阿司匹林抑制TMEM16A钙激活氯通道调控滋养细胞氧化应激预防子痫前期的机制研究

Trophoblast oxidative stress is one of the pathological mechanisms of hypertensive disorders during pregnancy, especially pre-eclampsia (PE). Aspirin can effectively reduce the risk of PE, however its target and mechanism remains unclear. Our previous study found that TMEM16A calcium-activated chloride channel involves in oxidative stress and promotes the incidence of hypertension. Simultaneously, in placental trophoblast, the expression of TMEM16A increased, antioxidant factors such as SOD and GPx decreased, and cell invasion decreased in PE. Aspirin reduced the expression of TMEM16A and inhibited above-mentioned process in trophoblast of PE. We hypothesize that aspirin may play a role in preventing PE by inhibiting TMEM16A calcium-activated chloride channel on regulation of trophoblast oxidative stress. This project aim to choose TMEM16A calcium-activated chloride channel as the target based on the previous works, and breed TMEM16A placenta-specific transgenic and knockout mice, to explore the prophylaxis mechanism of aspirin for PE by inhibiting trophoblast oxidative stress through TMEM16A, and to provide new target and theoretical basis for PE prevention, by clinical specimens, in vitro cell models and animal models of PE.

滋养细胞氧化应激是妊娠期高血压疾病尤其是子痫前期（PE）的病理机制之一。阿司匹林可有效降低PE的发生风险，其作用靶点及机制仍未阐明。我们前期研究发现，TMEM16A钙激活氯通道参与了氧化应激反应，促进了高血压的发病。而预实验发现子痫前期胎盘滋养细胞TMEM16A表达增加，抗氧化酶SOD，GPx活性下降，滋养细胞侵袭力下降，阿司匹林通过降低TMEM16A表达从而逆转上述过程。因此，我们设想阿司匹林通过抑制TMEM16A钙激活氯通道从而调控滋养细胞氧化应激水平并在预防PE中发挥作用。本项目拟在前期工作基础上以TMEM16A钙激活氯通道为切入点，并运用TMEM16A胎盘特异性转基因及敲除小鼠，从临床标本、体外细胞模型和PE动物模型多方面着手探讨阿司匹林通过TMEM16A钙激活氯通道抑制滋养细胞氧化应激从而预防PE的机制，为子痫前期的预防和治疗提供新的靶点及理论依据。

临床上运用最为广泛的预防子痫前期（Pre-eclampsia）的药物阿司匹林作用机制仍不明确。众所周知，PE的发病机制涉及氧化应激及滋养细胞过度凋亡。TMEM16A是Ca2+激活Cl-通道（CaCC）的分子基础，研究表明TMEM16A促进氧化应激及高血压的发生。CREB是一种广泛表达的环磷酸腺苷（cyclic adenosine monophosphate，cAMP）下游转录因子。CREB的磷酸化激活可促进Bcl-2蛋白的表达，抑制细胞凋亡，可能抑制高血压的发生发展。本研究采用免疫荧光，western blot，transwell，TUNEL细胞染色，免疫组化和流式细胞仪技术等实验手段；动物方面，我们构建了L-NAME诱导的PE大鼠；细胞方面，我们使用了滋养细胞系HTR-8/SVneo细胞，并给予TNF-α 进行刺激诱导氧化应激模型，H2O2进行刺激诱导凋亡模型，并使用低剂量阿司匹林（Low-dose aspirin，LDA）预处理。研究结果显示：（1）在TNF-α诱导的滋养细胞HTR-8/SVneo细胞模型中，LDA抑制氧化应激，促进Nrf2细胞核转位，促进细胞侵袭；（2）在人PE胎盘组织中，TMEM16A表达水平升高；（3）通过TMEM16A 过表达腺病毒转染HTR8/SVneo细胞发现TMEM16A促进氧化应激，LDA阻断TMEM16A对滋养细胞氧化应激的效应；（4）LDA 抑制人及大鼠PE胎盘滋养细胞线粒体途径凋亡；（5）在细胞实验中，LDA 抑制滋养细胞线粒体途径凋亡；（6）通过对人PE胎盘组织，PE大鼠胎盘组织，及CREB干扰链作用于HTR-8/SVneo细胞进行研究，发现LDA促进滋养细胞CREB磷酸化，抑制凋亡。该项目从滋养细胞的角度，探讨了TMEM16A及CREB在PE的氧化应激及凋亡中的作用，为PE的预防提供新的靶点及实验室依据。