目的 研究淫羊藿苷对衰老小鼠睾丸支持细胞胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor，GDNF）表达的作用及其机制。方法 将小鼠分为对照组、模型组及淫羊藿苷低、中、高剂量（2.5、5.0、10.0 mg/kg）组，给予药物干预后，统计性腺质量及指数，采用伊红染色法检测小鼠精子活率，血球板计数法统计精子数量，苏木素-伊红（hematoxylin-eosin，HE）染色评价睾丸组织损伤程度，Western blotting检测睾丸组织GDNF、Notch1、Notch1细胞内结构域（intracellular domain of Notch1，NICD）和HES1蛋白表达水平，免疫荧光和免疫组化分别检测睾丸组织Notch1和HES1表达。采用TM4细胞建立D-半乳糖诱导的衰老模型及HES1过表达模型，设置对照组、模型组和淫羊藿素低、中、高剂量（0.2、0.4、0.8 μmol/L）组，采用CCK-8法检测细胞活性，qRT-PCR检测GDNF mRNA表达水平，Western blotting检测GDNF、Notch1、NICD和HES1蛋白表达水平。结果 体内实验结果显示，与对照组比较，模型组小鼠性腺质量、性腺指数、精子活率和精子数量显著下降（P＜0.01），睾丸组织形态损伤，GDNF蛋白表达水平明显降低（P＜0.01），Notch1、NICD和HES1蛋白表达水平显著升高（P＜0.05、0.01）；与模型组比较，淫羊藿苷给药组小鼠性腺质量、性腺指数、精子活率和精子数量显著提高（P＜0.05、0.01），睾丸组织损伤明显改善，GDNF蛋白表达上调（P＜0.05、0.01），Notch1、NICD和HES1蛋白表达下调（P＜0.05、0.01）。体外实验结果显示，与对照组比较，模型组细胞活性明显下降（P＜0.01），GDNF mRNA和蛋白表达显著下降（P＜0.05、0.01），Notch1、NICD和HES1蛋白表达显著上升（P＜0.05、0.01）；与模型组比较，淫羊藿素可明显提高细胞活性（P＜0.05、0.01），促进GDNF mRNA和蛋白表达（P＜0.05、0.01），抑制Notch1、NICD和HES1蛋白表达（P＜0.05、0.01、0.001）；而HES1过表达可拮抗淫羊藿素对GDNF表达的促进作用（P＜0.01）。结论 淫羊藿苷能够有效促进衰老小鼠睾丸支持细胞GDNF表达，其机制可能与抑制Notch1/HES1通路有关。

Objective To study the effect and mechanism of icariin on expression of glial cell line derived neurotrophic factor (GDNF) in testicular Sertoli cells of aging mice. Methods Mice were divided into control group, model group, and icariin low-, medium-, and high-dose (2.5, 5.0, 10.0 mg/kg) groups. After drug intervention, the gonadal weight and index were measured. The sperm viability of mice was detected by eosin staining, the number of sperm was measured by hemocytometer counting, and the degree of testicular tissue damage was evaluated by hematoxylin-eosin (HE) staining. The expression levels of GDNF, Notch1, intracellular domain of Notch1 (NICD) and HES1 protein in testicular tissue were detected by Western blotting. Immunofluorescence and immunohistochemistry were used to detect the expressions of Notch1 and HES1 in testicular tissue, respectively. D-galactose-induced aging model and HES1 overexpression model were established using TM4 cells. Control group, model group, and icaritin low-, medium- and high-dose (0.2, 0.4, 0.8 μmol/L) groups were set up. The cell viability was detected using CCK-8 method. GDNF mRNA expression level was detected by qRT-PCR. GDNF, Notch1, NICD and HES1 protein expression levels were detected by Western blotting. Results The results of in vivo experiments showed that compared with control group, the gonadal weight, gonadal index, sperm motility and sperm count of mice in model group were significantly decreased (P < 0.01), testicular tissue morphology was damaged, GDNF protein expression level was significantly reduced (P < 0.01), Notch1, NICD, and HES1 protein expression levels were significantly increased (P < 0.05, 0.01). Compared with model group, mice in icariin treated group showed significant improvements in gonadal weight, gonadal index, sperm motility and sperm count (P < 0.05, 0.01), as well as significant improvement in testicular tissue damage. GDNF protein expression was up-regulated (P < 0.05, 0.01), while Notch1, NICD and HES1 protein expressions were down-regulated (P < 0.05, 0.01). The in vitro experimental results showed that compared with control group, the cell viability of model group was decreased significantly (P < 0.01), the expressions of GDNF mRNA and protein were significantly decreased (P < 0.05, 0.01), the expressions of Notch1, NICD, and HES1 proteins were significantly increased (P < 0.05, 0.01); Compared with model group, icaritin significantly increased cell viability (P < 0.05, 0.01), promoted GDNF mRNA and protein expressions (P < 0.05, 0.01), and inhibited Notch1, NICD and HES1 protein expressions (P < 0.05, 0.01, 0.001); Overexpression of HES1 could counteract the promoting effect of icaritin on GDNF expression (P < 0.01). Conclusion Icariin could effectively promote the expression of GDNF in testicular Sertoli cells of aging mice, and its mechanism may be related to the inhibition of Notch1/HES1 pathway.

R285.5

国家自然科学基金资助项目（82274191）；国家自然科学基金资助项目（82474321）