Phenylethanol glycosides from Cistanche tubulosa improved reproductive dysfunction by regulating testicular steroids through CYP450-3β-HSD pathway

Cistanche tubulosa (Schenk) R. Wight has been used frequently in traditional folk medicine for treatment of male sexual dysfunction (MSD). Phenylethanol glycosides, the main components of C. tubulosa, possess a variety of pharmacological activities due to their multiple properties. However, the underlying mechanism by which phenylethanol glycosides from C. tubulosa (CPhGs) regulates testicular steroids has not been elucidated to date. This study is to determine whether CPhGs promotes the reproductive functions of mice through CYP450-3β-HSD pathway of testosterone synthesis. The major compositions of C. tubulosa (CPhGs) were quantified by high performance liquid chromatography (HPLC). The model of reproductive injury in mice were induced by injection of hydrocortisone (HCT). Different doses of CPhGs (72, 145 and 289 mg/kg) and testosterone propionate (TP, positive control drug) were administrated intragastrically for 14 d. The reproductive functions (erectile incubation period, capture and ejaculation incubation period, number of captures and ejaculations) and organ weights (testicle, epididymis, seminal vesicle and penis) were then determined. The levels of luteinizing hormone and testosterone in serum were quantified by radioimmunoassay. The key enzymes in testosterone synthesis pathways such as steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc/CYP11A1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) in the testis were assessed by immunofluorescence (IF) staining or/and Western blot (WB) analysis. The results illustrated that the low dose of CPhGs (72 mg/kg) had no significant protective effect against the reproductive injury caused by HCT, while the moderate dose of CPhGs (145 mg/kg) improved the damaged reproductive ability and the declined levels of luteinizing hormone and testosterone in the model mice (P < 0.001, P < 0.05, respectively). In particular, high dose of CPhGs (289 mg/kg) was most effective in improving HCT-induced changes in body weight (P < 0.01), reducing the incubation period of the erectile (P < 0.001), capture (P < 0.05) and ejaculation (P < 0.01), and increasing the number of captures and ejaculations (P < 0.01, P < 0.05, respectively). The weights of testcle, epididymis, seminal vesicle and penis (P < 0.001, P < 0.01, P < 0.01, P < 0.001, respectively) were improved by high dose of CPhGs. The levels of testosterone and its upstream luteinizing hormone were up-regulated by high dose of CPhGs (P < 0.001). Meanwhile, the expressions of the key steroidogenic enzymes including CYP11A1 and 3β-HSD were significantly up-regulated after CPhGs treatment (P < 0.001), demonstrated that CPhGs exerted the effect through enhancing testosterone biosynthesis via CYP450-3β-HSD pathway. CPhGs could significantly protect against HCT-induced deleterious reproductive dysfunction and testis injury. The protective effects were exerted by up-regulating synthesis of testosterone via the CYP450-3β-HSD pathway in Leydig cells.