亚低温对心肺复苏大鼠海马神经细胞活性氧及半胱氨酸天冬氨酸蛋白酶3和自噬相关蛋白轻链3的影响

The impact of mild hypothermia on the ROS and expression of caspase-3mRNA and LC3 of hippocampus nerve cells in rats after cardiopulmonary resuscitation

目的：观察亚低温（MH）对心肺复苏（CPR）后海马神经细胞活性氧（ROS）产生量和半胱氨酸天冬氨酸蛋白酶3（caspase-3） mRNA和自噬相关蛋白轻链3（LC3）表达的影响。方法65只健康成年雄性SD大鼠随机数字法随机分为2组：空白对照组（BC，n＝5）；CPR组（n＝60）。CPR组制作窒息法心肺复苏模型，在自主循环恢复（ROSC）后再随机分为2组：常温CPR组（NT）和低温CPR组（HT）。NT组在ROSC后保持37℃恒温，HT组在ROSC后立即行低温32℃干预4 h。根据观察时点的不同，再将两组CPR组随机分2个亚组：即 ROSC后12 h和24 h组（NT-12，NT24，HT-12，HT-24）。到达观察时点先对存活大鼠进行神经功能缺损评分（NDS），然后立即取双侧海马组织，应用流式法检测大鼠海马单细胞悬液ROS水平。利用透射电镜观察海马神经细胞细胞核和线粒体的超微结构形态学变化。利用逆转录-聚合酶链反应（RT-PCR）技术测定海马神经细胞caspase-3mRNA的表达水平；蛋白免疫印迹法（WB）测定海马神经细胞LC3蛋白水平。应用SPSS 19.0软件包，计量数据以均数±标准差（x ±s）表示，两组间均数比较用成组t检验，多组间比较采用单因素方差分析。结果60只大鼠中有44只（73％）成功ROSC，存活到观察时点的有33只大鼠（55％）。NT和HT组各时点的NDS明显低于BC组（F＝8.107，P＜0.05），HT-12和HT-24组的NDS与相同时点的NT组比较有显著提高（t＝9.692，P＜0.01；t＝14.374，P＜0.01）。NT和HT组各时点的ROS产生量明显高于BC组（F＝16.824，P＜0.05），而HT-12和HT-24组的升高程度则明显低于相同时点的NT组大鼠（t＝9.836，P＜0.01；t＝7.499，P＜0.01）。NT和HT组各时点的caspase-3mRNA表达量明显高于BC组（F＝24.527，P＜0.05），而HT-12和 HT-24组的升高程度则明显低于相同时点的 NT 组大鼠（t ＝6.935，P＜0.01；t＝4.317，P＜0.01）。NT 和 HT 组各时点的 LC3B-Ⅱ/Ⅰ的表达量明显高于 BC 组（F＝6.584，P＜0.05），而 HT-12和 HT-24组的升高程度则明显低于相同时点的 NT 组大鼠（t＝10.836，P＜0.001；t＝2.653，P＝0.02）。NT组海马神经细胞核和线粒体的形态学变化较BC组有明显改变，HT组海马神经细胞核和线粒体与相同时点NT组比较损伤有所减轻。结论 MH能减轻ROSC后大鼠脑神经细胞损伤并提高其神经功能，其机制之一是减少了CPR后大鼠神经细胞ROS的产生，并通过抑制caspase-3和LC3的表达使神经细胞凋亡和自噬减少。

Objective To observe the impact of mild hypothermia （MH）on the reactive oxygen species （ROS）and expression of cacpase-3mRNA and light chain 3 （LC3,a subunit of immunoglobulin）in hippocampus nerve cells of rats after cardiopulmonary resuscitation （CPR）.Methods A total of 65 healthy male Sprague Dawley （SD）adult rats were randomly （random number）divided into 2 groups：blank control group （n =5）and CPR group （n =60）.Cardiac arrest （CA）was induced in rats of CPR group by asphyxia.The survival rats after CPR were randomly （random number）divided into 2 groups：normothermia CPR group （NT）and hypothermia CPR group （HT）.Homeothermia of 37 ℃ was maintained in NT group after restoration of spontaneous circulation （ROSC）,and hypothermal intervention to 32 ℃ was carried out in HT group for 4 hours immediately after ROSC.Both NT group and HT group were then randomly divided to 2 subgroups 12 hours and 24 hours after ROSC （NT-12,NT-24,HT-12,HT-24 subgroups）.During observation,the neurological deficit （NDS）of rats was scored,then the bilateral hippocampi were obtained from rats＆#39;head,and monoplast suspension of fresh hippocampus tissue was made immediately to determine the level of intracellular ROS by flow cytometry.Transmission electron microscope was used to observe the ultrastructure changes of cellular nucleus and mitochondria.Reverse transcription-polymerase chain reaction （RT-PCR）was used to determine the expression of caspase-3mRNA and Western-blotting （WB）was used to determine the level of LC3 in frozen hippocampus tissue.Measured data were analyzed with paired sample T test and One-Way ANOVA.Results Of 60 rats with CA,44 were successfully resuscitated （73%）and 33 survived until the end of the experiment （55%）.The NDSs of rats in NT and HT groups were significantly reduced in comparison with BC group （F=8.107,P〈0.05）,while the NDSs of rats in HT-12 subgroup and HT-24 subgroup were significantly increased in comparison with NDSs of rats in NT-12 subgroup and NT-24 subgroup,respectively （t=9.692,P〈0.01;t=14.374,P〈0.01 ）.The ROS in hippocampus nerve cells of rats in NT group and HT group were significantly increased compared to BC group （F=16.824,P〈0.05 ）,whereas the ROS in HT-12 and HT-24 subgroups were significantly reduced compared to ROS in NT-12 and NT-24 subgroups,respectively （t =9.836,P〈0.01;t =7.499,P〈0.01）.The expressions of caspase-3 mRNA in hippocampus nerve cells of rats in NT and HT groups were significantly increased compared to BC group （F=24.527,P〈0.05）,while the expressions of caspase-3 mRNA in rats of HT-12 and HT-24 subgroups were significantly reduced compared to NT-12 and NT-24 subgroups,respectively （t =6.935,P 〈0.01;t =4.317,P 〈0.01 ）.The level of LC3B-II/I in hippocampus nerve cells of rats in NT and HT groups were significantly increased compared to BC group （F=6.584,P〈0.05）,while the levels of LC3B-II/I in rats of HT-12 and HT-24 subgroups were significantly reduced compared to NT-12 and NT-24 subgroups,respectively （t=10.836,P〈0.001;t=2.653,P=0.02）.Ultrastructure damage of nucleus and mitochondria in NT group was more evident compared to BC group,and eumorphism of nucleus and mitochondria were maintained in rats of HT group compared to NT group.Conclusions The mild hypothermia reduced the injury of nerve cells and improved the neurological function of rats survived from cardiac arrest likely by reducing ROS production of nerve cells and inhibition the expression of caspase-3mRNA and lowering the level of LC3 leading to reducing cellular apoptosis and massive autophagy in rats survived from cardiac arrest after CPR.