锌联合白藜芦醇抑制1型糖尿病引起睾丸组织细胞凋亡的机制研究

Our previous work demonstrated that type 1 diabetes can induce testicular apoptosis predominantly by increasing oxidative stress. The testicular apoptosis probably is one of the important reasons for the infertility of male patients with diabetes. In our work, we also found that zinc deficiency exacerbated diabetic induction of testicular apoptosis, which was accompanied with testicular Akt dysfunction and significant down-regulation of Nrf2 and Sirt1 expression. Resveratrol is a natural anti-oxidant, whether zinc plus resveratrol supplementation could synergistically prevent type 1 diabetes-induced male testicular apoptosis? Our preliminary data phenomenonally supported this notion, therefore, we hypothesizes that the prevention of diabetes-induced testicular Nrf2 and Sirt1 down-regulation may contribute to the synergistical prevention of type 1 diabetes-induced testicular oxidative damage and apoptosis by zinc plus resveratrol supplementation. To test it, we will use Nrf2-KO mice and Sirt1-KO mice to further investigate whether Nrf2 and Sirt1 have direct protection against diabetes-induced testicular apoptosis after zinc plus resveratrol supplementation. This study will provide important experimental and theoretical evidence for clinical application of zinc and resveratrol to prevent diabetes-induced reproductive system damage and male infertility.

申请人前期工作已证实1型糖尿病（T1DM）通过诱导睾丸组织氧化损伤进而引起细胞凋亡，这可能是导致DM男性患者不育的重要原因之一；同时发现锌缺乏加剧细胞凋亡的过程中伴随着睾丸组织Nrf2和Sirt1蛋白的显著降低及Akt的功能异常。白藜芦醇（RSV）是天然的抗氧化剂，补充锌联合RSV能否抑制T1DM引起的睾丸组织细胞凋亡呢？前期工作已得出肯定答案。那么，具体机制如何呢？本研究提出科学假设：补锌联合RSV可能通过阻止T1DM睾丸组织Nrf2和Sirt1蛋白下调,进而抑制T1DM所致的睾丸组织氧化损伤及细胞凋亡。若假设成立，进一步应用Nrf2基因敲除小鼠和Sirt1基因敲除小鼠，对Nrf2和Sirt1在补锌联合RSV抵抗T1DM引起的睾丸组织细胞凋亡过程中是否发挥着直接作用加以验证。该研究将为临床应用锌联合RSV防治DM生殖系统损伤，进而预防DM引起的男性不育提供重要的实验及理论依据。

为了明确白藜芦醇（RSV）联合锌能否减轻1型糖尿病（T1DM）所致睾丸组织细胞凋亡，本课题组分别观察了补充RSV和/或锌对T1DM小鼠和同龄对照小鼠睾丸组织凋亡的影响。我们发现：与对照组相比，T1DM组小鼠睾丸组织中的细胞凋亡和氧化损伤程度明显升高；与此同时，在T1DM组睾丸组织中，糖代谢相关的关键蛋白Akt和GSK-3β功能显著下调，而Akt的负性调节蛋白PTEN, PTP1B和 TRB3的表达显著上调；给T1DM小鼠补RSV后能够部分阻断以上效应，补锌未能够阻断以上效应，补RSV联合锌未能够产生协同作用。进一步研究发现：给T1DM小鼠补RSV后能够激活睾丸组织中的Nrf2并上调其下游基因（如NQO1、HO-1、SOD、catalase及metallothionein）的表达。我们亦发现：RSV引起的Nrf2激活与Keap1的自噬降解密切相关，而Keap1的降解主要依赖于p62的表达上调。以上结果说明：RSV能够减轻T1DM所致的睾丸组织氧化损伤和细胞凋亡，其机制在于：RSV一方面能够通过上调p62促进Keap1的自噬降解、进而使Nrf2与Keap1解离，在进入细胞核；另一方面RSV能够活化Akt进而激活细胞核内的Nrf2，启动Nrf2下游抗氧化元件、发挥抗氧化损伤的作用。