A network pharmacology approach and validation experiments to investigate the mechanism of the Chinese medicine Wen-Dan Decoction in the treatment of steroid-induced femoral head necrosis

Abstract

Objective: The present work studied the mechanism of action of Wen-Dan Decoction in treating steroid-induced necrosis of the femoral head (SINFH) via network pharmacology and in vivo experiments.Methods: The microarray dataset GSE123568 (peripheral blood), which includes 30 SINFH patients and 10 non-SINFH samples (after using steroids), is included in the Gene Expression Omnibus (GEO) database. Differential expression analysis and weighted gene coexpression network analysis (WGCNA) were used to identify key SINFH-related genes. To investigate the functions of key SINFH-related genes, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed. We constructed a protein–protein interaction (PPI) network based on the STRING database. In addition, we screened active components in Wen-Dan Decoction and their possible role in treating hormonal osteonecrosis using the Traditional Chinese Medicine System Pharmacology (TCMSP) Database. Subsequently, the potential mechanisms of action of Wen-Dan Decoction in the treatment of SINFH obtained via network pharmacology analyses were validated in in vivo experiments.Results: A total of 608 DEGs were obtained (230 upregulated, 378 downregulated). In GO functional enrichment analysis, SINFH-related genes were mainly involved in neutrophil activation and immune response, and KEGG pathway analysis revealed that SINFH genes were mainly associated with cytokine receptor interactions, lipids and atherosclerosis, and tuberculosis. In addition, a total of 147 active ingredients were obtained for Wen-Dan Decoction, of which the core ingredient was quercetin, an active ingredient in licorice. Meanwhile, 277 target genes were identified for the treatment of SINFH with Wen-Dan Decoction, and NCF1, PTGS2, and RUNX2 were selected as core target genes. Furthermore, the results of quantitative real-time polymerase chain reaction (qRT–PCR) on peripheral blood and bone tissue samples from SINFH patients and non-SINFH samples showed that PGTS2 and NCF1 were upregulated in patient blood and necrotic bone tissues. In quercetin-treated mice, the appearance of femoral head necrosis was significantly improved, and micro-CT suggested that the bone density was significantly better. Finally, the results of qRT–PCR analysis of peripheral blood and femoral bone tissue from a mouse model of SINFH treated with quercetin showed that PGTS2 and NCF1 levels were downregulated compared with those of controls, which was consistent with the results of bioinformatics analysis.Conclusion: This study provides new insights into the genes and related pathways involved in SINFH and shows that PTGS2 and NCF1 could be used as potential drug targets in SINFH.