HO-1对铅所致神经细胞凋亡的保护作用及其分子机制研究

This study is designed to explore the dose-response characteristics and time-course characteristics of HO-1 in response to lead exposure by means of primary culture of the hippocampus, quantitative real-time RT-PCR and western blot. Cell transfection will be used to induce HO-1 over-expression in SH-SY5Y cell to study whether HO-1 offers protection effects against lead-induced neurons apoptosis. In addition, the down-stream gene products of HO-1 will be inhibited by means of specific enzyme inhibitors to explore the molecular mechanism through which HO-1 elicit protection against lead toxicity. On the other hand, HO-1 knockdown will be applied by siRNA to study whether the lead induced apoptosis is enhanced accordingly, thus to confirm that the protection effect is HO-1-dependant. HO-1 is actively induced by CoPP, an activator of HO-1, and is inhibited by ZnPP, to explore the protection effects of HO-1 against lead-caused damage in CNS in vivo by means of immunochemistry, in situ hybridization, atomic absorption spectrometer and flow cytometry technique,adding further evidence to support the results obtained in vitro. It is supposed to provide scientific basis on both the application of molecular intervene point to antagonize lead neurotoxicity and the deep elucidation of lead neurotoxicity through this study.

本项目拟采用海马神经细胞原代培养技术、Quantitative Real-time RT-PCR、western blot方法揭示铅暴露对海马神经细胞内HO-1转录和表达的剂量和时间效应特征。通过基因转染诱导HO-1过表达以研究其对铅所致SH-SY5Y细胞凋亡(流式细胞术)的保护作用，辅以特异性酶抑制剂抑制HO-1下游基因产物酶活性，研究该保护作用的分子机制。通过siRNA介导HO-1表达沉默是否加重铅毒效应来反证该保护作用的HO-1依赖性。通过HO-1的诱导剂（CoPP）和抑制剂(ZnPP)诱导SD大鼠体内HO-1水平改变，采用免疫组织化学、原位杂交、原子吸收和流式细胞术等方法研究体内条件下HO-1对铅致神经系统损伤的保护作用，并验证体外研究的结果。通过上述研究，可望为HO-1作为对抗铅神经毒作用的分子干预位点以及深入阐明铅神经毒作用机制提供科学依据。

铅是对人类危害最为严重的污染物之一，污染来源非常广泛。流行病学资料表明，慢性低水平的铅暴露不但影响儿童的智力发育，还影响行为能力和听觉等多方面的神经系统发育过程，其影响长远而严重。但迄今为止，对于铅所致包括神经细胞凋亡在内的神经毒性作用的确切机制以及可能的干预位点仍未完全明了。. 作为Nrf2下游调控蛋白之一的HO-1，在铅暴露过程中亦可受到其它细胞信号转导系统的调控，如P38, ERK1/2,和PI3K/AKT信号转导系统是以ROS依赖性方式介导铅所致HO-1的转录和表达异常，而JNK信号通路可能是以ROS非依赖性方式介导。诱导HO-1在细胞内过表达可明显减轻铅所致的神经细胞ROS的产生和细胞凋亡发生，相反地，通过敲降HO-1细胞内表达，铅所致细胞内ROS产生以及后续发生的细胞凋亡则更为严重，从而从正反两方面证实了HO-1在铅所致神经毒性中可起到重要的细胞保护作用。并且发现，HO-1的细胞保护作用是通过pre-Caspase 3、Bcl-2以及Bax等凋亡相关蛋白实现。后续动物实验中发现，经t-BHQ处理后的大鼠海马和皮层细胞中Nrf2的核转移明显增加并至少引发HO-1和NQO1的基因转录上升，该过程极可能介导了与此同时观察到t-BHQ可减轻铅所致海马和皮层氧化还原状态失衡以及细胞凋亡。此外还发现，HO-1的另一种生物激活剂Hemin亦可减轻PbAc导致的细胞死亡，从而从多角度证实了HO-1在铅神经毒作用中所扮演的重要角色，为铅中毒的分子干预位点探索提示了明确的方向。同时本研究结果也为铅神经毒作用机制的深入阐明提供了极有价值的科学依据。