Dysregulation Of Cystathionine γ-lyase (CSE)/hydrogen Sulfide Pathway Contributes To Ox-LDL-induced Inflammation In Macrophage

Objective:To investigate whether CSE/H2S pathway was involved in ox-LDL-triggeredinflammation in macrophage and the molecular mechanisms involved.Methods:Ox-LDL was used to stimulate and induce inflammatory responses in bothRaw264.7and primary macrophage.Reverse transcription PCR and western blot were applied to examine the mRNAand protein expression of CSE. The H2S production was measured by chemistry. CSEtransfection was used to overexpression and knockdown CSE. CSE inhibitors PAG andBCA were used to inhibit CSE activity respectively. The levels of proinflammatorycytokines such as TNF-α and ICAM-1in cell-free culture supernatant were determinedby ELISA. The expression of phospho-and total p38/ERK/JNK and IκBα were assayedby western blot analysis. The adhesion of macrophages to endothelial cells was detectedby adhesion assay. The NF-κB translocation was studied by immunfluorescence, andthe NF-κB activity was determined by ELISA.Results:Reverse transcription PCR showed that only CSE, instead of CBS RNA, wasdetected in Raw264.7cells. Reverse transcription PCR and western blot show that thelevels of CSE mRNA and protein expression were decreased, and H2S production whichwas measured by methylene blue method also decreased in ox-LDL-treated macrophage.CSE overexpression reduced the ox-LDL-stimulated tumor necrosis factor-α (TNF-α)generation in Raw264.7and primary macrophage while CSE knockdown enhanced it.Exogenous supplementation of H2S with NaHS and Na2S also decreased the productionof TNF-α and intercellular adhesion molecule-1(ICAM-1) in ox-LDL-stimulated macrophage, and alleviated the adhesion of macrophage to endothelial monolayer.Cysteine produced similar effects on the pro-inflammatory cytokine generation, whichwere reversed by CSE inhibitors PAG and BCA, respectively. Western blotting andimmunofluorescence study showed that NaHS and Na2S attenuated the phosphorylationand degradation of IκBα and p65nuclear translocation, as well as JNK activationcaused by ox-LDL. The JNK inhibitor suppressed the NF-κB transcription activity inox-LDL-treated cells. Furthermore, inhibitors of NF-κB (PDTC), ERK (U0126andPD98059) and JNK (SP600125) partially blocked the suppression by ox-LDL on theCSE mRNA levels.Conclusion:This findings demonstrate that ox-LDL may down-regulate the CSE/H2S pathway,which plays an anti-inflammatory role in ox-LDL-stimulated macrophage bysuppressing JNK/NF-κB signaling. The study reveals new therapeutic strategies foratherosclerosis, based on modulating CSE/H2S pathway.