中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2014年
6期
635-641
,共7页
陆件%钱会银%刘励军%周保纯%肖盐%毛锦宁%安国印%芮明忠%王涛%朱昌来
陸件%錢會銀%劉勵軍%週保純%肖鹽%毛錦寧%安國印%芮明忠%王濤%硃昌來
륙건%전회은%류려군%주보순%초염%모금저%안국인%예명충%왕도%주창래
亚低温%心肺复苏%活性氧%半胱氨酸天冬氨酸蛋白酶3%轻链3%自噬
亞低溫%心肺複囌%活性氧%半胱氨痠天鼕氨痠蛋白酶3%輕鏈3%自噬
아저온%심폐복소%활성양%반광안산천동안산단백매3%경련3%자서
Mild hypothermia%Cardiopulmonary resuscitation%Reactive oxygen species%Caspase-3%LC3%Autophagy
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。
目的：觀察亞低溫（MH）對心肺複囌（CPR）後海馬神經細胞活性氧（ROS）產生量和半胱氨痠天鼕氨痠蛋白酶3（caspase-3） mRNA和自噬相關蛋白輕鏈3（LC3）錶達的影響。方法65隻健康成年雄性SD大鼠隨機數字法隨機分為2組：空白對照組（BC，n＝5）；CPR組（n＝60）。CPR組製作窒息法心肺複囌模型，在自主循環恢複（ROSC）後再隨機分為2組：常溫CPR組（NT）和低溫CPR組（HT）。NT組在ROSC後保持37℃恆溫，HT組在ROSC後立即行低溫32℃榦預4 h。根據觀察時點的不同，再將兩組CPR組隨機分2箇亞組：即 ROSC後12 h和24 h組（NT-12，NT24，HT-12，HT-24）。到達觀察時點先對存活大鼠進行神經功能缺損評分（NDS），然後立即取雙側海馬組織，應用流式法檢測大鼠海馬單細胞懸液ROS水平。利用透射電鏡觀察海馬神經細胞細胞覈和線粒體的超微結構形態學變化。利用逆轉錄-聚閤酶鏈反應（RT-PCR）技術測定海馬神經細胞caspase-3mRNA的錶達水平；蛋白免疫印跡法（WB）測定海馬神經細胞LC3蛋白水平。應用SPSS 19.0軟件包，計量數據以均數±標準差（x ±s）錶示，兩組間均數比較用成組t檢驗，多組間比較採用單因素方差分析。結果60隻大鼠中有44隻（73％）成功ROSC，存活到觀察時點的有33隻大鼠（55％）。NT和HT組各時點的NDS明顯低于BC組（F＝8.107，P＜0.05），HT-12和HT-24組的NDS與相同時點的NT組比較有顯著提高（t＝9.692，P＜0.01；t＝14.374，P＜0.01）。NT和HT組各時點的ROS產生量明顯高于BC組（F＝16.824，P＜0.05），而HT-12和HT-24組的升高程度則明顯低于相同時點的NT組大鼠（t＝9.836，P＜0.01；t＝7.499，P＜0.01）。NT和HT組各時點的caspase-3mRNA錶達量明顯高于BC組（F＝24.527，P＜0.05），而HT-12和 HT-24組的升高程度則明顯低于相同時點的 NT 組大鼠（t ＝6.935，P＜0.01；t＝4.317，P＜0.01）。NT 和 HT 組各時點的 LC3B-Ⅱ/Ⅰ的錶達量明顯高于 BC 組（F＝6.584，P＜0.05），而 HT-12和 HT-24組的升高程度則明顯低于相同時點的 NT 組大鼠（t＝10.836，P＜0.001；t＝2.653，P＝0.02）。NT組海馬神經細胞覈和線粒體的形態學變化較BC組有明顯改變，HT組海馬神經細胞覈和線粒體與相同時點NT組比較損傷有所減輕。結論 MH能減輕ROSC後大鼠腦神經細胞損傷併提高其神經功能，其機製之一是減少瞭CPR後大鼠神經細胞ROS的產生，併通過抑製caspase-3和LC3的錶達使神經細胞凋亡和自噬減少。
목적：관찰아저온（MH）대심폐복소（CPR）후해마신경세포활성양（ROS）산생량화반광안산천동안산단백매3（caspase-3） mRNA화자서상관단백경련3（LC3）표체적영향。방법65지건강성년웅성SD대서수궤수자법수궤분위2조：공백대조조（BC，n＝5）；CPR조（n＝60）。CPR조제작질식법심폐복소모형，재자주순배회복（ROSC）후재수궤분위2조：상온CPR조（NT）화저온CPR조（HT）。NT조재ROSC후보지37℃항온，HT조재ROSC후립즉행저온32℃간예4 h。근거관찰시점적불동，재장량조CPR조수궤분2개아조：즉 ROSC후12 h화24 h조（NT-12，NT24，HT-12，HT-24）。도체관찰시점선대존활대서진행신경공능결손평분（NDS），연후립즉취쌍측해마조직，응용류식법검측대서해마단세포현액ROS수평。이용투사전경관찰해마신경세포세포핵화선립체적초미결구형태학변화。이용역전록-취합매련반응（RT-PCR）기술측정해마신경세포caspase-3mRNA적표체수평；단백면역인적법（WB）측정해마신경세포LC3단백수평。응용SPSS 19.0연건포，계량수거이균수±표준차（x ±s）표시，량조간균수비교용성조t검험，다조간비교채용단인소방차분석。결과60지대서중유44지（73％）성공ROSC，존활도관찰시점적유33지대서（55％）。NT화HT조각시점적NDS명현저우BC조（F＝8.107，P＜0.05），HT-12화HT-24조적NDS여상동시점적NT조비교유현저제고（t＝9.692，P＜0.01；t＝14.374，P＜0.01）。NT화HT조각시점적ROS산생량명현고우BC조（F＝16.824，P＜0.05），이HT-12화HT-24조적승고정도칙명현저우상동시점적NT조대서（t＝9.836，P＜0.01；t＝7.499，P＜0.01）。NT화HT조각시점적caspase-3mRNA표체량명현고우BC조（F＝24.527，P＜0.05），이HT-12화 HT-24조적승고정도칙명현저우상동시점적 NT 조대서（t ＝6.935，P＜0.01；t＝4.317，P＜0.01）。NT 화 HT 조각시점적 LC3B-Ⅱ/Ⅰ적표체량명현고우 BC 조（F＝6.584，P＜0.05），이 HT-12화 HT-24조적승고정도칙명현저우상동시점적 NT 조대서（t＝10.836，P＜0.001；t＝2.653，P＝0.02）。NT조해마신경세포핵화선립체적형태학변화교BC조유명현개변，HT조해마신경세포핵화선립체여상동시점NT조비교손상유소감경。결론 MH능감경ROSC후대서뇌신경세포손상병제고기신경공능，기궤제지일시감소료CPR후대서신경세포ROS적산생，병통과억제caspase-3화LC3적표체사신경세포조망화자서감소。
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'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.
