基于蛋白质组学的缺血预适应心肌保护相关机理研究  

Proteomic-based study to explore cardioprotective mechanism of ischemic preconditioning in mice

作　　者：尼鲁帕尔·谢甫开提 阿曼古丽·如则 唐婧[1] 赵翎 赵帮豪 高晓明[1,2] XIEFUKAITI Nilupaer;RUZE Amanguli;TANG Jing;ZHAO Ling;ZHAO Bang-hao;GAO Xiaoming(State Key Laboratory of Pathogenesis,Prevention and Treatment of High Incidence Diseases in Central Asia,Department of Cardiology,First Affiliated Hospital of Xinjiang Medical University,Xinjiang Key Laboratory of Medical Animal Model Research,Urumqi 830054,China;Clinical Medical Research Institute of Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]省部共建中亚高发病成因与防治国家重点实验室,新疆医科大学第一附属医院心内科,新疆医学动物模型研究重点实验室,新疆乌鲁木齐830054 [2]新疆医科大学临床医学研究院,新疆乌鲁木齐830054

出　　处：《中国病理生理杂志》2023年第2期212-219,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82060073;No.81870272);省部共建中亚高发病成因与防治国家重点实验室开放课题项目(No.SKL-HIDCA-2020-46;No.SKL-HIDCA-2021-XXG1);新疆重点实验室开放课题项目(No.2020D04027;No.2021D04020)。

摘　　要：目的:缺血预适应(IPC)是强有力的对抗心肌缺血/再灌注(I/R)损伤的心肌保护措施,但其保护作用相关的机制仍不完全清楚。本研究旨在识别缺血预适应处理后小鼠心肌差异表达蛋白,为缺血预适应进一步的基础及临床研究提供新思路。方法:用雄性成年C57BL/6小鼠建立心肌I/R损伤模型(缺血60 min/再灌注24 h)和IPC模型[3个(缺血5 min/再灌注5 min)循环的预适应+缺血60 min/再灌注24 h],用伊文思蓝和2,3,5-三苯基氯化四氮唑双重染色的方法测量心肌梗死面积。提取各组心肌总蛋白,通过将串联质谱标签、高效液相色谱分级技术以及基于质谱的定量蛋白质组学技术相结合,以差异表达量变化>1.2倍作为显著上调的阈值,<1/1.2作为显著下调的阈值,筛选差异表达蛋白。对差异表达蛋白进行同源蛋白簇功能分类统计。分别用qPCR和Western blot技术对差异显著的蛋白β-羟丁酸脱氢酶1(Bdh1)进行mRNA及蛋白水平上的验证。结果:与单纯I/R组相比,IPC干预后心梗面积降低33%(P<0.05)。定量蛋白质组学结果显示,与假手术组比较,I/R组表达下调的蛋白有91个,其中酮体氧化代谢关键酶Bdh1在下调蛋白中的表达倍数为0.708(<1/1.2);与I/R组相比,IPC组表达上调的蛋白有14个,其中Bdh1差异表达倍数最高(1.95倍)。qPCR及Western blot验证实验表明,与假手术组相比,I/R组Bdh1的mRNA及蛋白水平明显降低;IPC组Bdh1的mRNA及蛋白表达水平较I/R组明显升高,进一步验证了蛋白组学结果。结论:I/R损伤显著降低心肌Bdh1的mRNA及蛋白表达水平,而IPC处理显著恢复了Bdh1的mRNA及蛋白表达,表明酮体氧化代谢可能参与了缺血预适应介导的缺血心肌的保护。AIM: Ischemic preconditioning(IPC) is an endogenous protection strategy against myocardial ischemia/reperfusion(I/R) injury, but the mechanism related to its protective effect is still unclear. The purpose of this study is to identify the myocardial differentially expressed proteins after IPC and provide a new idea for further basic and clinical studies of IPC. METHODS: Male adult C57BL/6 mice were used to establish myocardial I/R injury(ischemia for 60 min and reperfision for 24 h) and IPC(3 cycles of ischemia for 5 min and reperfusion for 5 min, plus ischemia for 60 min and reperfision for 24 h) models. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride double staining. Tandem mass tag, high-performance liquid chromatography and mass spectrometry based quantitative proteomics were conducted to screen the differentially expressed proteins. The threshold to determine significantly up-or down-regulated proteins was defined as the expression level of proteins more than 1. 2-fold or less than 1/1. 2-fold between groups. The differentially expressed proteins were classified into clusters of orthologous proteins for functional analysis. Real-time quantitative PCR and Western blot were used to verify identified proteins. RESULTS: Compared with I/R group, IPC intervention significantly reduced the infarct size by 33%(P<0. 05). Quantitative proteomics results showed that 91 proteins were down-regulated in I/R group compared with sham group, among which β-hydroxybutyrate dehydrogenase 1(Bdh1), a key enzyme of ketone body oxidative metabolism, had 0. 708-fold decreased expression(<1/1. 2-fold). Fourteen proteins were up-regulated in IPC group compared with I/R group, among which Bdh1 had the highest expression(1. 95-fold). The qPCR and Western blot results showed that Bdh1 mRNA and protein expression levels were significantly lower in I/R group compared with sham group, and were significantly higher in IPC group compared with I/R group, further validating the proteomic results.

关 键 词：缺血预适应 缺血再灌注 蛋白质组学 β-羟丁酸脱氢酶1 

分 类 号：R541.4[医药卫生—心血管疾病] R363.2[医药卫生—内科学]