基于PERK/Nrf2/HO-1信号通路研究瑞马唑仑对心肌缺血再灌注损伤大鼠铁死亡的影响

摘要 目的：基于蛋白激酶R样内质网激酶（PERK）/核因子E2相关因子2（Nrf2）/血红素氧合酶-1（HO-1）信号通路探究瑞马唑仑对心肌缺血再灌注损伤（MIRI）大鼠铁死亡的影响。方法：将90只SD大鼠随机分为假手术（Sham）组、MIRI组、低剂量-瑞马唑仑组（L-瑞马唑仑组，5 mg/kg）、高剂量-瑞马唑仑组（H-瑞马唑仑组，20 mg/kg）、H-瑞马唑仑+PERK抑制剂组（瑞马唑仑20 mg/kg+GSK2606414 1 mg/kg），每组18只。采用结扎冠状动脉左前降支（LAD）0.5 h、再灌注2 h制备MIRI大鼠模型，于再灌注2 h后即刻尾静脉注射给药，再灌注24 h后进行组织取材。酶联免疫吸附（ELISA）法检测血清心肌损伤标志物肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白I（cTnI）]水平；HE染色观察心肌组织病理改变；Tunel染色检测心肌细胞凋亡；透射电镜观察心肌细胞超微结构变化；检测心肌组织中铁死亡相关标志物铁、活性氧（ROS）、谷胱甘肽（GSH）、丙二醛（MDA）]水平；蛋白质印迹法（Western Blot）检测心肌组织中PERK/Nrf2/HO-1信号通路相关蛋白表达。结果：与Sham组相比，MIRI组心肌结构受损，纤维排列紊乱，线粒体呈现显著的铁死亡特征（膜固缩，膜密度增加，嵴减少），血清中CK-MB、cTnI水平，心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平升高（P<0.05），心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达降低（P<0.05）；与MIRI组相比，L-瑞马唑仑组和H-瑞马唑仑组心肌组织上述病理改变明显减轻，血清CK-MB、cTnI水平，心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平降低（P<0.05），心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达升高（P<0.05）；与H-瑞马唑仑组相比，H-瑞马唑仑+PERK抑制剂组心肌组织上述病理改变加重，血清CK-MB、cTnI水平，心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平升高（P<0.05），心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达降低（P<0.05）。结论：瑞马唑仑可通过抑制铁死亡减轻大鼠MIRI，可能通过激活PERK/Nrf2/HO-1信号通路而实现。

Effect of Remazolam on Ferroptosis in Rats with Myocardial Ischemia-Reperfusion Injury was Studied Based on PERK/Nrf2/HO-1 Signaling Pathway

ABSTRACT Objective: To investigate the effect of remazolam on ferroptosis in myocardial ischemia-reperfusion injury (MIRI) rats based on protein kinase R-like ER kinase (PERK)/nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Methods: 90 SD rats were randomly grouped into sham operation (Sham) group, MIRI group, low-dose-remazolam group (L-remazolam group, 5 mg/kg), high-dose-remazolam group (H-remazolam group, 20 mg/kg), H-remazolam + PERK inhibitor group (remazolam 20 mg/kg + GSK2606414 1 mg/kg) with 18 rats in each group. The MIRI rat model was established by ligating the left anterior descending branch (LAD) of coronary artery for 0.5 h and reperfusion for 2 h. The drug was injected into the tail vein immediately after reperfusion for 2 h, and tissue samples were collected 24 h after reperfusion. Enzyme-linked immunosorbent assay (ELISA) method was applied to detect the levels of serum myocardial injury markers creatine kinase isoenzyme (CK-MB), cardiac troponin I (cTnI)]. HE staining was applied to observe the pathological changes of myocardial tissue. Tunel staining was applied to detect cardiomyocyte apoptosis. Transmission electron microscopy was applied to observe the ultrastructural changes of cardiomyocytes. The levels of ferroptosis-related markers iron, reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA)] in myocardial tissue were detected. Western blotting (Western Blot) was applied to measure the expression of PERK/Nrf2/HO-1 signaling pathway related proteins in myocardial tissue. Results: Compared with the Sham group, the MIRI group had damaged myocardial structure, disordered fiber arrangement, and mitochondria showed obvious ferroptosis (membrane pyknosis, increased membrane density, and decreased cristae), the levels of serum CK-MB and cTnI, the apoptosis rate of cardiomyocytes and the levels of iron, ROS and MDA in myocardial tissue were increased (P<0.05), the level of GSH and expressions of p-PERK/PERK, nuclear Nrf2/Nrf2 and HO-1 protein in myocardial tissue decreased (P<0.05). Compared with MIRI group, the above-mentioned pathological changes in myocardial tissue of L-Remazolam group and H-Remazolam group were significantly reduced, the levels of serum CK-MB and cTnI, the apoptosis rate of myocardial cells and the levels of iron, ROS and MDA in myocardial tissue were decreased (P<0.05), and the levels of GSH and the expressions of p-PERK/PERK, nuclear Nrf2/Nrf2 and HO-1 proteins in myocardial tissue were increased (P<0.05). Compared with H-Remazolam group, the above pathological changes in myocardial tissue were aggravated in H-Remazolam+PERK inhibitor group, the levels of serum CK-MB and cTnI, the apoptosis rate of myocardial cells and the levels of iron, ROS and MDA in myocardial tissue were increased (P<0.05), and the levels of GSH and the expressions of p-PERK/PERK, nuclear Nrf2/Nrf2 and HO-1 proteins in myocardial tissue were decreased (P<0.05). Conclusion: Remazolam can reduce MIRI rats by inhibiting ferroptosis, which may be achieved by activating PERK/Nrf2/HO-1 signaling pathway.