大鼠骨骼肌缺血后调适动态pH测量及其模拟酸灌注减轻缺血再灌注损伤研究

发布时间：2018-07-29 15:34

【摘要】：背景:近来有研究报道,心肌缺血后调适保护的重要机制是通过心肌缺血后再灌注初期维持3min的酸中毒状态起到作用。但其具体的p H变化如何以及模拟酸性灌注能否减轻缺血再灌注损伤未见深入研究。在骨骼肌方面的类似研究更亦未查见。目的:对大鼠骨骼肌缺血后调适过程进行动态p H测量,并模拟缺血后调适p H变化配置酸性灌注液输注大鼠体内,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优化方案,运用光纤p H仪于主缺血期、4个循环30s再灌/30s缺血后调适操作期及其后再灌注期对大鼠骨骼肌进行p H连续测量,并用乳酸及生理盐水配置与缺血后调适期等p H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2,3,5—氯化三苯基四氮唑(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:(1)缺血后调适于再灌注初期出现一个延长酸性平台,p H为6.81±0.133,时长为2min40s。(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、乳酸输注组均明显低于缺血再灌注组(P0.05);(3)Western Blot分析显示:p-Erk的表达缺血后调适组、乳酸输注组及生理盐水输注组均显著高于缺血再灌注组(P0.05)。(4)TTC染色检测显示,缺血后调适组及乳酸输注组梗死面积较缺血再灌注组明显减少(P0.05)。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,减轻大鼠骨骼肌缺血再灌注损伤。其机制可能直接作用于MPTP避免其即时开放,或通过Erk1/2磷酸化激活RISK信号通路而起到保护作用。
[Abstract]:Background: recently, it has been reported that the important mechanism of myocardial ischemic conditioning is to maintain the acidosis state of 3min in the early stage of myocardial ischemia and reperfusion. However, the specific pH changes and whether simulated acid perfusion can reduce ischemia reperfusion injury have not been further studied. Similar studies on skeletal muscle have also not been found. Aim: to measure the dynamic pH of skeletal muscle after ischemia in rats, and to investigate the effects of pH on ischemia reperfusion injury in rats. Methods: based on the modeling method of ischemic reperfusion injury of skeletal muscle in early stage and the optimized scheme of adjustment after ischemia in rat skeletal muscle. The pH of skeletal muscle of rats was continuously measured by fiber-optic pH instrument during the main ischemia period, 4 cycles 30 s / 30 s ischemia conditioning operation period and subsequent reperfusion period. The pH perfusion solution was prepared with lactic acid and physiological saline configuration and after ischemia adaptation period. Twenty-five adult male SD rats were randomly divided into sham-operation group, ischemia-reperfusion group, post-ischemic conditioning group, lactic acid perfusion group, physiological saline group, 5 rats in each group. According to the experimental plan, each group was tested for lactate dehydrogenase, the wet / dry weight ratio of gastrocnemius was measured by sampling gastrocnemius muscle, and the infarct area was measured by myeloperoxidase and (TTC) staining. The expression of Erk1/2, a key protein in the MAPK pathway, was detected by Western Blot in the right tibial anterior muscle. Results: (1) the pH of a prolonged acidic platform was 6.81 卤0.133, and the duration was 2 min 40 s. (2) lactate dehydrogenase, myeloperoxidase and wet / dry weight ratio were measured. The expression of p-Erk in the lactic acid infusion group was significantly lower than that in the ischemia reperfusion group (P0.05); (3) Western Blot analysis showed that the expression of p-Erk was significantly higher in the lactic acid infusion group and saline infusion group than in the ischemia reperfusion group (P0.05). (4) TTC staining. The infarct size of conditioning group and lactic acid infusion group was significantly lower than that of ischemia reperfusion group (P0.05). Conclusion: conditioning after ischemia can maintain the transient acidic state in the early stage of reperfusion. Acid perfusion solution can effectively simulate the effects of ischemic conditioning and alleviate the ischemia reperfusion injury of skeletal muscle in rats. The mechanism may play a direct role in MPTP to avoid its immediate opening, or through Erk1/2 phosphorylation to activate the RISK signaling pathway and play a protective role.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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