UCP2过表达抑制活性氧产生和炎症反应以减轻脓毒性心肌损伤  

Uncoupling protein 2 overexpression alleviates sepsis-induced myocardial injury via inhibiting reactive oxygen species production and inflammation

作　　者：耿争光 罗诗雨 李光素 陆钦菊 付豹 傅小云 Geng Zhengguang;Luo Shiyu;Li Guangsu;Lu Qinju;Fu Bao;Fu Xiaoyun(Department of Critical Care Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563003,Guizhou,China;Department of PICU,Guiyang Maternal and Child Health Care Hospital,Guiyang 550003,Guizhou,China;Department of Critical Care Medicine,Affiliated Hospital of Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]遵义医科大学附属医院重症医学科,贵州遵义563003 [2]贵阳市妇幼保健院PICU,贵州贵阳550003 [3]贵州医科大学附属医院重症医学科,贵阳550025

出　　处：《中华危重病急救医学》2020年第11期1346-1351,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81560308);贵州省普通高等学校科技拔尖人才支持项目(KY2016-078)。

摘　　要：目的探讨解耦联蛋白2(UCP2)过表达是否通过抑制活性氧(ROS)产生、降低炎症反应,发挥脓毒症心肌保护作用。方法将40只SD大鼠按随机数字表法分为假转染假手术组(Sham组)、假转染盲肠结扎穿孔术(CLP)致脓毒症组(CLP组)、单纯腺相关病毒(AAV)转染手术组(AAV组)和UCP2过表达手术组(UCP2组)4组,每组10只。UCP2组心肌注射UCP2腺相关病毒(AAV-UCP2;滴度1×1012 v.g/mL,每个点10μL,共60μL),3周后进行CLP;AAV组心肌转染AAV病毒,3周后进行CLP。制模后24 h评价模型是否制备成功并取材,荧光显微镜下观察心肌组织冰冻切片AAV病毒转染效果,蛋白质免疫印迹试验(Western blotting)检测心肌组织UCP2蛋白表达,二氢乙啶(DHE)染色检测心肌细胞ROS产生情况,酶联免疫吸附试验(ELISA)检测血清心肌标志物和炎性细胞因子水平。结果CLP术后24 h大鼠表现为立毛、眼鼻口分泌物增多,甚至出现脓尿、稀便及呼吸困难等症状;开腹后发现盲肠呈紫黑色,周围肠腔有脓性渗出;冰冻切片后荧光显微镜下可见病毒转染成功(转染部位呈绿色荧光)。进一步Western blotting检测显示,CLP组UCP2表达较Sham组增加(UCP2/β-tubulin:1.53±0.06比1,P<0.01);与AAV组相比,UCP2组UCP2表达进一步增高(UCP2/β-tubulin:1.96±0.22比1.59±0.07,P<0.01)。荧光显微镜下观察CLP组和AAV组ROS产生较Sham组明显增加;当UCP2过表达后,ROS产生较CLP组和AAV组明显减少(A值:1.03±0.10比1.81±0.13、1.67±0.08,均P<0.01)。ELISA结果显示,与Sham组相比,CLP组和AAV组乳酸脱氢酶(LDH)、肌酸激酶(CK)、心肌肌钙蛋白I(cTnI)、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平明显增加;当UCP2过表达后,以上心肌酶和炎性细胞因子分泌较CLP组和AAV组明显减少〔LDH(ng/L):48.97±1.04比56.85±1.36、57.08±1.54,CK(ng/L):235.23±20.33比306.34±25.93、304.76±25.29,cTnI(ng/L):199.79±18.27比241.88±14.32、243.33±23.79,TNF-α(ng/L):385.71±20.09比488.92±26.9Objective To investigate whether the overexpression of uncoupling protein 2(UCP2)can protect myocardium from sepsis by inhibiting the production of reactive oxygen species(ROS)and inflammatory response.Methods Forty Sprague-Dawley rats were divided into four groups according to random number table method(n=10):sham transfection and sham surgery group(Sham group),sham transfection and cecal ligation and perforation(CLP)group(CLP group),simple adeno-associated virus(AAV)transfection surgery group(AAV group),and UCP2 overexpression surgery group(UCP2 group).In UCP2 group,UCP2 adeno-associated virus(AAV-UCP2;titer 1×1012 v.g/mL,10μL per site,60μL in total)was injected into myocardium,and CLP was performed 3 weeks later.In AAV group,the myocardium was transfected with AAV virus and CLP was performed 3 weeks later.Twenty-four hours after modeling,whether the model was successfully prepared was evaluated.The transfection effect of AAV virus on the frozen sections of myocardial tissue was observed under fluorescence microscope,the expression of UCP2 protein was detected by Western blotting,ROS production was detected by dihydroethidine(DHE)staining,and serum myocardial markers and inflammatory cytokines were detected by enzyme linked immunosorbent assay(ELISA).Results Twenty-four hours after CLP,the rats showed stiff hair,increased secretions from eyes,nose and mouth,and symptoms of pyuria,loose stools,and dyspnea.After laparotomy,the cecum showed purple and black,and there was purulent exudation around the intestinal cavity.The virus was successfully transfected on frozen section under the fluorescence microscope(the site of the transfection was green fluorescence),and further Western blotting revealed that the expression of UCP2 in the CLP group was higher than that in the Sham group(UCP2/β-tubulin:1.53±0.06 vs.1,P<0.01).Compared with the AAV group,UCP2 expression was further increased in the UCP2 group(UCP2/β-tubulin:1.96±0.22 vs.1.59±0.07,P<0.01).Under the fluorescence microscope,ROS production in the CLP and

关 键 词：脓毒症 心肌保护 解耦联蛋白2 盲肠结扎穿孔术 

分 类 号：R459.7[医药卫生—急诊医学] R542.2[医药卫生—治疗学]