Study On The Mechanism Of Yang-xin-jie-yu Formula In Treating Adolescent Depression Based On The "Microbiome-gut-brain" Axis

The homeostasis of gut microbiota is closely related to the occurrence and development of Adolescent Major Depression（AMDD）.Intervention based on the "microbe gut brain" axis to target the onset of adolescent depression has become a new strategy for the treatment of adolescent depression.In clinical practice,our research team has found that the empirical formula Yang-Xin-Jie Yu Decoction（YXJYD）has a significant therapeutic effect on adolescent depression,and the appetite of adolescent depression patients significantly improved after taking YXJYD.Considering that the gut microbiota is closely related to the occurrence and development of depression,we performed chronic unpredictable mild stress（CUMS）induced depression model on young SD rats,take "Microbiota-Gut-Brain" axis（MGB）as the framework,and employed multi omics technology to study the mechanism of depression in adolescents,and based on the above results,explore the target and mechanism of YXJYD regulating the MGB in the treatment of adolescent depression.Firstly,UPLC-Q-TOF-MS technology was used to analyze the composition of YXJYD,identifying 23 chemical components,including DL-arginine,synephrine,magnolian alkaloid,naringin,quercetin,pogoslin V,icariin B,icariin C,icariin,genistein,icariin,schisandrin,schisandrin,hesperidin,shengchi-7-O-rutin,hyperin,isodendrobin,naringin-7-O-rutin Icariin II,icariin I,ginsenoside Rf,ginsenoside Rb1,and ginsenoside Rg3;Next,a adolescent depression model of rats induced by chronic unpredictable mild stress（CUMS）was constructed.Through behavioral experiments and determination of serum cortisol content,it was determined that YXJYD can significantly alleviate depression like behavior in the CUMS model rats（p<0.05）,and the effect is comparable to that of the fluoxetine treatment group;Subsequently,we used network pharmacology to construct the drug-component-target-pathway network for YXJYD,predicting that the mechanism of action of YXJYD mainly involves regulating mitochondrial energy metabolism pathway,GABA receptor pathway,glutamate receptor pathway,etc.In order to clarify the mechanism of YXJYD,we first used 16 S r RNA highthroughput sequencing technology and 1H NMR based metabonomics technology to analyze the effect of CUMS on the gut microbiota,host cecal contents,urine and serum metabolism of rats,and then used RNA seq technology to analyze the effect of CUMS on intestinal tissue and hippocampus,characterized the gut microbiota,metabolic network,intestinal gene expression,and hippocampus gene expression of adolescent depression rats,identify the gut microbiota,metabolites,metabolic pathways,and target genes closely related to the onset of adolescent depression,and in terms of the above multi-omics results to establish functional network of CUMS induced the occurrence and development of adolescent depression in young rats,and explore the antidepressant mechanism of YXJYD.The results showed that:（1）CUMS can lead to a decrease in gut microbiota diversity and significant changes in gut microbiota abundance.Six biomarker genus related to adolescent depression were identified,including Monoglobus,Lachnospiraceae_NK4A136_Group,g_<sub>norank_f<sub>norank_o<sub>Clostridia_UCG₀14,Bifidobacterium,Bacteroides,and Parabacteroides;YXJYD can significantly regulate disturbed gut microbiota that caused by CUMS,and up-regulated the abundance of Monolobus,Lachnospiraceae_NK4A136_Group,g_<sub>norank_f<sub>norank_o<sub>Clos tridia_UCG₀14 significantly,but has no effect on the abundance of Bifidobacterium;（2）Tax4Fun software was performed to predict the gut microbiota pathway,it was found that the tricarboxylic acid cycle,propionic acid metabolism pathway,and the α-Ketone isohexanoate,dehydrogenase,acetyl-coa-synthase,dihydrolipoate,dehydrogenase,l-lactate dehydrogenase,pyruvate dehydrogenase component α subunit,dihydrolipoamide dehydrogenase,pyruvate carboxylase,aconitic acid hydratase and pyruvate dehydrogenase E2 components were significantly related to the onset and development of adolescent depression;After administration of YXJYD,the expression abundance of pathways and enzymes was significantly rescued;（3）Metabolomics analysis found that CUMS caused two metabolites（L-isoleucine,succinic acid）in intestinal contents,nine metabolites（including taurine,dihydroxyacetone,L-malic acid,1-methylhistidine,pyroglutamic acid,dimethylglycine,2-octenoic acid,hippuric acid,and melatonin）in urine,and three metabolites（cysteine-S-sulfate,octanoic acid,and succinyl acetone）in the serum（| VIP |>1,Fold change>2,p<0.05）;After different doses of YXJYD were administered,the contents of L-isoleucine,succinic acid,taurine,dihydroxyacetone,L-malic acid,1-methylhistidine,hippuric acid,and dimethylglycine were significantly reduced,but the effect of YXJYD on 2-octenoic acid,pyroglutamic acid,and melatonin was not significant（p>0.05）;（4）The significant enrichment pathway of differentially expressed genes in intestinal tissue,the predictive pathway of gut microbiota function,and the interaction analysis of the results of differential metabolite enrichment pathway indicate that propionic acid metabolism and tricarboxylic acid cycle pathway are related to the occurrence of depression in young rats;However,no significant changes were found in the propionic acid metabolism pathway and tricarboxylic acid cycle pathway in the hippocampal tissue（FDR>0.05）;（5）The differentially expressed genes encoding malate dehydrogenase,2-oxoglutarate dehydrogenase,phosphoenolpyruvate carboxyl kinase,isocitric acid dehydrogenase,citrate synthase,malate dehydrogenase,2-oxoglutarate dehydrogenase,and aconitic acid hydratase in the tricarboxylic acid circulation pathway enriched by differentially expressed genes in the intestinal tissues of CUMS model rats were significantly differentially expressed,In propionic acid metabolic pathway,the genes encoding 3,2-trans-enoacyl-Co A isomerase,acyl coenzyme A oxidase,Llactate dehydrogenase,3-hydroxyisobutyryl-Co A hydrolase,and 4-aminobutyrate aminotransferase were significantly different in expression（p<0.05,| log2 FC | ≥ 1）;（6）Further interaction analysis revealed that CUMS could cause damage to the intestinal mucosal barrier function in rats（27 genes related to intestinal tight junction proteins were significantly differentially expressed）,mitochondrial energy metabolism abnormalities（174 genes related to energy metabolism were significantly differentially expressed）,and intestinal secretion peptide expression disorders（17 genes encoding secretion factors were significantly differentially expressed）.After YXJYD administration,The abnormal expression of genes has been significantly corrected.In summary,CUMS causes depression in young rats,which is related to its impact on gut microbiota,leading to disturbance in the host’s tricarboxylic acid cycle and propionic acid metabolism.YXJYD improves the abundance of Monog lobus,Lachnospiraceae_NK4A136_group,g_<sub>norank_f<sub>norank_o<sub>Clostridi a_UCG₀14,Bifidobacterium,Bacteroides,and Parabacteroides,affects the tricarboxylic acid cycle and propionate metabolic pathway,maintains the function albarrier of intestinal mucosal and mitochondrial energetic metabolism to play the antidepressant effect.In this way,we elucidate the occurrence and development of adolescent depression,the mechanism of YXJYD treatment for adolescent depression were explained from different aspects of "gut microbiota-host metabolism-host genes",providing a theoretical basis for clinical diagnosis and treatment of adolescent depression.