Network pharmacological analysis of the molecular targets and biological mechanisms associated with the treatment of oligospermic patients with RunJing formula

Background: Oligozoospermia is a disorder of the male reproductive system defined as the presence of <15 million sperm/ml in a semen sample. This condition is difficult to treat; previous work has shown that neither antioxidants nor antiestrogens can effectively improve sperm count. However, in China, traditional Chinese medicine (TCM) compound prescriptions have achieved good results in the clinical treatment of patients with oligozoospermia. Runjing formula (RJF), an empirical formula first prescribed by Professor Zeng, is widely used in the treatment of oligozoospermic patients. Preliminary unpublished data indicate that this formula can increase sperm count and improve sperm motility. However, the precise pharmacological mechanisms underlying these effects have yet to be elucidated. In this study, we aimed to investigate the molecular mechanisms underlying the effects of Runjing prescription in oligospermic patients. Our analysis focused specifically on network pharmacology and bioinformatic analysis.Methods: The active components and targets of RJF were screened and identified by means of network pharmacology and bioinformatic analysis, and differentially expressed genes (DEGs) were identified between patients with non-obstructive azoospermia and those with obstructive azoospermia. We then carried out protein-protein interaction (PPI) analysis. The PPI networks of presumptive RJF targets and oligospermia-related targets were visualized and combined to determine potential candidate targets for RJF in oligospermic patients. We also performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and constructed a gene pathway network. This allowed us to screen key target genes and analyze the degree of enrichment in specific signal pathways in order to explore their relevance to RJF and oligospermia.Results: Analysis showed that 29 target genes were related to the effects of RJF in oligospermic patients and led to the identification of 90 potential molecules. Our analysis showed that the functional annotation of these target genes was related to transcription, cytoplasm, and protein binding. Twenty-one pathways were particularly abundant, including apoptosis, the MAPK signaling pathway, and the Relaxin signaling pathway. AKT1 was identified as the core gene related to the effects of RJF in oligospermic patients, while JUN, FOS, NFKBIA, and TNFRSF represented the key genes in the network of gene pathways.Conclusion: RJF appears to exert influence on multi-components and multi-targets when used to treat oligozoospermia. The underlying molecular mechanisms appear to be complex and diverse and have direct or indirect relationships with multiple signaling pathways. In this study, bioinformatics and network pharmacology were used to extract the active components of RJF, and the differentially expressed genes associated with oligozoospermia were obtained from the gene expression omnibus (GEO) database. Finally, 29 target genes and 90 active components were identified. GO and KEGG enrichment analysis indicated that RJF mainly acts on oligozoospermia through MAPK and Relaxin signal pathways, and that the key genes involved are ATK1,JUN, FOS, NFKBIA, and TNFRSF. These results should help us to design new clinical and experimental research to further expand our understanding of oligospermia.