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Molecular hydrogen protects against oxidative stress-induced RAW 264.7 macrophage cells through the activation of Nrf2 and inhibition of MAPK signaling pathway

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Abstract

Background

Oxidative stress is involved in the development of many inflammatory, metabolic and aging diseases.

Objective

In this study we investigated, the protective effects of H2 on RAW 264.7 macrophage cell against LPS-and H2O2-induced oxidative stress by the inhibition of MAPK pathway and also activate the Nrf2 pathway.

Results

Our results showed H2 increased the macrophage cell proliferation and generated ROS and NO against LPS stimulation to exert an active immune response. Similarly, H2 protected the macrophage cell from H2O2-induced oxidative stress. H2 reduced the LPS-and H2O2-induced inflammatory cytokine production and intracellular calcium influxes. H2 inhibited the LPS-and H2O2-induced phosphorylation of MAPK pathway and its downstream signaling molecules. Furthermore, H2 protected the macrophage cell from mitochondrial apoptosis. H2 increased Nrf2 protein expression indicating its strong anti-oxidative effects against oxidative stress.

Conclusion

Collectively, our results indicate the strong antioxidant role of H2 against LPS-and H2O2-induced oxidative stress on macrophage cells by activating the Nrf2 pathway and inhibiting the MAPK-signaling pathway.

Graphic Abstract

Our results clearly showed that LPS increased the cellular ROS by recognizing the TLR4 and H2O2 rapidly increased the cellular (1) and mitochondrial (2) oxidative stress. Excessive ROS/NO molecules cause intracellular calcium influxes (3) As a results imbalance the cellular membrane homeostasis and activate the stress response MAPK signaling pathway with its downstream signaling protein and mitochondrial caspase protein (4) that collapse the anti-oxidant mechanisms and induced the inflammatory cytokine secretion; leads to cell apoptosis (5) Whether H2 reduced the cellular and mitochondrial oxidative stress, intracellular calcium influxes and inhibits the stress response MAPK, caspase cell signaling pathway through the activation of Nrf2/ARE signaling pathway (6) Consequently, increased the antioxidant enzymes and reduced the inflammatory cytokine that influences the macrophage cell proliferation (7) to protect the cell from apoptosis. The different effects of ROS and H2 used in this study are indicated in red and green, red arrow depicts ROS effects in the cell, green arrow depicts H2 effects.

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References

  • Arango Duque G, Descoteaux A (2014) Macrophage cytokines: involvement in immunity and infectious diseases. Front Immunol 5:491

    PubMed  PubMed Central  Google Scholar 

  • Benoit M, Desnues B, Mege JL (2008) Macrophage polarization in bacterial infections. J Immunol 181:3733–3739

    CAS  PubMed  Google Scholar 

  • Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O (2012) Oxidative stress and antioxidant defense. World Allergy Organ J 5:9–19

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chen HG et al (2013) Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS-stimulated RAW 264.7 macrophages. Int J Surg 11:1060–1066

    PubMed  Google Scholar 

  • Chuderland D, Seger R (2008) Calcium regulates ERK signaling by modulating its protein-protein interactions. Commun Integr Biol 1:4–5

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cohen J (2002) The immunopathogenesis of sepsis. Nature 420:885–891

    CAS  PubMed  Google Scholar 

  • Di Marzo N, Chisci E, Giovannoni R (2018) The role of hydrogen peroxide in redox-dependent signaling: Homeostatic and pathological responses in mammalian cells. Cells 7:156

    PubMed Central  Google Scholar 

  • Di Meo S, Reed TT, Venditti P, Victor VM (2016) Role of ROS and RNS sources in physiological and pathological conditions. Oxid Med Cell Longev 2016:1245049

    PubMed  PubMed Central  Google Scholar 

  • Dinarello CA (1997) Proinflammatory and anti-inflammatory cytokines as mediators in the pathogenesis of septic shock. Chest 112:321s–329s

    CAS  PubMed  Google Scholar 

  • Ding M, Zhao J, Bowman L, Lu Y, Shi X (2010) Inhibition of AP-1 and MAPK signaling and activation of Nrf2/ARE pathway by quercitrin. Int J Oncol 36:59–67

