Exploring the mechanism of Wuzi Yanzong Pill in treating menopausal osteoporosis based on bioinformatics and network pharmacology
Authors: Qiu Jingjing , Zhao Yu , Deng Zilan , Huang Juxiu , Zhang Yanlin , Zhao Beibei , Chen Lihua 
Published: September 2023

Abstract
Objective: To explore the pathogenesis of menopausal osteoporosis (MOP) based on bioinformatics. Furthermore, to explore the therapeutic mechanism of Wuzi Yanzong Pill(WZYZP) against MOP using network pharmacology and molecular docking.
Methods: The GEO datasets were applied to determine the differentially expressed genes (DEGs) between MOP and the controls. The Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP) database was used to obtain the main active components and corresponding targets penetrating the blood-brain barrier(BBB) in WZYZP. The targets related to MOP were obtained by GeneCards, Online Mendelian Inheritance in Man (OMIM), Drugbank, the Comparative Toxicogenomics Database (CTD), Therapeutic Target Database (TTD), and DisGeNET databases. The potential targets of WZYZP for the intervention of MOP were obtained by intersection with compound targets. Cytoscape software was used to construct the network diagram of WZYZP in the treatment of MOP. The protein-protein interaction (PPI) network was performed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The core targets were screened according to the node degree value. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of core targets were finished by Database for Annotation, Visualization, and Integrated Discovery (David) database platform, and molecular docking analysis was performed by AutoDock Vina software. The DEGs intersected with the hub genes of WZYZP against MOP.
Furthermore, the intersected hub genes were used to predict miRNAs in the Starbase and miRwalk database. The intersection was obtained by the mRNA-miRNA relationship. The intersected hub genes were placed in the AnimalTFdb and JASPAR to predict transcription factors (TFs) and took the intersection of mRNA-TFs. Cytoscape was used to draw the network diagram of mRNA-miRNA and mRNA-TFs relationship pairs.
Results: A total of 620 DEGs in GSE100609 were obtained. The top four genes (TP53, SMAD2, STAT1, CXCL8) were screened out as core genes. A total of 59 main active ingredients in WZYZP were screened, and the core active ingredients were quercetin, kaempferol, β-sitosterol, isorhamnetin, and glycitein. There were 131 target genes, including the top five hub genes: TP35, JUN, RELA, AKT1, and MAPK1. The biological process obtained from GO enrichment was mainly related to the regulation of osteoclast differentiation. The enrichment results of KEGG suggested that the effect of WZYZP in the treatment of MOP was focused on the signaling pathways such as estrogen, toll-like receptor, TNF, and osteoclast differentiation. A total of 4 intersections between DEGs and hub genes: TP53, CXCL8, SELE, and STAT1. The network diagram of mRNA-miRNA and mRNA-TFs. comprises 4 mRNAs, 47 TFs, and 66 miRNAs. Through the topology analysis of the network, four transcription factors (SPIB, MAX, NR2F1 and ELK1) significantly affected the expression of hsa-miR-19b-3p and hsa-miR-1294.
Conclusion: TP53, SMAD2, STAT1, and CXCL8 genes are of great value in the early diagnosis of MOP. The active ingredients of WZYZP may affect the expression of hsa-miR-19b-3p and hsa-miR-1294 by regulating four transcription factors (SPIB, MAX, NR2F1 and ELK1). These miRNAs may further regulate the expression of genes such as TP53, CXCL8 and STAT1.
Ethics and dissemination:
This study dose not require ethical approval of an ethical committee because it will not show personal information and will not infringe the privacy of the participants.