Mechanism Research Of Chang-An-Jun-Tai Decoction On IBS-D Rats With Liver Depression And Spleen Deficiency Based On PI3K/AKT Pathway

Objectives:Irritable bowel syndrome with predominant diarrhea(IBS-D)is a clinically common chronic functional gastrointestinal disease.At present,modern medical treatment lacks specific drugs.In the process of clinical practice,it has been discovered that Chang-An-Jun-Tai Decoction has a good therapeutic effect on IBS-D with liver depression and spleen deficiency,but its main therapeutic mechanism and effective ingredients are still unclear.1.Through the systematic analysis of the chemical components and blood components of " Chang-An-Jun-Tai Decoction ",identify the potential medicinal components of "Chang-An-Jun-Tai Decoction ".2.Using network pharmacology to predict the effective active ingredients and potential targets of Chang-An-Jun-Tai Decoction in the treatment of IBS.3.Use molecular docking technology to further screen out the targets and active ingredients of drugs that play a key role in the drug-target-disease network.4.The IBS-D rat model with liver depression and spleen deficiency syndrome was established through maternal separation + chronic restraint and acetic acid enema + senna gavage to study the pharmacodynamics of Chang-An-Jun-Tai Decoction in the treatment of IBS-D.5.To explore the potential mechanism of Chang-An-Jun-Tai Decoction in the treatment of IBS-D with liver depression and spleen deficiency syndrome based on the prediction target of network pharmacology.Methods:1.The components of ” Chang-An-Jun-Tai Decoction” in plasma were analyzed by UHPLC-QE-MS.2.Search for active pharmaceutical ingredients through TCMSP and BATMAN-TCM databases,and obtain compound targets through the Swiss target prediction database.Using OMIN,Gene Cards,and Dis Ge NET to predict IBS disease targets,and using STRING database and Cytoscape software to complete the molecular network architecture.DAVID,KEGG,GO database were used to perform the enrichment analysis,and preliminary analysis of key targets and pathways.3.Import the predicted potential active ingredients into the ZINC database to obtain the monomer structure diagram,obtain the structure diagram of the key target protein from the PDB database.Auto Dock software was used to complete the molecular docking of ligand and receptor,and use the Py Mol software to visually display the results.4.The IBS-D rat model with liver depression and spleen deficiency syndrome was established through maternal separation + chronic restraint and acetic acid enema + senna gavage.Sucrose preference test,open-field test,water avoid stress,forced swimming test,feces moisture content,food intake,abdominal withdrawal reflex test evaluation model to confirm that the successful establishment of the liver-stagnation and spleen-deficiency IBS-D rat model.5.IBS-D rat were divided into 8 groups: control group,model group,and CAJT high-dose group(3.4 g/kg/d),CAJT middle-dose group(1.7g/kg/d),CAJT low-dose group(0.85g/kg/d),pinaverium bromide group(15mg/kg/d),LY294002 group(0.75mg/kg/d+10ml/kg/d normal saline),LY294002+ CAJT group(LY294002 0.75mg/kg/d+CAJT 1.7g/ kg/d);kg/d);drug intervention for 2 weeks,during which the general condition and weight changes of the rats were observed and recorded.After the intervention,Sucrose preference test,open-field test,water avoid stress,forced swimming test,feces moisture content,food intake,abdominal withdrawal reflex test and HE staining of colon tissue were evaluated for pharmacodynamics.6.Colon tissues of rats were taken for bezylamine blue staining to determine the expression level of mast cells.The expression of rat serum histamine,interleukin-8 and?-HEX were detected by ELISA.Q-PCR and Western blotting was used to detect the expression of PI3K/AKT signaling pathway related targets in colon tissue and hippocampus.Results:1.To identify the blood components of "Changanjuntai",50 of blood components were identified.2.Using the network pharmacology method,94 co-action targets and 36 potential active components of the drug were screened out,which mainly involved the gene functions of cell proliferation,cell apoptosis,signal transduction,etc.The pathways involved mainly include Pathways in cancer,PI3K-Akt signaling pathways,Neuroactive ligand-receptor interaction,Calcium signaling pathway,etc.3.Molecular docking results showed that a total of 34 compounds screened by network pharmacology could bind to PI3 K receptor protein and form hydrogen bond with terminal amino acid.All the 36 compounds can bind to the AKT receptor protein and form hydrogen bond with the terminal amino acid.4.Compared with the control group,the general condition of the rats in model group:mental state,activity status,hair,food intake,feces status are all worse.Weight gain was significantly reduced(P<0.05).There were abnormal states similar to depression and anxiety: there was no difference in horizontal exercise