枸杞籽油激活SIRT3信号改善损伤睾丸线粒体功能及稳态的机制

With the effect of age, environment, diet and lifestyle, the male is suffering from infertility and early senescence,partly depending on mitochondrial dysfunction of the testis and imbalance of homeostasis.Precious researches have shown that SIRT3 pathway was associated with kinetic disorders of the mitochondria and degenerative diseases. And Lycium barbarum can improve spermic mitochondria amoung males, and lycium barbarum seed oil had an effect on anti-oxidation and anti-inflammation.The researches indicated that testosterone secretion can be improved in natural aging rat after gastric perfusion with lycium barbarum seed oil,and the drug-containing serum can act as a protective role in mitochondria in testicular spermatogenic cells, sertoli cells and leydig cells during the culture of these cells, and the SIRT3 expression was up-regulated by RNA-Seq and proteomic profiling.The SIRT3 expression was down-regulated and mitochondrial function was improved, after lycium barbarum seed oil was fed to natural aging and chronic stress of model rats. So the experimental results indicated that the activation of SIRT3 pathway by lycium barbarum seed oil can improve the mitochondrial homeostasis and mitochondrial function of the testis such as biosynthesis and occurrence , oxidative stress and autophagy and the likes .Based on the techniques and methods of Evening Primrose Seed Oil promoting the maturation of ovarian function in the last NFSC program,we will clarify the mechanism of mitochondrial function and homeostasis induced by activation of SIRT3 signal by lycium barbarum seed oil,due to the establishment of animal models such as aging rats,chronic stress rats,SIRT3-/-mice,and cell models of inflammation, oxidative stress and apoptosis.

男性随年龄、环境、饮食和生活方式的影响会出现不育和提前衰老，其部分原因是睾丸线粒体功能异常和稳态失衡。以往研究证明SIRT3通路与线粒体动力学障碍及退行性疾病相关，枸杞能改善男性精子线粒体；枸杞籽油具有抗氧化和抑炎症等的作用。前期研究提示：自然衰老大鼠枸杞籽油灌胃后改善睾酮分泌，其含药血清对培养睾丸生精细胞、支持细胞和间质细胞线粒体有保护作用，测序结果确定其SIRT3表达上调；枸杞籽油灌胃自然衰老和慢性应激慢性大鼠，结果显示SIRT3表达下调且改善线粒体功能。故推测：枸杞籽油激活SIRT3途径改善睾丸线粒体生物合成和发生、氧化应激和自噬等线粒体功能和稳态。课题组基于上一基金月见草油改善卵巢功能中成熟的研究技术和方法，提出以衰老大鼠、慢性应激大鼠模型和SIRT3-/-小鼠为动物模型及炎症、氧化应激以及凋亡细胞模型为研究对象，明确枸杞籽油激活SIRT3信号保护损伤睾丸线粒体功能和稳态的机制。

衰老、环境污染、社会压力、饮食结构调整以及生活方式的转变等多方面不良诱因，导致男性生殖健康障碍的发病率呈上升态势。男性随年龄、环境、饮食和生活方式的影响会出现不育和提前衰老，其部分原因是睾丸线粒体功能异常和稳态失衡。男性衰老作为一种不可避免的随时间变化的功能衰退，呈现在睾丸组织的衰老过程中。而线粒体自噬在维持器官稳态方面至关重要，参与抗衰老等多种机制。该项目开展的具体研究内容及重要结果：（1）在建立D-半乳糖诱导的亚急性衰老模型基础上，枸杞籽油通过激活大鼠睾丸组织SIRT3表达，保护大鼠睾丸抗氧化应，改善其睾丸生精功能和分泌功能；以200 mmol/L半乳糖干预M4细胞48h成功建立衰老模型，在此基础上，枸杞籽油干预TM4衰老细胞，阐明枸杞籽油通过激活SIRT3保护TM4细胞的抗氧化作用；为进一步探讨枸杞籽油抗氧化作用，TM4细胞沉默SIRT3后降低了枸杞籽油对其抗氧化的能力；枸杞籽油激活TM4细胞表达SIRT3，从而激活AMPK/PGC-1α通路稳定线粒体稳态保护细胞缓减氧化应激损伤。（2）0.9g/kg/d的六味地黄丸对D-半乳糖诱导亚急性衰老大鼠睾丸慢性炎症的作用，明确六味地黄丸通过抑制NF-κB活性缓解衰老大鼠睾丸炎症反应，其机制可能与通过激活AMPK/SIRT1通路有关。（3）2.5g/kg/d的金匮肾气丸通过激活睾丸线粒体自噬水平，增加睾酮合成关键酶的含量，调控线粒体自噬预防D-半乳糖诱导的亚急性衰老大鼠睾丸功能减退的分子效应机制以及在预防亚急性衰老雄性大鼠的睾丸功能减退方面发挥了保护作用，为临床金匮肾气丸预防睾丸老化提供实验依据。（4） 300mg/kg/d的二甲双胍能激活睾丸自噬水平，增加睾酮合成关键酶的水平，从而预防亚急性衰老大鼠睾丸功能减退，其机制可能与激活自噬,调节PINK1/Parkin信号通路有关。这为二甲双胍在PINK1/Parkin信号通路及作用效果的研究方面提供了一定的理论依据。研究结果证实揭示枸杞籽油激活 SIRT3 途径，改善线粒体功能和稳态的机制；明确六味地黄丸可以改善睾丸氧化应激和炎症反应；阐明金匮肾气丸调控线粒体自噬预防D-半乳糖诱导的亚急性衰老大鼠睾丸功能减退的分子效应机制；明确二甲双胍可以改善睾丸衰老、炎症和线粒体损伤等,为男性生殖健康障碍和部分男性生殖功能障碍患者提供了有益的治疗线索和科学依据.