L型钙离子通道在饮水型氟暴露致脑损伤过程中的作用及机制

Fluoride-induced oxygen radical damage theory and calcium contradiction Disease theory have been accepted nowadays, our former study results and relevant literature showed that the overload of Ca2+ within the neurons along with its trigger action of a series of adverse reactions in the downstream calcium signaling pathways was one of the keys in fluoride-induced brain damage, still the mechanism of how fluorosis brings about the overload of Ca2+ within the neurons remains unknown. On the basis of preliminary study and in combination with literature reports, this project first proposes the scientific hypothesis of “ L-type calcium channel might be one of the key links in fluoride-induced brain damage” and among the first to set L-type calcium channel as the observation point, also this study will combine chronic and subchronic fluorous exposure in SD rats in vivo with model of fluorosis cells in vitro, After the above experiments in vivo, using the open-field behavior reaction tank and Morris water maze to observe the alternations of the learning and memory behavior in all groups, and then we will combine western blot, RT-PCR with patch-clamp technique, detect the level of current intensity　of L-type calcium channel in hippocampal area nerve cells 、synaptosomes [Ca2+]、 the expression level, changes of Cav1.2 along with CaMKⅡ、c-Fos and Bel-2/Bax at both gene and protein 、 cell apoptosis and morphology, etc . In addition, we will further verify the scientific hypothesis by L-type calcium channel blocker nifedipinor and agonist BayK8644 in this project. We will detect the expression levels of some above-mentioned subunits and downstream genes/proteins in L-type calcium channel and cell apoptosis,etc. by experiments in vitro. This project attempts to explore the function and the mechanism of L-type calcium channel in fluoride-induced brain damage, evaluate the effect of the L-type calcium channel antagonist nifedipine on reducing fluoride-induced brain damage, provide basic data for the pathogenesis of fluoride-induced brain injury and prevention research, and further improve the fluoride-induced oxygen radical damage theory and calcium contradictory disease theory based on molecular level.

氟致氧自由基损伤和钙矛盾疾病学说已被公认，我们前期研究及相关文献均表明，神经元内Ca2+超载及其触发的下游钙信号通路上一系列有害反应可能是氟致脑损伤关键环节之一，但氟中毒致神经细胞内钙超载机理尚未明了。本项目首次拟以L型钙离子通道为观测点，将在体SD大鼠慢性、亚慢性氟暴露和体外海马神经元培养实验相结合，先观测在体动物脑功能变化，再率先应用膜片钳技术，结合蛋白印迹、RT-PCR等技术，检测氟致大鼠脑海马CA1区神经细胞L型钙离子通道电流强度、突触体内[Ca2+]、Cav1.2、CaMKⅡ、c-Fos和Bel-2基因/蛋白表达水平、神经细胞的凋亡和形态结构变化及L型钙离子通道拮抗剂/激动剂的影响，探讨L型钙离子通道在氟致脑损伤过程中的作用，评价L-型钙离子通道拮抗剂降低氟致脑损伤的效果，为氟致脑损伤发病机制和防治研究提供基础资料，从分子水平完善氟中毒致氧自由基损伤和钙矛盾疾病学说。

氟致钙矛盾疾病理论逐渐引起关注，但其具体分子机理尚未明了。本项目首次以L型钙离子通道为观测点，在体ICR小鼠氟暴露和体外PC12细胞培养实验相结合。在体ICR小鼠为慢/亚慢性氟暴露两部分，分对照、高氟(30/L)、低氟(5mg/L)、高/低氟+L-型钙通道激动剂注射组(FPL64176)、高/低氟+L-型钙通道拮抗剂注射组(Nifedipine)7组。结果表明，亚/慢性氟暴露能显著损伤小鼠学习记忆行为，并使其脑海马CA1区凋亡细胞数增加、脑海马内L-Ca2+通道Cav1.2基因/蛋白表达异常、抗凋亡Bcl-2基因/蛋白表达水平下降、促凋亡Bax基因/蛋白表达水平上升等，且上述分子表达水平与氟暴露剂量/时间呈正相关，而Nifedipine可逆转氟暴露致小鼠上述行为损伤及分子表达异常。PC12细胞[采用高氟(5.0mM)/低氟(0.5mM)联 合L型钙离子通道激 动剂FPL64176(0.5umol/L)/拮 抗剂Nifedipine(0.2umol/L)暴露]的实验结果表明，氟暴露可降低细胞存活率，使细胞内活性氧水平、Ca2+水平和细胞凋亡水平上升，并呈一定的剂量依赖性，氟暴露对PC12细胞L-钙离子通道Cav1.2、凋亡调节相关分子表达水平的影响基本与在体ICR小鼠实验结果一致。用脑片膜片钳技术检测了慢/亚慢性高氟(30/L)/低氟(5mg/L)暴露对ICR小鼠脑海马CA1区椎体神经元L型钙离子通道电流强度等的影响，结果表明，氟中毒小鼠脑海马CA1区锥体神经细胞L型钙离子通道电流峰值明显增加，电流相对值和稳态系数有显著性变化；电流失活时间与尾电流时间极显著增加等。上述结果提示，氟暴露致神经细胞L-Ca2+通道过度开放，引起细胞内钙超载，使其下游凋亡调节分子表达异常，导致细胞凋亡加剧，引起而神经细胞的损伤，最终引起脑功能(学习记忆行为)损伤，L-Ca2+通道抑制剂Nifedipine能够逆转上述氟暴露致神经细胞发生的分子事件而改善氟致神经细胞和脑功能损伤。Cav1.2分子可能是氟中毒治疗的潜在分子靶点，L型钙离子通道抑制剂Nifedipine可能是一种新型抗氟药物，0.2umol/L和5mg/kg•d分别是本研究得到的Nifedipine作用于PC12细胞培养和在体ICR小鼠的有效抗氟剂量。地氟病防治中要注意防范低氟暴露(5mg/L)脑健康风险。