Ethyl gallate, a scavenger of hydrogen peroxide that inhibits lysozyme-induced hydrogen peroxide signaling in vitro, reverses hypotension in canine septic shock

Abstract

Although hydrogen peroxide (H2O2) is a well-described reactive oxygen species that is known for its cytotoxic effects and associated tissue injury, H2O2 has recently been established as an important signaling molecule. We previously demonstrated that lysozyme (Lzm-S), a mediator of sepsis that is released from leukocytes, could produce vasodilation in a phenylephrine-constricted carotid artery preparation by H2O2 signaling. We found that Lzm-S could intrinsically generate H2O2 and that this generation activated H2O2-dependent pathways. In the present study, we used this carotid artery preparation as a bioassay to define those antioxidants that could inhibit Lzm-S's vasodilatory effect. We then determined whether this antioxidant could reverse the hypotension that developed in an Escherichia coli bacteremic model. Of the many antioxidants tested, we found that ethyl gallate (EG), a nonflavonoid phenolic compound, was favorable in inhibiting Lzm-S-induced vasodilation. In our E. coli model, we found that EG reversed the hypotension that developed in this model and attenuated end-organ dysfunction. By fluorometric H2O2 assay and electrochemical probe techniques, we showed that EG could scavenge H2O2 and that it could reduce H2O2 production in model systems. These results show that EG, an antioxidant that was found to scavenge H2O2 in vitro, was able to attenuate cardiovascular dysfunction in a canine in vivo preparation. Antioxidants such as EG may be useful in the treatment of hemodynamic deterioration in septic shock.

Mechanisms of systemic vasodilation by lysozyme-c in septic shock

“…We also examined whether EG could prevent the cellular lengthening caused by Lzm-S (see 5D), since EG was previously shown to prevent Lzm-S-induced vasodilation in an in vitro preparation and in an in vivo study (22). In the HAVSMC preparation, a dose-response effect of EG was observed.…”

“…The splanchnic arterial rings were constricted with phenylephrine (10 Ϫ5 mol/l), after which Lzm-S (10 Ϫ5 mol/l) or buffer solution was added to the preparation for 30 min. In addition, as part of this protocol, we determined whether pretreatment with EG (10 Ϫ5 mol/l), an inhibitor of Lzm-S's effects (22), could prevent Lzm-S-induced generation of cGMP. After 30 min of incubation with Lzm-S, the arterial samples were removed from the organ bath and frozen under liquid nitrogen.…”

“…In a third protocol, we added H2O2 (10 Ϫ8 to 10 Ϫ6 mol/l) to the HAVSMC preparation to see if the cells lengthened, since generation of H2O2 by Lzm-S is the mechanism by which lengthening would occur. In a fourth protocol, we examined whether EG (10 Ϫ5 to 10 Ϫ4 mol/l) could prevent Lzm-S-induced lengthening, since EG has been shown to scavenge H2O2 and has been found to prevent vasodilation in in vitro and in vivo preparations (22).…”

“…In this preparation, after induction with thiopental sodium (20 mg/kg, intravenously), the animals were continually infused with sufentanil citrate (0.05-0.3 g/min) and midazolam (5 g•kg Ϫ1 •min Ϫ1 ) over the course of the experiment (11,22). Drug rates were adjusted as required to abolish the palpebral reflex.…”

“…We also wanted to know whether the vasodilation caused by Lzm-S is reversible and whether Lzm-S's effect can be reversed by repeated washings in the organ bath preparation. In addition, we previously showed that ethyl gallate (EG), a nonflavonoid phenolic compound, could reverse Lzm-S-induced vasodilation by scavenging H 2 O 2 and by attenuating the reaction between H 2 O 2 and catalase to form compound I in in vivo and in vitro preparations (22). In the present study, to further substantiate that Lzm-S can directly cause smooth muscle cell lengthening, and moreover, to confirm that EG can block this effect, we investigated these questions in a human aortic vascular smooth muscle cell (HAVSMC) preparation.…”