Exploring The Study Of Zuogui Pill And Yougui Pill In Differential Treatment Of Diminished Ovarian Reserve Based On Metabolomics And Network Pharmacology

Objective:Based on the research methods of metabolomics and network pharmacology,the metabolite basis and related metabolic pathways of Zuogui Pills and Yougui Pills in the treatment of diminished ovarian reserve by syndrome differentiation were systematically analyzed,and their therapeutic mechanisms were further explored,which provides a basis for TCM syndrome differentiation and treatment of diminished ovarian reserve.Methods:1.Patients with kidney-yin deficiency syndrome with diminished ovarian reserve were collected from outpatient clinics of multi-center hospitals and given Zuogui Pill for 12 weeks;patients with kidney-yang deficiency with diminished ovarian reserve were treated with Yougui Pill for 12 weeks.Based on non-targeted metabolomics research technology Serum metabolites were analyzed before and after the Zuogui Pills or Yougui Pills for the treatment of diminished ovarian reserve to find the potential biomarkers,and metabolic pathway analysis of differential biomarkers before and after treatment with traditional Chinese medicine.Pregnancy rate,Kupperman score,bFSH,bLH,bE2,AMH,TCM syndrome score were counted.2.The potential biomarkers screened by metabolomics will be further verified by network pharmacology,and the molecular mechanisms and signaling pathways of possible targets in pathogenesis and treatment may be revealed.The target of chemical components of Zuogui Pill and Yougui Pill was screened through the database.Then find the target of the disease in the database.Finally,the PPI network diagram is obtained.The.tsv format file containing the relationship information of the role nodes exported from String is imported into Cytoscape software,and visualized,and the network feature index(Degree)and Betweenness(Betweenness)of the target are topologically performed using Network Analyzer and CytoNCA Parametric analysis.The online analysis database Metascape database was used to carry out GO and KEGG enrichment and visualization analysis of key targets,and predict the targets of Zuogui Pill in the treatment of kidney-yin deficiency syndrome with diminished ovarian reserve and Yougui Pill in the treatment of diminished ovarian reserve with kidney-yang deficiency syndrome The main active components of the drug and the screened target proteins were verified by molecular docking using AutoDock Tools 1.5.6,and the results were visualized using PyMol software.Results:1.Twenty-three patients with kidney-yin deficiency syndrome with diminished ovarian reserve were collected from outpatient clinics in multi-center hospitals,and given Zuogui Pill for 12 weeks;25 patients with diminished ovarian reserve with kidney-yang deficiency syndrome were treated with Yougui Pill for 12 weeks.Zuogui Pill can adjust 26 differential metabolites,including L-carnitine,acetyl-CoA,coenzyme A,stearic acid oleamide,cholesterol linoleic acid,cortisol,coenzyme Q10(CoQ10),etc.,a total of 12 metabolic pathways were mapped to steroid hormone biosynthesis,fatty acid degradation,citric acid cycle,pantothenic acid and CoA biosynthesis,Fatty acid elongation rate,propionic acid metabolism,tryptophan metabolism,thiamine metabolism,biosynthesis of unsaturated fatty acids,glycolysis/gluconeogenesis;Yougui Pills recall lin oil in patients with diminished ovarian reserve and kidney-yang deficiency syndrome 22 differences including amide,adipyl-CoA,L-lysine,lysine arginine,y-glutamyl-β-cyanoalanine,threonyl tyrosine,α-tocopherol,etc.The metabolites were mapped to 11 metabolic pathways,unsaturated fatty acid biosynthesis,citric acid cycle,sphingolipid metabolism,propionate metabolism,glycolysis/gluconeogenesis,aminoacyl-tRNA biosynthesis,thiamine metabolism,biotin metabolism.After treatment,the change in bFSH and bLH value is statistically significant(P<0.05),but the bE2,AMH value is not.The TCM syndrome type score and Kupperman score of patients with kidney-yin deficiency syndrome and kidney-yang deficiency syndrome patients with decreased ovarian reserve function after treatment with traditional Chinese medicine were significantly lower than those before treatment(P<0.05),and the difference was statistically significant.There were 2 cases of pregnancy in the diminished ovarian reserve kidney yin deficiency group,with a pregnancy rate of 8.7%,and 1 case of pregnancy in the diminished ovarian reserve kidney yang deficiency group,with a pregnancy rate of 4%.2.143 active ingredients of Zuogui Pill were identified,and 448 targets were predicted;159 active ingredients of Yougui Pill were predicted,and 420 targets were predicted.There were 113 genes in diminished ovarian reserve syndrome of kidney yin deficiency and 147 genes in diminished ovarian reserve syndrome of kidney yang deficiency.Finally,39 targets of Zuogui Pill for diminished ovarian reserve kidney yin deficiency and 41 targets for Yougui Pill to treat diminished ovarian reserve kidney yang deficiency were obtained.Zuogui Pill acts on core targets such as TP53,VEGFA,and ALB through a variety of active ingredients,involving biological processes such as response to steroid hormones and hormone response;including cell membranes,membrane rafts,organelle outer membranes and other cellular components;Molecular functions such as steroid binding,protein kinase activity,lipid binding,ATPase binding,phosphotransferase activity;PI3K-Akt signaling pathway,HIF-1 signaling pathway,estrogen signaling pathway,cellular senescence,JAK-STAT signaling pathway,Signal pathways such as MAPK signaling pathway.Yougui Pill acts on core targets such as AKT1,TP53,and VEGFA through a variety of active components,involving biological processes such as reproductive system development,reproductive structure development,embryo-placental development,and positive regulation of cell migration;membrane rafts,plasma membrane rafts,Cell components such as mitochondrial outer membrane and endoplasmic reticulum cavity;molecular functions such as G protein-coupled receptor binding,protein serine/threonine/tyrosine kinase activity;PI3K-Akt signaling pathway,HIF-1 signaling pathway,MAPK signaling pathway,cell senescence,IL-17 signaling pathway,JAK-STAT signaling pathway,tumor necrosis factor signaling pathway and other signaling pathways.The key active ingredients of Zuogui Pill and the core target of kidney-yin deficiency syndrome with diminished ovarian reserve,and the key active ingredients of Yougui Pill and the core target of diminished ovarian reserve and kidney-yang deficiency syndrome were all less than 0,indicating that the two stable combination between them.Conclusion:In this study,metabolomics and network pharmacology were used to preliminarily find that Zuogui Pill and Yougui Pill recall serum metabolites in patients with DOR kidney-yin deficiency syndrome and DOR kidney-yang deficiency syndrome,and play the role of syndrome differentiation and treatment mainly through core targets and signaling pathways.This research method can provide new ideas for exploring the objective basis of TCM syndrome differentiation and treatment.