Produit-Zengaffinen Nathalie, Pournaras Constantin J, Schorderet Daniel F
Institute for Research in Ophthalmology, Sion, Switzerland.
Mol Vis. 2009 Oct 19;15:2101-10.
Apoptosis is known to play a key role in cell death after retinal ischemia. However, little is known about the kinetics of the signaling pathways involved and their contribution to this process. The aim of this study was to determine whether changes in the expression of molecules in the mitochondrial apoptotic pathway might explain the progression of retinal damage following ischemia/reperfusion.
Retinal ischemia was induced by elevating intraocular pressure in the vitreous cavity to 150 mmHg for a period of 60 min. At time 0, 3 h (early phase), and 24 h (late phase) after reperfusion, the retinas were harvested and modifications in the expression of Bax, Bak, Bcl-2, and Bcl-x(L) as well as caspase-3 and -7, were examined by qPCR and, in some cases, by western blot.
qPCR analysis performed at the early phase after ischemia revealed a time dependent decrease in Bax, Bak, and Bcl-x(L) and no alteration in Bcl-2 mRNA expression in response to retinal ischemia. At the protein level, proapoptotic Bax and Bak were not modulated while Bcl-2 and Bcl-x(L) were significantly upregulated. At this stage, the Bax per Bcl-2 and Bax:Bcl-x(L) ratios were not modified. At the late phase of recovery, Bax and Bcl-x(L) mRNAs were downregulated while Bak was increased. Increased Bax:Bcl-2 and Bax:Bcl-x(L) ratios at both the mRNA and protein levels were observed 24 h after the ischemic insult. Analysis of caspases associated with mitochondria-mediated apoptosis revealed a specific increase in the expression of caspase-3 in the ischemic retinas 24 h after reperfusion, and a decrease in the expression of caspase-7.
This study revealed that Bcl-2-related family members were differently regulated in the early and late phases after an ischemic insult. We showed that the Bax:Bcl-2 and Bax:Bcl-x(L) balances were not affected in the initial phases, but the Bax:Bcl-x(L) ratio shifted toward apoptosis during the late phase of recovery. This shift was reinforced by caspase-3 upregulation.
已知细胞凋亡在视网膜缺血后的细胞死亡中起关键作用。然而，对于所涉及的信号通路的动力学及其对这一过程的贡献知之甚少。本研究的目的是确定线粒体凋亡途径中分子表达的变化是否可以解释缺血/再灌注后视网膜损伤的进展。
通过将玻璃体腔内眼压升高至150 mmHg持续60分钟来诱导视网膜缺血。在再灌注后的0小时、3小时（早期）和24小时（晚期），采集视网膜，并通过qPCR以及在某些情况下通过蛋白质印迹法检测Bax、Bak、Bcl-2和Bcl-x(L)以及caspase-3和-7表达的变化。
缺血后早期进行的qPCR分析显示，Bax、Bak和Bcl-x(L)随时间依赖性降低，而视网膜缺血后Bcl-2 mRNA表达无变化。在蛋白质水平上，促凋亡的Bax和Bak未被调节，而Bcl-2和Bcl-x(L)显著上调。在此阶段，Bax与Bcl-2以及Bax:Bcl-x(L)的比值未改变。在恢复的后期，Bax和Bcl-x(L)的mRNA下调，而Bak增加。在缺血损伤24小时后，在mRNA和蛋白质水平上均观察到Bax:Bcl-2和Bax:Bcl-x(L)比值增加。对与线粒体介导的凋亡相关的半胱天冬酶的分析显示，再灌注24小时后缺血视网膜中caspase-3的表达特异性增加，而caspase-7的表达降低。
本研究表明，Bcl-2相关家族成员在缺血损伤后的早期和晚期受到不同的调节。我们发现，在初始阶段Bax:Bcl-2和Bax:Bcl-x(L)的平衡未受影响，但在恢复后期Bax:Bcl-x(L)比值向凋亡方向转变。这种转变因caspase-3上调而加强。
