Skip to main content

Metabolomics Analysis of the Effect of Hydrogen-Rich Water on Myocardial Ischemia-Reperfusion Injury in Rats


To investigate the effect of hydrogen-rich water on myocardial tissue metabolism in a myocardial ischemia-reperfusion injury (MIRI) rat model. Twelve rats were randomly divided into a hydrogen-rich water group and a control group of size 6 each. After the heart was removed, it was fixed in the Langendorff device, and the heart was perfused with 37 °C perfusion solution pre-balanced with oxygen. The control group was perfused with Kreb’s-Ringers (K-R) solution, and the hydrogen-rich water group was perfused with K-R solution + hydrogen-rich water. Liquid Chromatograph Mass Spectrometer (LC-MS) analysis platform was used for metabolomics research. Principle component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), Variable importance in projection (VIP) value of OPLS-DA model (threshold value ≥1) were employed with independent sample T Test (p < 0.05) to find differentially expressed metabolites, and screen for differential metabolic pathways. VIP (OPLS-DA) analysis was performed with T test, and the metabolites of the control group and the hydrogen-rich water group were significantly different, and the glycerophospholipid metabolism was screened. Seven myocardial ischemia-reperfusion injury (MIRI)-related signaling pathways were identified, including glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI) anchored biosynthesis, and purine metabolism, as well as 10 biomarkers such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Hydrogen-rich water regulates the metabolic imbalance that could change MIRI myocardial tissue metabolism, and alleviate ischemia-reperfusion injury in isolated hearts of rats through multiple signaling pathways.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Data Availability

All data generated or analyzed during the present study are included in this published article.



Acute myocardial infarction




Liquid Chromatograph Mass Spectrometer


Myocardial ischemia-reperfusion injury

NAD+ :

Nicotinamide adenine dinucleotide


Orthogonal partial least squares discriminant analysis


Partial least squares discriminant analysis


Principle component analysis


Tricarboxylic acid


Total ion current


Variable importance in projection


  • Lansky AJ, Stone GW (2010) Periprocedural myocardial infarction: prevalence, prognosis, and prevention. Circ Cardiovasc Interv 3(6):602–610

    Article  Google Scholar 

  • Writing Group M, Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ et al (2016) Heart disease and stroke Statistics-2016 update: a report from the American Heart Association. Circulation. 133(4):e38–e360

    Google Scholar 

  • Visternichan O, Jalali SF, Taizhanova D, Muravlyova L, Igimbayeva G (2019) Dynamic changes in purine catabolism in patients with acute coronary syndrome that underwent percutaneous coronary intervention. Caspian J Intern Med 10(1):86–91

    PubMed  PubMed Central  Google Scholar 

  • Farhat N, Haddad N, Crispo J, Birkett N, McNair D, Momoli F, Wen SW, Mattison DR, Krewski D (2019) Trends in concomitant clopidogrel and proton pump inhibitor treatment among ACS inpatients, 2000-2016. Eur J Clin Pharmacol 75(2):227–235

    CAS  Article  Google Scholar 

  • Davidson SM, Ferdinandy P, Andreadou I, Bøtker HE, Heusch G, Ibáñez B, Ovize M, Schulz R, Yellon DM, Hausenloy DJ, Garcia-Dorado D, CARDIOPROTECTION COST Action (CA16225) (2019) Multitarget strategies to reduce myocardial ischemia/reperfusion injury: JACC review topic of the week. J Am Coll Cardiol 73(1):89–99

    Article  Google Scholar 

  • Bellanti F, Mirabella L, Mitarotonda D, Blonda M, Tamborra R, Cinnella G, Fersini A, Ambrosi A, Dambrosio M, Vendemiale G, Serviddio G (2016) Propofol but not sevoflurane prevents mitochondrial dysfunction and oxidative stress by limiting HIF-1α activation in hepatic ischemia/reperfusion injury. Free Radic Biol Med 96:323–333

    CAS  Article  Google Scholar 

  • Zhang L, Cao S, Deng S, Yao G, Yu T (2016) Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor. Peer J 4:e2612-e

