Argon Mediates Anti-Apoptotic Signaling and Neuroprotection via Inhibition of Toll-Like Receptor 2 and 4

Abstract

PurposeRecently, the noble gas argon attracted significant attention due to its neuroprotective properties. However, the underlying molecular mechanism is still poorly understood. There is growing evidence that the extracellular regulated kinase 1/2 (ERK1/2) is involved in Argon´s protective effect. We hypothesized that argon mediates its protective effects via the upstream located toll-like receptors (TLRs) 2 and 4.MethodsApoptosis in a human neuroblastoma cell line (SH-SY5Y) was induced using rotenone. Argon treatment was performed after induction of apoptosis with different concentrations (25, 50 and 75 Vol% in oxygen 21 Vol%, carbon dioxide and nitrogen) for 2 or 4 hours respectively. Apoptosis was analyzed using flow cytometry (annexin-V (AV)/propidiumiodide (PI)) staining, caspase-3 activity and caspase cleavage. TLR density on the cells’ surface was analyzed using FACS and immunohistochemistry. Inhibition of TLR signaling and extracellular regulated kinase 1/2 (ERK1/2) were assessed by western blot, activity assays and FACS analysis.ResultsArgon 75 Vol% treatment abolished rotenone-induced apoptosis. This effect was attenuated dose- and time-dependently. Argon treatment was accompanied with a significant reduction of TLR2 and TLR4 receptor density and protein expression. Moreover, argon mediated increase in ERK1/2 phosphorylation was attenuated after inhibition of TLR signaling. ERK1/2 and TLR signaling inhibitors abolished the anti-apoptotic and cytoprotective effects of argon. Immunohistochemistry results strengthened these findings.ConclusionThese findings suggest that argon-mediated anti-apoptotic and neuroprotective effects are mediated via inhibition of TLR2 and TLR4.

The Molecular Pathway of Argon-Mediated Neuroprotection

“…Moreover, the TREK-1 and the K ATP channel, which have been analyzed in vitro as well as in vivo, are not part of the argon-mediated effects. Ulbrich et al [38] analyzed six toll-like receptors, because the known intracellular pathway (including the obviously necessary phosphorylation of ERK1/2, as well as the suppression of heat-shock proteins and the balance of mitochondrial proteins due to argon administration) points to one common structure, i.e., the toll-like receptors. Among these six TLRs analyzed, only two of them showed specific extracellular modification and intracellular blocking of TLRs abandoned argon’s effects.…”

“…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]. …”

“…Ulbrich et al [38] performed investigations concerning argon’s mechanism in vivo and in vitro. In a neuroblastoma cell line, rotenone was used as an inhibitor of the mitochondrial respiratory chain, inducing apoptosis.…”

“…Recently, Zhao et al [22] were able to confirm the role of ERK1/2 in their 90 min OGD model in cortical neuronal cell culture of rat foetus. In addition, ERK inhibition using FR180204 directly suspended argon’s effects regarding ERK phosphorylation with subsequent increase in apoptosis in a neuroblastoma cell line, analyzed by Ulbrich et al [38]. In an in vivo model of retinal IRI, Ulbrich et al [45] were able to prove these effects in rats; an inhibition of ERK1/2 administering PD98059 intravenously significantly reduced argon’s neuroprotective effects.…”

Paclitaxel resistance development is associated with biphasic changes in reactive oxygen species, mitochondrial membrane potential and autophagy with elevated energy production capacity in lung cancer cells: A chronological study

“…For each sample, three independent readings were taken, then the average fluorescence of the samples at 460 nm was plotted. 36 …”