基于NADPH氧化酶研究脑Ang-(1-7)减轻多巴胺能神经元氧化损伤的作用机制

There is now unequivocal evidence that the Ang-(1-7) is a key component of the renin-angiotensin system（RAS）cascade, which counteracts the effects of Ang II. Accumulating evidence suggests that the Ang-(1-7), is an active member of the brain RAS. In our previous studies demonstrated that brain Ang II induce oxidative stress and contribute to dopaminergic neuronal apoptosis via AT1 receptor. Ang-(1-7) exhibited neuroprotective effects by reducing oxidative stress in the neurodegenerative disorder, however, the molecular events are currently unclear. NADPH oxidases are a major source of reactive oxygen species (ROS) and oxidative stress has been suggested to be key factors in the pathogenesis and the progression of the dopaminergic cell death. Ang II enhanced NADPH-derived OS and dopaminergic cell death. More recent in our laboratory, provided evidence that Ang-(1-7) might suppress NADPH-derived OS and attenuate dopaminergic cell death via Mas receptors. Emerging data indicate Ang-(1-7) could play critical roles in Parkinson’s Disease (PD). In the present study, we therefore investigated the cell viability, oxidative damage, NADPH oxidases, Hsp70, α-Synuclein and neuronal apoptosis on the rotenone-induced injury in vivo and in vitro. We evaluated the molecular mechanisms that exogenous Ang-(1-7) could participate in the regulation of NADPH oxidases by introducing several key antagonists on the PD models. The evidence collected in our present study will indicate that, Ang-(1-7) would be viewed as a promising therapeutic tool for the treatment of PD. Further insights in this regard would be obtained by studies aimed to clarify the significant variation in prevention and functional outcome on PD.

Ang-(1-7)是肾素-血管紧张素系统（RAS）系统旁代谢通路的重要活性因子，具有对抗平衡AngⅡ的不良作用。我们前期研究发现脑AngⅡ通过AT1受体激活氧化应激加重多巴胺能神经元凋亡，而Ang-(1-7)却有保护作用，具体机制不清。NADPH氧化酶与RAS密切相关，是活性氧的最主要来源，在神经变性级联反应中起关键性的作用。我们推测“Ang-(1-7)能够调节AngⅡ的毒性作用，参与帕金森病（PD）的发病机制与抑制氧化应激有关，而NADPH氧化酶是其主要的作用靶标”。为验证此假说，本课题采用鱼藤酮诱导损伤制作PD模型，利用免疫学、分子生物学等技术，分别观察Ang-(1-7)对神经元的存活率、胶质细胞活化、Hsp70、氧化损伤、α-Syn及神经元凋亡等的变化及其与NADPH氧化酶的关系。本课题分别从细胞和动物两个层次探求Ang-(1-7)对防治神经元变性损伤的机制，为PD的防治提供新思路。

帕金森病(Parkinson's disease, PD)是一种多见于中老年人的神经系统变性疾病。中枢神经系统存在相对独立的肾素-血管紧张素系统（RAS），随着对脑局部RAS的深入认识，证实脑内RAS与PD发病有密切关系。脑局部RAS发挥独立的功能作用，RAS与PD的发生与发展有密切的关系，参与DA神经元退行性变。血管紧张素-（1-7）[Ang-(1-7)]是RAS系统旁代谢通路的重要活性因子，具有对抗平衡AngⅡ的不良作用。我们前期研究发现脑AngⅡ通过AT1受体激活氧化应激加重多巴胺能神经元凋亡，而Ang-(1-7)却有保护作用，具体机制不清。NADPH氧化酶与RAS密切相关，是活性氧的最主要来源，在神经变性级联反应中起关键性的作用。我们推测“Ang-(1-7)能够调节AngⅡ的毒性作用，参与PD的发病机制与抑制氧化应激有关，而NADPH氧化酶是其主要的作用靶标”。为验证此假说，本课题采用鱼藤酮诱导损伤制作PD模型，利用免疫学、分子生物学等技术，分别观察Ang-(1-7)对神经元的存活率、胶质细胞活化、Hsp70、氧化损伤等的变化及其与NADPH氧化酶的关系。我们最近的研究发现， Ang II通过触发氧化应激参与了PD的发病机制。Ang-(1-7)已被证明可以减轻Ang II在许多疾病中的不良作用。本研究的目的是确定Ang-(1-7)是否对CATH.a神经元具有类似的作用。我们使用鱼藤酮诱导的神经元损伤模型来评估培养的CATH.a细胞系SOD、GSH和ROS水平的变化。我们还评估了单独和联合应用PBS、鱼藤酮、Ang-(1-7)或Mas受体拮抗剂A-779时AT1、AT2、Mas受体和Nox1、Nox2、P47phox、Hsp70的表达。采用qRT-PCR和western印迹法检测AT1、AT2、Mas受体和Nox1、Nox2、P47phox、Hsp70的mRNA和蛋白水平。发现Ang-(1-7)能显著降低鱼藤酮诱导的氧化损伤，提高SOD和GSH的生成。此外，Ang-(1-7)显著提高Mas受体表达，降低NADPH氧化酶活性，这些作用被A-779完全消除。我们的研究结果表明，Ang-(1-7)通过激活Mas受体表达和抑制NADPH氧化酶来减轻鱼藤酮诱导的CATH.a神经元的氧化损伤。本研究表明，Ang-(1-7)有望成为潜在PD的治疗靶标。