    Article  Google Scholar 

  • de Gusmão FMB, Becker C, Carvalho MHC, Barros LFM (2005) Angiotensin II inhibition during myocardial ischemia-reperfusion in dogs: effects on leukocyte infiltration, nitric oxide synthase isoenzymes activity and left ventricular ejection fraction. Int J Cardiol 100(3):363–370

    Article  Google Scholar 

  • Pei H-X, Hua R, Guan C-X, Fang X (2015) Ginkgolide B reduces the degradation of membrane phospholipids to prevent ischemia/reperfusion myocardial injury in rats. Pharmacology. 96(5–6):233–239

    CAS  Article  Google Scholar 

  • Kawai H, Chaudhry F, Shekhar A, Petrov A, Nakahara T, Tanimoto T, Kim D, Chen J, Lebeche D, Blankenberg FG, Pak KY, Kolodgie FD, Virmani R, Sengupta P, Narula N, Hajjar RJ, Strauss HW, Narula J (2018) Molecular imaging of apoptosis in ischemia reperfusion injury with radiolabeled Duramycin targeting Phosphatidylethanolamine: effective target uptake and reduced nontarget organ radiation burden. JACC Cardiovasc Imaging 11(12):1823–1833

    Article  Google Scholar 

  • Poulsen RH, Rasmussen JT, Bøtker HE, Waehrens LS, Falborg L, Heegaard CW, Rehling M (2014) Imaging the myocardium at risk with 99mTc-lactadherin administered after reperfusion in a porcine model. Nucl Med Biol 41(1):114–119.

    CAS  Article  PubMed  Google Scholar 

  • Song D, Liu X, Diao Y, Sun Y, Gao G, Zhang T, Chen K, Pei L (2018) Hydrogen-rich solution against myocardial injury and aquaporin expression via the PI3K/Akt signaling pathway during cardiopulmonary bypass in rats. Mol Med Rep 18(2):1925–1938.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • LeBaron TW, Kura B, Kalocayova B, Tribulova N, Slezak J (2019) A New Approach for the Prevention and Treatment of Cardiovascular Disorders. Molecular Hydrogen Significantly Reduces the Effects of Oxidative Stress. Molecules 24(11):2076

    CAS  Article  Google Scholar 

  • Yao L, Chen H, Wu Q, Xie K (2019) Hydrogen-rich saline alleviates inflammation and apoptosis in myocardial I/R injury via PINK-mediated autophagy. Int J Mol Med 44(3):1048–1062

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng X, Zhang F, Li J, Wang G (2019) Galuteolin attenuates cerebral ischemia/reperfusion injury in rats via anti-apoptotic, anti-oxidant, and anti-inflammatory mechanisms. Neuropsychiatr Dis Treat 15:2671–2680

    CAS  Article  Google Scholar 

  • Li L, Liu T, Li X, Liu X, Liu L, Li S, Li Z, Zhou Y, Liu F (2019a) Protein chip and bioinformatic analyses of differentially expressed proteins involved in the effect of hydrogen-rich water on myocardial ischemia-reperfusion injury. Int J Med Sci 16(9):1254–1259

    Article  Google Scholar 

  • Li X, Li L, Liu X, Wu J, Sun X, Li Z, Geng YJ, Liu F, Zhou Y (2019b) Attenuation of cardiac Ischaemia-reperfusion injury by treatment with hydrogen-rich water. Curr Mol Med 19(4):294–302

    CAS  Article  Google Scholar 

  • Li L, Li X, Zhang Z, Liu L, Liu T, Li S, et al. Effects of Hydrogen-rich Water on the PI3K/AKT Signaling Pathway in Rats with Myocardial Ischemia-reperfusion Injury. Current molecular medicine. 2019c:

  • Li L, Liu T, Liu L, Li S, Zhang Z, Zhang R, Zhou Y, Liu F (2019d) Effect of hydrogen-rich water on the Nrf2/ARE signaling pathway in rats with myocardial ischemia-reperfusion injury. J Bioenerg Biomembr 51(6):393–402

    CAS  Article  Google Scholar 

  • Cadenas S (2018) ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection. Free Radic Biol Med 117:76–89

