species as human,and save the obtained target information in CSV format;3.Using human Mendelian genetic database(OMIM)and Gene Cards database to search keywords of irritable bowel syndrome to find genes related to IBS,combined the results of this two databases,to obtain potential targets for CAJT treatment of IBS;4.The potential targets of CAJT and the IBS-related gene were subjected to Venn analysis,and the potential targets of CAJT in the treatment of IBS were obtained.Further,the cytoscape software was used to construct the network of “CAJT granules,active ingredients,targets,disease”;5.The potential targets of CAJT in the treatment of IBS obtained by Venn analysis was imported into String database,the research species was defined as human,the medium confidence was set as 0.4,the free protein was concealed,and the protein interaction diagram was obtained;6.Manually imported the potential targets of CAJT in the treatment of IBS to the Dis Ge NET database,select the gene for retrieval,and obtain the target type;7.Import the potential target of the obtained CAJT active ingredient into the DAVID online tool,define the study species as human,and perform the main functional annotation and pathways for the significant enrichment of the active ingredient target.Experiment 2: The anti-diarrhea,analgesic effects of CAJT Granules1.After 1 week of adaptive feeding of C57 mice,drug intervention was lasted for 7 days.The low dose of CAJT was 1.229 g/kg/day,the middle dose of CAJT was 2.457 g/kg/day,and the high dose of CAJT was 4.914g/kg/day,the dose of aspirin was 0.1g/kg/day,the dose of piperamine was 2mg/kg/day,and the dose of atropine was 1mg/kg/day;2.Hot plate analgesia: Before the intervention,mices were placed in a basin whose temputure was kept at(55 ± 0.5)°C(the mice were placed in a beaker in this basin),and each mouse were observed to the appearance of a nocifensive response(hind paw licking or jumping).Record the reaction time of nocifensive response at the 30 min,60min,and 90 min after the last administration of drugs.For those who did not respond for more than 60 s,the pain threshold was calculated as 60s;3.Acetic acid writhing: Thirteen minutes after the last administration of drugs,5 groups of mice were intraperitoneally injected with 0.6% acetic acid(0.1 ml/10g),and the number of writhing of ecah mice was recorded during the following 20 min;4.Diarrhea model: Each mouse was intragastrically administered with castor oil at the dose of 0.1 ml/10 g 30 min after the last administrationof drugs.The mice were placed in a metabolic cage desiged for mice and a qualitative filter paper was placed in the metabolic cage.After continuous observation for 6 hours,the defecation of the mice was observed,and the stool rate,loose stool level and diarrhea index were recorded.5.Small intestinal hyperfunction model: Each mouse was intraperitoneally injected with neostigmine mesylate(0.1mg/kg)after the last administrationof drugs.Thirty minutes later,each group of mice except the control group were intragastrically administered with 0.5% Evans blue and 5% neutral gum 0.2ml,15 min later obtain the entire small intestine,the distance between the Evans blue in the small intestine and the length of the whole small intestine were measured.Experiment 3: The efficacy of CAJT Granules in the treatment of IBS-D1.IBS-D rat model was established by the method of maternal separation and acetic acid enema.The model of SD rats with IBS-D was determined by abdominal withdrawal reflex score,water avoidance stress and fecal water content to make sure that visceral hypersensitivity was successfully made;2.SD rats were divided into 6 groups: control group,model group,CAJT low dose group,CAJT middle dose group,CAJT high dose group,and alosetron group;3.CAJT granules or Alosetron were intragastrically administered for 2weeks.The low,middle and high doses of CAJT granules were 0.85g/Kg/day,1.7g/Kg/day and 3.4g/Kg/day,respectively.The dose of alosetron is 200 u g/Kg/day;4.At the end of the drug intervention,behavioral observation was performed.Colorectal distention was performed for AWR score.Water avoidance stress was used to count the amount of defecationof the rats.The total field of the rats was measured from the open field test.The feces of each group were acquired,and the moisture content of the feces was assessed.HE staining of colon tissue in each group was observed.Experiment 4: The mechanism of Chang-An-Jun-Tai on the treatment of rats with diarrhea-predominant irritable bowel syndrome based on SCF/C-KIT/TRPV1The colon tissues of rats in each group were taken.The expression of mast cells was detected by benzylamine blue staining.The expressions of SCF,C-KIT,5-HTR1 