基于AMPK/SIRT1/HIF1a信号通路探讨金钗石斛多糖改善糖尿病大鼠睾丸生精功能的作用及机制研究

Research showed that 90 percent of men with diabetes are at risk of fertility decline, the prevalence was 10 times higher than in nondiabetic patients. Except for the the dendrobium nobile achieves hypoglycemic function, our previous study found that it could regulate reproduction-endocrine system, affect the process of testis glucose metabolism, which could also improve the spermatogenesis disturbance induced by diabetes, while its mechanism remains unclear. Based on the these results, we hypothesized that dendrobium nobile improves the spermatogenesis disturbance induced by diabetes mellitus, which by regulating the the process of testis glucose metabolism and promoting energy production by Sertoli cells through AMPK/SIRT1/HIF1a signaling pathway. In this proposal, an integral experiment will be adopted with that use dendrobium polysaccharide for treatment of diabetes rats and Sertoli cells cultured in vitro. On the one hand, the improvement of semen quality, testicular morphology, proferlization and aoptosis of spermatogenetic cells, as well as the oxidative damage were detected. Furthermore, the level and transportation of the product in testis glucose metabolism, detect the factors expression of AMPK/SIRT1/HIF1a signaling pathway, as well as the acetylation level of HIF1a were analyzed. On the other hand, the effect of glycolysis rate and molecular mechanism verification of dendrobium polysaccharide were analyzed at the cellular leve. This study will elucidate the pharmacological mechanism of dendrobium nobile in the improvement of the spermatogenesis disturbance induced by diabetes mellitus, which will provide new therapy idea and theory basis for male reproductive health combined with the fertility of diabetes patients in a clinical concerns.

研究显示，90%的糖尿病男性面临生育力下降，其不育症发病率是非糖尿病患者的10倍。我们前期研究发现金钗石斛除具备降血糖功能外，还具有调控生殖内分泌、影响睾丸糖代谢过程、改善睾丸生精功能的功效，但其具体机制未明。基于上述结果，我们推测金钗石斛通过AMPK/SIRT1/HIF1a信号通路调控睾丸糖代谢过程，促进睾丸能量产生与利用，改善糖尿病性睾丸生精障碍。本项目拟采用金钗石斛多糖治疗糖尿病大鼠模型和体外培养支持细胞的整体实验，一方面检测模型的精液质量、睾丸形态、生精细胞增殖与凋亡及氧化损伤的改善情况；进一步靶向分析睾丸糖代谢产物及转运情况，检测AMPK/SIRT1/HIF1a信号通路因子的表达和HIF1a蛋白乙酰化水平；另一方面在细胞水平阐明金钗石斛多糖对糖酵解速率的影响及分子机制验证，以明确金钗石斛改善糖尿病性睾丸生精障碍的药理机制，为临床上治疗男性糖尿病合并生精障碍提供新的手段和理论依据。

现阶段，国内外关于糖尿病的研究大都集中在糖尿病肝、肾、血管和神经系统的并发症之中，关于糖尿病所引发的生殖系统损伤的研究较为初级且并不深入和系统，特别是影响雄性生精功能的糖代谢机制还缺乏相关理论依据。研究发现金钗石斛除具备降血糖功能外，还可能具有调控生殖内分泌和改善睾丸生精功能的功效，但其具体机制未明。本项目通过水-醇沉淀的方法提取金钗石斛多糖（DNP），并进行了定性定量分析；在成功构建2型糖尿病大鼠模型的基础上，采用DNP干预8周。结果显示DNP可显著改善糖尿病大鼠的血糖水平和内分泌状态，改善胰岛素抵抗，并在一定程度上提高精子的数量和活力；显著改善睾丸生精小管的形态；增强睾丸生精细胞的增殖能力，抑制生精细胞的凋亡；提高能量代谢的关键调控因子SIRT1的表达。在成功构建1型糖尿病小鼠模型的基础上，DNP干预后可提高糖尿病小鼠的体重和睾丸重量、精子数量，并降低异常精子的比例。DNP可抑制糖尿病小鼠睾丸中Bax的表达，并上调Bcl-2、GATA-4、WT1、波形蛋白、LDHA、PKM2和HK2的表达；上述结果明确了DNP通过抑制细胞凋亡和激活睾丸糖酵解途径改善糖尿病性生精功能障碍的作用机制。此外，在本项目的资助下，亦探讨了DNP改善多囊卵巢综合征卵泡发育障碍，枸杞多糖、淫羊藿苷保护糖尿病大鼠睾丸生精功能等研究内容。但是，本项目探讨HIF1a乙酰化水平的变化调控睾丸糖酵解的分子机制还有待挖掘，对进一步明确金钗石斛在糖尿病性睾丸生精障碍中的药理机制，改善男性生殖健康合并糖尿病患者的生育能力还有待阐明。项目已经完成发表SCI论文7篇，北大核心论文2篇，培养硕士研究生1人，本科生3名，协助指导团队成员晋升中级职称2人，完成了项目预期研究成果要求。