自噬抑制剂3-甲基腺嘌呤在纳米氧化铝致斑马鱼幼鱼早期神经毒性中的作用

Role of Autophagy Inhibitor 3-Methyladenine in Early Neurotoxicity of A-lumina Nanoparticles to Zebrafish Larvae

由于纳米氧化铝(alumina nanoparticles,AlNPs)独特的理化性质,被广泛应用于医药、电子和化妆品等多个领域,但关于AlNPs的早期神经毒性效应及其潜在机制尚未完全阐明。为探讨AlNPs的毒作用机制,以及自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3MA)对AlNPs致斑马鱼幼鱼早期神经毒性的影响,将6 hpf(hours post-fertilization)的斑马鱼胚胎分为对照组、3MA组、AlNPs组和AlNPs+3MA组。观察胚胎和幼鱼的形态学变化以及幼鱼的一般毒性,并检测神经行为改变、氧化应激水平以及幼鱼体内自噬相关基因beclin1、lc3Ⅱ和vps34的表达情况。结果表明,各组幼鱼在死亡率、孵化率和畸形率方面无显著性差异。形态学观察结果显示,AlNPs组受精卵在12 hpf和24 hpf出现发育迟缓,加入3MA后,在24 hpf后受精卵发育好转。运动行为检测发现,AlNPs组幼鱼黑暗状态下的平均速度、移动距离和趋触性程度显著降低(P<0.05)。在强光刺激下的惊恐逃避反射实验中,AlNPs组幼鱼在光照时的速度显著降低(P<0.05)。氧化应激水平检测结果显示,AlNPs组超氧化物歧化酶(superoxide dismutase,SOD)活性显著降低(P<0.05),而AlNPs组乳酸脱氢酶(lactate dehydrogenase,LDH)活性显著升高(P<0.05)。自噬相关基因beclin1、lc3Ⅱ和vps34在AlNPs组表达均显著升高,加入3MA后,基因表达降低,并具有统计学意义(P<0.05)。上述结果表明,AlNPs诱导过度自噬的发生造成斑马鱼幼鱼早期神经毒性,而3MA可以减轻AlNPs引起的氧化损伤并下调自噬相关基因的表达,明显改善AlNPs暴露所致的早期神经毒性。

Alumina nanoparticles(AlNPs)are widely used in the medicine,electronics,and cosmetics due to its unique physical and chemical properties,but the neurotoxic effect and the potential mechanism of AlNPs has not yet been elucidated.In order to investigate the toxic mechanism of AlNPs and the effect of autophagy inhibitor 3-methyladenine(3MA)on the neurotoxicity of AlNPs to zebrafish larvae,zebrafish embryos were divided into controlgroup,3MA group,AlNPs group and AlNPs+3MA group at 6 hours post-fertilization(hpf).The morphological changes of embryos and larvae and the general toxicity of larvae were observed,and the neurobehavioral changes,oxidative stress levels and the transcriptional changes of autophagy-related genes(beclin1,lc3Ⅱand vps34)were detected.Our results showed that no significant differences were found for mortality,hatching rate,and deformity rate among groups.The results of morphological observation showed that the zebrafish embryos in AlNPs group developed slowly at 12 hpf and 24 hpf.After 3MA was added,the development of zebrafish embryos was im-proved after 24 hpf from developmental delay observed at 12 hpf in AlNPs+3MA group.The average swimming speed,moving distance and tactile degree of fish in the AlNPs group were significantly lower(P<0.05).In the pan-ic avoidance reflex test under strong light stimulation,the speed of larvae in AlNPs group was significantly de-creased(P<0.05).Superoxide dismutase(SOD)levels decreased,whereas the levels of lactate dehydrogenase(LDH)increased in AlNPs exposed fish(P<0.05).Moreover,the expression of beclin1,lc3Ⅱand vps34 gene was increased in AlNPs group,and decreased after adding 3MA(P<0.05).Our results suggest that neurotoxicity of ze-brafish larvae induced by AlNPs could be related to excessive autophagy,and 3MA could reduce the oxidative damage and down-regulate the expression of autophagy-related genes,and significantly decrease the neurotoxicity induced by AlNPs exposure.