A,SP,PKC and TRPV1 in intestinal mucosa were detected by western blot and QPCR.ResultsExperiment 1.A network pharmacology study to uncover the potential mechanism of CAJT granules1.A total of 165 potential active components of CAJT which including Berberine,coptisine,kaempferol,wogonin,tannic acid,scrophularin,scorpion were screened from TCMSP database,8 active components for ginseng ginseng,7 for atractylodes,15 for cockroaches,13 for white peony,18 for windproof,5 for tangerine peel,14 for berberine,6 for wood incense,and 92 for licorice;2.We obtain 286 predicted targets for all components,a total of 1705IBS-D-related gene targets were obtained from Genecard and OMIM,and 105 CAJT targets were figured out to treat IBS-D by Venn analysis;3.The network of “CAJT granules,active ingredients,targets,disease”showed that among all the potential active components of CAJT,sitosterol and leucovorin were most frequently linked.The top five linked of the targeted genes were MAPT,SL6A2,CYP19A1,ALOX5,ESR2;4.Protein interactions showed that AKT1 is the most interacting protein with other proteins(62),followed by PTGS2(47),HTR2A(11),and TRPV4(5);5.The results of the Dis Ge NET database showed that the most common target types are oxidoreductases(17),followed by transcription factors,receptors,and nucleic acid receptors(12);6.The KEGG pathway showed that the mechanism of CAJT in the treatment of IBS-D is mainly related to MARK pathway,neural activity ligand-receptor interaction,and calcium signaling pathway.Among them,there are 8 direct targets in the MARK pathway and neural ligands-receptor interaction pathway has 11 direct targets.Experiment 2: The anti-diarrhea,analgesic effects of CAJT Granules1.There was no significant difference in pain threshold between the hot plate analgesia group and the CAJT low dose group(P>0.05).There was a statistical difference between the normal group and the CAJT middle dose group,CAJT high dose,and aspirin group(P<0.05);There was no significant difference in the pain threshold between the aspirin group and the CAJT groups(P>0.05);2.There was no significant difference in pain threshold between the acetic acid writhing group and the low dose group of CAJT(P>0.05).There was significant difference in pain threshold between normal group and CAJT middle dose group,CAJT high dose and aspirin group(P<0.05);there was no significant difference in the pain threshold between the aspirin group and the CAJT groups(P>0.05);3.Compared with the normal group,the diarrhea model group showed an increase in the rate of loose stools(P<0.05).There was no difference between the model group and the low dose group of CAJT((P>0.05);Compared with the model group,the CAJT middle dose group,CAJT high dose group,and loperamide group had improved stool rate,the difference was statistically significant(P<0.05);Compared with the normal group,the stool index of the model group significantly increased,the difference was statistically significant(P<0.05);Compared with the model group,the stool index of the CAJT low dose group,the CAJT middle dose group,and the CAJT high dose group and the loperamide group were significantly improved,and the difference was statistically significant(P<0.05);4.Compared with the normal group,the small intestine propulsion rate of the model group was significantly higher,the difference was statistically significant(P<0.05);the small bowel propulsion rate of the CAJT low dose group was not statistically significant when compared with the model group(P>0.05);Compared with the model group,CAJT middle dose group,CAJT high dose group,atropine group significantly inhibited small bowel hyperfunction,the difference was statistically significant(P<0.05);There was no significant difference between the CAJT groups and the loperamide group(P>0.05).Experiment 3: the efficacy of CAJT Granules in the treatment of IBS-D1.General conditions of the rats in the normal group were active,their white hair is clean and shiny,and there is no contraction of the toes,abdominal muscles and arches.Rats in the model group were very active,frequently jumping up and down,fighting with each other,the hair of the rats was yellow,dry,rough and lack of luster.Occasionally the contraction of rats' toes and abdominal muscles were observed,the fecal quality was soft and thin,the litter smell was slightly obvious,and the rat cage was unclean.After treatment,the CAJT middle dose and CAJT high dose group were slightly active,chasing,their hair were neat and shiny,and the rat's metatarsophalangeal muscle contraction and arch-back up were not observed,while it was observed in the CAJT low dose group;2.Before treatment,the difference between the body weight of rats with