Shiting Yu1, Bing Han2, Xueyuan Bai3, Shichao Liu4, Xin Xing5, Daqing Zhao6, Meichen Liu7, Siming Wang8. 1. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: ysting0331@163.com. 2. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: 1403287499@qq.com. 3. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: baixy1212@163.com. 4. Academic Affairs Office, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: aralekyra@163.com. 5. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: 1043084272@qq.com. 6. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: zhaodaqing1963@163.com. 7. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: liumc0367@163.com. 8. Jilin Ginseng Academy, Changchun University of Chinese Medicine, 1035 Boshuo Road, Changchun, Jilin Province, 130117, China. Electronic address: lwsm126030@126.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Clinical applications and pharmacological research suggest that Dioscorea opposita Thunb. (Chinese yam), a well-known traditional Chinese medicine which has been used for more than 2000 years to nourish kidney-yang and protect the male reproductive system, might be efficacious for the treatment of erectile dysfunction (ED). AIM OF THE STUDY: This study aimed to investigate the active component extract of Chinese yam, determine its effectiveness in hydrocortisone-induced "kidney-yang deficiency syndrome" (KDS-Yang) rats and in oxidatively damaged TM3 cells and explore the underlying mechanism on restoring erectile function. MATERIALS AND METHODS: We clarified the Chinese yam cold-soaking extract (CYCSE) as the main active extract of Chinese yam by a CCK8 assay and further identified its composition. The KDS-Yang rats were induced by intragastric administration of hydrocortisone. After 10 d of CYCSE intervention, cavernous and testis morphology were stained with hematoxylin and eosin. Inducible nitric oxide synthase (iNOS), cyclic guanosine monophosphate (cGMP), testosterone, 8-hydroxy-2-deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay kits. Leydig cells were performed using immunohistochemistry. Reactive oxygen species were measured using a DCFH-DA fluorescent probe, and testicular collagenous fibers were stained with a Masson kit. Detection of testicular apoptosis was performed by a TUNEL assay. Nrf2 and NQO1 mRNA expression levels were measured by qRT-PCR. The protein expression levels of Nrf2, HO-1, TGF-β1 and SMAD2/3 were analyzed by Western blot. RESULTS: We demonstrated in KDS-Yang rats and oxidatively damaged TM3 cells that CYCSE successfully restored erectile function through ameliorating testicular function. Our data suggested that CYCSE can stimulate the NO/cGMP pathway and restore the cavernous morphology to protect against KDS-Yang-induced ED. It also protected testis morphology, increased Leydig cell proliferation and stimulated testosterone secretion. In the damaged testes, excessive increases in 8-OHdG and inhibition of SOD activity were ameliorated, and the Nrf2/HO-1 signaling pathway was enhanced after treatment with CYCSE, indicating that the antioxidant defense system was activated. These findings were also validated in vitro. Additionally, fibrosis of the testes and TM3 cells was reversed by CYCSE through the TGF-β1/SMAD2/3 pathway. CONCLUSION: CYCSE has a therapeutic effect on KDS-Yang-induced ED, and the mechanism includes stimulation of testosterone secretion, resistance to oxidative stress and prevention of fibrosis. These findings provide a new scientific verification for the application of Chinese yam in the treatment of KDS-Yang-induced ED.
ETHNOPHARMACOLOGICAL RELEVANCE: Clinical applications and pharmacological research suggest that Dioscorea opposita Thunb. (Chinese yam), a well-known traditional Chinese medicine which has been used for more than 2000 years to nourish kidney-yang and protect the male reproductive system, might be efficacious for the treatment of erectile dysfunction (ED). AIM OF THE STUDY: This study aimed to investigate the active component extract of Chinese yam, determine its effectiveness in hydrocortisone-induced "kidney-yang deficiency syndrome" (KDS-Yang) rats and in oxidatively damaged TM3 cells and explore the underlying mechanism on restoring erectile function. MATERIALS AND METHODS: We clarified the Chinese yam cold-soaking extract (CYCSE) as the main active extract of Chinese yam by a CCK8 assay and further identified its composition. The KDS-Yang rats were induced by intragastric administration of hydrocortisone. After 10 d of CYCSE intervention, cavernous and testis morphology were stained with hematoxylin and eosin. Inducible nitric oxide synthase (iNOS), cyclic guanosine monophosphate (cGMP), testosterone, 8-hydroxy-2-deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay kits. Leydig cells were performed using immunohistochemistry. Reactive oxygen species were measured using a DCFH-DA fluorescent probe, and testicular collagenous fibers were stained with a Masson kit. Detection of testicular apoptosis was performed by a TUNEL assay. Nrf2 and NQO1 mRNA expression levels were measured by qRT-PCR. The protein expression levels of Nrf2, HO-1, TGF-β1 and SMAD2/3 were analyzed by Western blot. RESULTS: We demonstrated in KDS-Yang rats and oxidatively damaged TM3 cells that CYCSE successfully restored erectile function through ameliorating testicular function. Our data suggested that CYCSE can stimulate the NO/cGMP pathway and restore the cavernous morphology to protect against KDS-Yang-induced ED. It also protected testis morphology, increased Leydig cell proliferation and stimulated testosterone secretion. In the damaged testes, excessive increases in 8-OHdG and inhibition of SOD activity were ameliorated, and the Nrf2/HO-1 signaling pathway was enhanced after treatment with CYCSE, indicating that the antioxidant defense system was activated. These findings were also validated in vitro. Additionally, fibrosis of the testes and TM3 cells was reversed by CYCSE through the TGF-β1/SMAD2/3 pathway. CONCLUSION: CYCSE has a therapeutic effect on KDS-Yang-induced ED, and the mechanism includes stimulation of testosterone secretion, resistance to oxidative stress and prevention of fibrosis. These findings provide a new scientific verification for the application of Chinese yam in the treatment of KDS-Yang-induced ED.