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摘要：
目的 探究七氟醚后处理（sevoflurane postconditioning, SpostC）是否通过调控缺氧诱导因子‑1α（hypoxia inducible factor‑1α, HIF‑1α）/B细胞淋巴瘤2/腺病毒E1B相互作用蛋白3（B cell lymphoma 2/adenovirus E1B protein‐interacting protein 3, BNIP3）通路，进而减轻小鼠心肌缺血再灌注损伤（myocardial ischemia reperfusion injury, MIRI）的分子机制。 方法 将40只小鼠按随机数字表法分为4组（每组10只）：假手术组（Sham组）、心肌缺血再灌注组（I/R组）、七氟醚后处理+I/R组（SpostC组）和七氟醚后处理+HIF‑1α抑制剂（2ME2）+I/R组（SpostC+2ME2组）。Sham组只穿线不结扎冠状动脉左前降支（left anterior descending coronary artery, LAD）。I/R组结扎LAD 30 min，再灌注120 min。SpostC组和SpostC+2ME2组在复灌即刻吸入1 MAC七氟醚15 min，然后再灌105 min。SpostC+2ME2组在LAD结扎前腹腔注射2ME2，剂量为15 mg/kg。采用心脏超声检测心脏功能，记录舒张期左心室前壁厚度（left ventricular end‑diastolic anterior wall thickness, LVAWd）、收缩期左心室前壁厚度（left ventricular end‑systolic anterior wall thickness, LVAWs）、射血分数（ejection fraction, EF）、左心室缩短分数（fractional shortening, FS）、舒张期左心室后壁厚度（left ventricular end‑diastolic posterior wall thickness, LVPWd）、收缩期左心室后壁厚度（left ventricular end‑systolic posterior wall thickness, LVPWs）、舒张期左心室内径（left ventricular internal dimension at end‑diastolic, LVIDd）和收缩期左心室内径（left ventricular internal dimension at end‑systolic, LVIDs）；H‑E染色观察心肌组织结构；TTC+伊文蓝染色法测定心肌梗死面积；ELISA法检测血清肌型肌酸激酶（creatine kinase M‑type, CKM）、肌钙蛋白T型2（troponin T‑type 2, TNNT2）、肌酸激酶同工酶（creatine kinase‑MB, CK‑MB）和乳酸脱氢酶（lactate dehydrogenase, LDH）的表达水平；透射电镜观察线粒体超微结构改变情况；Western blot法检测心肌组织中HIF‑1α、BNIP3、微管相关蛋白轻链3‑Ⅱ（microtubule‑associated protein light chain 3 Ⅱ, LC3‑Ⅱ）、自噬相关蛋白（Beclin1）、Toll样受体9（toll‑like receptor 9, TLR9）和IL‑6的蛋白水平；TUNEL染色法检测心肌细胞的凋亡情况。 结果 与Sham组比较，I/R组小鼠LVAWd、LVAWs、EF、FS、LVPWd和LVPWs明显降低（P<0.05），LVIDd和LVIDs明显升高（P<0.05），心肌组织损伤及线粒体损伤较重，心肌梗死面积较大，心肌细胞凋亡率较高，血清LDH、CKM、CK‑MB和TNNT2的表达水平升高（P<0.05），HIF‑1α、LC3‑Ⅱ、BNIP3、Beclin1、TLR9和IL‑6蛋白水平升高（P<0.05）。与I/R组比较，SpostC组小鼠LVAWd、LVAWs、EF、FS、LVPWd和LVPWs明显升高（P<0.05），LVIDd和LVIDs明显降低（P<0.05），心肌组织损伤及线粒体损伤减轻，心肌梗死面积减小，心肌细胞凋亡率降低，血清LDH、CKM、CK‑MB和TNNT2的表达水平明显降低（P<0.05），HIF‑1α、LC3‑Ⅱ、BNIP3、Beclin1和TLR9的蛋白水平明显升高（P<0.05），IL‑6蛋白水平明显降低（P<0.05）。与SpostC组比较，SpostC+2ME2组小鼠LVAWd、LVAWs、EF、FS、LVPWd和LVPWs明显降低（P<0.05），LVIDd和LVIDs明显升高（P<0.05），心肌组织损伤及线粒体损伤加重，心肌梗死面积增大，心肌细胞凋亡率升高，血清LDH、CKM、CK‑MB和TNNT2的表达水平明显升高（P<0.05），HIF‑1α、LC3‑Ⅱ、BNIP3、Beclin1和TLR9的蛋白水平明显降低（P<0.05），IL‑6蛋白水平明显升高（P<0.05）。 结论 SpostC可通过上调HIF‑1α/BNIP3通路激活线粒体自噬，减轻炎症反应，抑制心肌细胞凋亡，从而改善小鼠MIRI。
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Abstract: Objective To investigate whether sevoflurane postconditioning (SpostC) alleviates myocardial ischemia/reperfusion (I/R) injury in mice by regulating the hypoxia inducible factor‑1α (HIF‑1α)/B cell lymphoma 2/adenovirus E1B interacting protein 3 (BNIP3) pathway, in order to relieve myocardial ischemia reperfusion injury (MIRI) in mice. Methods According to random number table method, 40 mice were divided into four groups (n=10): a Sham group, a myocardial ischemia reperfusion (I/R) group, a sevoflurane postconditioning+I/R group (SpostC) group, a sevoflurane postconditioning group+HIF‑1α blocker (2ME2)+I/R (SpostC+2ME2) group. Sham group was only threaded without ligation of the left anterior descending coronary artery (LAD). LAD was ligated for 30 min and reperfused for 120 min in I/R group. In SpostC group and SpostC+2ME2 group, 1 MAC sevoflurane