the normal group and the model group and CAJT low dose group,CAJT middle dose group,CAJT high dose group,and alosetron group were statistically significant(P<0.05),there was no significant difference in body weight between the model group and each drug group(P>0.05);After intervention,there was no significant difference in body weight between the CAJT lowdose group,alosetron group and the model group(P>0.05);the CAJT middle dose group and the CAJT high dose group could increase body weight of rats significantly.The difference was statistically significant(P<0.05);3.Before treatment,compared with the normal group,the water content of feces in the model group and the CAJT low-dose group,the middle-dose group,the CAJT high-dose group and the alosetron group were higher than the normal group.The difference was statistically significant(P<0.05),there was no significant difference in the water content of rat feces between the model group and each medication group(P>0.05);After intervention,compared with the model group,the water content of feces in CAJT low dose group,CAJT middle dose group,CAJT high dose group,and alosetron group were significantly decreased,the difference was statistically significant(P<0.05);4.Before treatment,compared with the normal group,the fecal output of rats during water avoidance stress in the model group,the CAJT low dose group,the middle dose group,the CAJT high dose group,and the alosetron group were significantlyincreased(P<0.05);After intervention,compared with the model group,the fecal output of rats in the CAJT low dose group,the middle dose group,the CAJT high dose group,and the alosetron group during water avoidance stress were significantly reduced(P< 0.05);5.Before treatment,compared with the water injection volume when the AWR score was 3 in the the normal rats,the water injection volume of the model group,CAJT low dose group,CAJT middle dose group,CAJT high dose group,and the alosetron group rats were significantly decreased(P<0.05);After intervention,compared with the water injection volume when the AWR score was 3 in the the normal rats,the water injection volume of the model group,CAJT low dose group,CAJT middle dose group,CAJT high dose group,and the alosetron group rats were significantly elevated(P<0.05);6.After the drug intervention,the total distance of the open field test in the CAJT low dose group was reduced,but the difference was not statistically significant(P>0.05).The total distance of the open field test in the CAJT middle dose group,the high dose group of CAJT group and the alosetron group was significantly reduced,and the difference was statistically significant(P<0.05);7.HE staining of colon tissue showed that the intact mucosa,submucosa and muscle layer were observed in all groups.In the mucosa layer,columnar epithelial cells with a villus structure and regular arrangement of villus are formed,and blood vessels and lymphatic vessels can be observed in the submucosa,and lymphocytes can be observed in the lymphatic vessels.No mucosal edema,inflammatory cell infiltration was obversed.Experiment 4: The mechanism of Chang-An-Jun-Tai on the treatment of rats with diarrhea-predominant irritable bowel syndrome based on SCF/C-KIT/TRPV11.Metamine blue staining of mast cells showed that after intervention,compared with the normal group,the expression of colonic mast cells in the model group was significantly increased(P<0.05).Compared with the model group,the expression of colonic mast cells in the CAJT low dose group,the CAJT middle dose group,and the CAJT high dose group was significantly decreased(P<0.05),while the expression of mast cells in the alosetron group was decreased,but the difference was not statistically significant(P>0.05);2.Colonic expression of SCF protein showed that after intervention,compared with the normal group,the expression of SCF protein in the model group increased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of SCF protein in the colon of CAJT low dose group,CAJT middle dose group,CAJT high dose group and alosetron group was significantly decreased(P<0.05);3.Colonic expression of 5-HTR1 A protein in showed that after drug intervention,the expression of 5-HTR1 A protein in the model group was significantly increased when compared with the normal group(P<0.05);4.Colonic expression of SP protein showed that after intervention,the expression of SP protein in the model group was significantly increased compared with the normal group(P<0.05).Compared with the model group,the expression of SP protein in the colon of CAJT low dose group,CAJT middle dose group,CAJT high dose group and alosetron group was significantly decreased(P<0.05);5.Colonic expression of C-KIT