in the open field test(P>0.05),but vertical exercise was reduced(P<0.05).The sucrose preference rate was significantly reduced(P<0.05),and the number of feces increased during water avoid stress(P<0.05),and the floating time of the forced swimming test was prolonged(P<0.05).Intestinal mucosal permeability and visceral hypersensitivity increased: the feces water content increased(P<0.05).Abdominal withdrawal reflex test injection decreased(P<0.05).There were no obvious pathological changes in colon HE staining.5.Comparison of rats in each concentration of Chinese medicine group with the model group,General conditions: mental state,activity status,hair,food intake,feces status are all improved.Weight gain was significantly increased(P<0.05).Abnormal states similar to depression and anxiety Improvements: horizontal exercise and vertical exercise increased in the open field test(P<0.05).The sucrose preference increased significantly(P<0.05),and the number of feces decreased during water avoid stress(P<0.05).The floating time of the forced swimming test was shortened(P<0.05).Intestinal mucosal permeability and visceral hypersensitivity decreased: the feces water content increased(P<0.05).Abdominal withdrawal reflex test injection increased(P<0.05).6.After treatment,the number of toluidine blue staining of rat mast cells was significantly reduced(P<0.001).The CAJT-H can reduce the expression of PI3 K,AKT,m TOR,BDNF and Trk B m RNA in colon tissue(P<0.05);the CAJT-M can reduce the expression of PI3 K,m TOR,Trk B m RNA in colon tissue(P<0.05),AKT,BDNF,m RNA expression levels were not statistically different(P>0.05);the CAJT-L reduced colonic tissue PI3 K,AKT,Trk B m RNA expression levels(P<0.05),m TOR,BDNF,m RNA expression levels were not statistically significant Academic difference(P>0.05).The CAJT-H increased the expression of PI3 K,m TOR,BDNF and Trk B m RNA in the hippocampus(P<0.05),but there was no statistical difference in the expression of AKT m RNA(P>0.05);the CAJT-M increased the expression of hippocampal PI3 K,BDNF,and Trk B m RNA(P<0.05),and there was no statistical difference in the expression of AKT and m TOR m RNA(P>0.05);the CAJT-L had an effect on hippocampal PI3 K,AKT,and AKT m RNA expression.The m RNA expression of m TOR,BDNF,and Trk B had no significant effect(P>0.05).7.After treatment with Chang-An-Jun-Tai,the p-m TOR protein in the colon tissue of rats in the control group,the CAJT-H group,the CAJT-M group,the CAJT-L group,pinaverium bromide group,LY294002 group,and LY294002+CAJT group was relatively The expression level was reduced(P<0.05);the AKT protein expression of each group was not statistically different(P>0.05),the relative expression of p-AKT protein was reduced(P<0.05);the PI3 K protein expression of each group was not statistically different(P>0.05),except for the CAJT-L group,the relative expression of p-PI3 K protein in the other groups decreased(P<0.05).8.Compared with the model group: the expression of p-m TOR protein in the hippocampus of the control group,the CAJT-H group and pinaverium bromide group increased(P<0.05).There was no difference in the relative expression of p-m TOR protein in the CAJT-M group,the CAJT-L group,the LY294002 groupd andthe LY294002+CAJT group(P>0.05).There was no statistical difference in the expression of AKT protein in each group;the relative expression of p-AKT protein in the control group and each Chinese medicine group increased,however only the CAJT-H group was statistically significant(P<0.05).The relative expression of p-Akt protein in hippocampus of rats in LY294002 group and LY294002+CAJT group was decreased.There was no statistical difference in PI3 K protein expression in each group;the relative expression of hippocampal p-PI3 K protein in the control group and each Chinese medicine group increased,CAJT-H group,pinaverium bromide group There was statistical significance(P<0.05).The expression of p-PI3 K protein in the hippocampus of rats in the LY294002 group and LY294002+CAJT group decreased,and there was no statistical significance(P>0.05).9.Changanjuntai can reduce the expression of histamine,?-hexosaminidase and IL-8in serum of rats,and the difference is statistically significant(P<0.05).Conclusion:1.The active ingredients of Chang-An-Jun-Tai Decoction are mainly berberine,Ginsenoside Ro,kaempferol,Naringenin,atractylenolide II,wogonin and so on.2.The IBS-D rat model with liver depression and spleen deficiency syndrome was established through maternal separation + chronic restraint and acetic acid enema + senna gavage,which is consistent with its clinical manifestations.3.The PI3K/AKT pathway plays an important role in the occurrence,development and treatment of IBS-D with liver depression and spleen deficiency.4.Chang-An-Jun-Tai Decoction may play a role in the treatment of IBS-D with liver depression and spleen deficiency through PI3K/AKT signaling pathway and related downstream signaling proteins.