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BACKGROUND: Hydrogen sulfide (HS) has been demonstrated to mitigate ischemia-reperfusion injury (IRI) in multiple in vitro and in vivo models. Extensive analysis has demonstrated that its mechanism of protection, although incompletely understood, is complex and likely multifaceted. We therefore sought to further explore the downstream effects of HS administration in both in vitro and in vivo models of IRI.
METHODS: Murine myotubes were treated with either saline vehicle or HS 10μM. After 20 minutes of equilibration, cells underwent 3 hours of anoxia in a custom-made anoxia chamber (“ischemia”) or a parallel normoxic period of equivalent duration. Following anoxia, cells were returned to normoxic conditions (21% O2) for varying time intervals (“reperfusion”). Enzyme-linked immunosorbent assays (ELISA) were performed for phosphorylated Bad (phospho-Bad; serine 112) and heat shock protein-70 (HSP70). Next, C57BL/6 mice underwent delayed pharmacologic preconditioning with an intravenous dose of HS sufficient to raise bloodstream concentration by 10uM. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for HSP70, Bcl-XL, Bcl-2, Cox2, and fos. Values are expressed as relative mean±SEM.
RESULTS: Following 24h of “reperfusion” in vitro, phospho-Bad levels in all HS-treated (anoxic and non-anoxic) cells were greater than in non-treated cells (0.96±0.15 and 0.76±0.12 v. 0.44±0.07 and 0.62±0.09). At the same timepoint, phospho-Bad levels in anoxic HS-treated cells were significantly greater than non-treated anoxic controls (p=0.038). HSP70 levels had significantly increased following 24h of “reperfusion” in HS-treated cells compared with non-treated anoxic controls (5.12±1.05 v. 1.61±0.42, p=0.036). Three hours after in vivo HS administration, Bcl-XL levels had significantly increased compared with non-treated controls (2.05±0.13 v. 1.0, p=0.0002). Following 6h of HS preconditioning, Cox2 was similarly elevated (1.51±0.09, p=0.006). Fos was significantly increased within 1h of HS administration (2.66±0.51, p=0.03). Bcl-2 was increased at 3, 6, and 24h following treatment with HS, although this failed to reach statistical significance. HSP70 transcription was elevated by 24h (5.08±1.8).
CONCLUSION: The administration of HS results in a statistically significant increase in phospho-Bad (an inactivated form of the normally pro-apoptotic Bad that has been sequestered in the cytoplasm), as well as upregulation of Bcl-2 and Bcl-XL, protein members of the Bcl-2 family known to prevent apoptosis by inhibiting opening of the mitochondrial permeability transition pore. Furthermore, HS administration results in an increase in transcription and expression of HSP70, a chaperone protein typically upregulated in response to heat and oxidative-stress. Furthermore, preconditioning with HS results in an immediate increase in the anti-apoptotic fos. Lastly, HS administration increases the translation of protective Cox2, which acts via the inhibition of the tumor-suppressor p53 in some models. Thus, HS protects against IRI via downregulation of pro-apoptotic genes and a concurrent upregulation of anti-apoptotic genes as well as those genes responsible for molecular stabilization at times of stress. These findings provide important insights into the means by which HS confers protection against IRI.