SIRT1在氧化应激诱导蛋鸡卵泡闭锁中的作用与机制

Ovary atrophy in late laying phase or stressors resulted from oxidative stressed-induced follicular atresia is the main contributing factor for the inferior total laying performance and the early culling in practice. Previous study in our lab indicated that oxidative stress can reduce egg laying rate and follicle numbers. Sirtuin 1(SIRT1) as the primary factor to sense redox state, may play a main role in regulation granulosa cell apoptosis in mice and human, but its role in oxidative stress-induced follicular atresia is still not known. In this project, the effect of oxidative stress on follicular atresia and SIRT1 pathway will be investigated firstly to verify follicular atresia can be induced by oxidative stress. Then, the effect of oxidative stress on cell apoptosis, oxidative status and SIRT1 pathway in follicular granulosa cell will be investigated in vitro. Moreover, further experiment will be conducted to investigate the molecular mechanism of SIRT1 and its downstream signaling pathway mediated oxidative stress and cell apoptosis in granulosa cells of follicle by gene knockout and overexpression. At last, SIRT1 activator will be used in vivo to further testify the mechanism of SIRT1 and its down-strain signaling pathway in oxidative stress-induced follicular atresia. The results of this project will disclosure the relationship and molecular mechanism between oxidative stress and follicular atresia, and have a practical significance to improve the laying performance and fertility of layers and breeders in a short and lifetime manner.

生产中因卵巢机能衰退或应激等因素导致蛋鸡全期产蛋率低和淘汰过早，主要原因是在于氧化应激引起的卵泡闭锁。申请人前期研究发现，氧化应激可以降低蛋鸡产蛋率和卵泡数。鼠和人上的研究证实，感知氧化应激状态的关键蛋白SIRT1可参与调控卵泡颗粒细胞的凋亡，而SIRT1是否介导氧化应激引起卵泡闭锁仍未见报道。本项目首先考察氧化应激对蛋鸡卵泡闭锁和SIRT1的影响，探明氧化应激可以引起卵泡闭锁。在此基础上以蛋鸡卵泡颗粒细胞为材料，考察氧化应激对离体培养颗粒细胞凋亡和SIRT1的影响；再通过基因敲除和过表达等研究揭示SIRT1及其下游信号通路在氧化应激导致颗粒细胞凋亡中的作用机制；最后给蛋鸡使用SIRT1激活剂进一步在体外验证氧化应激通过SIRT1及其下游通路导致卵泡闭锁的机制。本研究对揭示氧化应激与卵泡闭锁的分子机制具有重大理论意义，并对提高蛋鸡和种鸡短期和长期生产性能和繁殖力具有重要的实践意义。

生产中因卵巢机能衰退或应激等因素导致蛋鸡全期产蛋率低和淘汰过早，主要原因是在于氧化应激引起的卵泡闭锁。因此本研究围绕科学假设：（1）氧化应激可以通过抑制SIRT1活性引起蛋鸡卵泡闭锁，降低蛋鸡产蛋率；（2）基因过表达SIRT1或其激活剂可以通过抑制其下游FoxO1和p53信号通路依赖的细胞凋亡，从而缓解氧化应激导致的卵泡颗粒细胞凋亡，进而降低卵泡闭锁。通过蛋鸡试验本项目成功在在体（蛋鸡）上证明了800µmol/kg的tBHP（叔丁基过氧化氢）腹腔注射可以造成蛋鸡卵巢氧化应激，进一步在颗粒细胞上证明了200µmol/L的H2O2处理24h后可获得理想的颗粒细胞氧化应激模型。在这两个应激模型的基础上，本项目通过细胞和动物试验两个层面研究并揭示了氧化应激可以通过引起颗粒细胞凋亡导致卵泡闭锁，进而降低产蛋率。同时通过使用SIRT1信号通路的抑制剂和激活剂，证明了SIRT1-FoxO1/P53是介导氧化应激诱导的颗粒细胞凋亡的关键信号通路。饲粮添加SIRT1激活剂600mg/kg白藜芦醇（RES）可缓解蛋鸡机体和卵巢的氧化应激和炎性反应，这与白藜芦醇通过激活SIRT1-FoxO1信号通路抑制卵泡颗粒细胞凋亡，缓解卵泡闭锁有关；此外这也与白藜芦醇可以激活Nrf2-ARE和NF-κB信号通路密切相关；研究还证明了由微生物介导的色氨酸-犬尿酸途径参与了氧化应激诱导的卵巢氧化应激和卵泡闭锁的过程。色氨酸代谢主要通过色氨酸-犬尿酸途径，也可以通过合成五羟色胺和褪黑素进而参与卵巢功能的调控。本研究发现氧化应激会改变色氨酸代谢，使之更倾向于色氨酸-犬尿酸代谢途径，从而降低血清五羟色胺和褪黑素水平，进一步加剧卵巢氧化应激。这些都为后续的研究和探索提供了新的思路。本研究对揭示氧化应激与卵泡闭锁的分子机制具有重大理论意义，并对提高蛋鸡和种鸡短期和长期生产性能和繁殖力具有重要的实践意义。