Hang Pengzhou, Zhao Jing, Su Zhenli, Sun Hanqi, Chen Tingting, Zhao Lihui, Du Zhimin
Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University (Institute of Clinical Pharmacy, the Heilongjiang Key Laboratory of Drug Research, Harbin Medical University), Harbin, China.
Department of Clinical Pharmacology, College of Pharmacy, Harbin Medical University, Harbin, China.
Cell Physiol Biochem. 2018;45(5):2136-2144. doi: 10.1159/000488049. Epub 2018 Mar 7.
Backgroud/Aims: Growing evidence suggests that both cardiomyocyte apoptosis and excessive autophagy exacerbates cardiac dysfunction during myocardial ischemia-reperfusion (IR). As a precursor of acetylcholine, choline has been found to protect the heart by repressing ischemic cardiomyocyte apoptosis. However, the relationship between choline and cardiomyocyte autophagy is unclear. The present study aimed to investigate whether autophagy was involved in the cardioprotection of choline during IR.
Rats were subjected to 30 min reversible ischemia by ligation of left anterior descending coronary artery followed by reperfusion for 2 h. Choline (5 mg/kg, i.v.) alone or along with rapamycin (5 mg/ kg, i.p.) were injected 30 min before ischemia. Transmission electron microscopy, hematoxylin and eosin (HE) and TUNEL staining were conducted to evaluate the effect of choline on cardiac apoptosis and autophagy. Protein levels of autophagic markers including LC3, beclin-1 and p62 as well as Akt and mammalian target of rapamycin (mTOR) were examined by Western blotting.
Myocardial IR-induced cardiac apoptosis and accumulation of autophagosomes was attenuated by choline. Choline treatment significantly ameliorated myocardial IR-induced autophagic activity characterized by repression of beclin-1 over-activation, the reduction of autophagosomes, the LC3-II/LC3-I ratio, and p62 protein abundance. In addition, IR-induced downregulation of p-Akt/mTOR cascade was increased by choline. However, the above functions of choline were abolished by rapamycin.
These findings suggest that choline plays a protective role against myocardial IR injury by inhibiting excessive autophagy, which might be associated with the activation of Akt/mTOR pathway. This study provides new mechanistic understanding of cardioprotective effect of choline and suggests novel potential therapeutic targets for cardiac IR injury.
背景/目的：越来越多的证据表明，心肌细胞凋亡和过度自噬都会加剧心肌缺血再灌注（IR）期间的心脏功能障碍。作为乙酰胆碱的前体，胆碱已被发现可通过抑制缺血心肌细胞凋亡来保护心脏。然而，胆碱与心肌细胞自噬之间的关系尚不清楚。本研究旨在探讨自噬是否参与了胆碱在IR期间的心脏保护作用。
通过结扎左冠状动脉前降支使大鼠经历30分钟可逆性缺血，随后再灌注2小时。在缺血前30分钟单独注射胆碱（5毫克/千克，静脉注射）或与雷帕霉素（5毫克/千克，腹腔注射）联合注射。进行透射电子显微镜检查、苏木精-伊红（HE）染色和TUNEL染色以评估胆碱对心脏凋亡和自噬的影响。通过蛋白质印迹法检测自噬标志物包括LC3、贝林1和p62以及Akt和雷帕霉素靶蛋白（mTOR）的蛋白质水平。
胆碱减轻了心肌IR诱导的心脏凋亡和自噬体积累。胆碱处理显著改善了心肌IR诱导的自噬活性，其特征为抑制贝林1过度激活、自噬体减少、LC3-II/LC3-I比值降低以及p62蛋白丰度降低。此外，胆碱增加了IR诱导的p-Akt/mTOR级联反应的下调。然而，雷帕霉素消除了胆碱的上述功能。
这些发现表明，胆碱通过抑制过度自噬对心肌IR损伤起保护作用，这可能与Akt/mTOR途径的激活有关。本研究为胆碱的心脏保护作用提供了新的机制理解，并为心脏IR损伤提出了新的潜在治疗靶点。
