【摘要】 目的 探讨核连蛋白1(NUCB1)在大鼠心肌梗死后炎症反应中的作用及机制。方法 收集2020年11月至2021年7月于武汉大学人民医院行冠状动脉造影的20例患者的血样，根据造影结果分为对照组（Control,n=10）和冠状动脉慢性闭塞型病变组（CTO,n=10），蛋白组学分析两组NUCB1的水平差异，蛋白质互作分析明确NUCB1与炎症通路蛋白的关联。运用左前降支动脉结扎术构建SD大鼠心肌梗死模型，随机分配为假手术组（Sham,n=8）和心肌梗死组（MI,n=8）。28 d后取材行苏木精-伊红（HE）及马松（Masson）染色评价心肌梗死组织的病理形态及变化；免疫荧光染色观察NUCB1的亚细胞定位及表达情况；荧光定量PCR(qPCR)、Western blot明确心肌梗死周边区域（BIZ）心肌组织中NUCB1、NOD样蛋白受体3(NLRP3)、诱导性环氧合酶（COX2）等炎症因子的表达水平。此外，构建H9c2心肌细胞缺氧模型，探究NUCB1及炎症因子在心肌细胞缺氧模型中的表达模式。结果 CTO组患者血清NUCB1水平显著上升，PPI分析发现NUCB1与炎症通路蛋白转化生长因子β诱导的分泌蛋白（TGFBI）、组织蛋白酶B(CTSB)、补体C4A存在相互关联。HE及Masson染色显示MI组大鼠心脏炎症细胞浸润明显，心肌细胞排列疏松，纤维化面积显著增加；qPCR、免疫荧光染色及Western blot显示与对照组相比，MI组BIZ心肌组织中NUCB1表达水平显著升高且相应促炎蛋白表达升高（P<0.05）。在H9c2心肌细胞缺氧模型中，qPCR及Western Blot显示NUCB1表达水平及促炎蛋白表达亦升高（P<0.05）。结论 NUCB1在大鼠心肌梗死及心肌细胞缺氧模型中表达水平升高，且可能通过NLRP3和COX2介导的炎症反应参与心肌梗死的发生发展。更多还原

【Abstract】 Objective To investigate the role and mechanism of nucleobindin 1(NUCB1) in inflammatory response in rats after myocardial infarction(MI). Methods The blood samples were collected from 20 patients underwent coronary angiography(CAG) from Renmin Hospital of Wuhan University from Nov. 2020 to July 2021. According to CAG results, the patients were divided into control group(n=10) and group of chronic total occlusion(CTO) of coronary artery(CTO group, n=10). The difference in NUCB1 level was analyzed by using proteomics, and correlation between NUCB1 and inflammatory pathway proteins was analyzed through protein-protein interaction(PPI). A MI model was established by using ligation of left anterior descending(LAD) in SD rats, and the rats were randomly divided into sham-operation group(Sham group, n=8) and MI group(MI group, n=8). After 28 d, the samples were stained with HE staining and Masson staining for reviewing morphopathology and pathological changes of MI tissue. The subcellular localization and expression of NUCB1 were observed by using immunofluorescence staining. Immunofluorescence PCR(qPCR) and Western blotting assay were used to determine the expression levels of inflammatory factors such as NUCB1, NOD-like receptor protein 3(NLRP3) and inducible cyclooxygenase(COX2) in myocardial tissue of border zone of infarction(BIZ). In addition, a hypoxia model of H9c2 cardiomyocytes was established to investigate the expression patterns of NUCB1 and inflammatory factors in the hypoxic model of cardiomyocytes. Results The serum level of NUCB1 was significantly elevated in CTO group. PPI analysis revealed that NUCB1 was correlated with inflammatory pathway proteins, including transforming growth factor-beta-induced protein(TGFBI), cathepsin B(CTSB), and complement C4A(C4A). HE staining and Masson staining showed that there was obvious inflammatory cell infiltration in the heart, loose arrangement of cardiomyocytes, and a significant increase in the area of myocardial fibrosis in MI group. The results of qPCR, immunofluorescence staining, and Western blotting assay showed that the expression level of NUCB1 was significantly increased in the myocardial tissue of BIZ, and the expression of corresponding proinflammatory proteins was also increased in MI group compared with control group(P<0.05). In the hypoxia model of H9c2 cardiomyocytes, qPCR and Western blotting assay showed that expression levels of NUCB1 and proinflammatory proteins were also increased(P<0.05). Conclusion The expression level of NUCB1 is increased in rat model of MI and hypoxic cardiomyocytes, and it may be involved in the occurrence and development of MI through inflammatory responses mediated by NLRP3 and COX2.更多还原