甲基苯丙胺肝脏毒性对血脑屏障通透性的影响及其机制研究

Methamphetamine (METH)-induced neurotoxicity is conventionally thought to originate in the brain which can also compromise blood-brain barrier (BBB) function and increase the BBB permeability. Considerable research has been conducted on the direct effect of METH on central neuronal system (CNS). Due to the metabolic dependence of the brain on peripheral organ function, peripheral organ damage may contribute to the neurological damage. METH has been demonstrated to produce liver damage. However, it is unknown if liver damage mediates the neurobiological actions of METH despite knowledge that it can compromise CNS function as evidenced in hepatic encephalopathy. Our previous study, using forensic autopsy cases, showed that higher expression of MMP9 and lower expression of claudin5 in fatal METH intoxication cases, indicating the increase in BBB permeability. In addition, fatal METH intoxication cases showed poor liver function. In this study, in vitro and in vivo methods will be utilized to exam the contribution of peripherally derived ammonia, MMP9 and TNF-α on BBB permeability. Furthermore, liver damage in fatal METH intoxication cases and the expression levels of MMPs, inflammatory cytokines and tight junction proteins in cerebral cortex, hippocampus and striatum will also be investigated. The aims of the proposal are to establish a novel theory of METH-induced BBB damage which will provide evidences for forensic practice and to provide a new potential target for clinical preventing METH-induced neurotoxicity.

传统观点认为METH直接作用于CNS，产生神经毒性，增加血脑屏障通透性；大多数研究集中于METH对CNS的直接损伤。因CNS对外周器官功能的代谢依赖，外周器官受损后CNS也受影响。METH对多个外周器官造成严重损害，其中肝脏损伤最为常见。前期实验发现，METH中毒死亡者肝功受损，且脑内MMP9升高，claudin5下降，提示血脑屏障通透性增加。通过查阅文献，分析认为METH的肝毒性引起的外周血中氨、MMP9及TNF-α升高可能参与了其对血脑屏障的毒性作用。本研究将进行动物和细胞实验，拟采用乳果糖改善METH引起的血氨升高（或加入外源性氨，模拟高血氨环境），SB-3CT阻断MMP9，infliximab阻断TNF-α，研究METH的肝毒性对血脑屏障毒性作用的影响及其分子机制，并用法医METH中毒死亡尸检材料验证假说。从而建立METH血脑屏障毒性作用的新学说，为法医学METH中毒提供新证据。

传统观点认为METH直接作用于CNS，产生神经毒性，增加血脑屏障通透性；大多数研究集中于METH对CNS的直接损伤。因CNS对外周器官功能的代谢依赖，外周器官受损后CNS也受影响。METH对多个外周器官造成严重损害，其中肝脏损伤最为常见。我们实验发现，METH中毒死亡者肝功受损，且脑内MMP9升高，claudin5下降，提示血脑屏障通透性增加。本研究使用乳果糖预处理，发现METH引起的肝脏毒性减轻，降低AST、ALT和血氨水平，METH处理组ZO-1、Claudin-5和Occludin降低，乳果糖预处理后有所恢复，上述结果提示乳果糖预处理可逆转METH导致的血脑屏障通透性的破坏。还发现METH处理组BECN1、LC3-Ⅱ、P62升高，Keap1 下降，总Nrf2升高，活化的caspase 3 和 PARP升高，核内Nrf2和HO-1下降，METH能诱导神经系统发生自噬、凋亡，且降低了抗氧化应激的能力。而用乳果糖预处理后，METH引起的肝毒性减轻，血氨降低，自噬、凋亡缓解，抗氧化应激能力增强，提示乳果糖有神经保护作用。并用法医METH中毒死亡尸检材料进行实验，发现METH中毒死亡者脑中TNF-α，IL-1β和iNOS升高，用法医尸体解剖材料证实了炎性细胞因子在METH中毒后脑中mRNA水平升高，而Nrf2下降，提示抗氧化应激能力下降。