FOXO4-DRI ALLEVIATES AGE-RELATED TESTOSTERONE SECRETION INSUFFICIENCY VIA TARGETING SENESCENT LEYDIG CELLS IN AGED MICE

Abstract

To investigate the effects and mechanism of targeted inhibition of FOXO4 -p53 interaction on testicular aging in mice. Animal study was designed. Three-month-old and 20-month-old male C57BL/6 mice were used as research objects. The mice were divided into FOXO4-DRI treated group, old control group and young control group, with 10 mice in each group. The FOXO4-DRI treated group were intraperitoneally injected with FOXO4-DRI (5 mg/kg/d) every other day for three administrations, and the old control group and young control group were injected with the same amount of normal saline, and the observation period is 30 days. Upon the endpoint of experiment, assess muscle performance of mice in each group through an upside down suspension test, and analyze testicular weight/body weight ratio and seminal vesicles weight/body weight ratio. Then, epididymal sperm counts and motility, serum testosterone, myocardial enzymes (CK and CK-MB), liver function (ALT and AST) and renal function (CREA and BUN) were measured. Immunohistochemical staining was performed to detect the expression of FOXO4 in testes. SA-β-Gal staining was performed to assess the quantity of senescent cells in testes. Western blot was performed to measure protein levels of testosterone synthesis-related enzymes (3β-HSD and CYP11A1), senescence-related proteins (p53, p21, and p16) and SASP factors (IL-1α, IL-1β, IL-6, IL-10, TNF-α, and TGF-β) in testes. Immunofluorescence staining was performed to detect the expression of meiotic marker SCP3. FOXO4 was expressed in LCs of the old mice, but not the young mice. Compared with old control group, the quantity of senescent LCs and expression of aging-related proteins (p53, p21 and p16) in the testes of FOXO4-DRI treatment group were significantly decreased (P <0.05). Levels of SASP factors including IL-1β, IL-10 and TGF-β were decreased (P<0.05). Testicular endocrine function-related indicators including serum testosterone concentration, expression of 3β-HSD and CYP11A1, and muscle performance were significantly improved (P<0.05). Spermatogenic function indicators including epididymal sperm counts and vitality (P<0.05) and expression of SCP3 were also improved. The testicular weight/body weight ratio (P >0.05) showed no significant changes, but the seminal vesicles weight/body weight ratio decreased significantly (P <0.05). Indicators of systemic organ function or injury including CK, CK-MB, ALT, AST, CREA and BUN showed no significant changes (P >0.05). Targeted inhibition of FOXO4-p53 interaction can safely and effectively eliminate senescent LCs in testes and repair testicular aging in mice, which may become a new strategy for the treatment of testicular aging.