The mechanism of the Wenshenyang recipe in the treatment of infertility by transcriptome analysis and network pharmacology

The Wenshenyang recipe (WSYR) has the effect of treating infertility, but the mechanisms underlying this activity have not been fully elucidated. In this study, network pharmacology and RNA sequencing were combined, with database-based “dry” experiments and transcriptome analysis-based “wet” experiments used conjointly to analyse the mechanism of WSYR in the treatment of infertility. In the dry analysis, 43 active compounds in WSYR and 44 therapeutic targets were obtained through a database search, 15 infertility pathways were significantly enriched, and key targets, such as ESR1, TP53, AKT1, IL-6, and IL-10 were identified. In the wet experimental analysis, HK-2 cells were treated with the three herbs of WSYR. Using the transcriptome sequencing platform, the expression level changes in 412 genes from 15 infertility pathways were observed, and 92 DEGs were finally obtained. Additionally, key targets, such as ESR2, STAT1, STAT3, and IL6, were identified. RT-qPCR experiments further verified that WSYR played an anti-inflammatory role by upregulating IL-4 and IL-10. By building the molecular docking model of ESR1, ESR2 and screening compounds, it was further found that xanthogalenol in Drynaria fortunei (Kunze) J.Sm. (Gusuibu), arachidonate in Cistanche deserticola Y.C.Ma (Roucongrong), and anhydroicaritin in Epimedium brevicornu Maxim. (Yinyanghuo) have good affinity for estrogen receptors. These findings provide evidence that WSYR exerts an estrogen-regulating role. By integrating the results of dry and wet experiments, it was found that the WSYR treats infertility by regulating hormone levels and inflammatory responses. HK-2 cells were cultured in a 384-well plate for 24 hours and treated with the three herbal extracts for 24 hours. Grouped into Gusuibu_control group, Gusuibu_experiment group, Roucongrong_control group, Roucongrong_experiment group, Yinyanghuo_control group, Yinyanghuo_experiment group and then subjected to RNAseq for differentially expressed gene determination.In the Gusuibu transcriptome experiment, there were 16 samples in the control group and 3 samples in the experimental group; in the Roucongrong transcriptome experiment, there were 16 samples in the control group and 3 samples in the experimental group; in the Yinyanghuo transcriptome experiment, there were 16 samples in the control group sample, 3 samples in the experimental group.