睾丸间质细胞自噬在精索静脉曲张导致的性腺功能低下中的作用及机制研究

One of the most important potential pathologic mediators of varicocele is hypoxia, which has been reported to induce autophagy as an independent factor. However, it is unknown about the role of autophagy in varicocele. Our preliminary work found in rat models that varicocele could induce autophagy in Leydig cells through the pathway “hypoxia/miR-96/mTOR/autophagy”which was reported early by us, and in vitro experiments demonstrated that regulation of autophagy changed the testosterone level that Leydig cells secreted to the supernatants. Therefore we assume that varicocele environment may modulate testosterone secretion through the miR-96 regulated autophagy. In order to verify the hypothesis, firstly, by in vivo and in vitro experiments, we intend to further confirm that varicocele can induce autophagy through the pathway “hypoxia/miR-96/mTOR/autophagy”, and the varicocele induced autophagy can regulate testosterone synthesis and secretion in Leydig cells. Secondly, in order to explore the detailed mechanisms that regulation of testosterone synthesis by varicocele induced autophagy, we plan to detect if autophagy affects the level of endoplasmic reticulum stress and oxidative stress, Leydig cell survival and the metabolism of cholesterol and androgen binding protein. Lastly, we aim to confirm the positive in vitro research results in varicocele rat models, and try to perform drug intervention by targeting miR-96 or autophagy in varicocele induced hypogonadism in the rat models. The results of this study are expected to provide the first evidence that the varicocele induced autophagy can regulate testosterone synthesis and secretion in Leydig cells, and to provide a new train of thought and a new target for the treatment of varicocele induced hypogonadism.

低氧是精索静脉曲张(Varicocele, VC)的重要病理因素之一。低氧可诱导细胞自噬，而目前关于自噬在VC发病中的作用尚未有报道。申请人前期研究发现了低氧调控自噬的新通路“低氧/miR-96/mTOR/自噬”，且预实验在大鼠模型中证实VC可能通过该通路诱导睾丸间质细胞自噬，并可进一步影响睾酮分泌。因此我们推测：“VC环境可能通过miR-96调控自噬进一步调节间质细胞分泌睾酮”。本课题拟首先验证VC确实可通过“低氧/miR-96/mTOR”诱导自噬，并影响睾酮分泌。其次，通过分析自噬在胆固醇代谢、内质网及线粒体保护、雄激素结合蛋白代谢及间质细胞存活过程中的作用，探索VC诱导的自噬在调控睾酮分泌中的具体机制。最后，验证上述结果，并尝试针对miR-96或自噬对VC诱导的性腺功能低下进行药物干预。本课题的实施有望首次阐明自噬在VC导致的性腺功能低下中的作用及机制，为该疾病的治疗提供一种新思路。

低氧是精索静脉曲张(Varicocele, VC)的重要病理因素之一，有研究表明低氧可诱导细胞自噬，而目前关于自噬在VC发病中的作用尚未有报道。近年的研究发现，自噬可以降解包括脂滴在内的多种细胞器，将胆固醇酯降解为游离胆固醇，而游离胆固醇是睾丸间质细胞合成睾酮的关键底物。为了解VC中自噬在睾酮合成中的作用，本项目进行了相关研究，发现如下：1.抑制自噬显著抑制了睾酮合成（p<0.05）。2.抑制自噬促进了脂滴和总胆固醇的累积，而游离胆固醇显著减少（p<0.05）。3.低氧诱导的自噬调节睾酮合成。我们发现短期低氧显著刺激睾酮合成（p<0.05），而抑制自噬后，低氧的刺激作用消失。同时短期低氧也显著减少了间质细胞中脂滴的大小（p<0.05）和数量（p<0.05），而抑制自噬能够很大程度上逆转这一变化。4.精索静脉曲张大鼠模型中，自噬抑制剂能够显著降低睾酮分泌（p<0.05）。上述数据首次证实，VC环境下自噬能够促进睾酮分泌，其中的机制可能是VC的低氧微环境诱导自噬发生，后者降解细胞内脂滴，为间质细胞提供游离胆固醇。我们的研究揭示了睾酮合成的一种自噬调节模式。