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摘要：
目的 评估O‑N‑乙酰葡糖胺（O‑N‑acetyl‑glucosamine, O‑GlcNAc）糖基化修饰转移酶（O‑GlcNAc transferase, OGT）介导的受体相互作用蛋白（receptor interacting protein kinase, RIPK）3糖基化在七氟醚后处理减轻离体大鼠心肌缺血再灌注（ischemia reperfusion, IR）损伤中的作用。 方法 取Langendorff离体灌注模型制备成功的大鼠心脏45个，采用随机数字表法分为5组（每组9个）：假手术组（SHAM组）、IR组、七氟醚后处理组（SPC组）、SPC+溶剂组［SPC+二甲基亚砜（dimethyl sulfoxide, DMSO）组］和SPC+OGT抑制剂组（SPC+OSMI‑1组）。建模完成后各组均平稳30 min，之后SHAM组给予K‑H液持续灌注150 min，其余各组大鼠心脏均经历停止灌注30 min后恢复灌注2 h的过程；SPC组、SPC+DMSO组和SPC+OSMI‑1组均于再灌注初给予含2.4%七氟醚的K‑H液持续灌注15 min；此外，在术前准备的K‑H液中，SPC+DMSO组给予50 μmol/L DMSO，SPC+OSMI‑1组给予50 μmol/L OSMI‑1。连续监测各组大鼠缺血前即刻（T0）、再灌注30 min（T1）、再灌注60 min（T2）、再灌注90 min（T3）、再灌注2 h（T4）的左室峰压（left ventricular peak pressure, LVSP）、左室舒张末压（left ventricular end‑diastolic pressure, LVEDP）、心率、左室压力升高最大速率（maximum rate of rise of left ventricular pressure, +dp/dtmax）与左室压力降低最大速率（maximum rate of drop of left ventricular pressure,−dp/dtmax）；在再灌注末，采用1%氯化三苯基四氮唑（triphenyl tetrazolium chloride, TTC）染色检测各组大鼠心肌梗死范围；采用ELISA法检测各组大鼠冠状动脉漏出液乳酸脱氢酶（lactate dehydrogenase, LDH）浓度；Western blot法检测各组大鼠心脏O‑GlcNAc、OGT、O‑糖基化糖苷酶（O‑GlcNAcase, OGA）、磷酸化受体相互作用蛋白1（phosphorylated RIPK1, p‑RIPK1）、磷酸化RIPK3（phosphorylated RIPK3, p‑RIPK3）和磷酸化混合系激酶结构域样蛋白（phosphorylated mixed lineage kinase domain‑like protein, p‑MLKL）蛋白水平。 结果 与T0比较，IR组、SPC组、SPC+DMSO组、SPC+OSMI‑1组T1~T4时心率、LVSP、+dp/dtmax、−dp/dtmax降低（P<0.05），LVEDP升高（P<0.05）；与SHAM组比较，IR组、SPC组、SPC+DMSO组、SPC+OSMI‑1组T1~T4时心率、LVSP、+dp/dtmax、−dp/dtmax降低（P<0.05），LVEDP升高（P<0.05）；与IR组比较，SPC组和SPC+DMSO组T1~T4时心率、LVSP、+dp/dtmax、−dp/dtmax升高（P<0.05），LVEDP降低（P<0.05）。与SHAM组比较：IR组、SPC组、SPC+DMSO组、SPC+OSMI‑1组心肌梗死范围和LDH浓度增加（P<0.05），OGT和OGA蛋白水平升高（P<0.05），p‑RIPK1蛋白水平升高（P<0.05）；IR组、SPC组、SPC+DMSO组O‑GlcNAc蛋白水平升高（P<0.05）；IR组、SPC+OSMI‑1组p‑RIPK3、p‑MLKL蛋白水平升高（P<0.05）。与IR组比较，SPC组、SPC+DMSO组心肌梗死范围和LDH浓度减少（P<0.05），O‑GlcNAc、OGT蛋白水平升高（P<0.05），p‑RIPK3、p‑MLKL蛋白水平降低（P<0.05）。与SPC组比较，SPC+OSMI‑1组心肌梗死范围和LDH浓度增加（P<0.05），O‑GlcNAc、OGT蛋白水平降低（P<0.05），p‑RIPK3、p‑MLKL蛋白水平升高（P<0.05）。 结论 七氟醚后处理可降低离体大鼠心肌IR损伤，其机制与激活OGT介导的RIPK3糖基化有关。
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Abstract: Objective To evaluate the effect of O‑N‑acetyl‑glucosamine (O‑GlcNAc) transferase (OGT) mediated receptor interacting protein kinase 3 (RIPK3) O‑GlcNAcylation on ischemia reperfusion injury (IR) in rats after sevoflurane postconditioning. Methods A total of 45 rat hearts were successfully prepared by Langendorff through the in vitro perfusion modeling method. According to the random table method, they were randomly divided into five groups (n=9): a sham operation group (group SHAM), an ischemia reperfusion group (group IR), a sevoflurane postconditioning group (group SPC), a SPC+solvent group [group SPC+dimethyl sulfoxide (DMSO)] and a SPC+OGT inhibitor group (group SPC+OSMI‑1). After modeling, each group kept stable for 30 min, then group SHAM was given K‑H solution for 150 min, and the rat hearts were