The Toxic Effect of ALLN on Primary Rat Retinal Neurons
The Toxic Effect of ALLN on Primary Rat Retinal Neurons
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ALLN 对原代大鼠视网膜神经元的毒性作用
DOI：
10.1007/s12640-016-9624-6
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发表时间：
2016-10-01
影响因子：
3.7
通讯作者：
Xiong, Kun
中科院分区：
文献类型：
--
作者：
Li, Na;Shang, Lei;Xiong, Kun
n-乙酰-亮氨酸-亮氨酸-去亮氨酸（ALLN）是一种蛋白酶体和钙蛋白酶的抑制剂，广泛用于减少蛋白酶体或钙蛋白酶介导的啮齿动物细胞死亡。然而，ALLN对视网膜神经元有一定的毒性。在10 μ M浓度下，ALLN对皮质神经元无毒性，但诱导视网膜神经元细胞死亡。ALLN对视网膜神经元的耐受浓度尚不清楚，ALLN诱导细胞死亡的确切机制尚不清楚。在本研究中，我们研究了ALLN对视网膜原代神经元的毒性作用。3-（4,5-二甲基噻唑-2-酰基）-2,5-二苯基溴化四唑（MTT）测定结果显示，1 μ M时细胞活力无显著变化，2.5、5和7.5 μ M ALLN处理后细胞活力下降，且在2.5、5和7.5 μ M处理时，乳酸脱氢酶（LDH）释放量显著升高，碘化丙啶（PI）阳性细胞显著增加。此外，在ALLN处理12和24 h后，caspase-3蛋白水平在5和7.5 μ M时上调。ALLN处理12和24 h后，5和7.5 μ M时Bax/Bcl-2比例升高，Annexin v阳性细胞增多。然而，微管相关蛋白1轻链3 (LC3) II/LC3 I的比例或单胺尸体碱（MDC）染色均无显著变化。我们的数据清楚地表明，在浓度等于或高于2.5 μ M时，ALLN可以诱导视网膜原代神经元细胞坏死和凋亡，而不是自噬。这些结果表明，原代视网膜神经元更容易受到ALLN的影响，并为ALLN损伤中ALLN敏感细胞的死亡提供了可能的机制。
N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of proteasomes and calpain, is widely used to reduce proteasomes or calpain-mediated cell death in rodents. However, ALLN is toxic to retinal neurons to some extent. At the concentration of 10 mu M, ALLN is non-toxic to cortical neurons, but induces cell death of retinal neurons in vitro. The tolerance concentration of ALLN for retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. In this study, we investigated the toxic effect of ALLN on primary retinal neurons. The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no significant changes of cell viability at 1 mu M but decreased cell viability after treatment of ALLN at 2.5, 5, and 7.5 mu M. Lactate dehydrogenase (LDH) release was highly elevated and propidium iodide (PI)-positive cells were significantly increased at 2.5, 5, and 7.5 mu M after all treatment times. Moreover, the protein levels of caspase-3 were up-regulated at 5 and 7.5 mu M after 12 and 24 h of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V-positive cells were increased at 5 and 7.5 mu M after 12 and 24 h of ALLN treatment. However, there were no significant changes in either the ratio of microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I or monodansylcadaverine (MDC) staining. Our data clearly show that at the concentrations equal to and higher than 2.5 mu M, ALLN may induce cell death of primary retinal neurons by necrosis and apoptosis, but not autophagy. These suggest that primary retinal neurons are more susceptible to ALLN treatment and provide a possible mechanism for the cell death of ALLN-sensitive cells in ALLN injury.