美托洛尔改善心肌缺血再灌注损伤的机制研究

Research on the mechanism of metoprolol in improving myocardial ischemia-reperfusion injury

摘要:

目的 探讨美托洛尔是否通过调控沉默信息调节因子1（SIRT1）/核转录因子相关因子2（Nrf2）信号通路抑制细胞焦亡来改善心肌缺血再灌注损伤（MIRI）。方法 选取32只8～10周龄健康雄性C57BL/6小鼠，复制MIRI模型，分为Sham组、MIRI组、MIRI+美托洛尔组（MIRI+美托洛尔）和MIRI+美托洛尔+EX527组（MIRI+美托洛尔+EX527），每组8只。采用超声心动图测量左心室短轴缩短率（LVFS）和射血分数（LVEF）评估各组小鼠的心脏功能；采用酶联免疫吸附试验测定小鼠血清乳酸脱氢酶（LDH）和cTnI水平，评估心肌细胞损伤程度；采用免疫荧光染色（PI染色法）检测小鼠心肌组织焦亡细胞数；Western blotting检测炎症因子白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18），焦亡相关蛋白半胱氨酸的天冬氨酸蛋白水解酶1（Caspase-1）、Caspase-11、活化胱天蛋白酶后裂解蛋白GSDMD，以及SIRT1、Nrf2蛋白的表达。结果 与Sham组比较，MIRI组小鼠的LVFS和LVEF降低（P <0.05）；与MIRI组比较，MIRI+美托洛尔组的LVFS和LVEF升高（P <0.05）；与MIRI+美托洛尔组比较，MIRI+美托洛尔+EX527组的LVFS和LVEF降低（P <0.05）。与Sham组比较，MIRI组小鼠的LDH和cTnI水平升高（P <0.05）；与MIRI组比较，MIRI+美托洛尔组LDH和cTnI水平降低（P <0.05）；与MIRI+美托洛尔组比较，MIRI+美托洛尔+EX527组LDH和cTnI水平升高（P <0.05）。与Sham组比较，MIRI组小鼠的心肌焦亡细胞数增加（P <0.05）；与MIRI组小鼠比较，MIRI+美托洛尔组心肌焦亡细胞数减少（P <0.05）；与MIRI+美托洛尔组小鼠比较，MIRI+美托洛尔+EX527组焦亡心肌细胞数升高（P <0.05）。与Sham组比较，MIRI组小鼠的IL-1β、IL-18、Caspase-1、Caspase-11、GSDMD蛋白相对表达量增加（P <0.05）；与MIRI组比较，MIRI+美托洛尔组IL-1β、IL-18、Caspase-1、Caspase-11、GSDMD蛋白相对表达量降低（P <0.05）；与MIRI+美托洛尔组比较，MIRI+美托洛尔+EX527组IL-1β、IL-18、Caspase-1、Caspase-11、GSDMD蛋白相对表达量升高（P <0.05）。与Sham组比较，MIRI组的SIRT1、Nrf2蛋白相对表达量降低（P <0.05）；与MIRI组比较，MIRI+美托洛尔组的SIRT1、Nrf2蛋白相对表达量升高（P <0.05）；与MIRI+美托洛尔组比较，MIRI+美托洛尔+EX527组的SIRT1、Nrf2蛋白相对表达量降低（P <0.05）。结论 美托洛尔通过调控Sirt1/Nrf2信号通路抑制细胞焦亡，减轻MIRI，具有显著的心脏保护作用，为临床治疗心肌缺血再灌注损伤提供了新的理论依据。

Abstract:

Objective To explore whether metoprolol ameliorates myocardial ischemia-reperfusion injury (MIRI) by inhibiting pyroptosis through regulating the silent information regulator 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway.Methods Thirty-two healthy male C57BL/6 mice aged 8 to 10 weeks were selected to establish the MIRI model and divided into Sham group, MIRI group, MIRI+ metoprolol group (MIRI + Met), and MIRI + metoprolol + EX527 group (MIRI + Met + EX527), with 8 mice in each group. The left ventricular fractional shortening (LVFS) and ejection fraction (LVEF) were measured by echocardiography to evaluate the cardiac function in each group. The levels of lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) in mouse serum were determined by enzyme-linked immunosorbent assay to evaluate the degree of myocardial cell injury. The number of pyroptotic cells in mouse myocardial tissue was detected by immunofluorescence staining (propidium iodide, PI staining). The expressions of inflammatory factors IL-1β and IL-18, as well as pyroptosis-related proteins Caspase-1, Caspase-11, and GSDMD were detected by Western blotting.Results Compared with the Sham group, LVFS and LVEF in the MIRI group decreased (P < 0.05); Compared with the MIRI group, the LVFS and LVEF in the MIRI + metoprolol group increased (P < 0.05); Compared with the MIRI + metoprolol group, the LVFS and LVEF in the MIRI + metoprolol + EX527 group were decreased (P < 0.05). Compared with the Sham group, the levels of LDH and cTnI in mice of the MIRI group increased (P < 0.05). Compared with the MIRI group, the levels of LDH and cTnI in the MIRI + metoprolol group decreased (P < 0.05). Compared with the MIRI + metoprolol group, the levels of LDH and cTnI in the MIRI + metoprolol + EX527 group were increased (P < 0.05). Compared with the Sham group, the number of myocardial pyroptotic cells in the MIRI group mice increased (P < 0.05); Compared with the mice in the MIRI group, the number of pyroptotic cells in the myocardium of the MIRI + metoprolol group decreased (P < 0.05). Compared with the mice in the MIRI + metoprolol group, the number of pyroptotic cardiomyocytes in the MIRI + metoprolol +EX527 group increased (P < 0.05). Compared with the Sham group, the levels of IL-1β, IL-18, Caspase-1, Caspase-11 and GSDMD in the MIRI group mice increased (P < 0.05) ; Compared with the MIRI group, the levels of IL-1β, IL-18, Caspase-1, Caspase-11 and GSDMD in the MIRI + metoprolol group decreased (P < 0.05). Compared with the MIRI + metoprolol group, the levels of IL-1β, IL-18, Caspase-1, Caspase-11 and GSDMD in the MIRI+ metoprolol + EX527 group increased (P < 0.05). Compared with the Sham group, the levels of SIRT1 and Nrf2 in the MIRI group mice decreased (P < 0.05) ; Compared with the MIRI group, the levels of SIRT1 and Nrf2 in the MIRI + metoprolol group increased (P < 0.05). Compared with the MIRI+ metoprolol group, the levels of SIRT1 and Nrf2 in the MIRI + metoprolol + EX527 group decreased (P < 0.05).Conclusion Metoprolol inhibits pyroptosis and reduces MIRI by regulating the SIRT1/Nrf2 signaling pathway, demonstrating a significant cardioprotective effect and providing a new theoretical basis for the clinical treatment of myocardial ischemia-reperfusion injury.