stopped perfusion for 30 min followed by 2 h reperfusion in all groups expect group SHAM. In group SPC, group SPC+DMSO and group SPC+OSMI‑1, K‑H solution containing 2.4% sevoflurane were continuously perfused at the first 15 min of reperfusion. In addition, in the K‑H solution prepared before surgery, group SPC+DMSO was given 50 μmol/L DMSO, while group SPC+OSMI‑1 was given 50 μmol/L OSMI‑1. Then, the left ventricular peak pressure (LVSP), left ventricular end‑diastolic pressure (LVEDP), heart rate, the maximum rate of rise of left ventricular pressure (+dp/dtmax), and the maximum rate of drop of left ventricular pressure (−dp/dtmax) immediately before ischemia (T0), after reperfusion for 30 min (T1), 60 min (T2), 90 min (T3), and 2 h (T4). At the end of reperfusion, the myocardial infarction area was measured by 1% 2,3,5 triphenyltetrazolium chloride (TTC) staining. The concentration of serum lactate dehydrogenase (LDH) was detected by enzyme‑linked immunosorbent assay (ELISA). The levels of O‑GlcNAc, OGT, O‑GlcNAcase (OGA), p‑RIPK1, p‑RIPK3 and phosphorylated mixed lineage kinase domain‑like protein (p‑MLKL) were measured by Western blot. Results Compared with those at T0, group IR, group SPC, group SPC+DMSO, and group SPC+OSMI‑1 showed remarkable decreases in heart rate, LVSP, +dp/dtmax and −dp/dtmax (P<0.05), and increases in LVEDP at T1 to T4 (P<0.05). Compared with group SHAM, group IR, group SPC, group SPC+DMSO and group SPC+OSMI‑1 showed significant decreases in heart rate, LVSP, +dp/dtmax and −dp/dtmax (P<0.05), and increases in LVEDP at T1 to T4 (P<0.05). Compared with group IR, group SPC and group SPC+DMOS showed obvious increases in heart rate, LVSP, +dp/dtmax and −dp/dtmax (P<0.05), and decreases in LVEDP (P<0.05). Compared with group SHAM, the myocardial infarction area and LDH content increased (P<0.05), the levels of OGT and OGA were up‑regulated and the levels of p‑RIPK1 were elevated in group IR, group SPC, group SPC+DMSO and group SPC+OSMI‑1 (P<0.05); the level of O‑GlcNAc increased in group IR, group SPC and group SPC+DMSO (P<0.05); and the levels of p‑RIPK3 and p‑MLKL were elevated in group IR and group SPC+OSMI‑1 (P<0.05). Compared with group IR, the myocardial infarction area and LDH content decreased (P<0.05), the levels of O‑GlcNAc and OGT were up‑regulated (P<0.05) and the levels of p‑RIPK3 and p‑MLKL were down‑regulated in group SPC and group SPC+DMSO (P<0.05). Compared with group SPC, the myocardial infarction area and LDH content increased (P<0.05), the levels of O‑GlcNAc and OGT were reduced (P<0.05) and the levels of p‑RIPK3 and p‑MLKL were up‑regulated in group SPC+OSMI‑1 (P<0.05). Conclusions Sevoflurane postconditioning can relieve myocardial IR injury in rats, which may be related to the activation of OGT mediated O‑GlcNAcylated RIPK3.
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