抑制HMGB1/STAT3轴活性对大鼠心肌缺血再灌注损伤的影响  

Knockdown of HMGB1 inhibits HMGB1-STAT3 binding and alleviates myocardial ischemia-reperfusion injury in rats

作　　者：宁萌[1] 齐炳才 冯建宇 龚义杰 高文卿[1] 李彤[1] Ning Meng;Qi Bingcai;Feng Jianyu;Gong Yijie;Gao Wenqing;Li Tong(Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases,Artificial Cell Engineering Technology Research Center,Tianjin Institute of Hepatobiliary Disease,Heart Central,The Third Central Hospital of Tianjin,Tianjin 300170,China;The Third Central Clinical College of Tianjin Medical University,Tianjin 300170,China)

机构地区：[1]天津市第三中心医院心脏中心,天津市重症疾病体外生命支持重点实验室,天津市人工细胞工程技术研究中心,天津市肝胆疾病研究所,天津300170 [2]天津医科大学三中心临床学院,天津300170

出　　处：《国际生物医学工程杂志》2024年第2期131-140,共10页International Journal of Biomedical Engineering

基　　金：天津市医学重点学科建设(TJYXZDXK-035A)。

摘　　要：目的研究抑制高迁移率族蛋白B1(HMGB1)/信号转导及转录激活因子3(STAT3)轴活性对大鼠心肌缺血再灌注损伤的影响。方法建立大鼠心肌缺血再灌注损伤的体内和体外模型,SD大鼠随机分为假手术组、模型组、甘草酸组和NSC74859组,每组6只。假手术组不结扎,假手术组和模型组不给药,甘草酸组和NSC74859组大鼠在缺血/再灌注前12 h 30 min和缺血后30 min分别尾静脉注射HMGB1拮抗剂甘草酸或STAT3抑制剂NSC748595 mg/kg。采用超声心动图评价心功能指标左心室缩短分数(FS)和左心室射血分数(EF),采用苏木精-伊红(HE)染色和TUNEL染色评估心肌细胞的凋亡,采用实时荧光定量PCR反应和Western Blot法检测HMGB1、STAT3和磷酸化STAT3(p-STAT3)的表达水平。MTS法测定H9C2细胞活力,通过细胞内腺苷三磷酸(ATP)含量测定和流式细胞术检测H9C2细胞的线粒体膜电位评估心肌细胞的存活。采用免疫沉淀法研究HMGB1/STAT3的作用方式。采用免疫染色法检测HMGB1/STAT3在细胞核和细胞质中的表达和迁移情况。结果抑制HMGB1或STAT3的表达后,大鼠EF和FS均升高,心肌细胞免疫浸润和凋亡下降;抑制HMGB1表达可降低STAT3的表达,但抑制STAT3表达不影响HMGB1的表达。缺氧导致HMGB1、p-STAT3表达升高,STAT3表达降低,在缺氧8 h时,STAT3表达水平突然升高。复氧后HMGB1、STAT3表达降低,p-STAT3表达升高,但p-STAT3(Ser 727)未参与该过程。缺血再灌注损伤后,HMGB1和STAT3在心肌细胞中牢固结合,但抑制STAT3或HMGB1会减弱这种结合。抑制HMGB1或STAT3表达可减轻心肌缺血再灌注损伤。缺氧后再复氧心肌细胞HMGB1表达增加,HMGB1从细胞核向细胞质迁移。结论抑制HMGB1/STAT3轴活性可有效降低大鼠心肌缺血再灌注损伤。Objective To investigate the effect of inhibitory activity of high mobility group protein B1(HMGB1),signal transduction and activator of transcription 3(STAT3)on myocardial ischemia-reperfusion injury in rats.Methods In vivo and in vitro models of MIRI were established.SD rats were randomly divided into a sham group,a model group,a glycyrrhizic acid group,and a NSC74859 group,with 6 rats in each group.Rats in the sham group were not ligation,and rats in the sham group and model group were not given medication.The rats in the glycyrrhizic acid group and the NSC74859 group were injected with HMGB1 antagonist glycyrrhizic acid or STAT3 inhibitor NSC748595 mg/kg in the tail vein at 12 h 30 min before ischemia/reperfusion and 30 min after ischemia,respectively.Left ventricular shortening fraction(FS)and left ventricular ejection fraction(EF)were evaluated by echocardiography,and apoptosis of cardiomyocytes was evaluated by hematoxylin-eosin(HE)and TUNEL staining.The expression levels of HMGB1,STAT3,and phosphorylated STAT3(p-STAT3)were detected by real-time fluorescence quantitative PCR and Western Blot.The viability of H9C2 cells was determined by the MTS assay,intracellular ATP content was determined,and the mitochondrial membrane potential of H9C2 cells was measured by flow cytometry to evaluate the survival of cardiomyocytes.The action mode of HMGB1/STAT3 was studied by the immunoprecipitation method.The expression and migration of HMGB1/STAT3 in the nucleus and cytoplasm were detected by immunostaining.Results After inhibiting the expression of HMGB1 or STAT3,EF and FS were increased,and immune infiltration and apoptosis of cardiomyocytes were decreased.Inhibition of HMGB1 expression could decrease the expression of STAT3,but inhibition of STAT3 expression didn’t affect the expression of HMGB1.Hypoxia could lead to increased expression of HMGB1 and p-STAT3,and decreased expression of STAT3.After 8 hours of hypoxia,the expression level of STAT3 suddenly increased.After reoxygenation,the expression of HMGB1 and S

关 键 词：缺血再灌注损伤 高迁移率族蛋白B1 信号转导及转录激活因子3 缺氧复氧损伤 

分 类 号：R542.2[医药卫生—心血管疾病]