Dai Chongshan, Tang Shusheng, Velkov Tony, Xiao Xilong
College of Veterinary Medicine, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, People's Republic of China.
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.
Mol Neurobiol. 2016 Sep;53(7):4685-700. doi: 10.1007/s12035-015-9396-7. Epub 2015 Aug 28.
Neurotoxicity remains a poorly characterized adverse effect associated with colistin therapy. The aim of the present study was to investigate the mechanism of colistin-induced neurotoxicity using the mouse neuroblastoma2a (N2a) cell line. Colistin treatment (0-200 μM) of N2a neuronal cells induced apoptotic cell death in a dose-dependent manner. Colistin-induced neurotoxicity was associated with a significant increase of reactive oxygen species (ROS) levels, with a concomitant decrease in the activities of superoxide dismutase (SOD), catalase (CAT), and the glutathione (GSH) levels. Mitochondrial dysfunction was evident from the dissipation of membrane potential and the increase of Bax/Bcl-2, followed by the release of cytochrome c (CytC). Caspase-3/7, -8, and -9 activations were also detected. Colistin-induced neurotoxicity significantly increased the gene expression of p53 (1.6-fold), Bax (3.3-fold), and caspase-8 (2.2-fold) (all p < 0.01). The formation of autophagic vacuoles was evident with the significant increases (all p < 0.05 or 0.01) of both of Beclin 1 and LC3B following colistin treatment (50-200 μM). Furthermore, inhibition of autophagy by pretreatment with chloroquine diphosphate (CQ) enhanced colistin-induced apoptosis via caspase activation, which could be attenuated by co-treatment with the pan-caspase inhibitor Z-VAD-FMK. In summary, our study reveals that colistin-induced neuronal cell death involves ROS-mediated oxidative stress and mitochondrial dysfunction, followed by caspase-dependent apoptosis and autophagy. A knowledge base of the neuronal signaling pathways involved in colistin-induced neurotoxicity will greatly facilitate the discovery of neuroprotective agents for use in combination with colistin to prevent this undesirable side effect.
神经毒性仍然是与黏菌素治疗相关的一种特征尚不明确的不良反应。本研究的目的是利用小鼠神经母细胞瘤2a（N2a）细胞系来探究黏菌素诱导神经毒性的机制。用黏菌素（0 - 200 μM）处理N2a神经细胞会以剂量依赖的方式诱导细胞凋亡。黏菌素诱导的神经毒性与活性氧（ROS）水平显著升高相关，同时超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性以及谷胱甘肽（GSH）水平下降。线粒体功能障碍表现为膜电位消散以及Bax/Bcl - 2升高，随后细胞色素c（CytC）释放。还检测到了半胱天冬酶 - 3/7、 - 8和 - 9的激活。黏菌素诱导的神经毒性显著增加了p53（1.6倍）、Bax（3.3倍）和半胱天冬酶 - 8（2.2倍）的基因表达（所有p < 0.01）。黏菌素处理（50 - 200 μM）后，Beclin 1和LC3B均显著增加（所有p < 0.05或0.01），自噬空泡的形成明显。此外，用二磷酸氯喹（CQ）预处理抑制自噬会通过半胱天冬酶激活增强黏菌素诱导的细胞凋亡，而用泛半胱天冬酶抑制剂Z - VAD - FMK共同处理可使其减弱。总之，我们的研究表明，黏菌素诱导的神经元细胞死亡涉及ROS介导的氧化应激和线粒体功能障碍，随后是半胱天冬酶依赖性凋亡和自噬。了解黏菌素诱导神经毒性所涉及的神经元信号通路将极大地促进用于与黏菌素联合使用以预防这种不良副作用的神经保护剂的发现。
