Cao Jianfang, Xie Hong, Sun Ying, Zhu Jiang, Ying Ming, Qiao Shigang, Shao Qin, Wu Haorong, Wang Chen
Department of Anesthesiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China.
Int J Mol Med. 2015 Dec;36(6):1529-37. doi: 10.3892/ijmm.2015.2366. Epub 2015 Oct 12.
The protective effects of sevoflurane post-conditioning against myocardial ischemia/reperfusion (I/R) injury (MIRI) have been previously reported. However, the mechanisms responsible for these protective effects remain elusive. In this study, in order to investigate the molecular mechanisms responsible for the protective effects of sevoflurane post-conditioning on isolated rat hearts subjected to MIRI, Sprague-Dawley rat hearts were randomly divided into the following 6 groups: i) the sham-operated control; ii) 2.5% sevoflurane; iii) ischemia/reperfusion (I/R); iv) 2.5% sevoflurane post-conditioning plus I/R; v) 2.5% sevoflurane post-conditioning + NG-nitro-L-arginine methyl ester (L-NAME) plus I/R; and vi) L-NAME plus I/R. The infarct size was measured using 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Additionally, the myocardial nitric oxide (NO), NO synthase (NOS) and nicotinamide adenine dinucleotide (NAD+) levels were determined. Autophagosomes and apoptosomes in the myocardium were detected by transmission electron microscopy. The levels of Bcl-2, cleaved caspase-3, Beclin-1, microtubule-associated protein light chain 3 (LC3)‑I/II, Na+/H+ exchanger 1 (NHE1) and phosphorylated NHE1 protein were measured by western blot analysis. NHE1 mRNA levels were measured by reverse transcription-quantitative polymerase chain reaction. Compared with the I/R group, 15 min of exposure to 2.5% sevoflurane during early reperfusion significantly decreased the myocardial infarct size, the autophagic vacuole numbers, the NHE1 mRNA and protein expression of cleaved caspase-3, Beclin-1 and LC3-I/II. Post-conditioning with 2.5% sevoflurane also increased the NO and NOS levels and Bcl-2 protein expression (p<0.05 or p<0.01). Notably, the cardioprotective effects of sevoflurane were partly abolished by the NOS inhibitor, L-NAME. The findings of the present study suggest that sevoflurane post-conditioning protects the myocardium against I/R injury and reduces the myocardial infarct size. The underlying protective mechanisms are associated with the inhibition of mitochondrial permeability transition pore opening, and with the attenuation of cardiomyoctye apoptosis and excessive autophagy. These effects are mediated through an increase in NOS and a decrease in phopshorylated NHE1 levels.
七氟醚后处理对心肌缺血/再灌注（I/R）损伤（MIRI）的保护作用此前已有报道。然而，这些保护作用的机制仍不清楚。在本研究中，为了探究七氟醚后处理对遭受MIRI的离体大鼠心脏保护作用的分子机制，将Sprague-Dawley大鼠心脏随机分为以下6组：i）假手术对照组；ii）2.5%七氟醚组；iii）缺血/再灌注（I/R）组；iv）2.5%七氟醚后处理+I/R组；v）2.5%七氟醚后处理+NG-硝基-L-精氨酸甲酯（L-NAME）+I/R组；vi）L-NAME+I/R组。采用2,3,5-三苯基氯化四氮唑（TTC）染色测量梗死面积。此外，测定心肌一氧化氮（NO）、一氧化氮合酶（NOS）和烟酰胺腺嘌呤二核苷酸（NAD+）水平。通过透射电子显微镜检测心肌中的自噬体和凋亡小体。采用蛋白质印迹分析检测Bcl-2、裂解的半胱天冬酶-3、Beclin-1、微管相关蛋白轻链3（LC3）-I/II、Na+/H+交换体1（NHE1）和磷酸化NHE1蛋白的水平。通过逆转录-定量聚合酶链反应测量NHE1 mRNA水平。与I/R组相比，早期再灌注期间暴露于2.5%七氟醚15分钟可显著减小心肌梗死面积、减少自噬泡数量、降低裂解的半胱天冬酶-3、Beclin-1和LC3-I/II的NHE1 mRNA和蛋白表达。2.5%七氟醚后处理还可增加NO和NOS水平以及Bcl-2蛋白表达（p<0.05或p<0.01）。值得注意的是，NOS抑制剂L-NAME部分消除了七氟醚的心脏保护作用。本研究结果表明，七氟醚后处理可保护心肌免受I/R损伤并减小心肌梗死面积。潜在的保护机制与抑制线粒体通透性转换孔开放以及减轻心肌细胞凋亡和过度自噬有关。这些作用是通过增加NOS和降低磷酸化NHE1水平介导的。
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