Lycium barbarum polysaccharides enhance testicular spermatogenesis in d‐galactose‐induced aging rats via calcium signaling

Abstract Background Lycium barbarum polysaccharide (LBP) has long been recognized as having a wide range of beneficial properties for improving proliferation. However, the protective effects and specific mechanisms of d ‐galactose‐induced testicular dysfunction in reproductively senescent rats are not fully understood. Materials and methods A d ‐galactose‐induced senescence model in male rats and a d ‐galactose‐induced TM3 cell model were used to investigate the effects of LBP. The protective effect on testicular spermatogenic function was assessed by histological analysis and SA‐β‐gal staining. In addition, key calcium signaling pathway alterations involved in LBP were assessed using a multi‐omics approach and validated by tissue. Single‐cell sequencing data were used to further analyze the cellular heterogeneity of calcium signaling. Results LBP significantly improved testicular structure, increased the number of spermatogonia in the seminiferous tubules, and significantly attenuated oxidative stress and testicular apoptosis. In addition, LBP restored the expression of key steroidogenic enzymes, as well as elevated levels of testosterone, follicle‐stimulating hormone (FSH), and estradiol (E2), and decreased levels of luteinizing hormone (LH). Mechanistically, LBP regulates key signaling pathways, including calcium homeostasis, Hippo and mTOR pathways, which play important roles in cell growth, apoptosis, and tissue regeneration. Single‐cell sequencing data show that calcium signaling is more active in the elderly compared to the young, mainly in Leydig cells, Round Spermatids, and Smooth Muscle Cells. In TM3 cell experiments, the LBP reduced SA‐β‐gal activity, downregulated aging markers (p16, p21, p53), and restored steroid production function. In addition, LBP regulated the Ca 2 ⁺/CaM/CaMKII signaling pathway, improved calcium homeostasis, and reduced apoptosis in rats and cells. Conclusion LBP improves d ‐galactose‐induced testicular spermatogenesis mainly by regulating calcium signaling and metabolic pathways and is closely related to elongating spermatids, round spermatids.