    CAS  Article  Google Scholar 

  • Yuan L, Dai X, Fu H, Sui D, Lin L, Yang L, Zha P, Wang X, Gong G (2018) Vaspin protects rats against myocardial ischemia/reperfusion injury (MIRI) through the TLR4/NF-κB signaling pathway. Eur J Pharmacol 835:132–139

    CAS  Article  Google Scholar 

  • Ji C, Song F, Huang G, Wang S, Liu H, Liu S, Huang L, Liu S, Zhao J, Lu TJ, Xu F (2018) The protective effects of acupoint gel embedding on rats with myocardial ischemia-reperfusion injury. Life Sci 211:51–62

    CAS  Article  Google Scholar 

  • Bainey KR, Armstrong PW (2014) Clinical perspectives on reperfusion injury in acute myocardial infarction. Am Heart J 167(5):637–645

    Article  Google Scholar 

  • Pell VR, Spiroski A-M, Mulvey J, Burger N, Costa ASH, Logan A, Gruszczyk AV, Rosa T, James AM, Frezza C, Murphy MP, Krieg T (2018) Ischemic preconditioning protects against cardiac ischemia reperfusion injury without affecting succinate accumulation or oxidation. J Mol Cell Cardiol 123:88–91

    CAS  Article  Google Scholar 

  • Liu N-B, Wu M, Chen C, Fujino M, Huang J-S, Zhu P et al (2019) Novel molecular targets participating in myocardial ischemia-reperfusion injury and Cardioprotection. Cardiol Res Pract 2019:6935147

    PubMed  PubMed Central  Google Scholar 

  • Han J-Y, Li Q, Pan C-S, Sun K, Fan J-Y (2019) Effects and mechanisms of QiShenYiQi pills and major ingredients on myocardial microcirculatory disturbance, cardiac injury and fibrosis induced by ischemia-reperfusion. Pharmacol Res 147:104386

    CAS  Article  Google Scholar 

  • Kim TT, Dyck JRB (2016) The role of CD36 in the regulation of myocardial lipid metabolism. Biochim Biophys Acta 1861(10):1450–1460

    CAS  Article  Google Scholar 

  • Robertson JL (2018) The lipid bilayer membrane and its protein constituents. J Gen Physiol 150(11):1472–1483

    CAS  Article  Google Scholar 

  • Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9(2):99–111

    CAS  Article  Google Scholar 

  • Glukhova A, Hinkovska-Galcheva V, Kelly R, Abe A, Shayman JA, Tesmer JJG (2015) Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase. Nat Commun 6:6250

    CAS  Article  Google Scholar 

  • Aghazadeh-Habashi A, Asghar W, Jamali F (2018) Drug-disease interaction: effect of inflammation and nonsteroidal anti-inflammatory drugs on cytochrome P450 metabolites of Arachidonic acid. J Pharm Sci 107(2):756–763

    CAS  Article  Google Scholar 

  • Tao L, Huang K, Wang J, Xue Y, Zhou Y, He F et al (2019) Retinol palmitate protects against myocardial ischemia/reperfusion injury via reducing oxidative stress and inhibiting apoptosis. American journal of translational research 11(3):1510–1520 33

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng P, Xie Z, Yuan Y, Sui W, Wang C, Gao X et al (2017) Plin5 alleviates myocardial ischaemia/reperfusion injury by reducing oxidative stress through inhibiting the lipolysis of lipid droplets. Scientific reports. 7:42574

    CAS  Article  Google Scholar 

  • Hu L, Cai N, Jia H (2017) Pterostilbene attenuates myocardial ischemia-reperfusion injury via the phosphatidylinositol 3’-kinase-protein kinase B signaling pathway. Experimental and therapeutic medicine 14(6):5509–5514

    CAS  PubMed  PubMed Central  Google Scholar 

  • Feng L, Liu W, Yang J, Wang Q, Wen S (2018) Effect of Hexadecyl Azelaoyl Phosphatidylcholine on Cardiomyocyte apoptosis in myocardial ischemia-reperfusion injury: a hypothesis. Medical science monitor: international medical journal of experimental and clinical research 24:2661–2667

