目的 从PI3K/Akt/mTOR通路角度,探讨七氟醚对小鼠心肌缺血再灌注损伤中自噬水平的影响。方法 小鼠24只随机分4组(n=6):假手术组,只开胸不结扎;缺血再灌注组,戊巴比妥钠麻醉下结扎冠状动脉左前降支行缺血/再灌注手术;七氟醚组,七氟醚麻醉下手术;七氟醚+LY294002组,术前一周每日注射PI3K阻断剂LY294002,余同七氟醚组。收集血浆检测心肌损伤标志物肌酸激酶-MB(CK-MB)和心肌肌钙蛋白Ⅰ(cTnI)水平。心肌组织用免疫印迹法检测自噬标志物 LC3、Beclin1,及信号通路磷脂酰肌醇3-磷酸激酶(PI3K)、蛋白激酶B(Akt)、雷帕霉素(mTOR) 总蛋白及磷酸化蛋白水平。结果 与假手术组相比,其余3组的血浆CK-MB、cTnI、心肌自噬标志蛋白LC3和Beclin1水平均增高(P<0.05),磷酸化PI3K、Akt、mTOR与总蛋白比值降低(P<0.05)。与假手术组相比,七氟醚组的PI3K、Akt、mTOR总蛋白水平差异无统计学意义;而缺血再灌注组PI3K、Akt水平增高(P<0.05),且mTOR蛋白水平降低(P<0.05);七氟醚+LY294002组PI3K水平增高(P<0.05),且Akt、mTOR蛋白水平降低(P<0.05)。与缺血再灌注组相比,七氟醚组和七氟醚+LY294002组血浆CK-MB和cTnI、LC3和Beclin1水平均降低(P<0.05)。与缺血再灌注组相比,七氟醚组的磷酸化PI3K、Akt、mTOR蛋白与总蛋白比值升高(P<0.05)。与七氟醚组相比,七氟醚+LY294002组血浆CK-MB、cTnI降低,LC3和Beclin1水平均增高(P<0.05);七氟醚+LY294002组的磷酸化PI3K、Akt、mTOR蛋白与总蛋白比值均降低(P<0.05)。结论 七氟醚抑制了缺血再灌注引发的过度自噬,可能的机制是上调了PI3K/Akt/mTOR通路蛋白磷酸化水平。
Abstract
Objective From the perspective of PI3K/Akt/mTOR pathway, to explore the potential effect of sevoflurane on autophagy levels in mice with myocardial ischemia-reperfusion injury. Methods Twenty-four mice were randomly divided into 4 groups (n=6): sham operation group(only chest opening without ligation), ischemia-reperfusion group (ligating the left anterior descending coronary artery as ischemia-reperfusion operation under pentobarbital anesthesia), sevoflurane group (surgery under sevoflurane anesthesia), and sevoflurane+LY294002 group (LY294002, PI3K blocker, was injected daily one week before ischemia-reperfusion surgery and the rest was the same as in Sevoflurane group). Plasma was collected to detect the levels of myocardial injury markers, creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Myocardial tissue was detected by western blot method to detect autophagy markers, LC3 and Beclin1, and signaling pathway proteins, phosphatase 3-kinase (PI3K), protein kinase B (Akt), rapamycin (mTOR) total protein and phosphorylated protein levels. Results Compared with sham operation group, the levels of plasma CK-MB, cTnI, markers of cardiac autophagy LC3 and Beclin1 in the other three groups were significantly increased (P<0.05), and the ratios of phosphorylated PI3K, Akt and mTOR to total protein significantly decreased (P<0.05). Compared with sham operation group, the total protein levels of PI3K, Akt and mTOR in sevoflurane group were not significantly different. The levels of PI3K and Akt significantly increased (P<0.05), and the level of mTOR protein significantly decreased (P<0.05) in ischemia-reperfusion group. The level of PI3K in sevoflurane +LY294002 group significantly increased (P<0.05), and the protein levels of Akt and mTOR significantly decreased (P<0.05). Compared with ischemia-reperfusion group, plasma LEVELS of CK-MB and cTnI, LC3 and Beclin1 in sevoflurane group and sevoflurane +LY294002 group significantly decreased (P<0.05). Compared with the ischemia-reperfusion group, the ratio of phosphorylated PI3K, Akt and mTOR protein to total protein levels in sevoflurane group significantly increased (P<0.05). Compared with sevoflurane group, plasma LEVELS of CK-MB, cTnI, LC3 and Beclin1 in sevoflurane +LY294002 group significantly increased (P<0.05). The levels of phosphorylated PI3K, Akt and mTOR protein to total protein in sevoflurane +LY294002 group significantly decreased (P<0.05). Conclusions Sevoflurane inhibits the excessive autophagy induced by ischemia-reperfusion, and the possible mechanism is that it upregulates the phosphorylation level of PI3K/Akt/mTOR pathway protein.
关键词
七氟醚 /
缺血再灌注 /
自噬 /
雷帕霉素
Key words
sevoflurane /
ischemia-reperfusion /
autophagy /
mTOR
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参考文献
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基金
山西省自然科学基金(201801D121226)
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