茯苓水提液对叠氮钠致原代培养的新生大鼠海马神经细胞线粒体损伤的影响

Effects of water extract from Poria cocos on mitochondrial impairment in hippocampal neurons induced by sodium azide

目的 从生化和形态学的角度观察茯苓水提液对叠氮钠致原代培养的新生大鼠海马神经细胞线粒体损伤的影响。方法 新生大鼠海马神经细胞原代培养至第 11天 ,吸弃培养液 ,加入MTT(0 .4mg·mL-1)温育 4h ,用自动酶标仪在 5 5 0nm处检测被神经细胞线粒体酶还原的MTT的量 ,以此作为评价细胞活力的指标。通过间接免疫荧光 (一抗为鼠抗α tubulin抗体 )的方法 ,利用激光扫描共聚焦显微镜观察来评价神经细胞骨架改变。结果 叠氮钠与培养细胞孵育能剂量依赖性地损伤细胞 ,6 4mmol·L-1孵育 4h时 ,细胞的线粒体酶还原MTT的能力为对照组的 (6 0 .73± 5 .13) % ,微管结构模糊、紊乱。茯苓水提液 (10～2 0mg·mL-1)与细胞预孵育 2 4h ,能明显抵抗叠氮钠引起的神经细胞线粒体还原MTT的能力下降 ,微管结构紊乱。结论 茯苓对维持神经细胞线粒体的功能及微管结构方面有重要作用 ,对防治某些神经退行性疾病如老年性痴呆、血管性痴呆及帕金森氏病等 。

OBJECTIVE: To investigate the effects of water extract from Poria cocos on the mitochondria impairment of primary cultured hippocampal neurons induced by sodium azide. METHODS: The hippocampal neurons from newborn rats were primarily cultured for 11 days. After the culture media was disposed, the neurons were incubated with MTT (0.4 mg&middotmL-1) for 4 h. The cell viability was judged by the percentage of MTT reduction which was measured by the enzyme immunity autoanalysor at the wavelength of 550 nm. The change of cytoskeleton labeled with (α-tubulin antibody) was observed by laser scanning confocal microscopy. RESULTS: Sodium azide injured hippocampal neurons dose-dependently. The cytoskeleton was significantly damaged when the hippocampal neurons were incubated with sodium azide at the dose of 64 mmol&middotL-1. The microtubule of neurons became disorder and blur, and the percentage of MTT reduction of the mitochondria in the neurons was (60.73 ±5.13) % compared with the control. However, after being incubated with neurons, the water extract from Poria cocos ( 10 ∼ 20 mg&middotmL-1) showed a significantly protective effect against the MTT reduction decrease in neurons induced by sodium azide. Furthermore, the intact structure of microtubule was seen clearly in primary cultured neurons. CONCLUSION: Poria cocos was demonstrated to play an important role in maintaining the function of mitochondria and normal morphology of neurons.