【摘要】 背景:研究报道,心肌缺血后调适的保护是通过心肌再灌注初期维持3 min的酸中毒起到作用,而在骨骼肌方面是否有此作用尚不清楚。目的:对大鼠骨骼肌缺血后调适过程进行动态pH测量,并模拟缺血后调适pH变化配置酸性灌注液输注,检测其对缺血再灌注损伤的影响。方法:基于前期大鼠骨骼肌缺血再灌注损伤建模方法和缺血后调适方案,运用光纤pH仪于主缺血期、4个循环30 s再灌/30 s缺血后调适操作期及其后再灌注期对大鼠骨骼肌进行pH连续测量,并用乳酸及生理盐水配置与缺血后调适期等pH灌注液备用。将25只健康成年雄性SD大鼠随机分为假手术组、缺血再灌注组、缺血后调适组、乳酸酸灌注组、生理盐水组5组,每组5只。各组按其实验方案进行相应实验并抽血检测乳酸脱氢酶,取样腓肠肌测算湿/干质量比值、髓过氧化物酶及TTC染色测算梗死面积,取样右侧胫前肌行Western Blot检测MAPK通路关键蛋白Erk1/2的表达。结果与结论:(1)缺血后调适于再灌注初期出现一个延长酸性平台,pH为6.81±0.133,时长为2 min 40 s。(2)乳酸脱氢酶、髓过氧化物酶、湿/干质量比值检测结果显示:缺血后调适组、乳酸酸灌注组明显低于缺血再灌注组(P<0.05);(3)Western Blot分析显示:p-Erk的表达缺血后调适组、乳酸酸灌注组及生理盐水组均显著高于缺血再灌注组(P<0.05);(4)TTC染色检测显示,缺血后调适组及乳酸酸灌注组梗死面积较缺血再灌注组明显减少(P<0.05);(5)结论:缺血后调适可维持再灌注初期短暂酸性状态;酸性灌注液可以有效模拟缺血后调适作用减少大鼠骨骼肌缺血再灌注损伤。其机制可能通过Erk1/2磷酸化激活RISK信号通路而起到保护作用。更多还原

【Abstract】 BACKGROUND: Ischemic postconditioning protects the myocardium from ischemia/recursion injury via maintaining 3-minute acidosis initially. But its effect on the skeletal muscle remains unclear. OBJECTIVE: To dynamically measure the pH values in rat skeletal muscle after ischemia, and then to simulate acidic perfusate infusion to investigate the effect of ischemic postconditioning on ischemia/reperfusion injury. METHODS: Based on the ischemia/reperfusion injury model and ischemic postconditioning protocol in previous study, dynamic measurement of pH values in rat skeletal muscle was conducted using pH instrument at the global ischemia, ischemic postconditioning(30/30 seconds) and reperfusion period, and then the acidic perfusate equivalent to pH in ischemic postconditioning period was prepared with lactic acid and normal saline. Twenty-five healthy adult male Sprague-Dawley rats were randomly divided into sham, ischemia/reperfusion, ischemic postconditioning, lactic acid, and normal saline groups(n=5 per group). Blood samples were collected to detect lactate dehydrogenase level. The samples from gastrocnemius were harvested to calculate the wet/dry ratio, level of myeloperoxidase, and infarct size through triphenyltetrazolium chloride staining. The samples from the right tibialis anterior muscle were taken to detect the expression level of Erk1/2 in the MAPK signaling pathway by western blot assay. RESULTS AND CONCLUSION: A prolonged acidic platform was detected in the early reperfusion during ischemic postconditioning, on which the pH value was 6.81±0.133, and the duration was 2 minutes and 40 seconds. The levels of lactate dehydrogenase and myeloperoxidase as well as the wet/dry ratio in the ischemic postconditioning and lactic acid groups were significantly lower than those in the ischemia/reperfusion group(P < 0.05). Western blot assay results showed that the expression level of p-Erk in the ischemic postconditioning, lactic acid and normal saline groups was significantly higher than that in the ischemia/reperfusion group(P < 0.05). Triphenyltetrazolium chloride staining results showed that compared with the ischemia/reperfusion group, the infarct area was significantly reduced in the postconditioning and lactic acid groups(P < 0.05). These findings suggest the existence of a short acidosis during ischemic postconditioning in the early reperfusion, and acidic perfusate can simulate the ischemic postconditioning and effectively attenuate ischemia/reperfusion injury in the rat skeletal muscle via activating Erk1/2 in RISK signaling pathway.更多还原