Metabolic profiling of hypoxia/reoxygenation injury in H9c2 cells reveals the accumulation of phytosphingosine and the vital role of Dan-Shen in Xin-Ke-Shu

• Published in: Phytomedicine (Stuttgart) 2018-10, Vol.49, p.83-94
• Main Authors: Sun, Lili, Jia, Hongmei, Ma, Liyan, Yu, Meng, Yang, Yong, Liu, Yang, Zhang, Hongwu, Zou, Zhongmei
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - drug effects; Cardioprotective effect; Cell Hypoxia; Cell Survival - drug effects; Drugs, Chinese Herbal - pharmacology; H9c2 cells; Hypoxia/reoxygenation injury; Metabolomics; Mitochondrial Membrane Transport Proteins; Myocardial Reperfusion Injury; Myocytes, Cardiac - drug effects; Oxidative Stress - drug effects; Rats; Salvia miltiorrhiza; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; UPLC-Q/TOF-MS; Xin-Ke-Shu
• ISSN: 0944-7113; 1618-095X
• DOI: 10.1016/j.phymed.2018.06.026

Xin-Ke-Shu (XKS), a patent medicine consisting of five commonly used traditional Chinese herbs, is used for the treatment of coronary heart diseases. A previous study showed that XKS has protective effects for ameliorating myocardial ischemia/reperfusion (I/R) injury. This study was aimed to deeply understand the mechanisms and compatible principle of XKS against hypoxia/reoxygenation (H/R) injury and the contribution of each single herb to the efficacy of XKS. An H/R model in H9c2 cardiomyocytes was applied to mimic I/R injury observed in vivo. The cell viability, the levels of LDH, MDA, SOD, and apoptosis were determined to evaluate the cardioprotection of XKS and its subtracted formula (knocked out one herb) in H/R injury. Cell metabolomics, combined with western blot analysis, was performed to uncover the inert molecular mechanism of XKS against H/R injury. Significant protective effects of XKS against oxidative stress and apoptosis induced by H/R injury were found in the pharmacodynamic evaluation. Moreover, the metabolic profile deviation of the H/R group from the control group was mainly ascribed to thirteen metabolites involved in four aberrant pathways, in which sphingolipid metabolism was revealed as the most relevant pathway involved in H/R injury (impact > 0.1). Notably, the accumulation of phytosphingosine (VIP = 5.84) was considered the most likely characteristic in H/R injury, which is well known to promote the opening of the mitochondrial permeability transition pore (mPTP) and activate cell apoptosis. Furthermore, XKS ameliorated all the abnormalities of the metabolic network in response to H/R injury. In agreement with this, a western blot analysis showed that XKS markedly regulated the over-expression of CaMK II and cleaved caspase-3. However, the subtracted formula showed no significant difference in comparison with the XKS group on protecting H/R injury except for QDS (subtracted Dan-Shen from XKS). The roots of Salvia miltiorrhiza Bge. (Dan-Shen) play an important role in the regulation of Ca2+ overloading, oxidative stress and apoptosis in H/R injury. Our study enabled information from holistic cell metabolomics to be used for mechanism and compatibility rule elucidations of TCMs. The significant cardioprotective effects of XKS against H/R injury in vitro were likely mediated by the inhibition of Ca2+ overloading, oxidative stress and cell apoptosis, wherein Dan-Shen plays an irreplaceable role. [Display omitted]