    CAS  PubMed  Google Scholar 

  • Ermak G, Davies KJ (2002) Calcium and oxidative stress: from cell signaling to cell death. Mol Immunol 38:713–721

    CAS  PubMed  Google Scholar 

  • Ge L, Yang M, Yang NN, Yin XX, Song WG (2017) Molecular hydrogen: a preventive and therapeutic medical gas for various diseases. Oncotarget 8:102653–102673

    PubMed  PubMed Central  Google Scholar 

  • Ichihara M, Sobue S, Ito M, Ito M, Hirayama M, Ohno K (2015) Beneficial biological effects and the underlying mechanisms of molecular hydrogen-comprehensive review of 321 original articles. Med Gas Res 5:1–21

    Google Scholar 

  • Ighodaro OM, Akinloye OA (2018) First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alex J Med 54:287–293

    Google Scholar 

  • Itoh T et al (2011) Molecular hydrogen inhibits lipopolysaccharide/interferon gamma-induced nitric oxide production through modulation of signal transduction in macrophages. Biochem Biophys Res Commun 411:143–149

    CAS  PubMed  Google Scholar 

  • Kawamura T et al (2010) Inhaled hydrogen gas therapy for prevention of lung transplant-induced ischemia/reperfusion injury in rats. Transplantation 90:1344–1351

    CAS  PubMed  Google Scholar 

  • Kensler TW, Wakabayashi N, Biswal S (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 47:89–116

    CAS  Google Scholar 

  • Kim EK, Choi EJ (2010) Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta 1(802):396–405

    Google Scholar 

  • Kura B, Bagchi AK, Singal PK, Barancik M, LeBaron TW, Valachova K, Šoltés L, Slezák J (2018) Molecular hydrogen: potential in mitigating oxidative-stress-induced radiation injury. Can J Physiol Pharm 97:287–292

    Google Scholar 

  • Kurokawa R, Seo T, Sato B, Hirano S, Sato F (2015) Convenient methods for ingestion of molecular hydrogen: drinking, injection, and inhalation. Med Gas Res 5:13

    PubMed  PubMed Central  Google Scholar 

  • Liguori I et al (2018) Oxidative stress, aging, and diseases. Clin Interv Aging 13:757–772

    CAS  PubMed  PubMed Central  Google Scholar 

  • McCoy CE, O’Neill LA (2008) The role of toll-like receptors in macrophages. Front Biosci 13:62–70

    CAS  PubMed  Google Scholar 

  • Muñoz Carrillo JL, Castro García FP, Gutiérrez Coronado O, Moreno García MA, Contreras Cordero JF (2017) In: Rezaei N (ed) Physiology and pathology of immunology. IntechOpen, London, pp 99–134

    Google Scholar 

  • Murakami Y, Ito M, Ohsawa I (2017) Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis. PLoS One 12:e0176992

    PubMed  PubMed Central  Google Scholar 

  • Nagai H, Noguchi T, Takeda K, Ichijo H (2007) Pathophysiological roles of ASK1-MAP kinase signaling pathways. J Biochem Mol Biol 40:1–6

    CAS  PubMed  Google Scholar 

  • Niture SK, Khatri R, Jaiswal AK (2014) Regulation of Nrf2-an update. Free Radic Biol Med 66:36–44

    CAS  PubMed  Google Scholar 

  • Ohno K, Ito M, Ichihara M, Ito M (2012) Molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases. Oxid Med Cell Longev 2012:353152

    PubMed  PubMed Central  Google Scholar 

  • Ohsawa I et al (2007) Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 13:688–694

    CAS  PubMed  Google Scholar 

  • Ohta S (2011) Recent progress toward hydrogen medicine: potential of molecular hydrogen for preventive and therapeutic applications. Curr Pharm Des 17:2241–2252

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ohta S (2014) Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharmacol Ther 144:1–11

    CAS  PubMed  Google Scholar 

  • Orrenius S, Gogvadze V, Zhivotovsky B (2015) Calcium and mitochondria in the regulation of cell death. Biochem Biophys Res Commun 460:72–81

    CAS  PubMed  Google Scholar 

  • Redza-Dutordoir M, Averill-Bates DA (2016) Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta 1863:2977–2992

