自噬在家兔心肺复苏后低温治疗脑保护中的作用机制

The brain protection mechanism of autophagy on the rabbits by hypothemia treatment after cardiopulmonary cerebral resuscitation

目的探讨自噬在低温治疗心肺脑复苏保护机制中对凋亡影响的研究。方法96只雄性新西兰家兔随机分为4组，空白对照（Sham）组、复苏对照（NT）组、低温治疗（PATH）组及自噬抑制剂LY294002（LY294002）组。除Sham组每组再分为3个亚组，即自主循环恢复（ROSC）后4、9、24h组，除Sham组为6只家兔外，另3组各时间点均为10只。模型采用经右心室致颤4min后复苏模型，PATH组在ROSC后立即予低温干预，目标温度34℃，维持4h；LY294002组用兔脑立体定位仪在复苏前20min给予脑室内注射溶解于二甲亚砜的LY294002（1．4mg／kg），并在复苏后予低温治疗。应用Westernblot方法测定脑组织Bcl-2、Caspase-9、Caspase-3以及LC3-Ⅱ和Beclin-1的表达。结果①NT组ROSC后9、24．hBel-2、Caspase-9、Caspase-3、LC3-Ⅱ和Beclin-1表达较Sham组明显增加（P〈0．05），且NT组24h较9h明显增加（P〈0．05）；②PATH组ROSC后9、24hBcl-2、Caspase-9、Caspase-3较NT组相应时间点减少（P〈0．05），LC3-Ⅱ和Beclin-1较NT组相应时间点增加（P〈0．05）；③LY294002组ROSC后9、24h Bcl-2、Caspase-9、Caspase-3较PATH组相应时间点增加（P〈0．05），LC3-Ⅱ和Beelin-1较PATH组相应时间点减少（P〈0．05）。结论低温治疗通过抑制凋亡减轻心肺复苏后脑水肿，其机制可能是增加自噬调节引起。

Ninety - six male New Zealand rabbits were randomly divided into 4 groups： a sham group, a normothermic treatment group （ NT group）, a post - arrest therapeutic hypothermia treatment group （ PATH group） and an autophagy - suppressive LY294002 group（ LY294002 group）. Aside from a sham group of 6 rabbits, each group was divided into 3 sub- groups of 10 rabbits： 4, 6 or 24 hours after return of spontaneous circulation （ROSC）. The NT group had only a right ventricular fibrillation for 4 minutes and was allowed to recover to ROSC. In the PATH group, the hypothermia intervention occurred immediately after ROSC. The target temperature was 34 ℃ and was maintained for 4 hours. In LY294002 group, an injection of LY294002 （1.4 mg/kg）, which dissolves in dimethyl sulfoxide, into the rabbit＇s cerebral ventricle was made 20 minutes before ROSC occurred. The correct insertion location was determined by using a rabbit stereotaxic apparatus. Hypothermia intervention was also applied, occurring immediately after ROSC. The Western- blotting method is applied to determine the brain tissue Bcl - 2, Caspase - 9, Caspase - 3 and LC3 - II and Beclin - 1 expression. Results For the NTgroup both 9 and 24 hours after ROSC all results significantly increased over the sham group （P 〈 0. 05）, and within the NT group the 24 - hour subgroup was obviously increased over the 9 - hour subgroup（P 〈0.05 ）. For the PATH group both 9 and 24 hours after ROSC Bcl -2, Caspase -9, Caspase - 3 results decreased compared to the corresponding time point in the NT group （ P 〈 0.05 ）, whereas the LC3 - Ⅱ and Beclin - 1 results increased （ P 〈 0.05 ）. For the LY294002 group both 9 and 24 hours after ROSC Bcl -2, Caspase -9, Caspase -3 results increased compared to the corresponding time point in the PATH group （ P 〈 0.05 ）, whereas the LC3 - Ⅱ and Beelin - 1 results decreased （P 〈 0. 05 ）. Conclusion Hypothermia treatment reduces brain edema after cardiopulmonary resuscitation by inhibiting apoptosis, the mechanism may be related to increased autophagy adjustment.