环孢素A保护人滋养细胞免于氧化损伤的分子机制

In a normal pregnancy, the oxides and antioxidants remain in balance. When the balance is disrupted towards an overabundance of oxides, the oxidative stress occurs. Oxidative stress affects multiple physiological processes from oocyte maturation to fertilization, embryo development and pregnancy. There is growing literature on the effects of oxidative stress in female reproduction with involvement in the pathophysiology of miscarriage, preeclampsia, fetal growth restriction and other situations such as free radical-induced birth defects. Thereby, strategies to overcome oxidative injury are very important for maintaining a viable pregnancy. Our previous studies in vitro have shown that Cyclosporin A (CsA) at low concentrations can promote the proliferation and invasiveness of human first-trimester trophoblast cells through the epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK) signaling pathway. Our recent work demonstrated that focal adhesion kinase (FAK) pathway was also involved in CsA-enhanced trophoblast migration and invasion. In our current experiments, we found that CsA could decrease the serum starvation and hydrogen peroxide-induced trophoblast apoptosis, and restore hydrogen peroxide-reduced FAK and Src activation in JEG-3 cells. However, the molecular mechanism of CsA protecting trophoblast cells from oxidative injury remains unclear. In this study, we plan to explore whether CsA could enhance the viability of human trophoblast cells by reducing the oxidative stress level, whether CsA could improve the function of mitochondria to reduce trophoblast reactive oxygen species (ROS) production, regualte the expression of apoptosis and proliferation related proteins to reduce trophoblast apoptosis and improve proliferation through FAK, Src and the downstream ERK and PI3K/AKT signaling pathways under oxidative stress, and further verify the protective effect of CsA via murine abortion-prone matings in vivo. This study will help to enrich the understanding of the intracellular signalling pathways of CsA, and at the same time provide the theoretical basis for developing new strategies to prevent and treat the oxidative injury related pregnancy disorders.

妊娠时机体氧化物和抗氧化物间维持的平衡状态若被打破，则会发生流产、子痫前期等疾病。因此，克服氧化损伤对正常妊娠的维持具有重要意义。本课题组首次发现，低浓度环孢素A（CsA）通过增强表皮生长因子受体（EGFR）、粘着斑激酶（FAK）信号通路升高ERK活化水平，促进滋养细胞增殖与侵袭。我们新近研究发现，CsA可减少血清饥饿和过氧化氢诱导的滋养细胞凋亡，升高FAK、Src活化水平。但是，CsA减轻滋养细胞氧化损伤的分子机制还不明确。本课题以氧化应激为切入点，研究CsA是否通过降低氧化应激水平改善滋养细胞增殖；是否通过FAK、Src及下游的ERK、PI3K/AKT通路改善线粒体功能减少滋养细胞活性氧（ROS）产生、调节凋亡增殖相关蛋白表达减少细胞凋亡、改善增殖，并应用小鼠模型验证CsA的保护作用。本课题将有助于丰富CsA细胞内信号通路的认识，同时为制定氧化损伤相关妊娠疾病的防治新策略提供理论依据。

正常妊娠过程中，机体的氧化系统和抗氧化系统保持相对平衡，如果这种稳态被打破则会导致氧化应激损伤。近年来研究发现，氧化损伤参与多种病理性妊娠的发病，与流产、先兆子痫、胎儿生长受限等妊娠并发症密切关联。因此，控制氧化损伤对于正常妊娠的维持具有重要意义。环孢素A（CsA）是具有划时代意义的免疫抑制剂，其临床应用可显著改善实体器官移植的近期存活率，是器官移植后免疫抑制和抗排斥反应的首选药物。我们课题组既往研究发现，低浓度CsA可提高原代人正常滋养细胞的增殖与侵袭力，抑制低血清培养诱导的滋养细胞凋亡，并改善小鼠流产模型的妊娠结局，提示CsA可能为滋养细胞功能障碍导致的妊娠疾病的潜在治疗药物。因此，本课题以氧化应激为切入点，进一步研究CsA在氧化应激状态下是否减轻滋养细胞的氧化损伤，并展开对其分子机制的研究。我们发现：（1）JEG-3细胞经500μM H2O2处理24 h可建立滋养细胞氧化损伤的体外模型；（2）粘着斑激酶（FAK）和Src信号通路及MAPK信号通路参与滋养细胞氧化应激损伤；（3）低浓度CsA可以通过促进H2O2处理后JEG-3细胞FAK和Src之间的相互活化缓解滋养细胞氧化损伤；（4）不明原因自然流产妇女血清的氧化水平升高，CsA可减少自然流产妇女血清诱导的滋养细胞凋亡。此外，我们还发现：（1）环孢素A可促进人滋养细胞侵入并取代活化的人脐静脉内皮细胞，从而可能参与改善子宫螺旋动脉的重塑；（2）环孢素A通过活化粘着斑激酶（FAK）信号通路促进人滋养细胞的体外生长；（3）CD147在不明原因自然流产患者绒毛组织的表达水平下降，可能通过参与调控滋养细胞的侵袭能力维持正常妊娠。这些研究结果有助于拓展CsA细胞内信号通路和临床应用范围的认识，同时为制定氧化损伤相关妊娠疾病的防治新策略提供理论依据。