Comparative Researches On The Antidepressant Effects Of Xiaoyaosan And Its Efficacy Groups Based On Gut Microbiota And Fecal Metabolomics

Background:Xiaoyaosan(XYS),a classic and famous traditional Chinese medicine(TCM)prescription,has the functions of soothing the liver and strengthening the spleen according to TCM theory.The antidepressant effects of XYS have been confirmed both clinically and pharmacologically.At present,the researches on the antidepressant effects and mechanisms of XYS are mainly focused on the whole prescription.XYS contains eight herbs and its compatibility is complex.Disassembling a prescription is one of the effective approaches to study the effects and mechanisms of TCM prescriptions.It has been found that the antidepressant effects of XYS are closely related to the different therapeutic effects of individual herbs contained in XYS.Therefore,it is consistent with the composition principle of XYS to explain the antidepressant effects of XYS from the roles of different functions of herbs contained in XYS.However,the current studies on the antidepressant effects of XYS have been focused on the core mental symptoms of depression.Moreover,most of the studies just descriptively and qualitatively described the changes of depressive behaviors before and after the compatibility of herbs.The antidepressant effects of XYS and its disassembled groups need to be characterized in-depth and quantitatively from multiple perspectives and levels.Clinically,patients with depressive disorders are accompanied by significant physical symptoms,especially gastrointestinal symptoms.The disorder of gastrointestinal functions has great influences on the occurrence,development and prognosis of depression.In addition,gastrointestinal dysfunctions will lead to abnormal structures of gut microbiota,which will aggravate the patient’s depressive symptoms.However,it is still lacking to study the antidepressant effects of XYS from the perspective of depression-related gastrointestinal functions.Based on the above problems,in the present study,firstly,a depression model was applied by using the chronic unpredictable mild stress(CUMS)method.According to the efficacies of eight herbs contained in XYS,XYS was divided into two“Efficacy Compositions”groups,i.e.,the Shugan(SG)and the Jianpi(JP).And then,the antidepressant effects of XYS and its efficacy groups were analyzed and compared from the aspects of depressive behaviors,gastrointestinal functions,microbiome,and metabolomics.The results of this study will provide a new perspective for understanding the different therapies of XYS from the perspective of improving gastrointestinal functions and will provide new ideas and approaches to study the compatibility of other TCM prescriptions with anti-depressant effects.This study will also offer a theoretical and experimental basis for the selections of corresponding therapies for depression in the clinic.Objective:To clarify the contribution of different efficacy groups to Xiaoyaosan in regulating depression-related gastrointestinal functions based on the analysis of intestinal microflora and fecal metabolomics.Methods:1.After one-week adaptation,all rats were randomly assigned into six groups(n=6):the negative control group(NC),the CUMS model group(MS),the venlafaxine group(PC),the Xiaoyaosan group(XYS),the Shugan group(SG),and the Jianpi group(JP).The CUMS procedure was performed on MS and all the treatments.The antidepressant effects of PC,XYS,SG,and JP on the depressed rats were analyzed by determining the indexes of body weight,sucrose preference rate,crossing numbers,rearing numbers in the OFT,and immobility time in the FST.We integratively compared the effects of PC,XYS,SG,and JP on behaviors of CUMS depressed rats.2.The effects of PC,XYS,SG,and JP on the gastrointestinal functions of CUMS model rats were analyzed by measuring the gastric residual rates,intestinal propulsive rates,the levels of gastrointestinal hormones including GAS,MTL and SS and digestive enzymes includingα-amylase,trypsin and cellulase.We integratively compared the effects of PC,XYS,SG,and JP on gastrointestinal functions of CUMS depressed rats.3.The 16S r RNA analysis of cecum samples was performed.The effects of XYS,SG,and JP on the cecum microbiota of CUMS model rats were evaluated by species composition,α-diversity,andβ-diversity.Potential microbial biomarkers associated with depression at the phylum,family,and genus levels were further screened.The differences in the number and callback degrees of potential microbial markers(EI)regulated by XYS,SG,and JP were compared.4.Metabolomics analysis was performed using the ~1H-NMR technique and