二甲双胍通过AMPK-自噬信号通路促进脑保护作用的研究  

Study on the mechanism of metformin promoting brain protection via AMPK autophagy signal pathway

作　　者：覃智芳 王燕泽 陈碧华 周满红 QIN Zhi-fang;WANG Yan-ze;CHEN Bi-hua;ZHOU Man-hong(Department of Cardiology,Kweichow Moutai Hospital,Renhuai564500 Guizhou Province,China;Department of Emergency,Afiliated Hospital of Zunyi Medical University,Zunyi 563003,Guizhou Province,China;Department of Biomedical Engineering and Imaging,Army Military Medical University,Chongqing 400000,China)

机构地区：[1]贵州茅台医院心血管内科,贵州仁怀564500 [2]遵义医科大学附属医院急诊科,贵州遵义563003 [3]陆军军医大学生物医学工程与影像学系,重庆400000

出　　处：《中国临床药理学杂志》2023年第23期3424-3428,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金青年科学基金资助项目(81701300)。

摘　　要：目的 探讨二甲双胍(Met)预处理能否对心搏骤停/心肺复苏(CA/CPR)后脑损伤起到保护作用及其可能的机制及信号通路。方法 以电刺激法构建CA模型。将大鼠随机分为假手术组(0.9%NaCl灌胃14 d,不诱导CA)、模型组(0.9%NaCl灌胃14 d后造模)、实验组(200 mg·kg^(-1)Met+0.9%NaCl灌胃14 d后造模)、Cc组[200 mg·kg^(-1)Met+0.9%NaCl灌胃14 d,造模前1 h经腹膜腔注射5′-腺嘌呤核苷酸依赖的蛋白激酶(AMPK)抑制药Cc]、氯喹组[200 mg·kg^(-1)Met+0.9%NaCl灌胃14 d,造模前1 h腹膜腔注射自噬抑制药氯喹]和AICAR组(200 mg·kg^(-1)Met+0.9%NaCl灌胃14 d,造模前1 h经腹膜腔注射AMPK激动药AICAR)。以生存曲线记录CA/CPR后7 d生存率,以神经病残疾评分表对CA/CPR后7 d神经功能进行评分,以考马斯亮蓝法测定丙二醛(MDA)水平;以活性氧簇(ROS)荧光探针DHE检测脑组织ROS水平,以蛋白质印迹法测定脑组织中AMPK、p-AMPK、微管相关蛋白轻链3Ⅰ(LC3Ⅰ)、LC3Ⅱ、p62蛋白表达水平。结果 假手术组、模型组、实验组、Cc组、氯喹组、AICAR组大鼠的7 d生存率分别100%、40%、70%、40%、40%和65%,7 d神经功能评分分别为(78.35±1.65)、(46.50±4.41)、(67.93±4.64)、(49.50±3.53)、(52.00±2.83)和(68.33±1.53)分,ROS水平分别为(417.60±8.37)、(748.60±36.05)、(575.80±10.73)、(713.80±18.85)、(668.20±9.58)和(566.00±24.48)U·mg^(-1),MDA水平分别为(4.38±0.33)、(8.06±0.76)、(5.50±0.48)、(7.18±0.29)、(6.82±0.31)和(5.20±0.34)nmol·mg^(-1),p-AMPK/AMPK水平分别为0.74±0.04、0.87±0.01、1.61±0.01、0.55±0.05、1.09±0.09和1.27±0.07,p62相对表达水平分别为0.42±0.02、0.86±0.05、0.61±0.04、0.98±0.04和0.78±0.03,LC3Ⅱ/LC3Ⅰ水平分别为0.29±0.32、0.37±0.26、0.96±0.78、0.58±0.26、0.43±0.03和1.40±0.10。实验组、AICAR组的上述指标与模型组比较,Cc组、氯喹组的上述指标与实验组比较,差异均有统计学意义(均P<0.05)。结论 Met预处理通过激活AMPK信号通�Objective To explore whether metformin preconditioning can protect brain injury after cardiac arrest/cardiopulmonary resuscitation(CA/CPR)and its possible mechanisms and signaling pathways.Methods Constructed a CA model by using electrical stimulation.Rats were randomly divided into the sham group(0.9%NaCl gavage daily for14 days,without inducing CA),model group(CA model was induced after 0.9%NaCl gavage daily for 14 days),experimental group(CA model was induced after 200 mg·kg^(-1)Met+0.9%NaCl gavage daily for 14 days),Cc group[200 mg·kg^(-1)Met+0.9%NaCl gavage daily for 14 days,adenosine 5'-monophosphate kinase-activated protein(AMPK)inhibitor Compoud C(Cc)was injected intraperitoneally at 1 hour before modeling],chloroquine(CQ)group(daily 200 mg·kg^(-1)Met+0.9%NaCl gavage for 14 days,intraperitoneal injection of autophagy inhibitor CQ at 1 hour before modeling)and AICAR group(continuous daily 200 mg·kg^(-1)Met+0.9%NaCl gavage for 14 days,intraperitoneal injection of AMPK agonist AICAR at 1 hour before modeling).The 7-day survival rate after CA/CPR via using survival curves was recorded;the neurological function 7-days after CA/CPR was scored by using the neurological disability scale;the protein malondialdehyde(MDA)level were measured using the Coomassie Brilliant Blue method;detection of reactive oxygen species(ROS)levels in brain tissue by using ROS fluorescent probe DHE;Western blotting was used to determine the expression levels of related proteins such as AMPK,p-AMPK,microtubule associated protein 1 light chain 3Ⅰ(LC3Ⅰ),LC3Ⅱ,and p62 in brain tissue.Results The 7-day survival rates of sham,model,experimental,Cc,CQ and AICAR groups rats were 100%,40%,70%,40%,40%and 65%,respectively;the 7-day neurological function scores were 78.35±1.65,46.50±4.41,67.93±4.64,49.50±3.53,52.00±2.83 and 68.33±1.53,respectively;the ROS levels were(417.60±8.37),(748.60±36.05),(575.80±10.73),(713.80±18.85),(668.20±9.58)and(566.00±24.48)U·mg^(-1),respectively;the MDA levels were(4.38±0.33),(8.06±0.76),(5.50

关 键 词：二甲双胍 腺苷5′-单磷酸激酶活化蛋白 自噬 心搏骤停/心肺复苏 脑功能保护 

分 类 号：R977.15[医药卫生—药品]