纳米氧化铝亚慢性染毒致小鼠神经细胞线粒体损伤机制

Mechanism of the mitochondria damage of the nerve cells in mouse after chronic exposure to nano-alumina

目的探讨纳米氧化铝对ICR小鼠神经细胞线粒体形态及功能的损伤作用。方法健康成年ICR小鼠分别滴鼻暴露纳米氧化铝(<50 nm)25,50和75 mg·kg-1,连续1个月。电镜观察海马CA3区神经细胞线粒体的超微结构,并测量线粒体直径;采用定磷法测定脑皮质Na+-K+-ATPase与Ca2+-Mg2+-ATPase活力;Western蛋白质印迹法检测细胞色素c氧化酶亚单位Ⅳ(COXⅣ),Bcl-2结合蛋白1(Beclin 1)和微管相关蛋白1轻链3Ι蛋白(LC3Ι)和LC3Ⅱ蛋白的表达。结果与正常对照组相比,50 mg·kg-1组海马CA3区神经元线粒体明显肿胀,内嵴结构排列稀疏,核周线粒体呈空泡化;线粒体平均直径明显增加〔(0.49±0.02)μm,P<0.05〕;75 mg·kg-1组线粒体大多呈微球形,嵴结构排列紧密,线粒体平均直径显著降低〔(0.36±0.02)μm,P<0.05〕。脑皮质线粒体酶活力随着染毒剂量的增加而降低,与正常对照相比,50和75 mg·kg-1组的Na+-K+-ATPase与Ca2+-Mg2+-ATPase活力显著降低,分别为6.37±0.22,5.48±1.53和3.21±0.99,(3.28±0.15)kU·g-1蛋白(P<0.05);50和75 mg·kg-1组的Ca2+-Mg2+-ATPase活力显著降低(P<0.05)。75 mg·kg-1组COXⅣ蛋白表达量为1.35±0.66,显著低于其他2个剂量组(P<0.05)。与正常对照相比,75 mg·kg-1组Beclin表达量为2.23±0.20,自噬特异蛋白LC3Ⅱ/LC3Ⅰ的比例为0.45±0.10,均显著高于正常对照组(P<0.05)。结论线粒体功能紊乱可能是纳米氧化铝神经毒性机制之一,而神经细胞可能通过自噬清除受损线粒体。

OBJECTIVE To explore the potential neurotoxicity of nano-alu mina （〈50 n m）in vivo, we treated the ICR mouse with the nano-alu mina to investigate the mitochondrial da mage of nerve cells on morphology and function.METHODS Adult male mice were exposed to nano-alu mina （〈50 n m）of 0,25,50 and 75 mg·kg -1 by nasal instillation for 1 month.Then we observed the mitochondrial ultra-structure of the nerve cells in CA3 region of hippoca mpus,and measured the mean dia meter in every group.The activities of Na ＋-K ＋-ATPase and Ca2 ＋-Mg2 ＋-ATPase were tested by the determination of the inorganic phosphorus,which was the deco mposition product of ATPase.Western blot analysis was used to detect the expression of COX-Ⅳ,Beclin1 ,LC3Ιand LC3Ⅱ.RESULTS Co mpared with 0 and 25 mg·kg -1 groups exposed to Al2 O3 nanopartilces （Al2 O3 NPs）,the mitochondria of CA3 region in hip-poca mpus in 50 mg·kg -1 group beca me ede matous and swollen with sparse and broken cristae sur-rounding the nuclear,and the mean dia meter was higher（0.49 ±0.02 μm,P 〈0.05）.But co mpared with 50 mg·kg -1 group,the mitochondria in 75 mg·kg -1 group beca me s maller with inner cristae of high density,and the mean dia meter was lower（0.36 ±0.02 μm,P〈0.05）.The enzy me activity of the mito-chondria in cerebral cortex decreased dose-dependently with exposure,the activities of Na ＋-K ＋-ATPase in 50 and 75 mg·kg -1 groups（6.37 ±0.22 kU·g -1 protein,5.48 ±1 .53 kU·g -1 protein）and Ca2 ＋-Mg2 ＋-ATPase in 50 and 75 mg·kg -1 groups （3.21 ±0.99 kU·g -1 protein,3.28 ±0.15 kU·g -1 protein）were lower than the 0 mg·kg -1 group（P〈0.05）.Meanwhile,the Ca2 ＋-Mg2 ＋-ATPase in 50 and 75 mg·kg -1 groups showed lower activities in co mparison with the 25 mg·kg -1 group.The 75 mg·kg -1 group expressed higher level of the COX-Ⅳ protein 1 .35 ±0.66（P〈0.05）than other groups.Both expression of Beclin1 protein and rate of LC3Ⅱ/LC3Ⅰin 75 mg·kg -1 group were more than the 0 mg·kg -1 group. CONCLUSION The mitochondrial dysfunction may be the potential neurotoxicity of nano-alu mina,and the da maged mitochondria were cleared by autophagy.