Argon mediates protection by interleukin‐8 suppression via a TLR2/TLR4/STAT3/NF‐κB pathway in a model of apoptosis in neuroblastoma cells in vitro and following ischemia‐reperfusion injury in rat retina in vivo

Abstract

Argon has recently come into scientific focus as a neuroprotective agent. The underlying neuroprotective mechanism remains unknown although toll-like receptors were recently suggested to play an important role. We hypothesized that TLR-associated downstream transcription factors are responsible for argon's effects, leading to anti-apoptotic and antiinflammatory properties. Apoptosis was induced in human neuroblastoma cells. Immediately afterwards, argon treatment (75 Vol% for 2 h) was initiated. Cells were analyzed, measuring mitochondrial membrane potential, reactive-oxygen-species, annexin-V/propidium iodide staining, transcription factor phosphorylation and binding activity as well as protein and mRNA expression of interleukins. Argon's in vivo effects were analyzed by quantification of retinal ganglion cell density, mRNA expression, serum cytokine analysis and immunohistochemistry after retinal ischemia reperfusion injury (IRI) in rats. Argon diminished rotenone-induced kappa-lightchain-enhancer' of activated B-cells (NF-jB) and signal transducer and activator of transcription 3 (STAT3) but not Cerebral ischemia and reperfusion injury -for example, stroke -is associated with destruction of neuronal tissue and subsequent increased mortality or functional disability. At a molecular level inflammation, oxidative stress and apoptosis play an important role in this detrimental process (Ahmad et al. 2014). As a result of the fact that neurons have a limited potential to regenerate, ischemia and reperfusion injury is a major cause of morbidity and mortality (Ng et al. 2011 Abbreviations used: DMSO, dimethylsulfoxid; ERK-1/2, extracellular regulated kinase-1/2; IL, interleukin; IRI, ischemia reperfusion injury; NF-jB, kappa-light-chain-enhancer' of activated B-cells; STAT3, signal transducer and activator of transcription 3.

The Molecular Pathway of Argon-Mediated Neuroprotection

“…Furthermore, the authors demonstrated that IRI-induced caspase-3 cleavage was suppressed by argon 75%, if administered immediately after injury. Extended FACS analysis by Zhao et al and Ulbrich et al confirmed the anti-apoptotic properties of argon, as well as results, proving a reduction in reactive oxygen species (ROS) formation and normalization of mitochondrial membrane potential after argon treatment [38,39,47]. …”

“…These findings were then translated and confirmed in vivo: In a standardized model of retinal ischemia-reperfusion injury argon inhalation for 2 h (75% argon, 21% O 2 , remainder N 2 ) protected retinal ganglion cells und decreased retinal TLR2 and 4 expression. Inhibition of TLR2 and TLR4 signaling attenuated argon’s neuroprotective effects completely [39]. The authors concluded that TLRs are responsible for argon-mediated effects.…”

“…Ulbrich et al [39] were able to demonstrate that the inhibition of TLR2, TLR4 or both significantly inhibited argon-mediated protective effects in vitro. Of note, argon’s effects were accompanied by a suppression of interleukin-1-receptor-associated kinase 4 (IRAK4), but no alteration was found in myeloid differentiation primary response gene 88 (MyD88), while both proteins are thought to be a crucial part of TLR downstream signaling.…”

“…This argon-mediated suppression of NF-κB and STAT3 signaling was counteracted by TLR inhibition in vitro, rendering these possible pathways. Interestingly, no effects were detectable for signal transducer and activator of transcription (STAT) 5 and cAMP responsive element binding protein (CREB), although these two transcription factors are known to play a role in neuroregeneration [39]. It is more than likely that even more transcription factors are involved in the pathway from surface receptor to intra- and extracellular effect-sides of argon-mediated neuroprotection, which remain part of future investigation.…”

“…Ulbrich et al [39] investigated argon’s effect on TLR-dependent inflammatory cytokines in human neuroblastoma cells (SY5Y) after rotenone treatment. While rotenone was able to induce several inflammatory cytokines like TNF-α, IL-1β, IL-6, IL-8 und IL-10, only IL-8 was affected by argon treatment.…”