Neural Regeneration Research ›› 2023, Vol. 18 ›› Issue (6): 1371-1377.doi: 10.4103/1673-5374.355978

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Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

Stefanie Scheid1, Adrien Lejarre1, Jakob Wollborn1, 2, Hartmut Buerkle1, Ulrich Goebel3, Felix Ulbrich1, *   

  1. 1Department of Anesthesiology and Critical Care, Medical Center - University of Freiburg, Faculty of Medicine, Freiburg, Germany; 2Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 3Department of Anesthesiology and Critical Care Medicine, St. Franziskus-Hospital, Muenster, Germany
  • Online:2023-06-15 Published:2023-01-05
  • Contact: Felix Ulbrich, MD, felix.ulbrich@uniklinik-freiburg.de.
  • Supported by:
    This work was financially supported by the Department of Anesthesiology and Critical Care, Medical Center – University of Freiburg, Germany. The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding program Open Access Publishing.

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Abstract: The noble gas argon has the potential to protect neuronal cells from cell death. So far, this effect has been studied in treatment after acute damage. Preconditioning using argon has not yet been investigated. In this study, human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon (25%, 50%, and 74%; 21% O2, 5% CO2, balance nitrogen) at different time intervals before inflicting damage with rotenone (20 µM, 4 hours). Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining. Surface expressions of Toll-like receptors 2 and 4 were also examined. Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins, such as extracellular-signal regulated kinase (ERK1/2), nuclear transcription factor-κB (NF-κB), protein kinase B (Akt), caspase-3, Bax, Bcl-2, interleukin-8, and heat shock proteins. Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8. Cells were also pretreated with OxPAPC, an antagonist of TLR2 and 4 to elucidate the molecular mechanism. Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells. Preconditioning with 74% argon for 2 hours was used for further experiments showing the most promising results. Argon decreased the surface expression of TLR2 and 4, whereas OxPAPC treatment partially abolished the protective effect of argon. Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt. Preconditioning inhibited mitochondrial apoptosis and the heat shock response. Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8. Immunohistochemistry confirmed the alteration of TLRs and interleukin-8. OxPAPC reversed the argon effect on ERK1/2, Bax, Bcl-2, caspase-3, and interleukin-8 expression, but not on NF-κB and the heat shock proteins. Taken together, argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors. Argon may represent a promising therapeutic alternative in various clinical settings, such as the treatment of stroke.

Key words: apoptosis, inflammation, interleukin-8, neuroprotection, rotenone, SH-SY5Y, Toll-like receptor 2, Toll-like receptor 4