Study On The Compatibility Mechanism Of Radix Bupleuri-Radix Paeoniae Alba Antidepressant Effect Based On Cerebral Cortical Purine Metabolism In CUMS Model Rats

Rationales:As an important part of compound Chinese herbal medicine,herb pair is the bridge between single medicine and compound prescription.The key to the effect of herb pair lies in its precise compatibility.Reasonable compatibility can reduce toxicity and increase efficacy.Therefore,it is of great significance to study the compatibility mechanisms of the herb pair.Radix Bupleuri,Xinsan,Shugan jieyu,and Radix Paeoniae Alba,Suanlian,Yangxue rougan,they are central herbs of tiaochang qingzhi prescriptions,which was used in multiple antidepressant prescriptions,such as Xiaoyao San Chaihu Shugan San,Sini San,etc.In the early stage,we have conducted research on the contribution of Radix Bupleuri-Radix Paeoniae Alba herb pair to the Xiaoyao San,the representative prescription of Shugan Jieyu,changes of chemical compositions before and after compatibility and the pharmacokinetic mechanisms of the compatibility,but the compatibility mechanisms of its antidepressant pharmacological effects was still unclear.Therefore,in this study,a multi-technology platform of behavior,metabolomics,and molecular biology were used to compare the effects of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility,so as to clarify the compatibility mechanism of antidepressantObjective:1.To evaluate the antidepressant synergistic effects of the compatibility of Radix Bupleuri-Radix Paeoniae Alba by the chronic unpredictable mild stress（CUMS）rats model.2.Based on the non-targeted metabolomics research to compare the regulatory effects of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility on the cortical metabolic profile of CUMS rats and to focus on the key metabolic pathways that the herb pair regulates the cortex of depressed rats.3.Based on the results of targeted quantitative research,to analyze the regulatory effects of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility on the metabolites in the cortical purine metabolism pathway of CUMS rats.4.To study the effects of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility on purine metabolism-related enzymes by molecular biological means,and to analyze its effects on the expression of NOD-like receptor protein 3（NLRP3）inflammasome-related proteins and inflammatory factors in the inflammatory signaling pathway.Methods:1.First,the rats were randomly divided into 9 groups:the control group（C,Control）,the model group（M,Model）,the positive drug（Venlafaxine）group(P,Positive,35 mg·kg^-1).low-dose and high-dose of Radix Bupleuri group(CL,CH crude drug amount 7.5,15 g·kg^-1),low-dose and high-dose of Radix Paeoniae Alba group(BL,BH crude drug amount 7.5,15 g·kg^-1),Low-dose and high-dose of Radix Bupleuri-Radix Paeoniae Alba compatibility group(CBL,CBH crude drug amount 7.5,15 g·kg^-1),CUMS modeling and administration lasted for 28 days,and through the traditional pharmacodynamic indicators of depression（weight,open field test,sucrose preference test,and forced swimming test）to evaluate the efficacy of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility.2.The rats’cortex samples of control group（C,Control）,model group（M,Model）,Radix Bupleuri low-dose group(CL,crude drug amount 7.5 g·kg^-1),Radix Paeoniae Alba low-dose group(BL,crude drug amount 7.5 g·kg^-1)and Radix Bupleuri-Radix Paeoniae Alba compatibility group(CBL,crude drug amount 7.5 g·kg^-1)were collected,UPLC-MS/MS non-targeted metabolomics was used to screen the endogenous metabolites in the cortex of CUMS rats that can be adjusted after the compatibility of Radix Bupleuri-Radix Paeoniae Alba.The Metabo Analyst webpage was used to find the key metabolic pathways where the herb pair were effective.3.On the basis of the key metabolic pathways screened by non-targeted metabolomics,the contents of 11 metabolites（AMP,IMP,GMP,adenine,hypoxanthine,xanthine,guanine,adenosine,inosine,xanthosine,guanosine）of purine metabolism in cortex of depressed rats were analyzed by quantitative analysis,and to clarify the regulatory effects of Radix Bupleuri-Radix Paeoniae Alba before and after compatibility.4.Enzyme linked immunosorbent assay（ELISA）kit were used to compare the regulatory effects of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility on the the levels of xanthine,xanthine oxidase（XOD）,xanthine dehydrogenase（XDH）in purine metabolism and inflammatory factors interleukin-1β（IL-1β）,interleukin-6（IL-6）,tumor necrosis factor（TNF-α）.Western blot were used to compare the influences of Radix Bupleuri-Radix Paeoniae Alba before and after the compatibility on the expression of inflammation-related proteins,including NLRP3,caspase-1,IL-1βand IkB-α.Results:1.The CUMS rats behavior results showed that compared with the control group,the model group rats’body weight,sucrose