β-淀粉样蛋白对原代培养大鼠海马神经细胞钙稳态和线粒体线粒体通透性转运孔道的影响

Effect of β-amyloid on calcium homeostasis and mitochondrial permeability transition pore proteins in primary cultured rat hippocampal neurons

目的研究β-淀粉样蛋白（β-amyloid）对原代培养的大鼠脑海马神经细胞内钙稳态和线粒体通透性转运孔（mltoehortrial permebility transitionpore，MPTP）的影响，探讨其神经毒性作用的机制。方法原代培养8d后的Wistar大鼠脑海马神经细胞，用老化的Aβ25-35，对其进行1，10和20μmol／L剂量的染毒，培养24和48h后观察海马神经细胞形态、游离钙浓度、Ca^2＋-ATP酶活性、线粒体膜电位，应用Western blot方法分析MPTP组成蛋白表达情况。结果随着Aβ25-35剂量的增加和染毒时间的延长，可以观察到原代培养的海马神经细胞呈现出弥漫性肿胀、开始变形、出现空泡、胞体边缘模糊的形态变化；原代培养海马神经细胞内的游离钙离子的浓度也呈逐渐上升趋势，其中Aβ25-35 20μmol／L组在24h和48h时，细胞内钙离子浓度分别为（31．04±4．16）和（32．80±0．43），均显著高于对照组（20．95±4．04）和（22．23±0．49）（P〈0．05）；Ca2＋-ATP酶活性随着染毒剂量和染毒时间增加明显降低（P〈0．05），其中Aβ25-3520μmol／L组在24h和48h时，细胞内Ca2＋ATP酶活性为（2．14±0．01）和（1．10±0．05）U／mgprol，均显著低于对照组（5．17±0．08）和（4．57±0．06）（P〈0．05）；线粒体膜电位Aβ25-3520μmol／L组在24h和48h时，细胞内线粒体膜电位分别为（20．34±7．05）和（19．05±6．15），均显著低于对照组（38．47±0．72）和（40．07±1．26）（P〈0．05）；有明显剂量反应关系；MPTP孔道蛋白的WB结果显示蛋白表达量随着染毒剂量增加而增加。结论Aβ25-35,通过破坏细胞的钙稳态激活MPTP孔道影响线粒体功能从而发挥神经毒性作用。

Objective To study the effects of β-amyloid （Aβ25-35） on calcium homeostasis and mitochondrial permeability transition pore （MPTP） in primary cultured rat hippocampal neurons, and explore the mecha- nism of neurotoxicity of Aβ25-35- Methods After 8 days of primary culture, hippocampal neurons were exposure with different doses（ 1 μmol/L, 10 μmol/L, and 20 μmol/L, respectively） of Aβ25-35, and 0 μmol/L dose as control group. After cultured 24 h and 48 h, observed hippocampal neuronal morphology,intracellular free calcium concentration, activity of Ca -ATPase, mltochondrial membrane potential, and MPTP protein expression by Western Blotting method. Results With the increase of Aβ25-35 dose and exposure time,morphological changes of hippoeampal neurons in primary culture were observed, showing a diffuse swelling, deformation, vacuoles, and cell edge fuzzy;The concentration of free caleiumions of Aβ25-3520 μmol/L group were （31.04 ± 4.16 ） and （ 32.80±0.43 ） compared the control group with the （20.95 ± 4.04） and（22.23± 0.49 ） 24 h and 48h after the exposure of Aβ25-35 , the concentration showed a gradual upward trend （ P 〈 0.05 ）. The activity of Ca2 ＋ -ATPase Aβ25-3520 μmol/L group were （2.14±0.01 ） and （1.10± 0.05 ）U/mgprol, compared the control group with the （5.17 ±0.08） and （4.57 ±0.06） （P 〈0.05）24 h and 48 h after the exposure of Aβ25-35. The membrane potential of mitoehondrial decreased significantly in the ap groups compared with the control group. （P 〈 0. 05） ,The membrane potential of mitoehondrial Aβ25-35 20 μmol/L group were （20.34 ±7. 05） and （ 19. 05 ± 6. 15 ） , compared the control group with the （ 38.47 ±0.72 ） and （40.07 ±1.26 ） （ P 〈 0.05 ） 24 h and 48 h after the exposure of Aβ25-35. With the increase of exposure dose and time, the activity of Ca2 ＋ -ATPase and mitochondrial, and there was a significant dose-response relationship. The results of WB of MPTP showed that the protein expression was increased with the increase of dose. Conclusion Aβ25-35 exhibits a neurotoxicity effect by disturbing the calcium homeostasis, and affecting mitochondrial function by means of activating of MPTP channels.