Integrating network pharmacology and in vitro experiment to investigate the protective effects of semen cuscutae flavonoid on sertoli cells and blood-testis barrier in male infertility under heat stress

Background: Semen cuscutae is one of the common drugs used in treating male infertility (MI) in Traditional Chinese Medicine (TCM), and semen cuscutae flavonoid (SCF) is the main active component of semen cuscutae. The mechanism of SCF on sertoli cells (SCs) and blood-testis barrier (BTB) in male infertility under heat stress have not been clarified yet. Methods: : Here, network pharmacology were used to explore the targets and signaling pathways of SCF in the treatment of MI. Then, in vitro experiments were integrated to verify the results of network pharmacology. In the in vitro experiments, the cell viability was measured by performing the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Ki67 and AR expression were detected by performing immunohistochemistry staining, and AR, Akt, CK-18, and Occludin levels were measured using western blotting. The Akt inhibitor assay was further performed to elucidate the signaling pathway underlying the function of SCF. Result: The data showed a total of 136 potential therapeutic targets of SCF, including HPS90AA1, SRC, JUN, TP53, MAPK1, AKT1, etc., were identified. In vitro experiments suggested that SCF significantly improved the viability of SCs, upregulated the expression of AR, Akt, Occludin, and Ki67, and downregulated the expression of CK-18. After adding the Akt inhibitor to block the PI3K-Akt signaling pathway, the effect of SCF decreased, indicating that SCF regulates the PI3K-Akt signaling pathway via AR, and then regulates occludin, Ki67, and CK-18. Gene ontology (GO) enrichment and Kyoto Encyclopedia of genes and genomes (KEGG) pathway analyses also suggested that SCF regulates cell proliferation and differentiation mainly via the PI3K-Akt signaling pathway. Conclusions: : We suggested that the active components of SCF can promote AR expression, and regulate the PI3K-Akt signaling pathway and its downstream factors (occludin, Ki67, and CK-18), to regulate the viability, proliferation, differentiation, and BTB permeability of SCs, ultimately protecting reproductive ability.