Nrf2通路在MA与HIV-Tat蛋白协同诱导多巴胺能神经元自噬中的作用及机制

Methamphetamine (MA) abuse and human immunodeficiency virus (HIV) infection are the most serious public health problems all over the world. Recent studies have indicated that MA and HIV-Tat protein have obvious synergistic effects on neuronal damage. HIV-Tat protein and methamphetamine abuse can lead to degeneration, necrosis and apoptosis of neurons. Nuclear factor-E2 - related factor (Nrf2) plays a critical role in the regulation of cellular redox status. The Nrf2-ARE pathway is considered as the new antioxidant pathway, and the antioxidant’s effects involve in antioxidative stress, regulating inflammatory injury, anti-apoptosis and so on. Our previous studies found that methamphetamine and HIV-1 Tat protein could induce synergistically dopaminergic neuron's autophagy, and the oxidative stress might be the basis of autophagy, but the synergistic mechanisms are not clear yet. In vivo and in vitro, the present study aims to detect the level of oxidative stress, autophagy and autophagic signaling pathway changes, as well as to observe the downstream target genes of Nrf2-ARE signaling pathway and regulation effect by electron microscope, double immunohistochemical staining, confocal fluorescence microscopy, flow cytometry, HPLC, RT-PCR and Western blotting. The aim is to clear out the role of oxidative stress, the signal rugulation mechanism and protective effect of Nrf2 pathway in dopaminergic neuron's autophagy induced synergistically by methamphetamine and HIV-1 Tat protein. We are looking forward to find a new target in the methamphetamine abuse and HIV (AIDS Dementia Complex, ADC).

甲基苯丙胺（MA）滥用和HIV感染是当今全球最严重两大公共卫生问题，MA与HIV-Tat蛋白对神经元损害有着显著的协同作用，均能导致神经元变性、坏死和凋亡。Nrf2-ARE是一种新型抗氧化信号通路，具有抗氧化应激、调节炎症损伤、拮抗细胞凋亡等作用。我们前期研究发现MA与HIV-Tat蛋白能协同诱导多巴胺能神经元自噬，氧化应激可能是其自噬的基础，但协同机制尚不明确。本研究采用综合方法包括电镜、免疫双标、共聚焦荧光显微镜、流式细胞仪、高效液相、RT-PCR及蛋白印迹等技术，从体内和体外水平检测氧化应激因子、自噬及相关自噬信号通路改变，明确氧化应激的作用，并观察Nrf2-ARE信号通路及调控的下游靶基因影响，以阐明Nrf2在MA与HIV-Tat蛋白协同诱导多巴胺神经元自噬的保护作用及其信号调控机制，为毒品滥用的HIV感染者（特别是艾滋病痴呆）提供新的干预靶标。

甲基苯丙胺（Meth）滥用和HIV感染是当今全球最严重两大公共卫生问题。云南滥用毒品人数逐年增加，毒品滥用群体常伴HIV高发。研究表明，甲基苯丙胺可显著增加HIV感染者神经症状及HIV痴呆发生率，二者对神经系统损伤具有协同作用，均能导致神经元变性、坏死和凋亡。Nrf2是一种新型抗氧化信号通路，具有抗氧化应激、调节炎症损伤、拮抗细胞凋亡等作用。我们前期研究发现Meth与HIV-Tat蛋白能协同诱导多巴胺能神经元自噬，但协同机制尚不明确。本项目从体内和体外水平检测氧化应激因子、自噬及相关自噬信号通路改变，明确氧化应激的作用，阐明Nrf2信号通路在调控Meth与HIV-Tat蛋白协同诱导多巴胺能神经元自噬中的机制。我们发现，Meth与HIV-Tat蛋白协同诱导多巴胺能神经元凋亡和自噬，自噬蛋白LC3Ⅱ与Beclin-1表达增加，且具有时间和剂量依赖性；3-MA能够抑制两者诱导的细胞自噬，而Rapa则促进了两者诱导的细胞自噬，早期自噬在两者协同诱导细胞凋亡过程中起到保护的作用；Meth与HIV-Tat蛋白协同诱导细胞氧化应激损伤，ROS水平升高，SOD1和GPX1蛋白水平降低，表明氧化应激在Meth与HIV-Tat蛋白诱导的多巴胺能神经元自噬中扮演重要作用；明确了自噬信号通路ATG5、ATG7及p-mTOR参与了两者协同诱导的自噬的机制；Meth与HIV-Tat蛋白作用细胞后Nrf2蛋白表达下调，并转移入核内，阐明了Nrf2信号通路参与抗氧化应激反应来调控Meth与HIV-Tat蛋白协同诱导多巴胺能神经元自噬。提前使用抗氧化抑制剂N-乙酰半胱氨酸酰胺（NACA）和人参皂苷Rb1，发现ROS水平降低，SOD1和GPX1蛋白水平升高，表明NACA和人参皂苷Rb1可以调控Meth与HIV-Tat蛋白协同诱导多巴胺能神经元自噬。上述研究对揭示毒品危害与HIV感染者的神经生物学机制、预防和干预具有重要的科学意义和社会价值。