Epimedin C promotes mitochondrial transfer and delays thymus atrophy in 4-VCD induced mimetic-menopausal mice

Prevention of thymus atrophy during menopause is of great significance for improving the immune function and overall health of menopausal women. Epimedin C is one of the major bioactive compounds in Herba Epimedii, a traditional herbal medicine for the treatment of menopausal syndrome in China, but the action and mechanism of Epimedin C in the treatment of menopausal thymus atrophy remains unclear.

To study the effect of Epimedin C on thymus atrophy in 4-vinylcyclohexene diepoxide (4-VCD) induced mimetic-menopausal mice and explore its mechanism from new perspectives of tunneling nanotubes (TNTs) formation and mitochondrial transfer (MitoT).

The effects of Epimedin C in the 4-VCD induced menopause-like phenotype in mice were observed, and the thymic output function was evaluated by the quantitative detection of T cell receptor excision circles (TRECs). The structure of the thymus was observed by H&E. The arrangement and quantity of different cell subpopulations of thymic epithelial cells (TECs) and thymocytes were detected by multiple fluorescent staining and flow cytometry. Mitochondrial morphology was observed with transmission electron microscopy. LC-MS/MS was used to analyze and identify the differential protein expression in thymus before and after Epimedin C treatment. Actin polymerization inhibitor was used to verify the possible mechanism of Epimedin C. The treadmilling-balance of actin, TNTs formation, and MitoT processes were observed by specific fluorescent probe labeling. The interaction between G-actin, Thymosin β4 (Tβ4), and Epimedin C were studied by protein cross-linking assay.

Epimedin C significantly increased the thymus weight and the area of the thymus medulla, increased the grip strength and bone strength in 4-VCD induced mimetic-menopausal mice, and enhanced ovarian secretion function. It could affect the thymus output, increase CK5 and CK8 expression, maintain the reticular structure of TECs, inhibit the differentiation of thymocytes into double positive cells (CD4⁺CD8⁺) and CD4SP (CD3⁺TCR β⁺CD4⁺CD8^-) cells. Epimedin C promoted the conversion of G-actin to F-actin and accelerated MitoT via stimulating the TNTs formation, which related with the downregulation of Tβ4 and obstruction to the formation of Tβ4-G-actin complex.

Epimedin C can promote TEC activity in 4-VCD induced mimetic-menopausal mice by decreasing the expression of Tβ4, inhibiting the binding of Tβ4 to G-actin, promoting the F-actin polymerization and the TNTs-depended MitoT.