Xu Yang, Wang Jingye, Song Xinghui, Wei Ruili, He Fangping, Peng Guoping, Luo Benyan
Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 89 Qingchun Road, Hangzhou 310003, China.
Department of Neurology, First Affiliated Hospital, Anhui Medical University, 218 Jixi Road, Hefei 230022, China.
Brain Res Bull. 2016 Jan;120:97-105. doi: 10.1016/j.brainresbull.2015.11.007. Epub 2015 Nov 10.
Many studies have demonstrated the key role of lysosomes in ischemic cell death in the brain and have led to the "lysosomocentric" hypothesis. In this hypothesis, the release of cathepsin-B due to a change of lysosomal membrane permeabilization (LMP) or rupture is critical, and this can be prevented by its inhibitors CA074 and CA074-me. However, the role of CA074-me in neuronal death and its effect on the change of lysosomal membrane integrity after global cerebral ischemia/reperfusion (I/R) injury is not clear, so we investigated this here. Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me (1 μg, 10 μg) were given intracerebroventricularly 1h before ischemia or 1h post reperfusion. The changes of heat shock protein 70 (Hsp70), cathepsin-B, lysosomal-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and the change of lysosomal pH were evaluated respectively. Hippocampal CA1 neuronal programmed necrosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD(+) level after I/R injury were also inhibited, while the upregulation of Hsp70 was strengthened by CA074-me pre-treatment. Delayed fulminant leakage of cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture.
许多研究已证明溶酶体在脑缺血性细胞死亡中起关键作用，并由此产生了“以溶酶体为中心”的假说。在该假说中，由于溶酶体膜通透性（LMP）改变或破裂导致组织蛋白酶B的释放至关重要，而其抑制剂CA074和CA074-me可预防这种情况。然而，CA074-me在全脑缺血/再灌注（I/R）损伤后神经元死亡中的作用及其对溶酶体膜完整性变化的影响尚不清楚，因此我们在此进行了研究。在20分钟全脑I/R损伤后评估大鼠海马CA1神经元死亡情况。在缺血前1小时或再灌注后1小时经脑室内给予CA074-me（1μg、10μg）。分别评估热休克蛋白70（Hsp70）、组织蛋白酶B、溶酶体相关膜蛋白1（LAMP-1）、受体相互作用蛋白3（RIP3）的变化以及溶酶体pH值的变化。全脑I/R损伤诱导的海马CA1神经元程序性坏死在预处理和后处理时均被CA074-me所预防。再灌注2天后，弥漫性细胞质组织蛋白酶B和LAMP-1免疫染色与固缩核变化同步，并且在氧糖剥夺（OGD）后检测到溶酶体pH值升高以及溶酶体染料DND-153泄漏。这两种变化均表明溶酶体膜破裂和组织蛋白酶B泄漏，而CA074-me预处理可强烈抑制这种情况。I/R损伤后RIP3的过表达和核转位以及NAD(+)水平的降低也受到抑制，而CA074-me预处理可增强Hsp70的上调。由于溶酶体破裂导致的组织蛋白酶B延迟暴发性泄漏是20分钟全脑I/R损伤诱导的神经元程序性坏死中的关键有害因素。除了作为组织蛋白酶B的抑制剂外，CA074-me可能通过维持溶酶体膜完整性和防止溶酶体破裂而具有间接神经保护作用。
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