potential biomarkers of feces for CUMS were screened.The shared and differential regulatory effects of PC,XYS,SG,and JP were demonstrated from the perspective of metabolite levels,including their effects on metabolic profiles,potential biomarkers,and corresponding metabolic pathways.The differences in the callback degrees of potential microbial markers(EI)in feces regulated by XYS,SG,and JP were compared.Results:1.The results of depressive behavior indexes:After 28-day CUMS modeling,as compared with NC,the body weights,the sucrose preference rates,the crossing numbers,and the rearing numbers of the CUMS model rats were significantly decreased(P<0.01)while the immobility time was significantly increased(P<0.01),indicating a successful CUMS model.The weights and the sucrose preference rates significant increased(P<0.01)in XYS as compared with MS.XYS had no significant effects on crossing numbers,and rearing numbers of MS rats(P>0.05).Compared with MS,SG significantly increased the sucrose preference rates,the body weights,the crossing numbers,and the rearing numbers(P<0.05,P<0.01).JP significantly increased the weights,the crossing numbers and the rearing numbers(P<0.01),but could not reverse the sucrose preference rates(P>0.05).All the treatments notably shortened the immobility time of MS rats(P<0.01,P<0.05).The integrated scores of depressive behavior indexes of all the groups were ranked as follows:NC(0.95)>PC(0.58)>SG(0.52)>XYS(0.13)>JP(-0.36)>MS(-1.83).2.The results of gastrointestinal functions indexes:Compared with NC,the gastric residual rates,and the activity ofα-amylase of MS significantly decreased(P<0.01),while the intestinal propulsive rates and the levels of GAS,MTL,and SS of MS significant increased(P<0.01),suggesting that CUMS depressed rats showed gastrointestinal dysfunctions.All the treatments remarkable increased the gastric residual rate and reduced the intestinal propulsive rates and the levels of GAS and SS as compared with MS(P<0.01).Only PC(P<0.05)could significantly decrease the level of MTL as compared with MS.Compared with MS,the level ofα-amylase was significantly increased in SG(P<0.01).There were no significant differences in the activities of trypsin and cellulase among the six groups.The integrated scores of gastrointestinal functions indexes of all the groups were ranked as follows:NC(-1.07)<PC(-0.43)<SG(-0.22)<XYS(0.17)<JP(0.61)<MS(0.94).3.The results of gut microbiota analysis:the results of theα-diversity analysis showed that there was no significant difference in the abundance and diversity of microbial communities among NC,MS,XYS,SG,and JP.The results ofβ-diversity showed that there were significant differences in the community distribution between MS and NC.There were significant differences in the community distribution among the five groups(P<0.01).The microbiota composition of SG was the most similar to that of NC,followed by XYS,and the last was JP.At phylum,family,and genus levels,a total of 31 potential microbial biomarkers associated with CUMS were identified.20 biomarkers were significantly reversed by SG while 16 and 11 biomarkers were reversed by XYS and JP,respectively.The EI on the potential microbial biomarkers at the phylum level of XYS,SG,and JP was 196.35,172.19,and 72.81,respectively.At the family level,the EI of XYS,SG,and JP was 414.27,620.45,and 309.85 respectively.At the genus level,the EI of XYS,SG,and JP was 770.38,976.45,and 499.62,respectively.4.The results of fecal metabolomics analysis:multivariate statistical analysis showed that the metabolic profile of MS was significantly changed compared with that of NC.Ten metabolites were identified as potential biomarkers for the CUMS-induced depression.Eight potential biomarkers were significantly reversed by XYS while 5 and 4 biomarkers were reversed by SG and JP,respectively.The EI of XYS,SG,and JP on metabolites are ranked as follows:XYS(900.00)>SG(479.50)>JP(371.63).Concerning the corresponding metabolic pathways,XYS significantly regulated all the 7 metabolic pathways associated with the CUMS,while SG and JP regulated 6 and 3 pathways,respectively.Conclusions:This study investigated and compared the antidepressant effects of XYS and its efficacy groups i.e.,SG and JP,from the aspects of behaviors,gastrointestinal functions,gut microbiome,and fecal metabolomics.Based on the results of this study,for the pharmacological effects of each treatment on depression-related behaviors,gastrointestinal dysfunctions,and gut microbiota disorders,SG was similar to XYS,and both of them were superior to JP.For the pharmacological effects of depression-related fecal metabolites,XYS was better than SG and JP.