| Objective:Depression is a common clinical psychiatric disorder that seriously threatens human health.Yulangsan is an ethnic medicine practiced in Zhuang and Yao medicine in Guangxi that has not been fully exploited.Yulangsan has various pharmacological effects.Studies have confirmed that the antidepressant effect of its main active component Yulangsan Polysaccharide(YLSPS)is clear,but the underlying mechanism anti-depression of YLSPS is still pooly unknown.Methods:Replication of a chronic unpredictable stress(CUMS)depression rat model to validate the antidepressant effect of YLSPS.A non-targeted metabolomics approach was established by UPLC-Q-TOF/MS technology to examine the regulatory effects of YLSPS on urinary metabolic profiles and metabolites in depressed rats.Reverse transcription-polymerase chain reaction(RT-PCR)was used to investigate the effect of YLSPS on the genes related to tryptophan-kynurenine(TRP-KYN)metabolic pathway in depressed rats’brain.A plasma TRP-KYN targeted metabolomics method was established based on UFLC-Q-Trap/MS technology to study the regulatory effects of YLSPS on the main metabolites tryptophan(TRP),kynurenine(KYN),5-Hydroxyindole-3-aceticacid(5-HIAA),kynurenine acid(KYNA),5-Hydroxytryptamine(5-HT)in this pathway.16S r RNA sequencing technology was applied to study the effect of YLSPS on the gut microbiota structure of depressed rats.The spearman correlation analysis method was used to screen out the"depression phenotype variables～TRP-KYN metabolism～depression-related gut bacteria"correlation pairs.Results:The results of pharmacodynamics showed that both the low and high dose groups of YLSPS could significantly improve the depression-induced slow weight gain,decreased curiosity and exploration ability,and loss of pleasure in depressed rats.Urinary untargeted metabolomics results showed that YLSPS significantly ameliorated depression-induced alterations in the urinary metabolic profile of depressed rats,modulating 13 urinary metabolites such as KYN,with the main metabolic pathway regulated being TRP-KYN.RT-PCR results revealed that the expression of enzyme genes related to TRP-KYN metabolic pathway was disturbed in depressed rat brain tissue,and YLSPS could significantly regulate the expression of these enzyme genes and bring them back close to the normal group.The established TRP-KYN targeted metabolomics method complied with the pharmacopoeia,where the linearity(r~2>0.9900),intra-day precision RSD ranged from 1.66%to 8.80%,24 h stability RSD of samples and standards ranged from 1.51%to 9.84%,spiked recoveries ranged from 85%to 114.25%,and RSD values ranged from 1.68%to 14.45%.The results showed that the contents of TRP,KYN,5-HT,5-HIAA and KYNA in plasma in the model group were significantly lower than those in the blank group,and the contents of TRP,KYN,5-HIAA and KYNA were significantly increased after treatment with YLSPS.The results of gut microbiota sequencing showed that the composition and structure of fecal gut microbiota of depressed rats changed significantly at the phylum,family,genus and species levels,and on the phylum level,the abundance of Patescibacteria was significantly decreased in the model group compared with the blank group(p<0.01),while the Bacteroidota and Firmicutes both showed an increasing trend but no statistical difference;on the genus level,compared with the blank group,17 different genera were found in the model group,of which the YLSPS could adjust the abundance of 12 genera,such as Candidatus-Ssccharimonas、Ruminococcus、Romboutsia、Negativibacillus、Allobaculum、Roseburia,et al.The results of Spearman correlation analysis showed that there were 3 pairs of strong correlation(r>|0.75|)for“tryptophan and its metabolites～intestinal bacteria”,which were Romboutsia～5-HT(r=-0.83),Turicibacter～5-HT(r=-0.76)and Negativibacillus～KYNA(r=0.75).5 pairs of"biochemical index～intestinal bacteria genus"(r>|0.7|)were found,which were Dubosiella～5-HT(r=0.77),Negativibacillus～TNF-α(r=0.76),Paludicola/Ruminococcus～IL-18(r=0.75,r=0.72),Roseburia～TNF-α(r=-0.73)and Eubacterium-siraeum-group～IL-1β(r=-0.77).Conclusion:YLSPS may exert antidepressant effects by regulating the composition of gut microbiota,increasing the abundance of beneficial bacteria and decreasing the proportion of harmful bacteria,and by regulating enzymes such as TDO,KMO,KYNU and metabolites such as TRP,KYN,5-HIAA and KYNA in the tryptophan-kynurenine metabolic pathway. |
