纳米氧化铝暴露致斑马鱼神经发育毒性和帕金森样症状作用研究

Neurodevelopmental toxicity and Parkinsonism-like symptoms induced by nano-alumina exposure in zebrafish

[背景]纳米氧化铝(nano-Al_(2)O_(3))是一种广泛应用的纳米材料,其对环境和生物的影响引起了广泛关注。斑马鱼是一种常用的模式生物,其基因组与人类有较高的同源性,被广泛用于毒性研究。[目的]探究nano-Al_(2)O_(3)暴露对斑马鱼的发育毒性和神经毒性及相应的作用机制。[方法]将受精后6 h(6 hpf)的斑马鱼胚胎随机分为空白对照组和不同剂量nano-Al_(2)O_(3)暴露组(200、400、600、800和1000μg·mL^(-1))。每组30颗胚胎,每24 h更换一次培养液,培养至144 hpf。统计48和72 hpf时斑马鱼孵化率和累计至144 hpf的畸形率。144 hpf时利用斑马鱼行为分析仪记录斑马鱼的运动轨迹,分析各组斑马鱼运动的总距离和平均速度。144 hpf时,在荧光显微镜下观察转基因斑马鱼vmat2:GFP的多巴胺能神经元、vegf:GFP的脑部血管和elavl3:EGFP的中枢神经系统神经元的发育状况,并用Image Pro Plus进行相应的统计分析。利用实时荧光定量PCR检测144 hpf时斑马鱼神经元发育相关基因(syn2α、gap43、dat)、路易小体形成相关基因(α-syn)和自噬相关基因(pink1、parkin)的表达情况。[结果]与空白对照组相比,nano-Al_(2)O_(3)暴露组在48 hpf时孵化率降低,累计畸形率升高(P<0.05),出现鱼鳔发育迟缓、鱼鳔缺失、身体弯曲等现象。自主行为学检测结果显示,与空白对照组相比,nano-Al_(2)O_(3)暴露组斑马鱼游动的总距离降低(P<0.001),平均速度明显减小。荧光观察结果显示,与空白对照组相比,nano-Al_(2)O_(3)暴露组vmat2:GFP转基因斑马鱼多巴胺能神经元长度减小(P<0.001)。此外,vegf:GFP转基因斑马鱼脑部血管明显缺失,elavl3:EGFP转基因斑马鱼中枢神经系统神经元荧光强度减弱(P<0.001),中枢神经系统神经元长度减小(P<0.01)。实时荧光定量PCR分析结果显示,与空白对照组相比,不同浓度nano-Al_(2)O_(3)暴露组斑马鱼的α-syn、syn2α、dat和gap43(除400μg·mL^(-1)暴露组外)基因表达上调(P<0.01),而600和800μg·mL^(-1)nano-Al_(2)O_(3)暴露组斑马鱼的parkin和除200μg·mL^(-1)暴露组外的所有暴露组pink1基因均表达出现下调(P<0.05)。[结论]Nano-Al_(2)O_(3)暴露对斑马鱼幼鱼具有发育毒性,并诱导斑马鱼出现帕金森病样症状,初步推测其机制可能与nano-Al_(2)O_(3)诱导线粒体自噬障碍有关。

[Background]Nano-alumina(nano-Al_(2)O_(3))is a widely utilized nanomaterial.Its impacts on the environment and biological systems have garnered significant attention.Zebrafish serves as a common model organism in scientific research due to its high homology with the human genome and is extensively used in toxicity studies.[Objective]To investigate the developmental toxicity and neurotoxicity of nano-Al_(2)O_(3) exposure in zebrafish and the corresponding mechanisms of action.[Method]Zebrafish embryos at 6 h post-fertilization(hpf)were randomly assigned to a control group and five dose groups exposed to nano-Al_(2)O_(3) at concentrations of 200,400,600,800,and 1000μg·mL^(−1),respectively.Thirty embryos were included in each group,and the culture medium was replaced every 24 h until 144 hpf.The hatching rates at 48 and 72 hpf and the cumulative malformation rates up to 144 hpf were calculated.At 144 hpf,a zebrafish behavior analyzer was used to record the movement trajectories of the zebrafish,and the total distance traveled and average speed were analyzed for each group.At 144 hpf,the development of dopaminergic neurons in transgenic zebrafish expressing vmat2:GFP,brain vessels in those expressing vegf:GFP,and central nervous system neurons in those expressing elavl3:EGFP were observed under a fluorescence microscope,and statistical analysis was conducted using Image Pro Plus.Realtime quantitative PCR was employed to detect the expression levels of neuron development-related genes(syn2α,gap43,dat),Lewy body formation-related gene(α-syn),and autophagy-related genes(pink1,parkin)at 144 hpf.[Results]Compared to the control group,the nano-Al_(2)O_(3) exposed groups exhibited reduced hatching rates at 48 hpf and increased cumulative malformation rates(P<0.05),with phenomena such as delayed development,absence of the swim bladder,and body curvature.The autonomous behavioral tests revealed that the nano-Al_(2)O_(3) exposed zebrafish showed a decrease in the total distance swum(P<0.001)and a significant reduction in average speed compared to the control group.The fluorescence observations indicated that the length of dopaminergic neurons in vmat2:GFP transgenic zebrafish was reduced in the nano-Al_(2)O_(3) exposed groups(P<0.001).Additionally,vegf:GFP transgenic zebrafish exhibited a significant absence of brain vessels,while elavl3:EGFP transgenic zebrafish showed a weakened fluorescence intensity of central nervous system neurons(P<0.001)and a decreased length of these neurons(P<0.01).The real-time quantitative PCR analysis revealed that compared to the control group,the gene expression levels ofα-syn,syn2α,dat,and gap43 were upregulated in the zebrafish exposed to nano-Al_(2)O_(3)(except for the 400μg·mL^(−1)exposure group)(P<0.01),while the expression levels of parkin were downregulated in the 600 and 800μg·mL^(−1)nano-Al_(2)O_(3) exposed groups,and the expression levels of pink1 were downregulated in all exposure groups except for the 200μg·mL^(−1)group(P<0.05).[Conclusion]Exposure to nano-Al_(2)O_(3) exhibits developmental toxicity in zebrafish larvae and induces Parkinsonism-like symptoms in zebrafish.The preliminary speculation of the mechanism suggests that it may be related to nano-Al_(2)O_(3)-induced mitochondrial autophagy impairment.