was inhaled for 15 min immediately after reperfusion, and then reperfused for 105 min. In SpostC+2ME2 group, 2ME2 was injected intraperitoneally at a dose of 15 mg / kg before LAD ligation. Cardiac ultrasound was utilized to evaluate cardiac function, including left ventricular end‑diastolic anterior wall thickness (LVAWd), left ventricular end‑systolic anterior wall thickness (LVAWs), ejection fraction (EF), fractional shortening (FS), left ventricular end‑diastolic posterior wall thickness (LVPWd), left ventricular end‑systolic posterior wall thickness (LVPWs), left ventricular internal dimension at end‑diastolic (LVIDd), and left ventricular internal dimension at end‑systolic (LVIDs). The structure of myocardial tissue was observed by H‑E staining. The myocardial infarction size was measured by TTC+Evans Blue double staining. The levels of serum lactate dehydrogenase (LDH), creatine kinase M‑type (CKM), creatine kinase isoenzyme (CK‑MB), and troponin T‑type 2 (TNNT2) were detected by enzyme‑linked immunosorbent assay (ELISA). The changes of mitochondrial ultrastructure were observed by transmission electron microscopy. The levels of HIF‑1α, BNIP3, microtubule‑associated protein light chain 3 Ⅱ (LC3‑Ⅱ), autophagy‑related protein (Beclin1), Toll‑like receptor 9 (TLR9) and interleukin (IL)‑6 proteins expression were detected by Western blot. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase‑mediated dUTP‑biotin nick end labeling (TUNEL) staining. Results Compared with the Sham group, the I/R group showed significant decreases in LVAWd, LVAWs, EF, FS, LVPWd, and LVPWs (P<0.05), and increases in LVIDd and LVIDs (P<0.05), as well as aggravated myocardial tissue and mitochondrial damage, enlarged infarct area and an increased myocardial cell apoptotic rate, increased contents of serum LDH, CKM, CK‑MB and TNNT2 (P<0.05), and increased protein expression of HIF‑1α, LC3‑Ⅱ, BNIP3, Beclin1, TLR9, IL‑6 (P<0.05). Compared with the I/R group, the SpostC group presented significant increases in LVAWd, LVAWs, EF, FS, LVPWd, and LVPWs (P<0.05), and decreases in LVIDd and LVIDs (P<0.05), as well as relieved myocardial tissue and mitochondrial damage, reduced infarct area and an decreased myocardial cell apoptotic rate, reduced contents of serum LDH, CKM, CK‑MB and TNNT2 (P<0.05), increased protein expression of HIF‑1α, LC3‑Ⅱ, BNIP3, Beclin1 and TLR9 (P<0.05), and decreased IL‑6 levels (P<0.05). Compared with the SpostC group, the SpostC+2ME2 group showed significant decreases in LVAWd, LVAWs, EF, FS, LVPWd and LVPWs (P<0.05), and increases in LVIDd and LVIDs (P<0.05), as well as aggravated myocardial tissue and mitochondrial damage, enlarged infarct area and an increased myocardial cell apoptotic rate, increased contents of serum LDH, CKM, CK‑MB and TNNT2 (P<0.05), and decreased protein expression of HIF‑1α, LC3‑Ⅱ, BNIP3, Beclin1 and TLR9 (P<0.05), and increased IL‑6 levels (P<0.05). Conclusions SpostC can relieve MIRI in mice through up‑regulating the HIF‑1α/BNIP3 signaling pathway to activate mitochondrial autophagy, alleviate inflammatory response and myocardial apoptosis.
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