Solving the Question of Protection: Hydrogen Sulfide Confers Protection from Ischemia Reperfusion Injury via Activation of the JAK-STAT Pathway
Natalia Jimenez, B.A., Peter Henderson, M.D., M.B.A, Allie Sohn, B.S., Yoann Millet, B.A., Jason Spector, M.D..
Weill Cornell Medical College, New York, NY, USA.
Although there is an increasing body of evidence that demonstrates that hydrogen sulfide (HS) provides significant protection against ischemia-reperfusion injury (IRI), the mechanism by which this protection is conferred remains poorly understood. The JAK-STAT signaling pathway is known to regulate multiple cell processes including proliferation, differentiation and apoptosis via modulation of nuclear gene expression. Previous work has shown that this pathway is activated by mechanical preconditioning, which is known to provide protection against IRI. We hypothesize that this critical cell survival pathway would be similarly activated in the setting of IRI by pharmacologic preconditioning via treatment with HS.
Myotubes treated with media containing no HS (control) or 10 μM HS, were then allowed to equilibrate for 20 minutes before being exposed to anoxia (0% O2) for 3 hours, and then placed in normoxic conditions (21% O2) to simulate ischemia-reperfusion. Thirty seconds, 5, 15, or 30 minutes after reperfusion, cells were lysed with RIPA buffer and the isolated protein concentrations were assessed through BCA protein analysis. A western blot was performed using monoclonal rabbit pSTAT 1(Tyr701), pSTAT 3(Tyr705), and GAPDH antibodies. Band intensities were quantified, normalized to anti-GAPDH bands, and analyzed with student’s t-tests (statistical significance set at α=0.05).
Western analysis of proteins specific for anti-pSTAT 3(Tyr705) antibodies demonstrated that treatment with HS led to increased phosphorylation of this protein beginning at 30 seconds of reperfusion, and then returning to baseline after 30 minutes of reperfusion. The most notable and significant increase was seen immediately after exposing cells to normoxic conditions, 111% increase, compared to non-HS treated cells at the same time point (p=.029), and a 124% increase above the non-HS-treated normoxic control. Western analysis of protein specific for anti-pSTAT1(Tyr701) antibodies demonstrated a similar trend, increasing phosphorylation immediately after reperfusion, peaking a few minutes later, and then returning to baseline 15 minutes after reperfusion.
The increased activation of STAT3 in the presence of HS in this in vitro model of skeletal muscle IRI suggests that the cytoprotective action of HS is mediated, in part, through the JAK-STAT pathway. Because phosphorylation of JAK-STAT is implicated in the survival pathway in mechanical ischemic preconditioning and is known to activate downstream cell survival genes (such as Bcl-2 and iNOS), these data provide crucial insights into the mechanism by which therapeutic delivery of HS confers protection against IRI. Understanding the intracellular signaling pathways activated by pharmacologic pre- and post-conditioning with HS is crucial to making this an efficacious translational therapy.
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