纳米氧化铝对新生Wistar乳大鼠皮质神经元线粒体自噬的影响

Effect of nano-alumina on mitophagy in primary cortical neuronal cells from Wistar newborn rats

目的研究纳米氧化铝对新生Wistar乳大鼠皮质神经元线粒体自噬的影响。方法神经元原代培养4 d进行纯度鉴定,13 nm纳米氧化铝0.5 mmol·L-1染毒12,24和48 h,检测培养液上清中乳酸脱氢酶(LDH)活性;流式细胞仪检测线粒体膜电位(MMP);透射电镜观察线粒体的超微结构和线粒体自噬体;单丹酰戊二胺(MDC)荧光染色观察自噬小体的形成;Western蛋白质印迹法检测Bcl-2结合蛋白1(Beclin1)和微管相关蛋白1轻链3(LC3)Ⅱ/Ⅰ蛋白的表达;溶酶体红色荧光探针Lysotracker和线粒体绿色荧光探针Mitotracker共染色观察溶酶体与线粒体的融合。结果神经元培养4 d,经纯度鉴定95%以上为神经元。与正常对照组相比,纳米氧化铝0.5 mmol·L-1染毒12和24 h组上清液中LDH活性均明显增加(P<0.05),但随着染毒时间的延长LDH活性呈下降趋势。MMP测定结果显示,随着染毒时间的延长,MMP逐渐降低(P<0.01)。细胞超微结构观察发现,纳米氧化铝组有线粒体肿胀和空泡形成,并可检测到线粒体自噬体。MDC荧光染色显示,正常对照组仅见极少量的MDC阳性荧光颗粒,纳米氧化铝组可见明显的MDC阳性荧光颗粒。自噬相关蛋白Beclin1和LC3Ⅱ/Ⅰ的表达随着染毒时间的延长逐渐增加(P<0.05)。荧光共染色显示,纳米氧化铝组线粒体与溶酶体融合。结论纳米氧化铝可引起神经元发生自噬以及线粒体损伤,受损的线粒体可能通过线粒体自噬作用清除。

OBJECTIVE To study the effect of nano-alumina（nano-Al2O3 ）on mitophagy in primary cortical neuronal cells from Wistar newborn rats. METHODS The purity of neuronal cells was detected by immunohistochemistry,and the lactate dehydrogenase（LDH）assay was performed to determine the viability of the cells treated with 13 nm nano-Al2O3 0.5 mmol·L-1 for 12,24 and 48 h,respectively. The mitochondrial membrane potential（MMP）was detected by flow cytometry analysis . The ultrastructure of mitochondria and mitophagy vacuoles was observed by transmission electron microscopy（TEM）. Auto-phagic vacuoles were observed by dansylpentanediamine（MDC）staining and the expression of autoph-agy related protein Beclin1 and LC3Ⅱ/ Ⅰ was determined by Western blotting. Mitophagy was observed by Lysotracker and Mitotracker staining respectively. RESULTS More than 95% cells were neuronal cells. The activity of LDH in the supernatant liquid exposed to nano-Al2O3 for 12 and 24 h groups was sig-nificantly increased compared with the control group（P﹤0.05）. After exposure to nano-Al2O3 ,the mito-chondrial membrane potential was significantly decreased compared with the control group（ P ﹤0.01）. The results of TEM displayed mitochondrial swelling and the formation of vacuoles and mitophagy in nano-Al2O3 groups. MDC positive fluorescence particles were observed and the expression of autophagy related protein Beclin1 and LC3Ⅱ/ Ⅰ was increased in nano-Al2O3 groups compared with the control group（ P ﹤ 0. 05 ）. The result of Lysotracker and Mitotracker colocalization showed the fusion of mitochondria and lysosomals. CONCULSION Nano-Al2O3 may induce autophagy and mitochondria damage in neuronal cells while the damaged mitochondria may be removed by mitophagy.