Siqi Guan1, Yutian Zhu2, Jingshang Wang3, Lei Dong4, Qi Zhao5, Lu Wang6, Bin Wang7, Haisong Li8. 1. First Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, China; TCM Department, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, 100026, China. Electronic address: janlynn@163.com. 2. Urology Department, Peking University Third Hospital, Beijing, 100191, China. Electronic address: 262971254@qq.com. 3. TCM Department, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, 100026, China. Electronic address: wangjingshang108325@126.com. 4. Beijing University of Chinese Medicine, Beijing, 100029, China. Electronic address: 120087007@qq.com. 5. First Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, China. Electronic address: 13520029590@163.com. 6. First Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, China. Electronic address: 1533985316@qq.com. 7. First Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, China; Andrology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China. Electronic address: dayiwangbin@sina.com. 8. First Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, China; Andrology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China. Electronic address: lihs369@163.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae is the seed of Cuscuta japonica Choisy, and Fructus Lycii is the mature fruit of Lycium barbarum L. (Solanaceae). Semen Cuscutae and Fructus Lycii (SC-FL) are well-known Chinese medicine which have been used to tonify the kidney and replenish the essence for thousands of years. Chinese physicians prefer to prescribe them for treating male infertility. Recent studies have found that SC-FL repair spermatogenic dysfunction, however, the therapeutic mechanism has yet to be clearly elucidated. AIM OF THE STUDY: This study aimed at evaluating the therapeutic effect of SC-FL in glucosides of Tripterygium wilfordii Hook. f (GTW)-induced dyszoospermia rats and elucidating the underlying molecular mechanism. MATERIALS AND METHODS: Seventy-eight Sprague-Dauley (SD) rats were randomly divided into five groups: normal control (treated with saline), GTW (treated with saline), GTW + levocarnitine (treated with levocarnitine), GTW + SCFL (treated with SC-FL), and LY (LY294002, the PI3K inhibitor) +SCFL (treated with SC-FL). GTW (40 mg/kg/d) was intragastrically administered for 4 weeks to establish dyszoospermia model. From the start of the study, LY was additionally injected into the tail vein of rats of the LY + SCFL group once a week. After 8 weeks, semen quality and organ coefficient were determined and sex hormone, inhibin B, and epididymal carnitine levels were measured. Testicular tissue and its ultrastructure were observed using H&E (hematoxylin-eosin) staining and electron microscopy. Immunohistochemistry, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to determine the protein and mRNA expression of SCF, c-kit, PI3K, p-Akt, Bad, Bcl-2, and Bax in rat testis. RESULTS: Compared with the GTW group, semen quality, the organ coefficient, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and epididymal carnitine levels were significantly improved in the GTW + SCFL group (P < 0.05 or P < 0.01). Histomorphology and testicular ultrastructural evaluation showed that in the GTW + SCFL group, the structure and arrangement of seminiferous tubules were better, the amount of spermatogenic cells increased significantly, the morphology of spermatogenic cells improved, and the mitochondria increased, compared to those in the GTW group. Immunohistochemistry, western blotting, and qRT-PCR results showed that compared with the GTW group, the expression of SCF, c-kit, PI3K, p-Akt, and Bcl-2 in the GTW + SCFL group was increased, while that of Bax and Bad was decreased. The expression of p-Akt and Bcl-2 decreased, while that of Bad and Bax increased in the LY + SCFL group compared with the SCFL group. CONCLUSION: SC-FL can effectively inhibit spermatogenic cell apoptosis and promote their proliferation, and the mechanism may be related to the regulation of the SCF/c-kit--PI3K--Bcl-2 pathway.
ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae is the seed of Cuscuta japonica Choisy, and Fructus Lycii is the mature fruit of Lycium barbarum L. (Solanaceae). Semen Cuscutae and Fructus Lycii (SC-FL) are well-known Chinese medicine which have been used to tonify the kidney and replenish the essence for thousands of years. Chinese physicians prefer to prescribe them for treating male infertility. Recent studies have found that SC-FL repair spermatogenic dysfunction, however, the therapeutic mechanism has yet to be clearly elucidated. AIM OF THE STUDY: This study aimed at evaluating the therapeutic effect of SC-FL in glucosides of Tripterygium wilfordii Hook. f (GTW)-induced dyszoospermia rats and elucidating the underlying molecular mechanism. MATERIALS AND METHODS: Seventy-eight Sprague-Dauley (SD) rats were randomly divided into five groups: normal control (treated with saline), GTW (treated with saline), GTW + levocarnitine (treated with levocarnitine), GTW + SCFL (treated with SC-FL), and LY (LY294002, the PI3K inhibitor) +SCFL (treated with SC-FL). GTW (40 mg/kg/d) was intragastrically administered for 4 weeks to establish dyszoospermia model. From the start of the study, LY was additionally injected into the tail vein of rats of the LY + SCFL group once a week. After 8 weeks, semen quality and organ coefficient were determined and sex hormone, inhibin B, and epididymal carnitine levels were measured. Testicular tissue and its ultrastructure were observed using H&E (hematoxylin-eosin) staining and electron microscopy. Immunohistochemistry, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to determine the protein and mRNA expression of SCF, c-kit, PI3K, p-Akt, Bad, Bcl-2, and Bax in rat testis. RESULTS: Compared with the GTW group, semen quality, the organ coefficient, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and epididymal carnitine levels were significantly improved in the GTW + SCFL group (P < 0.05 or P < 0.01). Histomorphology and testicular ultrastructural evaluation showed that in the GTW + SCFL group, the structure and arrangement of seminiferous tubules were better, the amount of spermatogenic cells increased significantly, the morphology of spermatogenic cells improved, and the mitochondria increased, compared to those in the GTW group. Immunohistochemistry, western blotting, and qRT-PCR results showed that compared with the GTW group, the expression of SCF, c-kit, PI3K, p-Akt, and Bcl-2 in the GTW + SCFL group was increased, while that of Bax and Bad was decreased. The expression of p-Akt and Bcl-2 decreased, while that of Bad and Bax increased in the LY + SCFL group compared with the SCFL group. CONCLUSION: SC-FL can effectively inhibit spermatogenic cell apoptosis and promote their proliferation, and the mechanism may be related to the regulation of the SCF/c-kit--PI3K--Bcl-2 pathway.