preference rate,number of crossing and rearing reduced on the 28th day（P<0.001）,whereas the immobility time of forced swimming increased（P<0.001）.Compared with the model group,low-dose compatibility group of the Radix Bupleuri-Radix Paeoniae Alba can significantly reverse the weight of CUMS rats（P<0.001）,sucrose preference rate（P<0.01）,the number of crossing and rearing in the open field test（P<0.001）,and forced swimming immobility time（P<0.001）.The effects of the low-dose compatibility group of Radix Bupleuri and Radix Paeoniae Alba were stronger than the low-dose single-administered group;high-dose compatibility group of the Radix Bupleuri-Radix Paeoniae Alba group can significantly improve the body weight of CUMS rats（P<0.001）,the number of crossing（P<0.001）and rearing（P<0.05）,the immobility time of forced swimming（P<0.01）,and the regulatory effects were stronger than the high-dose single-administered group.2.22 different metabolites related to depression were screened between the control group and the model group by the non-targeted metabolomics.Compared with the control group,there are 12 elevated metabolites and 10decreased metabolites in the cortex of the model group.Among them,the Radix Bupleuri single-administered group can significantly adjust 13 species,the Radix Paeoniae Alba single-administered group can significantly adjust 14species,and the compatibility of Radix Bupleuri-Radix Paeoniae Alba in the same dose can adjust 16 species.Among the 7 metabolic pathways affected by CUMS,purine metabolism was most related to depression.Six purine metabolites were disordered in depressed rats,and three of them can be significantly regulated after the compatibility of Radix Bupleuri-Radix Paeoniae Alba3.In the targeted metabolomics analysis of purine metabolism in rat cortex,compared with the control group,there are 11 purine metabolites had significant differences in in the CUMS model group.Among them,the elevated metabolites were:IMP,adenine,xanthine,and guanine;the decreased metabolites were:AMP,GMP,adenosine,inosine,xanthosine,guanosine,and hypoxanthine.After Radix Bupleuri was administered,only IMP reduced significantly（P<0.01）;after administration of Radix Paeoniae Alba,inosine increased significantly（P<0.01）and IMP decreased significantly（P<0.01）,After the compatibility administration of Radix Bupleuri-Radix Paeoniae Alba,the contents of inosine（P<0.01）and adenosine（P<0.01）increased significantly,and it can significantly reduce the contents of IMP（P<0.001）,adenine（P<0.01）,xanthine（P<0.01）and guanine（P<0.001）.The reversed effects of Radix Bupleuri-Radix Paeoniae Alba compatibility were better than single-administration.4.After CUMS modeling,the contents of xanthine（P<0.001）and XOD（P<0.05）in rats cortex increased significantly,the content of XDH（P<0.001）decreased significantly,and the protein expressions of NLRP3,IL-1β,caspase-1 and IκB-αin rats cortex increased（P<0.05）,accompanied by the levels of inflammatory factors IL-1β,IL-6 and TNF-αincreased significantly（P<0.001）.Compared with the model group,the Radix Bupleuri-Radix Paeoniae Alba compatibility group can significantly reduce the contents of xanthine（P<0.001）,XOD（P<0.01）,increase the content of XDH（P<0.001）,and the regulatory degree was better than single administration.Regarding the regulation of inflammatory signal pathway related proteins and inflammatory factors,the Radix Bupleuri-Radix Paeoniae Alba compatibility group can significantly inhibit the protein expression of NLRP3（P<0.05）,IL-1β（P<0.05）,caspase-1（P<0.01）and IκB-α（P<0.01）in the cortex of depressed rats.And it can significantly reduce the levels of IL-1β,IL-6 and TNF-α（P<0.001）.The reversed degree of Radix Bupleuri-Radix Paeoniae Alba compatibility was stronger than the single-administration.Conclusion:In the behavioral field,the efficacy of the Radix Bupleuri-Radix Paeoniae Alba high and low-dose compatibility group were better than the equal-dose Radix Bupleuri and Radix Paeoniae Alba single-administered group,and the low-dose compatibility group was better than the high-dose compatibility group.The results of metabolomics studies suggested that in the process of depression,a variety of metabolites of purine metabolism were disordered in the rat cortex.The compatibility of Radix Bupleuri-Radix Paeoniae Alba had a significant regulatory effect on purine metabolism,and the reversed degree was stronger than the single-administration.In addition,the compatibility of Radix Bupleuri-Radix Paeoniae Alba can regulate the content of key enzymes in purine metabolism,inhibit the activation of NLRP3inflammasomes and the occurrence of inflammation.The improvement effects were better than the single-administered group.In summary,the synergistic antidepressant effect of the compatibility of Radix Bupleuri-Radix Paeoniae Alba was related to the regulation of purine metabolism and the improvement of inflammation.