线粒体融合分裂异常在PBDE-47致PC12细胞线粒体质量改变中的作用

The role of abnormal mitochondrial fusion and fission in PBDE-47-induced change in mitochondrial mass in PC12 cells

目的探讨2,2’,4,4’-四溴联苯醚(PBDE-47)对大鼠肾上腺嗜铬细胞瘤(PC12)细胞线粒体质量的影响及其机制。方法不同浓度PBDE-47处理高分化的PC12细胞24h,实验分为对照组、1、10、20μmol/LPBDE-47处理组,采用透射电子显微镜观察细胞内线粒体的结构和数量变化;线粒体膜心磷脂特异性染料壬基吖啶橙(NAO)染色后,利用流式细胞术检测线粒体质量;Westernblot技术检测线粒体融合蛋白1(Mfn1)和分裂蛋白1(Fis1)的表达水平。为了进一步探究线粒体融合分裂改变在PBDE-47致PC12细胞线粒体质量变化中的作用,采用线粒体融合促进剂M1进行处理,实验分为对照组、5μmol/LM1处理组、20μmol/LPBDE-47处理组和5μmol/LM1+20μmol/LPBDE-47联合处理组,并检测NAO荧光强度和Mfn1及Fis1的表达水平。结果透射电子显微镜显示,对照组PC12细胞线粒体数量丰富,膜结构正常,随着PBDE-47染毒剂量的提高,线粒体数量减少,其中10μmol/L和20μmol/LPBDE-47处理组可见线粒体嵴消失、线粒体肿胀甚至线粒体空泡化改变;流式细胞术结果显示,与对照组比较,10μmol/L和20μmol/LPBDE-47处理组PC12细胞NAO荧光强度明显减弱,差异均有统计学意义(P<0.05);Westernblot结果显示,与对照组比较,10μmol/L和20μmol/LPBDE-47处理组Mfn1和Fis1蛋白表达水平均明显降低,差异均有统计学意义(P<0.05)。与20μmol/LPBDE-47处理组比较,5μmol/LM1+20μmol/LPBDE-47联合处理组中PC12细胞的Mfn1和Fis1蛋白表达水平以及NAO荧光强度均明显升高,差异均有统计学意义(P<0.05)。结论PBDE-47可通过抑制PC12细胞线粒体融合和分裂过程损伤线粒体质量。

Objective To investigate the effect of 2, 2', 4, 4'-tetrabromodiphenyl ether (PBDE-47) on the mitochondrial mass in rat adrenal pheochromocytoma (PC12) cells and the potential mechanisms. Methods Highly differentiated PC12 cells were divided into control, 1, 10 or 20 μmol/L PBDE-47-treated groups and cultured for 24 h. Transmission electron microscopy was employed to observe the changes in mitochondrial morphology and quantity in PC12 cells. Flow cytometry was used to measure the fluorescence intensity of Nonyl Acridine Orange (NAO), a fluorescent indicator of mitochondrial membrane cardiolipin, to reflect mitochondria mass. Western blotting was used to determine the expression levels of Mitofusion 1 (Mfn1) and Fission 1 (Fis1) proteins. To further explore the role of abnormal mitochondrial fusion and fission in PBDE-47-induced mitochondrial mass changes, PC12 cells were divided into control group, 5 μmol/L M1 treatment group, 20 μmol/L PBDE-47 treatment group and 5 μmol/L M1+20 μmol/L PBDE-47 combined treatment group and cultured for 24 h, then the fluorescence intensity of NAO and expression levels of Mfn1 and Fis1 proteins were detected. Results The control group showed numerous mitochondria with normal morphology, while the number of mitochondria decreased after PBDE-47 treatment. Especially, the disappeared cristae, swelling and vacuoles of mitochondria and decreased fluorescence intensity of NAO (P<0.05) were observed in 10 and 20 μmol/L PBDE-47-treated groups. Meanwhile, the expression levels of Mfn1 and Fis1 proteins in the 10 and 20 μmol/L PBDE-47-treated groups were significantly decreased compared with control group (P<0.05). However, 5 μmol/L M1 co-treatment with 20 μmol/L PBDE-47 significantly increased the levels of Mfn1 and Fis1 proteins and fluorescence intensity of NAO compared with the 20 μmol/L PBDE-47 group (P<0.05). Conclusion PBDE-47 can inhibit the mitochondrial fusion and fission process, thus leading to damage of mitochondria mass in PC12 cells.