摘要/Abstract
摘要： 目的·探讨心搏骤停复苏大鼠实行低温治疗后，不同复温速率对神经元自噬的影响。方法·将雄性SD大鼠随机分为假手术组、常温组、慢速复温组和快速复温组；除假手术组外，其他3组建立5 min窒息性心搏骤停模型后再行心肺复苏。常温组维持大鼠体温(37.0±0.5) ℃；慢速复温组和快速复温组给予持续4 h的低温治疗（34 ℃）后，慢速复温组以0.5 ℃ /h，快速复温组以4 ℃ /h复温至(37.0±0.5) ℃。2个复温组再各分出1个复温氯喹组，术前1 h腹腔注射氯喹10 mg/kg。采用尼式染色观察皮质运动神经元形态变化，Western blotting及免疫荧光法检测不同时间点自噬及溶酶体相关蛋白——微管相关蛋白1轻链3- Ⅱ（microtubule-associated protein 1 light chain 3- Ⅱ，LC3- Ⅱ）、溶酶体相关膜蛋白2（lysosome-associated membrane protein 2，LAMP2）、P62、Beclin1（Bcl 2-interacting protein-1）、组织蛋白酶D（cathepsin D）、泛素蛋白（ubiquitin）的表达量。结果·与常温组相比，慢速复温组于低温治疗结束后6 h时LC3- Ⅱ和Beclin1表达量显著增加（均P=0.000）。与慢速复温组相比，快速复温组皮质神经元活细胞数量显著减少（P=0.000），LAMP2及cathepsin D的表达量显著降低（均P=0.000），ubiquitin（P=0.007）和P62（P=0.000）表达量增加。与未加氯喹组相比，慢速复温氯喹组ubiquitin（P=0.000）和P62（P=0.001）表达量增加，而快速复温氯喹组ubiquitin（P=0.000）和P62（P=0.007）表达量减少。结论·心搏骤停复苏实行低温治疗后快速复温可导致大鼠神经元溶酶体功能障碍、自噬流受损，神经元损伤增加；而慢速复温过程中自噬激活，神经元损伤减少。
关键词: 心搏骤停, 低温治疗, 复温, 自噬, 神经元, 溶酶体
Abstract:
Objective · To investigate the effects of different rewarming rates on neuron autophagy in the rats with therapeutic hypothermia following cardiac arrest and cardiopulmonary resuscitation. Methods · The SD rats were randomized into 4 groups, i.e., sham operation group (Sham group), normothermia group (Normo group), slow rewarming group (SR group) and fast rewarming group (FR group). Except Sham group, the other three groups were given cardiopulmonary resuscitation after the establishment of 5-min asphyxial cardiac arrest model. The rats in Normo group were maintained at (37.0±0.5) ℃ ; SR group and FR group were treated with hypothermia (34 ℃ ) for 4 h, and SR group was rewarmed at 0.5 ℃ /h and FR group was rewarmed at 4 ℃ /h to (37.0±0.5) ℃ . SR+chloroquine group and FR+chloroquine group were isolated from the two rewarming groups, and the rats in these two groups were injected intraperitoneally with chloroquine 10 mg/kg 1 h before operation. Morphological changes of cortical motor neurons were detected by Nissl staining. The expressions of autophagy- and lysosomal-related proteins at corresponding time points, i.e., microtubule-associated protein 1 light chain 3- Ⅱ (LC3- Ⅱ ), lysosome-associated membrane protein 2 (LAMP2), P62, Bcl 2-interacting protein-1 (Beclin1), cathepsin D, and ubiquitin, were examined by Western blotting and immunofluorescence. Results · The expressions of LC3- Ⅱ and Beclin1 increased significantly in SR group 6 h after hypothermia treatment compared to Normo group (both P=0.000). Compared with SR group, FR group showed an obviously lower number of viable cortical neurons (P=0.000), significantly decreased expressions of LAMP2 and cathepsin D (both P=0.000), and increased expressions of ubiquitin (P=0.007) and P62 (P=0.000). Compared with SR group, the expressions of ubiquitin (P=0.000) and P62 (P=0.001) in SR+chloroquine group increased, while the expressions of ubiquitin (P=0.000) and P62 (P=0.007) decreased in FR+chloroquine group compared with FR group. Conclusion · Fast rewarming after therapeutic hypothermia for cardiac arrest and cardiopulmonary resuscitation can lead to neuron lysosomal dysfunction, impaired autophagy flux, and increased neuronal damage; however, slow rewarming activates autophagy and reduces neuronal injury.
Key words: cardiac arrest, hypothermia therapy, rewarming, autophagy, neuron, lysosome
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