环境激素壬基酚以GPR30受体部分激动剂方式通过Ca2+信号途径干扰雌激素心肌保护作用的研究

Nonylphenol (NP), one of the representative environmental endocrine disrupting chemicals (EDCs), can mimic the effects of estrogen. Estrogen has cardioprotective effect, however, the underlying mechanism is still not very clear, and it is also not known whether NP can interrupt the cardioprotective effect of estrogen. Our previous studies have found that estradiol (E2) inhibited the L-type Ca2+ channel current (ICa) of cardiomyocytes, and that in rat isolated heart ischemia reperfusion (I/R) injury model, E2 showed an obvious cardioprotective effect. Meanwhile, similar to E2, NP also inhibited the ICa. Although both E2 and NP could inhibit ICa, surprisingly, when we applied the mixture of NP and E2 to the perfusion solution of isolated heart I/R model, the cardioprotective effect of E2 was significantly inhibited, indicating that there may be some kinds of interactive relationship between the actions of NP and E2. By comparing the dose-dependent relationship curves of NP and E2, we noticed that NP had a similar effect to E2, but the maximal effect (Emax) obtained with NP is smaller than that with E2. NP is also known as a weak agonist of estrogen receptor (ER) in many reports, therefore we propose a hypothesis that NP may act as a partial agonist of ER to interrupt the effect of E2. Thus, when E2 is absent or at low concentration, NP may act as a ER agonist; while, when E2 is present, NP may act as an antagonist to block the action of E2. There are three kinds of estrogen receptors (ERalpha, ERbelta and GPR30), among which GPR30 is thought to be the major receptor which mediates the nongenomic rapid response, especially in the acute response in the I/R model of isolated heart. It was reported that during I/R injury the phosphorylation level of calmodulin-dependent kinase II (CaMKII) is increased and intracellular Ca2+ is over loaded. One of the mechanisms underlying the cardioprotective effect of E2 is to reduce the phosphorylation level of CaMKII and to inhibit ICa. In the present research, by employing rat isolated heart I/R injury model and cultured neonatal rat cardiomyocyte hypoxia reoxygenation model, we will observe the changes of CaMKII phosphorylation level, ICa and [Ca2+]i to obtain the dose-response relationships of the effect of E2 and the interrupting effect of NP on them. We will also observe the effects of GPR30 receptor agonist and antagonist, as well as CaMKII inhibitors. Finally, we will check the binding affinity of E2 and NP with GPR30 receptor by using radio-ligand binding assay and estimate the efficacy of E2 and NP by [Ca2+]i level in HEK293 cells transfected with GPR30 cDNA. Based on the experiments designed above, we will try to verify the hypothesis that acting as a partial agonist of GPR30 receptor, NP may interrupt the cardioprotective effect of E2 via the intracellular Ca2+ signaling system. The proposed partial agonist view point will also help us understand the mechanism of EDCs' action.

环境激素壬基酚(NP)有拟雌激素样作用，雌激素有心肌保护作用，然而我们发现NP却能拮抗雌激素的心肌保护作用，因此我们提出了对NP更确切的描述应该是“雌激素受体部分激动剂”的新观点。GPR30受体是介导雌激素非基因组快速反应的主要受体，雌激素的心肌保护作用与下调细胞Ca2+信号系统功能有关。最近我们又通过分子对接模拟，首次发现NP可以与GPR30的中心部位结合。本课题将采用缺血再灌注损伤及细胞缺氧再复氧损伤模型，观察心肌损伤程度、Ca2+电流、细胞[Ca2+]i、钙调蛋白激酶磷酸化水平等的变化，探讨NP对E2心肌保护作用的干扰，并将在多层次的观察指标上绘制量效关系曲线，结合应用计算机辅助分子对接模拟、配体结合实验和动物整体实验，验证我们提出的NP以GPR30受体部分激动剂方式通过细胞内Ca2+信号系统干扰雌激素心肌保护作用的假说，为更确切地认识NP的心血管毒性作用机制提供实验和理论依据。

本课题研究了环境激素壬基酚(Nonylphenol，NP)的心脏毒性作用，并通过观察其对GPR30受体的作用特点探讨了其毒性作用机制。采用心肌缺血再灌注损伤模型和心肌细胞缺氧再复氧模型观察了NP对雌激素（estradiol，E2)心肌保护作用的干扰现象，并通过检测CaMKII磷酸化水平等指标的变化，探讨了NP毒性作用与信号转导变化的关系。采用G protein coupled receptor 30 （GPR30）受体激动剂G1和抑制剂G15探讨了上述作用与GPR30受体的关系；采用分子对接模拟及工具细胞验证了NP对E2与GPR30受体相互作用量效关系的影响。课题基本验证我们提出的NP以GPR30受体部分激动剂方式干扰雌激素心肌保护作用的假说。本课题在现有的基础上，也进行了一定的拓展研究，观察了其他类型环境激素如Bisphenol A（双酚A，BPA）对雌激素心脏保护作用的干扰及机制；研究了NP和BPA在诱导变应性鼻炎中的作用及其对GPR30受体的作用。研究结果表明NP的作用具有GPR30受体半激动剂的特点，是其作为环境激素发挥干扰作用的重要机制，为深入理解NP毒性特点提供了实验数据和理论依据。