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摘要：
目的 探讨磷脂酰肌醇‑3‑激酶/蛋白激酶B（phosphoinositide‑3‑kinase/protein kinase B, PI3K/Akt）信号通路在微RNA（microRNA, miR）‑27a调控大鼠心肌缺血再灌注损伤（myocardial ischemia reperfusion injury, MIRI）中的作用机制，并进一步明确miR‑27a对大鼠心肌产生的影响是否与抑制氧化应激反应相关。 方法 清洁级雄性SD大鼠，体重220~280 g，8~10周龄，96只大鼠按随机数字表分为6组（每组16只）：假手术组（Sham组）、心肌缺血再灌注组（IR组）、腺相关病毒（adeno‑associated virus, AAV）空白对照+IR组（AVV‑NC组）、过表达miR‑27a+IR组（AAV‑miR‑27a组）、低表达miR‑27a+IR组（AAV‑miR‑27a‑antago组）、低表达miR‑27a+IR+PI3K抑制剂组（LY组）。AAV⁃miR⁃27a组于造模前14 d尾静脉注射AAV⁃miR⁃27a进行过表达干预，AAV⁃miR⁃27a⁃antago组与LY组造模前14 d尾静脉注射AAV⁃miR⁃27a⁃antago进行低表达干预。结扎大鼠冠状动脉左前降支（left anterior descending, LAD）30 min再灌注120 min制备MIRI模型。LY组于造模前30 min腹腔注射PI3K抑制剂LY294002（1.5 mg/kg）。再灌注120 min后，取血样，采用ELISA法检测各组大鼠血清中肌钙蛋白T（cardiac troponin T, cTnT）、肌酸激酶同工酶（creatine kinase MB, CK‑MB）的浓度；比色法检测血清中乳酸脱氢酶（lactate dehydrogenase, LDH）活性；处死大鼠，摘取心脏切片进行H‑E染色观察心肌组织病理学变化，伊文蓝‑2，3，5‑氯化三苯基四氮唑（2, 3, 5‑triphenyl tetrazolium chloride, TTC）法检测大鼠心肌梗死面积；比色法检测心肌组织超氧化物歧化酶（superoxide dismutase, SOD）、谷胱甘肽（glutathione, GSH）和丙二醛（malondialdehyde, MDA）水平；采用Western blot法测定心肌组织PI3K、Akt及磷酸化（phospho, p）‑PI3K、p‑Akt的蛋白水平。 结果 与Sham组比较：IR组心肌梗死面积百分比增加（P<0.05）；血清cTnT、CK‑MB浓度及LDH活性增加（P<0.05）；心肌组织MDA水平增加，SOD、GSH水平降低，p‑PI3K、p‑Akt表达上调（P<0.05）。与IR组比较：AAV‑miR‑27a组心肌梗死面积百分比增加（P<0.05）；血清cTnT、CK‑MB浓度及LDH活性增加（P<0.05）；心肌组织MDA水平增加，SOD、GSH水平降低，p‑PI3K、p‑Akt表达下调（P<0.05）。与IR组比较：AAV‑miR‑27a‑antago组心肌梗死面积百分比降低（P<0.05）；血清cTnT、CK‑MB浓度及LDH活性降低（P<0.05）；心肌组织MDA水平降低，SOD、GSH水平增加，p‑PI3K、p‑Akt表达上调（P<0.05）。与AAV‑miR‑27a‑antago组比较：LY组心肌梗死面积百分比增加（P<0.05）；血清cTnT、CK‑MB浓度及LDH活性增加（P<0.05）；心肌组织MDA水平增加，SOD、GSH水平降低，p‑PI3K、p‑Akt表达下调（P<0.05）。 结论 下调miR‑27a表达可能通过激活PI3K/Akt信号通路以及抑制氧化应激反应减轻大鼠MIRI。
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Abstract: Objective To explore the mechanism of the phosphatidylinositol‑3‑kinase/protein kinase B (PI3K/Akt) signaling pathway by which microRNA (miR)‑27a regulates myocardial ischemia reperfusion injury (MIRI) in rats, and to further determine whether the effect of miR‑27a on rat myocardial production is associated with the inhibition of oxidative stress response. Methods A total of 96 specific pathogen‑free male SD rats, weighing 220‒280 g, aged 8 to 10 weeks old, were selected. According to the random number table method, they were divided into six groups (n=16): Sham operation group (group Sham), myocardial ischemia reperfusion group (group IR), adeno‑associated virus (AAV) blank control+IR group (group AVV‑NC), overexpressed miR‑27a+IR group (group AAV‑miR‑27a), down‑expressed miR‑27a+IR group (group AAV‑miR‑27a‑antago) and down‑expressed miR‑27a+IR+PI3K inhibitor group (group LY). Rats in group AAV⁃miR⁃27a were injected with AAV⁃miR⁃27a by the tail vein for overexpression intervention 14 days before molding, while those in groups AAV⁃miR⁃27a⁃antago and LY were injected with AAV⁃miR⁃27a⁃antago for down-expression intervention 14 days before molding. A MIRI model was established by occlusion of the left coronary anterior descending artery for 30 min followed by reperfusion for 120 min. Rats in group LY were intraperitoneally injected with the PI3K inhibitor, LY294002 (1.5 mg/kg) 30 min before modeling. After reperfusion for 120 min, blood samples were collected. The concentrations of cardiac troponin T (cTnT) and creatine kinase MB (CK‑MB) were detected by enzyme‑linked immunosorbent assay (ELISA). The activity of lactate dehydrogenase (LDH) was detected by colorimetry. The rats were then sacrificed to collect the heart were collected for observation of the pathological changes by hematoxylin‑eosin (H‑E) staining and measurement of myocardial infarct size by Evans blue‑2, 3, 5‑triphenyltetrazolium chloride (TTC) staining. The contents of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the myocardium were detected by colorimetry. The protein levels of PI3K, Akt, p‑PI3K, and p‑Akt were determined by Western blot. Results Compared with group Sham, group IR showed increases in the percentage of myocardial infarct size (P<0.05), increases in the levels of cTnT and CK‑MB and LDH activity in serum (P<0.05); increases in the content of MDA in myocardial tissue, decreases in the levels of SOD and GSH, and up-regulation of the expression of p‑PI3K and p‑Akt (P<0.05). Compared with group IR, group AAV‑miR‑27a presented increases in the percentage of myocardial infarct size (P<0.05), increases in the levels of cTnT and CK‑MB and LDH activity in serum (P<0.05); increases in the content of MDA in myocardial tissue, decreases in the levels of SOD and GSH, and down-regulation of the expression of p‑PI3K and p‑Akt (P<0.05). Compared with group IR, group AAV‑miR‑27a‑antago showed decreases in the percentage of myocardial infarct size (P<0.05), decreases in the levels of cTnT and CK‑MB and LDH activity in serum (P<0.05); decreases in the content of MDA in myocardial tissue, increases in the levels of SOD and GSH, and up-regulation of the expression of p‑PI3K and p‑Akt (P<0.05). Compared with group AAV‑miR‑27a‑antago, group LY presented increases in the percentage of myocardial infarct size (P<0.05), increases in the levels of cTnT and CK‑MB and LDH activity in serum (P<0.05); increases in the content of MDA in myocardial tissue, decreases in the levels of SOD and GSH, and down-regulation of the expression of p‑PI3K and p‑Akt (P<0.05). Conclusions Down‑regulation of miR‑27a expression may alleviate MIRI in rats by activating the PI3K/Akt signaling pathway, and inhibiting oxidative stress.
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