内质网应激在砷致神经细胞毒性中的作用机制及干预研究

Arsenic is a naturally occurring element that is ubiquitously present in the environment. High concentration of naturally occurring arsenic in drinking water is a major health problem in different parts of the world. Arsenic is also a neurotoxical substance. The adverse effect of acute and sub-chronic exposures to arsenic on the nervous system has been receiving more and more attention. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and caspase-12. GRP are the most abundant glycoprotein in ER and play a critical role in regulation of ER. Over-expression antisense and ribozyme approaches in tissues culture systems have directly demonstrated that GRP78 can protect cells against death. The ER is an essential intracellular organelle involved in intracellular calcium homeostasis, in folding and processing of proteins and in cell death activation. Ca2+ release from the ER is mediated through ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R). Changes in ER Ca2+ homeostasis induce the accumulation of unfolded proteins and activate the ER-stress-induced apoptosis pathway.The purpose of the present study was to elucidate the role of ER-mediated apoptotic pathway in the neurotoxic effects induced by sodium arsenite.

砷中毒是一种严重危害人类健康的地方性疾病，新疆是我国砷中毒的高发区之一。针对砷的神经毒性作用，目前尚无有效治疗方法。砷可引发内质网应激，胞内钙离子失衡与砷的细胞毒性密切相关。改变GRP78表达水平，可以阻止疾病的进程。为了深入揭示砷的神经细胞毒性机制，本研究采用神经细胞来源N2a细胞，建立亚砷酸钠染毒细胞模型，Western-blot检测内质网应激相关蛋白表达，在证实砷诱导神经细胞产生内质网应激，通过改变GRP78表达水平，探讨GRP78对砷诱导的神经细胞毒性作用及其分子机制；并进一步探讨阻止内质网Ca2+释放是否能降低亚砷酸钠的神经细胞毒性作用。研究结果对揭示砷的神经毒性作用有重要意义，为地方性砷中毒的防治提供新思路和新靶点。

本研究采用稳定表达EYFP-ER的CHO细胞做为研究对象，将pEYFP-ER质粒转染CHO细胞后，内质网可被EYFP-ER标记。将NaAs02作用于稳定表达EYFP-ER的CHO细胞后，观察NaAs02对内质网结构的影响，NaAs02处理的细胞中可见内质网呈空泡状或颗粒状改变。将NaAs02作用于N2a细胞不同时间后，免疫印迹方法检测NaAs02染毒不同时间(12h、24h、36h、48h)后GRP78和激活型caspase-12表达水平，染砷24h的细胞，GRP78表达水平升高，染砷48h后，GRP78表达水平进一步升高，并可检测到激活型caspase-12。证实NaAs02引发内质网应激。以此为基础，通过建立大鼠染砷模型，使用内质网Ca2+通道IP3R阻断剂2-APB和RyR阻断剂Dantrolene作用大鼠，明确2-APB和Dantrolene对砷的神经毒性和骨质疏松的保护作用｡10mg/kg NaAsO2染毒组的海马神经元出现核膜凹陷，并有线粒体肿胀，Dantrolene与2-APB药物处理组恢复明显｡与染砷组相比，Dantrolene与2-APB治疗组海马组织Bcl-2 蛋白表达增加，Bax ､GRP78及Caspase-12的蛋白表达降低｡与对照组相比，各剂量NaAsO2染毒组大鼠的海马组织的ROS水平，SOD 活力，GSH和MDA 含量均出现显著变化，Dantrolene与2-APB治疗组，海马组织的抗氧化酶水平逐渐恢复，差异具有统计学意义｡与正常对照组相比，NaAsO2可激活海马Caspase-3 酶活性，使用Dantrolene与2-APB药物后，Caspase-3 酶活性显著下降｡Micro-CT检测各项骨小梁指标显示，染毒组与正常组相比骨质疏松明显，使用Dantrolene与2-APB药物后，大鼠骨质疏松程度明显减轻。IP3R阻断剂2-APB和RyR阻断剂Dantrolene能缓解NaAsO2中毒所致的神经损伤和骨质疏松｡究其机制，2-APB和Dantrolene可能通过内质网应激及氧化应激减轻NaAsO2致大鼠的神经毒性作用。