睾丸支持细胞缝隙连接蛋白Cx43在糖尿病诱发生精功能障碍中的调控机制及桃叶珊瑚苷保护作用研究

Diabetic male fertility is a common chronic complication of diabetes. Spermatogenesis dysfunction is one of the main factors of male fertility induced by diabetes. Illustrating the pathogenesis of diabetic male fertility and seeking the more effective therapeutic method is a common academic focus of the diabetes and male reproduction health fields. Sertoli cells, as the only somatic cell contacted with germ cells in seminiferous tubule, provide structural and nutritional support to germ cells as well as maintain the spermatogenous microenvironment. Sertoli cells play an irreplaceable role in spermatogenesis process. Therefore, Sertoli cells have been regarded as a primary protective barrier or major target cells for spermatogenesis dysfunction induced diabetes. Connexins 43(Cx43) of Sertoli cells is a promising candidate that regulates the integrity of blood-testis barrier (BTB), Sertoli cells metabolism and oxidative stress. The previous studies indicated that hyperglycemia induced disruption of BTB, abnormal Sertoli cells metabolism and oxidative stress injury would be closely related to the down-regulation of Cx43. Hence, the current study was performed at molecular, cellular and whole level to reveal roles of abnormal expression and distribution of Cx43 induced diabetes by measuring the integrity of BTB, Sertoli cells metabolism, sperm quality and antioxidation using metabolomic approach and molecular biological technology. Importantly, the key pathogenesis of spermatogenesis dysfunction induced diabetes were fully explored by Sertoli cells as an entry points and Cx43 as a target. On that basis, we further investigate mechanism of action that Aucubin regulates Cx43 to protect against hyperglycemia induced spermatogenesis dysfunction involved in by using cell and animal model. The topic will successfully provide the theoretic and experimental basis for treatment of male fertility induced diabetes.

糖尿病所致男性不育是其常见的慢性并发症，生精功能障碍被认为是糖尿病诱发男性不育的主要因素之一。探明其发病机制、寻求有效防治方案是糖尿病和男性生殖健康领域共同关注的热点。睾丸支持细胞（SCs）是生精小管内唯一与生精细胞接触的体细胞，为精子发生提供微环境和物质支持，是糖尿病损伤生精功能的第一靶细胞。SCs缝隙连接蛋白Cx43在调控血睾屏障完整性，SCs能量代谢，氧化应激中发挥着重要作用。前期研究发现：糖尿病导致SCs结构和功能的损伤均伴随着Cx43表达的下调。因此，本研究以SCs为切入点，以Cx43为靶点，从整体-细胞-分子水平，综合细胞代谢组学和分子生物学技术，探讨高血糖下Cx43异常表达和分布对血睾屏障，SCs能量代谢，精子质量及氧化应激的影响，揭示糖尿病诱发生精功能障碍的发病机制。并在此基础上研究桃叶珊瑚苷通过调控Cx43发挥对疾病的逆转作用，为糖尿病性男性不育的防治奠定理论和实验基础。

糖尿病男性不育症是糖尿病和男性生殖领域共同关注的焦点，但其发病机制尚不明确。睾丸支持细胞（SCs）在维系精子赖以生存的微环境，主导有序的精子生成过程中具有不可替代的作用。SCs表达的Cx43 是维持支持细胞间以及支持细胞-生殖细胞-间质细胞间细胞通讯的重要连接蛋白，对于介导精子发生过程至关重要。因此，本研究以SCs为切入点，以Cx43为靶点，探讨高血糖下Cx43异常表达和分布对血睾屏障，精子质量及氧化应激的影响，揭示糖尿病诱发生精功能障碍的发病机制。并在此基础上研究桃叶珊瑚苷通过调控Cx43发挥对疾病的逆转作用。本课题首先研究了睾丸支持细胞Cx43在糖尿病状态下的变化。体内实验结果显示：糖尿病导致的血睾屏障通透性增加， Cx43表达下降，睾丸组织萎缩，精子形态异常，提示糖尿病导致了睾丸生精功能障碍。体外研究发现：高糖降低了SCs与SCs间的跨膜电阻值，增加了细胞层的通透性，下调Cx43的表达，抑制了细胞间连接通讯功能，诱导睾丸支持细胞氧化应激损伤。并且提示血睾屏障通透性增加与Cx43表达的降解密切相关。随后通过操纵Cx43过表达和沉默，验证其在糖尿病状态下对血睾屏障完整性和生精功能的调控作用。结果显示：高糖环境下Cx43的过表与沉默可以正反调控体外血睾屏障的完整性、多种紧密连接蛋白的表达，同时调节细胞内氧化应激的状态。同时，上调睾丸组织的Cx43可以有效逆转糖尿病导致的血-睾屏障通透性增加，恢复生精功能，提示Cx43可能成为改善糖尿病睾丸损伤和精功能障碍的潜在靶点。最后，在此基础上，本课题研究了中药单体桃叶珊瑚苷治疗糖尿病睾丸损伤的作用及分子机制。结果显示：桃叶珊瑚苷可以有效的逆转糖尿病导致的睾丸生精功能障碍，并且其上调睾丸支持细胞Cx43的表达，是逆转II型糖尿病损伤血-睾屏障完整性，恢复生精功能的主要原因之一。本课题的顺利完成为阐述糖尿病诱发男性不育症的发病机制，寻找新的治疗策略提供靶点和理论基础。