Hydrogen sulfide inhibits nitric oxide production and nuclear factor-κB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Abstract

Hydrogen sulfide (H 2S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine γ-lyase (CSE) and/or cystathionine β-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H 2S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H 2S solution prepared by bubbling pure H 2S gas and NaSH, a H 2S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H 2S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H 2S, but enhanced by the selective CSE inhibitor β-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H 2S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H 2S. In addition, LPS-induced nuclear factor (NF)-κB activation was diminished in RAW264.7 macrophages preincubated with H 2S. Interestingly, the inhibitory effect of H 2S on NF-κB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-κB. Collectively, our results suggest that H 2S can inhibit NO production and NF-κB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.