| Rationales:Depression is one of the affective mental disorders,which has the characteristics of high rate of recurrence,disability and suicide.The clinical manifestations are mainly core symptoms such as low mood,loss of pleasure,loss of interest,mental retardation and anxiety;cognitive symptoms such as inattention and memory loss;as well as physical symptoms such as poor appetite,fatigue and loss of libido.The treatment of depression is mainly based on drug,however,some existing chemical antidepressants have side effects and the patient compliance is poor,while traditional Chinese medicine not only significantly improve the core symptoms of depression,but also improve some of its complications and has less adverse reactions,which has gradually attracted the attention of scholars at home and abroad.Therefore,it is of great significance to develop a new generation of antidepressant traditional Chinese medicine.In the early days,our team screened the antidepressant components of Xiaoyao San,combined with the chemical composition analysis,the optimization of Xiaoyao San and the extraction process,Sheng Jiang and Fu Ling in the original side were removed,and Chaigui Fang were obtained.The Chaigui Fang extract was prepared by the process of combining water extraction and ethanol precipitation,and the auxiliary materials were added to make Chaigui granules.At present,the new drug clinical trial approval has been obtained(2018L03149).In our previous study,the antidepressant effect of Chaigui granules through the depressant model of behavioral despair,reserpine antagonism,chronic mild unpredictable stress(CUMS)and serotonin(5-HT)induced taro have already studied,and we have also discussed its antidepressant effect to improve anxiety,learning and memory disorders,gastrointestinal discomfort,decreased sexual function and other symptoms associated with depression.The regulatory effects on serum metabolites,inflammatory cytokines,neurotransmitters,neuroendocrine hormones,gut hormones and sex hormones,and mRNA and protein of key targets of hippocampal c AMP cell signaling pathway was used to study the antidepressant mechanism of Chaigui granules.However,the research on the antidepressant effect of Chaigui granules on the signaling pathway is limited to the c AMP signaling pathway,and whether it can affect other signaling pathways to exert antidepressant effects is still unclear.Therefore,the combination of network pharmacology method and molecular biology(Real Time qPCR and Western blot)method were used to further study the antidepressant mechanism of Chaigui granules.Objective:This study used network pharmacology to study the antidepressant mechanism of Chaigui granules,and obtained an important signaling pathway for Chaigui granules to exert its antidepressant effects.The prediction results of network pharmacology were detected by molecular biology(Real Time qPCR and Western blot)techniques.The results of this study were integrated with the results of previous studies to comprehensively explain the antidepressant mechanism of Chaigui granules.Methods:1.The antidepressant mechanism of Chaigui granules based on network pharmacology: In this study,the antidepressant mechanism of Chaigui granules was investigated from the main prescription of Chaigui granules(Bupleurum)and compound by network pharmacology.The two were mutually verified to obtain important signaling pathways for Chaigui granules to exert its antidepressant effects.(a)The main active ingredients of Bupleuri radix were obtained by TCMSP,literature excavation and the study of our lab.Based on the DRAR-CPI,Gene Cards and OMIM to predict and screen the active components of Bupleuri radix.The Cytoscape software was used to construct the active components-antidepressant targets network of Bupleuri radix.The protein interactions network of antidepressant targets was constructed by using the String database and Cytoscape software.The relation of the main active ingredients and the targets were validated by Systems Dock Web Site.The Dis Ge NET database was used to attribute the type of the targets.The GO and KEGG pathways involved in the targets were analyzed by DAVID software,and then the important signaling pathway was obtained.