The Effect And Underlying Mechanisms Of Statins On Cystathionine γ-lyase-H₂S Pathway In Macrophages

Objective: Hydrogen sulfide（H₂S）, the third gaseous transmitter, is implicated in various pathophysiologic processes. In the cardiovascular system, H₂S exerts cardioprotective effects, regulates vascular tone, and inhibit atherogenesis. Recent studies reported that atorvastatin, the inhibitor of 3-hydroxyl-3-methyl coenzyme A reductase, affected H₂S formation in kidney and other organs in mice. However, the underlying mechanisms are not fully understood. In this study, we examined the effects and underlying mechanisms of three different statins（fluvastatin, atorvastatin and pravastatin） on H₂S formation in raw264.7 macrophages.Methods: Murine raw264.7 macrophages were cultured and seeded into 96-well microplates and treated with various concentrations of fluvastatin（1.25, 2.5, 5 and 10μM）atorvastatin（10, 25, 50 and 100μM）or pravastatin（25, 50 and 100μM）for different time periods. Cell viability was determined with MTT assay. The H₂S production was indicated by extracellular sulfide level as determined by modified methylene blue method. Intracellular H₂S levels was also assessed and visualized by a fluorescent H₂S probe WSP-1 under a fluorescent microscope. The m RNA and protein expressions of H₂S synthases cystathionine γ-lyase（CSE）and 3-mercaptopyruvate sulfurtransferase（3-MST） were examined by quantitative polymerase chain reaction and western blotting, respectively. CSE activity was detected using an enzyme-coupled assay with lactate dehydrogenase（LDH）. CSE si RNAs were transfected using lipofectamine 2000. The knockdown efficiency was determined at 24 h after transfection with western blotting. The IL-1β and MCP-1 m RNA levels were determined by quantitative PCR.Results: Three tested statins fluvastatin, atorvastatin and pravastatin differentially decreased the cell viability of macrophages in concentration-dependent manners after 24 h treatment. Specifically, 5μM fluvastatin and 100μM atorvastatin and pravastatin began to diminish the cell viability significantly. Notably, there was a remarkable rise in H₂S level in fluvastatin and atorvastatin-stimulated macrophages, while pravastatin failed to show any significant effect on it. Moreover, fluvastatin and atorvastatin enhanced the m RNA and protein expression of CSE in dose- and time-dependent manners. However, fluvastatin did not alter the m RNA or protein expression of another H₂S-producing enzyme 3-MST. Blockade of CSE with its inhibitor DL-propargylglycine（PAG）or si RNA markedly reduced the H₂S level in fluvastatin-stimulated macrophages. Moreover, treatment with 2.5μM fluvastatin for 48 h enhanced the CSE activity by 4.4-fold in macrophages. In addition, fluvastatin elevated Akt phosphorylation, which occurred as early as 15 min after treatment, peaked at 1h, and lasted at least 3h. Both PI3 K inhibitor LY294002（10μM）and Akt inhibitor perifosine（10μM） were able to reverse the increases of CSE m RNA and H₂S production in fluvastatin-stimulated macrophages. Last, we showed that fluvastatin reduced the m RNA levels of pro-inflammatory molecules such as IL-1β and MCP-1 in LPS-treated macrophages, which were completely reversed by CSE inhibitor PAG.Conclusion: In summary, our findings demonstrate that lipophilic fluvastatin and atorvastatin, rather than hydrophilic pravastatin, elevated the H₂S production in raw264.7 macrophages by stimulating CSE expression and its activity. The Akt signaling pathway may play an important role in this process. Taking into account the anti-atherosclerotic and anti-inflammatory actions of H₂S, the modulation of CSE/ H₂S pathway may thus also underlie the cholesterol-independent effect of statins.