Kesaraju Shailaja, Schmidt-Kastner Rainald, Prentice Howard M, Milton Sarah L
Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida 33431, USA.
J Neurochem. 2009 Jun;109(5):1413-26. doi: 10.1111/j.1471-4159.2009.06068.x. Epub 2009 Mar 26.
Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.
淡水龟通过一些已被充分描述的生理过程，能够在长时间缺氧和复氧的情况下存活，且不会出现明显的脑损伤，但对于与缺氧存活相关的分子变化，目前的研究还很少。我们研究了乌龟在早期（1小时）和长期缺氧（4、24小时）及复氧过程中的应激蛋白和凋亡调节因子。蛋白质免疫印迹分析显示，在缺氧的第一个小时内就出现了变化；多种应激蛋白（热休克蛋白72、葡萄糖调节蛋白94、热休克蛋白60、热休克蛋白27和血红素加氧酶-1）增加，而凋亡调节因子（Bcl-2和Bax）减少。内质网应激蛋白葡萄糖调节蛋白78的水平没有变化。在长期缺氧过程中，应激蛋白水平仍然升高，而Bcl-2/Bax比值没有改变。在缺氧过程中，未观察到裂解的半胱天冬酶3水平的变化，而凋亡诱导因子显著增加。此外，我们没有发现Bax从细胞质向线粒体缺氧转位的证据，也没有发现凋亡诱导因子在线粒体和细胞核之间移动的证据。除了血红素加氧酶-1外，复氧并没有导致应激蛋白或凋亡调节因子的进一步增加。免疫组织化学证实了对细胞损伤的明显保护作用，这表明经历缺氧和复氧的乌龟大脑没有明显损伤。结果表明，分子适应性增强了促生存机制并抑制了凋亡途径，从而赋予淡水龟缺氧耐受性。
