Study On The Enhanced Antidepressant Effects And Synergistic Mechanisms Of Chaihu-baishao Herb Pair And The Combination Of Its Ingredients

Background:Traditional Chinese medicine(TCM)prescriptions possess the characteristics of complex prescription(sovereign,minister,assistant,guide)and diverse action rules(xiang xu,xiang shi,xiang wu),which can achieve satisfactory curative effects without serious side effects,that reflects the therapeutic advantages of TCM.However,how to interpret the scientific connotation of the compatibility of TCM and use the common language of the scientific community to characterize the rationality of the compatibility of TCM is a major challenge.The herb pair is a fixed combination of two or three herbs under the guidance of TCM theory.Compared with the TCM prescriptions,the herb pair with a simplified composition,and it is easy to clarify the medicinal substances and the mechanism.As the most common form of the TCM compatibility application,herb pair as well as a small prescription,which can reflect the special rules and internal connections of TCM prescriptions.The Bupleurum chinense DC(Chaihu)-Paeonia lactiflora Pall(Baishao)herb pair is the basic medicine of many famous antidepressant prescriptions such as Xiaoyao San,Chaihushugan San,Sini San,and so on.Chaihu could soothe the live and relieve depression;Baishao could nourish blood and soften the liver,the two herbs were combined to form the core herb pair for regulating emotions.Modern pharmacological research showed that this herb pair possessed a definite antidepressant effect.However,whether Chaihu-Baishao herb pair compatibility could enhance the antidepressant effect? What is the synergistic mechanism on enhanced antidepressant effect? Whether the combination of this herb pair key ingredients possess a synergistic antidepressant effect? What is the synergistic antidepressant mechanism? Those questions remain unclear.Aims:(1)To clarify whether Chaihu-Baishao herb pair compatibility possesses enhanced antidepressant effect;to systematically clarify the synergistic mechanism of ChaihuBaishao herb pair compatibility on enhanced antidepressant effect.(2)To screen the key antidepressant active ingredients of Chaihu-Baishao herb pair,and clarify whether the combination of key ingredients possesses the synergistic antidepressant effect;to systematically clarify the synergistic antidepressant mechanism of the compatibility of the key ingredients.Methods:(1)By network pharmacology methods to predict the antidepressant ingredients,antidepressant targets,and antidepressant pathways of Chaihu-Baishao herb pair.Constructing an ingredient-target-pathway network and conducting network topology analysis to find the regulation targets and pathways of Chaihu,Baishao,and ChaihuBaishao herb pair;revealing the targets and pathways co-regulation by Chaihu and Baishao,as well as the targets and pathways were regulated independently by Chaihu and Baishao.From the perspective of the network pharmacology clarifying the synergistic mechanism of Chaihu-Baishao herb pair compatibility on antidepressant effect.In addition,the key antidepressant ingredients of Chaihu-Baishao were screen by the analysis of the ingredient contribution index.(2)By chronic unpredictable mild stress,rat body weight and behavior(sugar water preference,open field crossing grid number,forced swimming immobility time)as indicators to evaluate and compare the effect of Chaihu,Baishao,and Chaihu-Baishao herb pair on the depression symptoms of rats at the same dosage of low and high respectively,and evaluating the enhanced antidepressant effect of Chaihu-Baishao herb pair.Plasma,peripheral blood mononuclear cells(PBMCs),and hippocampus as samples;by LC-MS metabolomics analysis technology to find the differential metabolites and metabolic pathways regulated by Chaihu,Baishao,and ChaihuBaishao herb pair;reveal the differential metabolites and metabolic pathways coregulated by Chaihu and Baishao,as well as the differential metabolites and metabolic pathways were regulated independently by Chaihu and Baishao.From the perspective of metabolomics clarifying the synergistic mechanism of Chaihu-Baishao herb pair on enhanced antidepressant effect.Correlation analysis of the results of network pharmacology and metabolomics,and combining the number of differential metabolites contained in metabolic pathways to screen main metabolic pathways.The main metabolic pathways and related signaling pathways were verified by western blotting and enzyme-linked immunosorbent assay to clarify the key synergistic mechanism of Chaihu-Baishao herb pair on enhanced antidepressant effect.