    CAS  Article  Google Scholar 

  • Pepe S (2019) Raising the dead: mitochondrial Cardiolipin as a key target for post-cardiac arrest resuscitation, Ischaemia-reperfusion injury and cardiomyopathy. Heart Lung Circ 28(3):360–363

    Article  Google Scholar 

  • Wu S-Z, Tao L-Y, Wang J-N, Xu Z-Q, Wang J, Xue Y-J, et al. Amifostine Pretreatment Attenuates Myocardial Ischemia/Reperfusion Injury by Inhibiting Apoptosis and Oxidative Stress Oxidative medicine and cellular longevity 2017; 2017: 4130824

  • Zálešák M, BlaŽíček P, Pancza D, Gablovský I, Štrbák V, Ravingerová T (2016) Hyperosmotic environment blunts effectivity of ischemic preconditioning against ischemia-reperfusion injury and improves ischemic tolerance in non-preconditioned isolated rat hearts. Physiol Res 65(6):1045–1051

    Article  Google Scholar 

  • Ke-Wu D, Xu-Bo S, Ying-Xin Z, Shi-Wei Y, Yu-Jie Z, Dong-Mei S, Yu-Yang L, de-An J, Zhe F, Zhi-Ming Z, Hai-Long G, Zhen-Xian Y, Chang-Sheng M (2015a) The effect of exogenous creatine phosphate on myocardial injury after percutaneous coronary intervention. Angiology. 66(2):163–168

    Article  Google Scholar 

  • Paradies G, Paradies V, Ruggiero FM, Petrosillo G (2018) Mitochondrial bioenergetics and cardiolipin alterations in myocardial ischemia-reperfusion injury: implications for pharmacological cardioprotection. Am J Physiol Heart Circ Physiol 315(5):H1341–H1H52

    Article  Google Scholar 

  • Zhai M, Li B, Duan W, Jing L, Zhang B, Zhang M, et al. (2017) Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis. J Pineal Res. 63(2):

  • Ke-Wu D, Xu-Bo S, Ying-Xin Z, Shi-Wei Y, Yu-Jie Z, Dong-Mei S, Yu-Yang L, de-An J, Zhe F, Zhi-Ming Z, Hai-Long G, Zhen-Xian Y, Chang-Sheng M (2015b) The effect of exogenous creatine phosphate on myocardial injury after percutaneous coronary intervention. Angiology. 66(2):163–168

    Article  Google Scholar 

Download references


The authors are grateful for the support provided by the Affiliated Hospital of Hebei University of China. The authors are grateful for the experimental hydrogen-rich water provided by Professor Zhilin Li from the College of Chemistry, Hebei University (patent number ZL102557227B).


This research was financially supported by the Medical Science Research Key Project Foundation of Hebei Province (No. 20130369).

Author information

Authors and Affiliations



Liangtong Li, Tongtong Liu, Li Liu and Zhe Zhang performed the LC-MS analyses. Liangtong Li and Tongtong Liu collected the data and performed the statistical analyses. Shaochun Li and Zhiling Zhang participated in sample collection and the signaling pathway analysis. Yujuan Zhou and Fulin Liu designed the study. Liangtong Li and Tongtong Liu wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yujuan Zhou or Fulin Liu.

Ethics declarations

Conflict of interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

The present study was performed at the Central Laboratory of Affiliated Hospital of Hebei University. All animal experiments were approved by the Animal Ethical and Welfare Committee of Hebei University (Baoding, China, Approval No. 2017010) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hebei University.

Patient consent for publication

Not applicable.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Liangtong Li and Tongtong Liu, should be regarded as co-first authors.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, L., Liu, T., Liu, L. et al. Metabolomics Analysis of the Effect of Hydrogen-Rich Water on Myocardial Ischemia-Reperfusion Injury in Rats. J Bioenerg Biomembr 52, 257–268 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Hydrogen-rich water
  • Myocardial ischemia-reperfusion injury
  • Metabolomics
  • Biomarker
  • Signaling pathway