protein showed that after intervention,the expression of C-KIT protein in the model group was significantly increased when compared with the normal group(P<0.05).Compared with the model group,the expression of C-KIT protein of CAJT middle dose group and high dose CAJT group decreased significantly(P<0.05),Although the expression of C-KIT protein in the CAJT low dose group and the alostron group were relatively decreased,the difference was not statistically significant(P>0.05);6.Colonic expression of PKC protein showed that after intervention,compared with the normal group,the expression of PKC protein in the model group increased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of PKC protein in colonic cells of CAJT low-dose group,CAJT middle-dose group,CAJT high-dose group and alosetron group was significantly decreased,and the difference was statistically significant(P<0.05);7.Colonic expression of TRPV1 protein showed that after intervention,the expression of TRPV1 protein in the model group was significantly increased compared with the normal group(P<0.05).Compared with the model group,the expression of TRPV1 protein in the colon of CAJT middle dose group and CAJT high dose group was significantly decreased(P<0.05),although the expression of TRPV1 protein of the CAJT low dose group and alostron group were relatively decreased,the difference was not statistically significant(P>0.05);8.Colonic expression expression of SCF m RNA showed that after intervention,the expression of SCF m RNA in the model group was significantly increased when compared with the normal group,the difference was statistically significant(P<0.05).Compared with the model group,the expression of SCF m RNA in the colon of CAJT low dose group,CAJT middle dose group,CAJT high dose group and alosetron group were significantly decreased(P<0.05)9.Colonic expression of of 5-HTR1 A m RNA showed that after intervention,the expression of 5-HTR1 A m RNA in the model group was significantly increased when compared with the normal group(P<0.05).Compared with the model group,the expression of 5-HTR1 A m RNA in the colon of CAJT low dose group,CAJT middle dose group,CAJT high dose group and alosetron group were significantly decreased(P<0.05);10.Colonic expression of SP m RNA showed that after intervention,the expression of SP m RNA in the model group was significantly increased when compared with the normal group(P<0.05).Compared with the model group,the expression of SP m RNA in the colon of CAJT low-dose group,CAJT middle-dose group,CAJT high-dose group and alosetron group were significantly decreased(P<0.05);11.Colonic expression of C-KIT m RNA expression showed that after intervention,the expression of C-KIT m RNA in the model group was significantly increased compared with the normal group(P<0.05).Compared with the model group,the expression of C-KIT m RNA in the colon of CAJT middle dose group and high dose CAJT group were significantly decreased(P<0.05),although the expression of C-KIT m RNA of rats in the CAJT low dose group and the alosetron group were relatively decreased,the difference was not statistically significant(P>0.05);12.Colonic expression of PKC m RNA showed that after intervention,the expression of PKC m RNA in the model group was significantly increased when compared with the normal group(P<0.05).Compared with the model group,the expression of PKC m RNA in the colon of CAJT low dose group,CAJT middle dose group,CAJT high dose group and alosetron group were significantly decreased(P<0.05);13.Colonic expression of TRPV1 m RNA expression showed that after intervention,the expression of TRPV1 m RNA in the model group was significantly increased compared with the normal group(P<0.05).Compared with the model group,the expression of TRPV1 m RNA in the colon of CAJT middle dose group and CAJT high dose group were significantly decreased(P<0.05),although the colonic TRPV1 m RNA in CAJT low dose group and alosetron group rats were relatively decreased,the difference was not statistically significant(P>0.05).Conclusion1.Active ingredients of CAJT such as berberine,copperine,wogonin,kaempferol,hesperidin and citric acid focous on PKC,HTR,TRPV1 to treat IBS;2.CAJT middle dose and CAJT high dose have obvious analgesic effect;CAJT middle dose and CAJT high dose can significantly inhibit the gastrointestinal motility to exert its antidiarrheal effect;3.CAJT middle dose and CAJT high dose significantly improved intestinal hypersensitivity in rats and reduced the moisture content of rat feces;4.CAJT middle dose and CAJT high dose can reduce the expressions of MC,5-HTR1 A,SP and PKC by inhibiting SCF/C-KIT signaling system,and reducing the expression of TRPV1 to treat IBS-D.