(b)The TCM database@Taiwan,TCMID,Batman-TC database and literature mining method were used to collect the chemical constituents of the single-flavored drugs of Chaigui granules The Batman-TCM database was used to select the highly reliable protein as a candidate target,combined with CTD,TTD,Pharm GKB,OMIM,Gencards and Drugbank database the targets of antidepressant effect of Chaigui granules were obtained;the String database and Cytoscape softeware were used to construduct protein interaction network and further obtain the TOP20 targets;the algorithm of MCODE in Cluster Viz plug-in of Cytoscape software and the algorithm of MCL in the Cluster Maker plug-in of Cytoscape software were used to respectively module analyze of the protein interaction network,and obtain the Score≥4 modules and the TOP1 module.The DAVID database was used to perform KEGG pathway analysis of the targets of Chaigui granules in the TOP20 of protein interaction,the TOP1 module and the Score ≥ 4 modules based on the MCODE algorithm,the TOP1 module and the Score ≥ 4 modules based on the MCL algorithm,in which the important singnaling pathway for Chaigui granules to excert its antidepressant effects was obtained.2.Effect of Chaigui granules on PI3K/Akt Signaling Pathway in Hippocampus of CUMS Model Rats :(a)The CUMS model was replicated,and the antidepressant effects of Chaigui granules were verified by the indicators of body weight,Open field(escape from the central grid time,crossing number,number of erects)and sucrose preference rate.Hippocampal samples were obtained for subsequent experimental verification.(b)The mRNA levels of PI3 K,Akt,m TOR,Foxo3 a,GSK-3β,and β-catenin on the PI3K/Akt signaling pathway in hippocampus were detected by the method of Real Time qPCR;(c)The protein levels of Akt,m TOR,Foxo3 a and GSK-3β were detected by the method of Western blot.3.Integrated analysis of antidepressant mechanism of Chaigui granules: The methed of network pharmacology was used to integrated analyze the key targets of the PI3K/Akt signaling pathway and the results of the differential metabolites,neurotransmitters and the key targets of c AMP signaling pathways.Result:1.The results of Network pharmacology: The KEGG pathway analysis of the antidepressant target of Bupleurum suggests that the PI3K/Akt signaling pathway may play an important role in the antidepressant process of the main prescription Chaigui granules(Bupleurum).The KEGG Pathway analysis results of antidepressant targets for protein interaction of TOP20 targets,TOP1 module and Score≥4 modules based on MCODE algorithm,TOP1 module and Score≥4 modules based on MCL algorithm of Chaigui granules indicate that PI3K/Akt signaling pathway may play an important role in the antidepressant process of Chaigui granules.2.The results of Real Time qPCR: Compared with the blank group,the mRNA levels of PI3 K and Akt in the model group were significantly lower(P<0.05,P<0.01),which were significantly increased after the administration of Chaigui granules extract(P<0.05,P<0.05).The mRNA levels of m TOR and β-catenin were significantly decreased(P<0.01,P<0.01),which were significantly increased after the administration of Chaigui granules extract(P<0.01,P<0.01).The mRNA levels of GSK-3β and Foxo3 a in the model group were significantly increased(P<0.05,P<0.05),which were significantly decreased after the administration of Chaigui granules extract(P<0.01,P<0.01).The results showed that Chaigui granules can exert antidepressant effects by regulating the mRNA expression of key targets in PI3K/Akt signaling pathway.3.The results of Western blot: Compared with the blank group,the protein level of Akt in the hippocampus of the model group was significantly decreased(P<0.01),which was significantly increased after the administration of Chaigui granules extract(P<0.01).The protein level of m TOR in the model group was decreased,which was increased after the administration of Chaigui granules extract.The protein levels of GSK-3β and Foxo3 a in the model group were significantly increased(P<0.05,P<0.05),which were significantly decreased after the administration of Chaigui granules extract(P<0.01,P<0.05).The results showed that Chaigui granules can exert antidepressant effects by regulating the expression of key proteins in PI3K/Akt signaling pathway.4.The results of integrated analysis: Chaigui granules may exert its antidepressant effects by affecting glucose metabolism,lactic acid metabolism and choline metabolism through regulating PI3K/Akt signaling pathway.This regulation may be caused by the changes of enzymes such as HK1,HK2,G6 PC,PLD1,PLD2,and LDHA.Chaigui granules may exert antidepressant effects by affecting NE and 5-HT metabolism through regulating PI3K/Akt signaling pathway.This regulation may be caused by the changes of enzymes such as YARS,TH,BRPF1,TYR,and TDO2.Chaigui granules exert its antidepressant effects by regulating PI3K/Akt signaling pathway and c AMP signaling pathway,which may be mainly related by CREB and BDNF.Conclusion:1.Network pharmacology method predicts that Chaigui granules may exert its antidepressant effects by regulating PI3K/Akt signaling pathway.2.Molecular biology techniques have verified that Chaigui granules can regulate the mRNA levels of PI3K/Akt,m TOR,Foxo3 a,GSK-3β,β-catenin and the protein levels of Akt,m TOR,Foxo3 a and GSK-3β in PI3K/Akt signaling pathway to play its antidepressant effect.3.Integrated analysis indicated that Chaigui granules can exert its antidepressant effects at multiple levels by regulating metabolites,monoamine neurotransmitters and the key targets of signaling pathways. |
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