Multi-omics and network pharmacology approaches reveal Gui-Ling-Ji alleviates oligoasthenoteratozoospermia by regulating arachidonic acid pathway.

BACKGROUND

Gui-Ling-Ji (GLJ) described in the ancient medical book 'Yunji Qijian' is a traditional Chinese medicine formula used to improve male fertility. It is now available for the treatment of oligoasthenoteratozoospermia (OAT). However, the active ingredients and mechanism of GLJ are not clear.

PURPOSE

The aim of this study was to clarify the active ingredients and mechanism of GLJ in OAT.

METHODS

Firstly, the cyclophosphamide-induced OAT rat model was established to evaluate the efficacy of GLJ. Secondly, serum/urine-based metabolomics and lipidomics and tissue-based transcriptomics were performed to discover the differential metabolites and genes in rats. Furthermore, network pharmacology was constructed to explore the associated mechanisms based on the results of multi-omics analysis. Finally, cellular experiment on testicular mesenchymal stromal cells (TM3) was used to validate the active ingredients and the key metabolic pathway.

RESULTS

Rats were administered GLJ by gavage every day for 3 weeks. Testicular damage and weight loss caused by cyclophosphamide were restored in rats, the sperm count and motility were improved, and levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) secretion were also elevated. Compared to the metabolites of OAT rats, 51 and 37 differential metabolites regulated by GLJ were identified from serum and urine respectively, 54 lipid differential metabolites regulated by GLJ were identified by lipidomics. At the same time, 23 of the 258 differential genes were found to be regulated by OAT rats and then reverse-regulated by GLJ. Network pharmacology has identified 13 pathways (Steroid hormone biosynthesis, Taurine and hypotaurine metabolism, Primary bile acid biosynthesis, Linoleic acid metabolism, Retinol metabolism, Glycerophospholipid metabolism, Ether lipid metabolism, Sphingolipid metabolism, Arachidonic acid metabolism, Glutathione metabolism, Arginine biosynthesis, Arginine and proline metabolism, D-Arginine and D-ornithine metabolism), four metabolites (arachidonic acid, oestrone sulphate, phosphatidylglycerol choline and sphingomyelin) and 15 targets (ABCB11, ALDH18A1, CCL3, CD244, CIITA, CYP2C8, DLL1, ITGA4, ESR1, AR, ABCB1, ABCC1, ALB, PLA2G1B and NOS2). GLJ, psoralen, isopsoralen, liquiritin, isoliquiritin, liquiritigenin, and ginsenoside Ro could significantly promote T secretion from TM3 cells. Additionally, arachidonic acid metabolism particularly the cyclooxygenase pathway, is closely related to the promotion of testosterone secretion by GLJ in TM3.

CONCLUSION

GLJ has a therapeutic efficacy in cyclophosphamide-induced OAT rats, which can modulate the disorders of lipid metabolism and amino acid metabolism. Arachidonic acid metabolism may be a key pathway, and six prototype compounds are potential key active ingredients for GLJ.