消退素D1抑制自噬减轻猪心肺复苏后脑损伤的机制研究

Study on the mechanism of resolvin D1 in alleviating brain injury after cardiopulmonary resuscitation through the inhibition of autophagy in pigs

目的探讨消退素D1(resolvin D1,RvD1)通过调控自噬途径减轻猪心肺复苏(cardiopulmonary resuscitation,CPR)后脑损伤的作用机制。方法国产雄性白猪19头,体质量30~41 kg。应用随机数字表法,将实验动物分为3组,即假手术(sham,S)组(n=5)、CPR组(n=7)与RvD1组(n=7)。S组动物仅进行气管插管、血管置管等操作准备,CPR组和RvD1组动物经右心室电刺激诱发心脏骤停8 min后CPR 5 min的方法建立实验模型。于复苏后5 min时,RvD1组动物经股静脉注射RvD10.6μg/kg,S组和CPR组动物给予等量溶媒。于复苏后1、3、6和24 h时,采集静脉血标本,应用ELISA法检测神经元特异度烯醇化酶(neuron specific enolase,NSE)和S100β蛋白(S100βprotein,S100β)。复苏后24 h进行神经功能缺损评分(neurological deficit score,NDS),然后安乐死并获取大脑皮层组织,应用western blot法检测磷酸化磷酸腺苷活化蛋白激酶(phosphorylated AMP-activated protein kinase,p-AMPK)、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,p-mTOR)、微管相关轻链蛋白3 II(microtubule-associated protein light chain 3,LC3Ⅱ)和p62。三组间比较应用单因素方差分析和Bonferroni事后检验。结果与S组相比,CPR组和RvD1组动物复苏后24 h NDS显著升高,血清NSE和S100β显著增加(均P<0.05)。然而,与CPR组相比,RvD1组动物复苏后24 h NDS显著降低[(182±34)和(124±18),P<0.05],在复苏3 h后血清NSE和S100β浓度显著减少[NSE(ng/mL):3 h为(23.1±3.8)和(18.0±2.2),6 h为(27.3±2.9)和(19.8±1.4),24 h为(28.1±1.3)和(15.1±2.1);S100β(pg/mL):3 h为(1611±208)和(1322±100),6 h为(1825±197)和(1410±102),24 h为(1613±138)和(1183±139),均P<0.05]。与S组相比,CPR组和RvD1组动物在复苏后24 h脑皮层p-AMPK与LC3II表达显著增加,p-mTOR与p62表达显著减少(均P<0.05)。然而,与CPR组相比,RvD1组动物在复苏后24 h脑皮层p-AMPK与LC3Ⅱ表达显著减少、同时p-mTOR与p62表达显著增加[p-AMPK:(0.28±0.08)和(0.17±0.03);LC3Ⅱ:(0.33±0.09)和(0.21±0.04);p-mTOR:(0.13±0.02)和(0.16±0.02);p62:(0.16±0.05)和(0.22±0.02),均P<0.05]。结论RvD1减轻猪CPR后脑损伤的保护机制可能与抑制AMPK/mTOR通路介导的细胞自噬有关。

Objective To investigate the mechanism of resolvin D1(RvD1)in alleviating brain injury after cardiopulmonary resuscitation(CPR)through regulating autophagy pathway in pigs.Methods Nineteen male domestic pigs,weighing 30-41 kg,were divided into 3 groups using a random number table method:sham group(S group,n=5),CPR group(n=7),and RvD1 group(n=7).In the S group,the animals only experienced general preparation.In the CPR and RvD1 groups,the pig CPR model was established by 8 min of cardiac arrest caused by electrically induced ventricular fibrillation,and followed by 5 min of CPR.At 5 min after resuscitation,a dose of 0.6μg/kg of resolvin D1 was injected via femoral vein in the RvD1 group,and the same amount of vehicle was similarly administered in the other two groups.At 1,3,6,and 24 h after resuscitation,blood samples were collected from the femoral vein to measure serum concentrations of neuron specific enolase(NSE)and S100βprotein by ELISA.At 24 h after resuscitation,neurological function was evaluated by neurological deficit score(NDS),and then the animals were euthanized to obtain cerebral cortex for measuring the expressions of phosphorylated AMP-activated protein kinase(p-AMPK),phosphorylated mammalian target of rapamycin(p-mTOR),microtubule-associated protein light chain 3(LC3 II)and p62 by Western blot.The variables were compared with One-way analysis of variance and then the Bonferroni test among the three groups.Results During 24 h after resuscitation,the NDS was significantly increased accompanied with significantly greater concentrations of NSE and S100βin serum in the CPR and RvD1 groups compared to the S group(all P<0.05).However,the NDS was significantly decreased at 24 h after resuscitation[(182±34)vs.(124±18),P<0.05],and serum NSE and S100βwere significantly reduced starting 3 h after resuscitation in the RvD1 group compared to the CPR group[NSE(ng/mL):(23.1±3.8)vs.(18.0±2.2)at 3 h,(27.3±2.9)vs.(19.8±1.4)at 6 h,and(28.1±1.3)vs.(15.1±2.1)at 24 h;S100B(pg/mL):(1611±208)vs.(1322±100)at 3 h,(1825±197)vs.(1410±102)at 6 h,and(1613±138)vs.(1183±139)at 24 h,all P<0.05].The expression levels of p-AMPK and LC3 II were significantly increased while the expression levels of p-mTOR and p62 were significantly decreased at 24 h after resuscitation in the CPR and RvD1 groups compared to the S group(all P<0.05).However,the expression levels of p-AMPK and LC3 II were significantly lower and the expression levels of p-mTOR and p62 were significantly higher at 24 h after resuscitation in the RvD1 group compared to the CPR group[p-AMPK:(0.28±0.08)vs.(0.17±0.03);LC3 II:(0.33±0.09)vs.(0.21±0.04);p-mTOR:(0.13±0.02)vs.(0.16±0.02);p62:(0.16±0.05)vs.(0.22±0.02),all P<0.05].Conclusions The protective mechanism by which RvD1 alleviates brain injury after CPR in pigs might be related to the inhibition of neuronal autophagy mediated by AMPK/mTOR pathway.