(3)By corticosterone-induced injury PC12 cell model,the cell viability rate as an indicator to screen the antidepressant ingredients of Chaihu-Baishao herb pair predicted by the network pharmacology,and select the key antidepressant active ingredients of Chaihu and Baishao respectively.Three synergistic evaluation mathematical models of Chou-Talalay,Loewe,and HAS were used to evaluate the synergistic antidepressant effects of key ingredients combination.PC12 cells as samples,by LC-MS metabolomics analysis technology,to find the differential metabolites and metabolic pathways regulated by single ingredients and ingredients combination;reveal the differential metabolites and metabolic pathways co-regulated by the two ingredients,as well as the differential metabolites and metabolic pathways were regulated independently by single ingredients.From the perspective of metabolomics clarifying the synergistic mechanism of ingredients combination on synergistic antidepressant effect.Correlation analysis of the results of network pharmacology and metabolomics,and combining the number of differential metabolites contained in metabolic pathways to screen main metabolic pathways.The main metabolic pathways and related signaling pathways were verified by western blotting and enzyme-linked immunosorbent assay to clarify the key synergistic mechanism of ingredients combination on synergistic antidepressant effect.Results:(1)The network pharmacology prediction results shown: there were 19 antidepressant ingredients of Chaihu,which could regulate 179 depression targets and20 pathways,and there were 11 antidepressant ingredients of Baishao,which could regulate 155 depression targets and 20 pathways.The ingredient-target-pathway network topology analysis result shown: there were 135 targets and 17 pathways coregulated by Chaihu and Baishao;Chaihu could regulate 44 targets and 3 pathways and Baishao could regulate 20 targets and 3 pathways independently,which reflect the Chaihu-Baishao herb pair with the synergistic mechanism of multi-ingredient-multitarget-multi-mechanism.The ingredients contribution index screening result shown:there were three key antidepressant ingredients of Chaihu(saikosaponin A,quercetin,and isorhamnetin),and two key antidepressant ingredients of Baishao(paeoniflorin,albiflorin).(2)The evaluation results of the antidepressant effect on the depressed rat model shown: at the same low and high doses of 7.5 g/kg,15 g/kg Chaihu-Baishao herb pair compatibility could enhance the antidepressant effect.The metabolomics results showed: in plasma,Chaihu could regulate 10 differential metabolites and 4 metabolic pathways,Baishao could regulate 9 differential metabolites and 4 metabolic pathways,after compatibility,Chaihu-Baishao herb pair could regulate 14 differential metabolites and 5 metabolic pathways;in PBMCs,Chaihu could regulate 8 differential metabolites and 6 metabolic pathways,and Baishao could regulate 9 differential metabolites and 5metabolic pathways,after compatibility,Chaihu-Baishao herb pair could regulate 11 differential metabolites and 8 metabolic pathways;in the hippocampus,Chaihu could regulate 12 differential metabolites and 6 metabolic pathways,and Baishao could regulate 10 differential metabolites and 9 metabolic pathways,after compatibility,Chaihu-Baishao herb pair could regulate 16 differential metabolites and 11 metabolism pathways.In the three samples,the number of differential metabolites and metabolic pathways regulated by the herb pair compatibility was more than that of the single herb.The key metabolic pathway screening results showed: regulation of arachidonic acid metabolism,tryptophan metabolism,and purine metabolism were the main synergistic mechanisms of Chaihu-Baishao herb pair on enhanced antidepressant effect.Experimental verification results showed: regulation of arachidonic acid metabolism,purine metabolism,MAPK/NF-?B/NLRP3 signaling pathway were the key synergistic mechanisms of Chaihu-Baishao herb pair on enhanced antidepressant effect.(3)The evaluation results of the antidepressant effect on the cell model shown:saikosaponin A and albiflorin were the key antidepressant active ingredients of ChaihuBaishao respectively.The synergy evaluation results showed: the compatibility of 2.5?mol/L saikosaponin A and 25 ?mol/L albiflorin possessed the best synergistic antidepressant effect;the compatibility of 1.25-2.5 ?mol/L saikosaponin A and 12.50-25 ?mol/L albiflorin was a synergistic antidepressant dose range.The metabolomics results shown: in PC12 cells,saikosaponin A could regulate 12 differential metabolites and 3 metabolic pathways;albiflorin could regulate 10 differential metabolites and 5metabolic pathways,after compatibility,saikosaponin A-albiflorin ingredients compatibility could regulate 16 differential metabolites and 7 metabolic pathways.The number of differential metabolites and metabolic pathways regulated by the ingredient compatibility was more than that of the single ingredients.The key metabolic pathway screening results showed: regulation of tryptophan metabolism,purine metabolism,TCA cycle,glutamate metabolism were the main synergistic mechanisms of saikosaponin A-albiflorin ingredients compatibility on synergistic antidepressant effect.Experimental verification results showed: regulation of tryptophan metabolism,purine metabolism,TCA cycle,glutamate metabolism,MAPK/NF-?B/NLRP3 signaling pathway was the key synergistic mechanism of saikosaponin A-albiflorin ingredient pair on synergistic antidepressant effect.Conclusions:(1)Chaihu-Baishao herb pair compatibility and its key antidepressant ingredients saikosaponin A-albiflorin compatibility could enhance antidepressant effects.(2)Chaihu-Baishao herb pair compatibility and its key antidepressant ingredients saikosaponin A-albiflorin compatibility possessed the synergistic mechanism of multiingredient-multi-target-multi-mechanism,the regulation of tryptophan metabolism,purine metabolism,TCA cycle,glutamate metabolism,MAPK/NF?B/NLRP3 signaling pathway were the key synergistic mechanisms.