    CAS  PubMed  Google Scholar 

  • Senger DR, Li D, Jaminet SC, Cao S (2016) Activation of the Nrf2 cell defense pathway by ancient foods: disease prevention by important molecules and microbes lost from the modern western diet. PLoS One 11:e0148042

    PubMed  PubMed Central  Google Scholar 

  • Shao A et al (2016) Hydrogen-rich saline attenuated subarachnoid hemorrhage-induced early brain injury in rats by suppressing inflammatory response: possible involvement of NF-κB pathway and NLRP3 inflammasome. Mol Neurobiol 53:3462–3476

    CAS  PubMed  Google Scholar 

  • Shaughnessy LM, Swanson JA (2007) The role of the activated macrophage in clearing Listeria monocytogenes infection. Front Biosci 12:2683–2692

    CAS  PubMed  PubMed Central  Google Scholar 

  • Son Y et al (2011) Mitogen-activated protein kinases and reactive oxygen species: how can ROS activate MAPK pathways? J Signal Transduct 2011:792639

    PubMed  PubMed Central  Google Scholar 

  • Son Y, Kim S, Chung HT, Pae HO (2013) Reactive oxygen species in the activation of MAP kinases. Methods Enzymol 528:27–48

    CAS  PubMed  Google Scholar 

  • Sun Q et al (2009) Hydrogen-rich saline protects myocardium against ischemia/reperfusion injury in rats. Exp Biol Med (Maywood) 234:1212–1219

    CAS  Google Scholar 

  • Tian Y et al (2017) Effects of hydrogen-rich saline on hepatectomy-induced postoperative cognitive dysfunction in old mice. Mol Neurobiol 54:2579–2584

    CAS  PubMed  Google Scholar 

  • Xie K et al (2010) Protective effects of hydrogen gas on murine polymicrobial sepsis via reducing oxidative stress and HMGB1 release. Shock 34:90–97

    CAS  PubMed  Google Scholar 

  • Yuan J, Wang D, Liu Y, Chen X, Zhang H, Shen F, Liu X, Fu J (2018) Hydrogen-rich water attenuates oxidative stress in rats with traumatic brain injury via Nrf2 pathway. J Surg Res 228:238–246

    CAS  PubMed  Google Scholar 

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Author notes

  1. Rahima Begum and Cheol-Su Kim equally contributed to this work.

Authors and Affiliations

  1. Department of Environmental Medical Biology, Wonju College of Medicine Yonsei University, Wonju, Gangwon, 26426, Republic of Korea

    Rahima Begum, Cheol-Su Kim, Ailyn Fadriquela, Johny Bajgai, Xingyu Jing, Dong-Heui Kim & Kyu-Jae Lee

  2. Department of Global Medical Science, Wonju College of Medicine Yonsei University, Wonju, Gangwon, 26426, Republic of Korea

    Rahima Begum & Xingyu Jing

  3. Department of Microbiology, Wonju College of Medicine Yonsei University, Wonju, Gangwon, 26426, Republic of Korea

    Soo-Ki Kim

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  1. Rahima Begum
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  2. Cheol-Su Kim
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Contributions

RB and CSK contributed equally to this work; RB performed most of the experiments, analyzed the results, interpreted the data, wrote and revised the whole manuscript; CSK planned, supervised the whole study, performed the experiment, interpreted the results and revised the manuscript; AF and JB performed the experiments, analyzed the results, and revised the manuscript; XJ performed the experiment and revised the manuscript; DHK revised the manuscript; SKK suggested the plan and revised the manuscript; KJL conceived, supervised and supported the whole study, and revised the whole manuscript.

Corresponding author

Correspondence to Kyu-Jae Lee.

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Conflict of interest

Rahima Begum, Cheol-Su Kim, Ailyn Fadriquela, Johny Bajgai, Xingyu Jing, Dong-Heui Kim, Soo-Ki Kim and Kyu-Jae Lee that they have no conflict of interest.

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The article does not contain any studies with human and animal and this study was performed following institutional and national guidelines.

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Begum, R., Kim, CS., Fadriquela, A. et al. Molecular hydrogen protects against oxidative stress-induced RAW 264.7 macrophage cells through the activation of Nrf2 and inhibition of MAPK signaling pathway. Mol. Cell. Toxicol. 16, 103–118 (2020). https://doi.org/10.1007/s13273-020-00074-w

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