Impaired Autophagosome Clearance Contributes to Local Anesthetic Bupivacaine-induced Myotoxicity in Mouse Myoblasts
Impaired Autophagosome Clearance Contributes to Local Anesthetic Bupivacaine-induced Myotoxicity in Mouse Myoblasts
复制标题
自噬体清除受损导致局麻药布比卡因诱导小鼠成肌细胞的肌肉毒性
DOI：
10.1097/aln.0000000000000568
复制
发表时间：
2015-03-01
期刊：
影响因子：
8.8
通讯作者：
Ding, Zhengnian
中科院分区：
文献类型：
--
作者：
Li, Rongrong;Ma, He;Ding, Zhengnian
背景：本研究探讨了自噬在布比卡因诱导的小鼠成肌细胞C2c12细胞毒性中的作用(S)。方法：用布比卡因处理C2c12细胞。用3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium溴化法(n=3~30)、活体/死亡法(n=3~4)和形态改变法(n=3)评价心肌毒性。微管相关蛋白轻链3转化(n=4~12)和轻链3标点(n=4~5)反映自噬小体的形成。通过p62蛋白水平(n=4)和自溶酶体生成(n=3)来评价自噬小体清除情况。结果：布比卡因可引起明显的细胞损伤。值得注意的是，在布比卡因处理的细胞中，自噬小体的生成显著增加，轻链3点的形成(72.7±6.9比2.1±1.2)和轻链3转换(2.16±0.15比0.33±0.04)证明了这一点。布比卡因使蛋白激酶B/哺乳动物靶标雷帕霉素/p70核糖体蛋白S6信号失活。然而，经布比卡因处理后，细胞中p62蛋白的表达水平显著升高(1.29±0.15比1.00±0.15)，表明该药物损害了自噬小体的清除。进一步检查发现，布比卡因阻断自噬小体-溶酶体融合(10.87%±1.48%vs.32.94%±4.22%)。给予雷帕霉素增加了自噬小体的清除，最重要的是，改善了布比卡因处理的细胞的存活率。然而，自噬相关蛋白5(ATG5)的敲除加剧了布比卡因引起的自噬小体清除和肌肉毒性的损害。结论：在布比卡因攻击后，自噬小体的形成是一种应激反应机制；然而，由于自噬小体-溶酶体融合不充分，自噬小体清除受到损害。因此，自噬小体清除功能受损似乎是布比卡因引起肌肉毒性的一种新机制。
Background:The current study examined the role(s) of autophagy in myotoxicity induced by bupivacaine in mouse myoblast C2c12 cells. Methods:C2c12 cells were treated with bupivacaine. Myotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (n = 3 to 30), live/dead assay (n = 3 to 4), and morphological alterations (n = 3). Autophagosome formation was reflected by microtubule-associated protein light chain 3 conversion (n = 4 to 12) and light chain 3 punctation (n = 4 to 5). Autophagosome clearance was evaluated by p62 protein level (n = 4) and autolysosomes generation (n = 3). Results:Bupivacaine induced significant cell damage. Notably, there was a significant increase in autophagosome generation as evidenced by light chain 3 puncta formation (72.7 ± 6.9 vs. 2.1 ± 1.2) and light chain 3 conversion (2.16 ± 0.15 vs. 0.33 ± 0.04) in bupivacaine-treated cells. Bupivacaine inactivated the protein kinase B/mammalian target of rapamycin/p70 ribosomal protein S6 kinase signaling. However, cellular levels of p62 protein were significantly increased upon bupivacaine treatment (1.29 ± 0.15 vs. 1.00 ± 0.15), suggesting that the drug impaired autophagosome clearance. Further examination revealed that bupivacaine interrupted autophagosome–lysosome fusion (10.87% ± 1.48% vs. 32.94% ± 4.22%). Administration of rapamycin increased autophagosome clearance and, most importantly, improved the survival in bupivacaine-treated cells. However, knockdown of autophagy-related protein 5 (atg5) exacerbated bupivacaine-induced impairment of autophagosome clearance and myotoxicity. Conclusions:The data suggest that autophagosome formation was induced as a stress response mechanism after bupivacaine challenge; however, autophagosome clearance was impaired due to inadequate autophagosome–lysosome fusion. Therefore, impairment of autophagosome clearance appears to be a novel mechanism underlying bupivacaine-induced myotoxicity.