Rodrigues C M, Solá S, Silva R, Brites D
Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, Portugal.
Mol Med. 2000 Nov;6(11):936-46.
The pathogenesis of bilirubin encephalopathy and Alzheimer's disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-beta (Abeta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Abeta-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring.
Primary rat neuron and astrocyte cultures were incubated with UCB or Abeta peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot.
Incubation of neural cells with both UCB and Abeta induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by > 50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Abeta (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Abeta, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05).
UCB and Abeta peptide activate the apoptotic machinery in neural cells. Toxicity occurs through a mitochondrial-dependent pathway, which in part involves opening of the permeability transition pore. Furthermore, membrane permeabilization is required for cytochrome c release from mitochondria and can be prevented by UDC or TUDC. These data suggest that the mitochondria is a pharmacological target for cytoprotection during unconjugated hyperbilirubinemia and neurodegenerative disorders, and that UDC or TUDC may be potential therapeutic agents.
胆红素脑病和阿尔茨海默病的发病机制似乎分别源于未结合胆红素（UCB）和β淀粉样蛋白（Aβ）肽的积累，这可能导致细胞凋亡。线粒体膜通透性增加以及膜间蛋白的释放与细胞死亡密切相关。抑制线粒体通透性是熊去氧胆酸（UDC）和牛磺熊去氧胆酸（TUDC）在肝细胞和非肝细胞中防止细胞凋亡的一条途径。在本研究中，我们进一步描述了UCB和Aβ在分离的神经细胞中诱导的细胞毒性，并研究了分离的线粒体与这两种物质孵育期间的膜扰动情况。此外，我们评估了抗凋亡药物UDC和TUDC是否能阻止任何变化的发生。
将原代大鼠神经元和星形胶质细胞培养物单独或在UDC存在的情况下与UCB或Aβ肽一起孵育。通过DNA片段化和细胞核形态变化评估细胞凋亡。将分离的线粒体单独或与UDC、TUDC或环孢素A联合用每种毒素处理。用分光光度法测量线粒体肿胀，并通过蛋白质印迹法测定细胞色素c蛋白水平。
用UCB和Aβ孵育神经细胞均诱导了细胞凋亡（p<0.01）。与UDC共同孵育可使细胞凋亡减少>50%（p<0.05）。两种毒素均导致分离的线粒体膜通透性增加（p<0.001）；而用UDC预处理具有保护作用（p<0.05）。TUDC在预防由Aβ介导的基质肿胀方面甚至更有效（p<0.01）。UDC和TUDC分别显著减少了与UCB和Aβ诱导的线粒体通透性增加相关的细胞色素c释放（p<0.05）。此外，环孢素A显著抑制了由UCB介导的线粒体肿胀和细胞色素c外流（p<0.05）。
UCB和Aβ肽激活神经细胞中的凋亡机制。毒性通过线粒体依赖性途径发生，这部分涉及通透性转换孔的开放。此外，细胞色素c从线粒体释放需要膜通透性增加，而UDC或TUDC可以阻止这种情况发生。这些数据表明线粒体是未结合胆红素血症和神经退行性疾病期间细胞保护的药理学靶点，并且UDC或TUDC可能是潜在的治疗药物。
