Dl-3-n-butylphthalide protects the heart against ischemic injury and H9c2 cardiomyoblasts against oxidative stress: involvement of mitochondrial function and biogenesis

Tian et al. Journal of Biomedical Science (2017) 24:38 DOI 10.1186/s12929-017-0345-9 RESEARCH Open Access Dl-3-n-butylphthalide protects the heart against ischemic injury and H9c2 cardiomyoblasts against oxidative stress: involvement of mitochondrial function and biogenesis Xiaochao Tian1*, Weiliang He2, Rong Yang1 and Yingping Liu3 Abstract Background: Myocardial infarction (MI) is an acute and fatal condition that threatens human health. Dl-3-nbutylphthalide (NBP) has been used for the treatment of acute ischemic stroke. Mitochondria may play a protective role in MI injury. However, there are few reports on the cardioprotective effect of NBP or the potential mitochondrial mechanism for the NBP-induced protection against cardiac ischemia injury. We investigated the therapeutic effects of NBP in an in vivo MI model and an in vitro oxidative stress model, as well as the potential mitochondrial mechanism. Methods: This study comprised two different experiments. The aim of experiment 1 was to determine the protective effects of NBP on MI and the underlying mechanisms in vivo. In part 1, myocardial infarct size was measured by staining with 2,3,5-triphenyltetrazoliumchloride (TTC). Myocardial enzymes and mitochondrial enzymes were assayed. The aim of experiment 2 was to investigate the role of NBP in H2O2-induced myocardial ischemic injury in H9c2 cells and to determine the potential mechanism. In part 2, H9c2 cell viability was evaluated. ROS levels, mitochondrial morphology, and mitochondrial membrane potential of H9c2 cells were measured. ATP levels were evaluated using an assay kit; mitochondrial DNA (mtDNA), the expressions of NRF-1 and TFAM, and mitochondrial biogenesis factors were determined. Results: NBP treatment significantly reduced the infarct ratio, as observed by TTC staining, decreased serum myocardial enzymes in MI, and restored heart mitochondrial enzymes (isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and a-ketoglutarate dehydrogenase (a-KGDH) activities after MI. Moreover, in in vitro studies, NBP significantly increased the viability of H9c2 cells in a dose-dependent manner, reduced cell apoptosis, protected mitochondrial functions, elevated the cellular ATP levels, and promoted H2O2-induced mitochondrial biogenesis in H9c2 cardiomyoblasts. Conclusion: Collectively, the results from both the in vivo and in vitro experiments suggested that NBP exerted a cardioprotective effect on cardiac ischemic injury via the regulation of mitochondrial function and biogenesis. Keywords: Myocardial infarction, Dl-3-n-butylphthalide, Mitochondrial function, Mitochondrial biogenesis * Correspondence: 1 Department of Cardiology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tian et al. Journal of Biomedical Science (2017) 24:38 Background Myocardial infarction (MI), the most common cardiovascular complaint, is an acute and fatal condition of the cardiovascular system that threatens human health [1]. The ischemic heart experiences cardiomyocyte loss, with subsequent reparative fibrotic healing, decreased cardiac function, which plays an important role in progression of cardiac hypertrophy, and arrhythmia [2]. Although myocardial protection has recently improved, there are still a limited number of effective therapies to improve myocardial resistance to ischemic injury [3]. Thus, the identification of novel drug targets for the treatment of MI is imperative. The abundance of mitochondria in the heart is well known; they are known shown to provide continuous energy. It has become increasingly clear that mitochondrial function is involved in ischemic injury. Impaired mitochondria disturb ATP generation [4]. A close association has been identified between altered mitochondrial morphology and dynamics and MI injury [5]. It has also been reported that the protection of mitochondria in the post-infarct myocardium resulted in decreased mitochondrial dysfunction and cardiomyocyte apoptosis [6]. In addition, oxidative stress occurred mainly in the mitochondria; damaged cells attack the mitochondria [7]. Researchers have shown that oxidative stress plays an important role in myocardial ischemic injury [8, 9]. Ischemic oxidative stress results in the reduction of myocardial antioxidants, loss of mitochondrial membrane potential, and release of superoxide [10, 11]. Therefore, the identification of a pharmacological agent that exerts protective effects in mitochondria and alleviates MI injury might be an ideal cardioprotective strategy. Dl-3-n-butylphthalide (NBP), a small molecule extracted from a Chinese herb (Chinese celery), has been approved for the treatment of acute ischemic stroke by the State Food and Drug Administration of China [12]. Previous studies have shown that NBP conferred neuroprotective effects via the improvement of microcirculation dysfunction during ischemia [13], decreased the cerebral infarct area in brain ischemia models [14], protected neuron activity in stroke [15], and attenuated inflammatory responses [16] in cultured astrocyte models. In addition, NBP has preventive and therapeutic effects on the improvement of outcomes after cardiac arrest and resuscitation [17]. Recent studies have indicated that NBP ameliorated oxidative stress and mitochondrial damage, which reduced endothelial cell death after oxygen glucose deprivation in vitro [18]. It has been reported that oxidative stress affected the mitochondrial apoptotic pathway [19]. However, at present, the effect of NBP on mitochondrial function and energy metabolism in MI have not been comprehensively described. Page 2 of 10 Therefore, the aims of this study were to investigate the therapeutic effects of NBP on in vivo MI models, as well as to determine the potential mechanism with a focus on the mitochondrion in vitro oxidative stress models. Methods Animals and ethical statement The experimental protocols used in this study were in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. All animal manipulations were performed in accordance with the recommendations of the Committee of the Care and Use of Laboratory Animals at Hebei Medical University. Preparation of rat MI model Ra (...truncated)