| Part One Mechanism of Acute Sevoflurane Preconditioning in Myocardial ProtectionObjectiveTo investigate the acute phase of sevoflurane preconditioning (APC) against myocardial ischemia/reperfusion (I/R) injury and the mechanism of nuclear factor-κB (NF-κB) pathway induced apoptosis and anti-apoptosis in rat myocardium.MethodsIn this in vivo study, the animals were anesthetized with intraperitoneal pentobarbital, intubated and mechanically ventilated. 143 adult male Sprague Dawley (SD) rats were randomly divided into 8 groups: (1) Control (CON) group received sham operation; (2) I/R group was produced by 30 min occlusion of the left anterior descending branch of coronary artery followed by 2 h reperfusion; (3) APC group, rats were exposed to 30 min of 2.5% sevoflurane followed by 15 min washout before I/R; (4) SEVO group, rats were exposed to 30 min of 2.5% sevoflurane followed by a 165 min washout period; (5) PTN group received NF-κB inhibitor parthenolide (PTN); (6) DMSO group received the PTN solvent dimethylsulfoxide; (7) PTN+APC group and (8) APC+PTN group, PTN was used before or after exposure to sevoflurane respectively.Left ventricular (LV) samples were obtained to measure myocardial infarct size by using triphenyl tetrazolium chloride (TTC) staining. Myocardial apoptosis was determined by TUNEL assay and apoptosis index. LV tissues were collected before ischemia and at the end of 2 h reperfusion for determining the expression of NF-кB (p65, p50), inflammatory factors intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-α(TNF-α), anti-apoptosis factor B-cell lymphoma-2 (Bcl-2) and apoptosis factor Caspase-3 in myocardium by Western blot. ResultsSevoflurane preconditioning significantly decreased myocardial infarct size and the myocardium apoptosis in APC group compared with I/R group. PTN administered before or after APC abolished this effect (P<0.05).APC induced up-regulation of NF-κB (p65, p50) before ischemia (P<0.05). At the end of 2 h reperfusion, NF-κB (p65, p50) were significantly up-regulation in I/R and APC groups, but these levels were lower in APC group than in I/R group. Before ischemia, Bcl-2 was significantly elevated in APC and SEVO groups (P<0.05). At the end of 2 h reperfusion, ICAM-1, TNF-αand Caspase-3 expressions were significantly increased in I/R group compared with CON group (P<0.05); These increases were blunted in APC group.ConclusionThe results of our in vivo study indicated that APC produced acute myocardial protection against I/R injury. NF-κB acted not only as a trigger but also as a mediator that played an important role in APC through upregulation of NF-κB and the anti-apoptosis protein Bcl-2 during the preconditioning period and then through down-regulation of the inflammatory proteins ICAM-1 and TNF-αduring reperfusion, decreasing Caspase-3 expression and apoptosis in myocardium. APC ultimately protected myocardium against I/R injury by reducing infarct size and improving cardiac function. Part Two Mechanism of Delayed Cardioprotective of Sevoflurane PreconditioningObjectiveTo investigate the delayed protection of sevoflurane preconditioning (SWOP) against myocardial ischemia/reperfusion (I/R) injury and the mechanism of nuclear factor-κB (NF-κB) pathway induced autophagy and apoptosis during the cardioprotection in rats.MethodsIn this in vivo study, 24 hours before coronary artery occlusion (30 min) followed by 2 h reperfusion, 140 adult male Sprague Dawley (SD) rats were randomly assigned to receive 33% oxygen (I/R group) or 2.5% sevoflurane for 2 h (SWOP group). Rats received 33% oxygen or 2.5% sevoflurane without subsequent I/R were served as the control (CON) group or sevoflurane-control (SEVO) group respectively. The NF-κB inhibitor parthenolide (PTN) was administered intraperitoneal before sevoflurane exposure (PTN+SWOP group) or given alone (PTN group). DMSO group received the PTN solvent dimethylsulfoxide.Triphenyl tetrazolium chloride (TTC) staining method was used to measure left ventricular (LV) infarct size. The myocardium tissue injury and autophagosome were observed by optical microscope or transmission electron microscopy respectively. After reperfusion technetium-99m-labeled annexin-Ⅴwas administered intravenously for noninvasive identification of apoptotic cell death by using radionuclide imaging. LV tissues were collected before ischemia and at the end of 2 h reperfusion for determining the expression of NF-кB (p65, p50, p-IκBα), inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin 1β(IL-1β), autophagy protein microtubule associated protein 1 light chain3-Ⅱ(LC3-Ⅱ) and Cathepsin B, apoptosis factor Caspase-3 in myocardium by Western blot.ResultsSevoflurane preconditioning significantly decreased infarct size and marked necrosis of myocardial tissue in SWOP group compared with I/R group. However, this protection was abolished by administration of PTN before sevoflurane preconditioning (P<0.05). The ultrastructural analysis of the myocardium showed that autophagosome were detected in SWOP group before ischemia. Radionuclide imaging showed that the target/background (T/B) ratio of radioactivity at the infarct areas in SWOP group was lower than in I/R group after reperfusion (P<0.05).SWOP induced an up-regulation of NF-κB (p65, p50, p-IκBα), TNF-α, IL-1β, LC3-Ⅱand Cathepsin B before ischemia (P<0.05). At the end of 2 h reperfusion, NF-κB (p65, p50, p-IκBα), TNF-α, IL-1β, LC3-Ⅱ, Cathepsin B and Caspase-3 were significantly increased in I/R and SWOP groups, but they were lower in SWOP group than in I/R group except the expression of Cathepsin B (P<0.05).ConclusionThe results of our in vivo study indicated that SWOP produced delayed myocardial protection against I/R injury. NF-κB pathway acted as a trigger by negative feedback mechanism that played an important role in SWOP. SWOP upregulated the expression of NF-κB (p65, p50, p-IκBα), inflammatory factors TNF-αand IL-1β, and then activated autophagy to a less extend during the preconditioning period. Moreover, SWOP down-regulated the expression of NF-κB (p65, p50, p-IκBα), inflammatory factors (TNF-α, IL-1β) during reperfusion, decreased autophagy and apoptosis. SWOP ultimately protected myocardium against I/R injury by reducing infarct size at the end of reperfusion. |
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