褪黑素通过Nrf2/ARE信号通路保护大鼠完整冷冻移植卵巢的功能及其作用机制

Whole ovarian cryopreservation and transplantation has been suggested as a future effective way of fertility preservation in young patients with cancer. However, the research is still in the initial stage, and the successful rate is low. Melatonin, a powerful antioxidative hormone, has protective effects on ischemia/reperfusion injury of organ transplantation. Our researches improved cryopreservation protocols and successfully performed whole ovarian transplantation to chemotherapy-induced premature ovarian insufficiency model rats, with recovery endocrine function and successful delivery postoperatively. It was found that melatonin could enhance the antioxidant defense of transplanted ovary. And melatonin could increase the Nrf2 mRNA expression in ovarian granulosa cells induced by H2O2. MT1 receptor was found in ovarian tissue. But the mechanism of melatonin is unclear. We intend to inject melatonin into the rats with whole ovarian transplantation in perioperative period. Ovarian hormones and oxidative stress indicators are being detected to explore the effect of melatonin against oxidative stress. MT antagonist and Nrf2 inhibitor are being applied to explore the mechanism of melatonin based on Nrf2 signaling pathway. H2O2 induced oxidative stress mode are being built in vitro to investigate the role of melatonin in protecting oxidative stress via MT1/Nrf2/ARE signaling pathway, in order to provide a theoretical basis for reserving reproductive endocrine in cancer patients.

完整卵巢冷冻移植是年轻肿瘤患者恢复卵巢功能的一种极具潜力的方法，但目前成功率较低。褪黑素可效保护器官移植，且可能通过MT/Nrf2/ARE信号通路发挥抗氧化应激作用。课题组前期研究优化冷冻方案，成功建立大鼠完整卵巢冷冻移植模型，在术后恢复内分泌及生育功能；研究发现褪黑素可提高冷冻移植卵巢的抗氧化能力，并提高卵巢颗粒细胞氧化应激后的Nrf2表达；卵巢组织中存在MT1受体。但褪黑素在卵巢冷冻移植中的作用机制不明，因此本课题拟在大鼠完整卵巢冷冻移植前后给予褪黑素，通过检测卵巢内分泌功能、氧化应激指标探讨褪黑素对完整卵巢移植的保护作用，给予MT拮抗剂、Nrf2抑制剂探讨褪黑素通过MT1/Nrf2/ARE信号通路发挥作用的机制；并应用H2O2诱导构建大鼠卵巢颗粒细胞氧化应激模型，体外探讨褪黑素通过MT1/Nrf2/ARE信号通路抗氧化应激的作用机制，为保留肿瘤患者生殖内分泌提供理论依据。

完整卵巢冷冻移植是年轻肿瘤患者恢复卵巢生殖内分泌功能的一种极具潜力的方法，但目前成功率较低。本项目前期研究成功建立优化的大鼠完整卵巢冷冻移植模型，在术后恢复内分泌功能并分娩；并且褪黑素可以提高冷冻移植卵巢组织的抗氧化应激指标，有效保护大鼠完整冷冻移植卵巢，促进大鼠术后生殖内分泌功能的恢复。因此本项目进一步研究褪黑素的作用机制，发现在完整卵巢冷冻移植大鼠中注射褪黑素，可使卵巢组织中氧化应激指标MDA、ROS表达水平降低，羟自由基清除率升高，SOD、GSH表达水平升高，并且随着时间延长、剂量增大，褪黑素对完整卵巢移植的保护作用也显著提高。另外，高剂量褪黑素还可以使大鼠卵巢组织中MT1受体表达显著增加，胞浆Nrf2蛋白表达水平降低，而胞核Nrf2表达水平升高。但给予非选择性褪黑素受体拮抗剂则降低褪黑素对卵巢组织的保护作用，血清ROS水平显著提高，HO-1表达显著降低，雌激素水平显著降低；但给予MT2特异性拮抗剂并不影响褪黑素对卵巢组织的保护作用。给予Nrf2特异性抑制剂也降低褪黑素对卵巢组织的保护作用，SOD1、HO-1基因表达降低，HO-1蛋白表达降低，胞核Nrf2蛋白表达水平下降。应用H2O2诱导构建大鼠卵巢颗粒细胞氧化应激模型的体外试验得到类似的结果。因此，本项目提示褪黑素通过MT1/Nrf2/ARE信号通路发挥抗氧化应激作用，降低完整卵巢移植的缺血再灌注损伤，保护完整卵巢冷冻移植的生殖内分泌功能恢复，提高移植成功率，这为肿瘤患者行完整卵巢冷冻移植以保留卵巢的生殖内分泌功能提供了理论依据。