Pharmacologic Pre-conditioning with Hydrogen Sulfide Mitigates Ischemia-Reperfusion Injury via a Xanthine Oxidase-Mediated 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.
Hydrogen sulfide (HS) has been shown to significantly mitigate the damage caused by ischemia-reperfusion injury (IRI), but the mechanism of action remains poorly understood. As a partial inhibitor of oxidative phosphorylation, HS reduces ATP synthesis with a resultant increase in the activity of xanthine oxidase (XO) and the generation of reactive oxygen species (ROS). We hypothesize that the low level of ROS result in the upregulation of natural antioxidant and antiapoptotic pathways, thereby conferring protection against a subsequent profound ischemic event. As such, we sought to determine whether the XO inhibitor allopurinol would negate the protective effects of pharmacologic preconditioning with HS in both in vitro and in vivo models of muscle IRI.
In vitro, myotubes were treated with either no HS or allopurinol (non-treated control), 10μM HS alone, 10μM allopurinol alone, or both 10μM HS and 10μM allopurinol, then 20 minutes later were exposed to 3 hours of anoxia (0% O2) followed by 3 hours of normoxia (21% O2). A TUNEL assay was used to determine the apoptotic index (AI) for each group. In vivo, 12 C57/BL6 mice were injected with saline, 10μM HS alone, 10μM allopurinol alone, or both 10μM HS and 10μM allopurinol, then 20 minutes later underwent 3 hours of tourniquet-induced hindlimb ischemia followed by 3 hours of reperfusion. The bilateral gastrocnemious and soleus muscle were harvested, stained with H&E, and subjected to a TUNEL assay to determine the AI. Values are presented as mean AI ± standard error, and statistical analysis was performed using a series of four pairwise t-tests (family error α=0.05).
In vitro, myotubes treated with allopurinol alone had an AI (10.5%±1.5%) that was similar to that of the non-treated control (10.8%±1.6%, p=0.641). Myotubes treated with HS alone resulted in a significant decrease in AI (2.1%±1.0%) compared to non-treated controls (p<0.001). Treatment of myotubes with both allopurinol and HS resulted in a loss of protective effect (8.1%±1.1%), with the AI being significantly greater than that of HS alone (p=0.010), and not significantly different than that of the non-treated control (p=0.3019). In vivo, the ischemic muscle of mice treated with allopurinol alone had an AI (8.7%±0.8%) similar to the non-treated control (9.2%±1.6%, p=0.199). Mice treated with HS alone resulted in a significant decrease in AI (2.5%±0.4%) compared to non-treated control (p<0.001). Treatment with both allopurinol and HS resulted in a loss of the protective effect (6.4%±2.0%), with an AI being significantly greater than that of HS alone (p=0.002), and significantly less than the non-treated control (p=0.034).
Our results demonstrate that inhibition of XO with allopurinol negates the protective effects of pharmacologic pre-conditioning with HS in both in vitro and in vivo models of IRI. These data indicate that HS exerts its protective effect at least partially through a xanthine oxidase-mediated pathway. These findings offer important insights into the mechanism by which HS confers protection against IRI, and therefore have significant implications for the understanding and defining the means by which HS may be clinically applied to the prevention of IRI.
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