基于镉诱导氧化应激的神经细胞自噬体积累介导凋亡及调控分子机理研究

Oxidative stress-induced neuronal cell apoptosis is closely associated with neurodegenerative diseases such as Parkinson’s disease, Alzheimer's disease, etc. The previous work from our Lab found that cadmium (Cd)-induced oxidative stress results in reactive oxygen species (ROS) generation and activates mammalian target of rapamycin (mTOR)/mitogen-activated protein kinases (MAPKs) pathways contributing to neuronal apoptosis; Cd-induced autophagosome accumulation was associated with apoptosis of neuronal cells. The aim of this study is, by experimental models for Cd exposure, to deeply investigate the relation of Cd-induced autophagosome accumulation to core autophagy protein expression, autophagy flux and their role in cell apoptosis, and to dissect manifestations of Cd-induced autophagosome, autophagy flux and apoptosis post upregulation or downregulation of NOX2 and its regulatory proteins including p22phox、p40phox、p47phox、p67phox、Rac1 in NADPH oxidases 2 (NOX2) complex, as well as signal molecules such as Akt, protein phosphatase 2A (PP2A), protein phosphatase 5 (PP5), extracellular signal-regulated kinase (Erk1/2) and c-Jun N-terminal kinase (JNK) in neuronal cells, respectively, to elucidate moleculer mechanism of autophagosome accumulation mediatation of apoptosis in neuronal cells of Cd-induced oxidative stress. Then, the study on signal transduction pathways involved in rapamycin prevention of Cd-induced neuronal ROS production and hence intervening autophagy accumulation against apoptosis are conducted to reveal molecular mechanism (s) for its regulative actions, as well as its significance and applied value, thereby giving new idea and theory for prevention of Cd-induced neurodegenerative diseases.

氧化应激诱导神经细胞凋亡与神经变性相关疾病如PD、AD等发生有密切关系。本课题组前期工作发现，镉诱导氧化应激ROS产生和激活mTOR/MAPKs通路致神经细胞凋亡；镉诱导神经细胞自噬体积累与凋亡有关系。本项目拟通过镉实验模型，深入探索镉诱导神经细胞自噬体积累与核心自噬相关蛋白表达、自噬流的关系及其在细胞凋亡中的作用；分别解析氧化应激NOX2复合物中NOX2及其调节蛋白p22phox、p40phox、p47phox、p67phox、Rac1以及信号分子Akt、PP2A、PP5、Erk1/2、JNK在下调或上调后，镉诱导神经细胞自噬体、自噬流和凋亡表现，阐明镉诱导氧化应激的神经细胞自噬体积累介导凋亡分子机理。然后，研究雷帕霉素抑制镉诱导神经细胞氧化应激干预自噬体积累抗凋亡涉及的信号转导通路，揭示其发挥调控作用的分子机制及其理论意义和应用价值，为防止镉诱导的神经变性相关疾病提供新思路和理论依据。

氧化应激诱导神经细胞凋亡与神经变性相关疾病如PD、AD等发生有密切关系。本项目通过镉染毒神经细胞毒性实验模型，并以PD诱导剂（6-羟多巴胺、MPP+、鱼藤酮）染毒神经细胞PD模型作为阳性对照实验，综合研究了镉/PD诱导剂诱导神经细胞自噬体积累/减少与核心自噬相关蛋白表达、自噬流的关系及其在细胞凋亡中的作用；解析了氧化应激NOX2复合物中NOX2及其调节蛋白p22phox、p40phox、p47phox、p67phox、Rac1以及信号分子Akt、mTOR、AMPK、PP2A、PP5、JNK、XIAP在下调或上调后，镉/PD诱导剂诱导神经细胞自噬体、自噬流和凋亡表现；开展了镉/PD诱导剂诱导钙信号转导和/或ROS/H2O2产生激活或抑制AMPK、PP5、JNK和/或XIAP通路关联自噬/自噬流异常导致神经细胞凋亡的分子机理研究。其中每个环节包含了探讨雷帕霉素、NAC、二甲双胍、白藜芦醇的调控作用及其机理。结果显示：镉通过激活Akt介导Beclin 1磷酸化损伤自噬流导致自噬体积累依赖的神经细胞凋亡；镉通过Ca2+依赖激活JNK通路损伤自噬呈现自噬体增加和Atg表达异常，进而导致神经细胞凋亡；在镉模型中，镉诱导神经细胞NOX2及其调节蛋白p22phox、p40phox、p47phox、p67phox、Rac1上调引发ROS通过PP2A/PP5-JNK信号转导阻断自噬流导致自噬体积累依赖的凋亡，这被ROS清除剂NAC有力地阻滞；而在PD模型中，6-OHDA、MPP+或鱼藤酮上调NOX2、p22phox、p40phox、p47phox、p67phox、Rac1诱导H2O2通过妨碍AMPK/Akt-mTOR信号通路导致神经细胞死亡、或激活PTEN和抑制Akt致神经细胞自噬抑制依赖的凋亡；镉诱导线粒体ROS抑制XIAP通路致神经细胞凋亡。研究证明，雷帕霉素调控XIAP通路改善镉诱导神经细胞自噬流损伤抗凋亡；二甲双胍通过抑制镉诱导神经细胞ROS依赖的PP5/AMPK-JNK通路以及通过改善镉诱导神经细胞自噬体积累抗凋亡。这些证据强烈地呈现了靶向调控ROS/H2O2、Ca2+信号、AMPK、Akt/mTOR、PP2A、PP5、JNK和/或XIAP信号转导网络，应用NAC、雷帕霉素、二甲双胍、白藜芦醇改善镉诱导氧化应激和/或自噬异常可以为防止镉/氧化应激诱导的神经变性疾病开发利用。