Study Of Effects Of Cystathionine/hydrogen Sulfide On Inflammatory Responses Induced By Homocysteine In Macrophages And Related Mechanisms

Part ⅠEffects of CSE/H2 S signaling on inflammatory responses inducedby homocysteine in macrophagesObjective: To investigate whether CSE/H2 S signaling was involved in Hcy-triggered inflammatory responses in C57BL/6 and Raw264.7 macrophages.Methods: In vivo experiments, C57BL/6 mice were treated with methionine, GYY4137 or PAG, and were sacrificed two weeks after methionine treatment. The level of plasma Hcy was determined by Hcy assay kits. The methylene blue method was applied to determine H2 S level in the plasma. The levels of pro-inflammatory cytokines TNF-α and IL-1β in the plasma were determined by enzyme linked immunoabsorbent assay(ELISA). Quantitative PCR and western blot were applied to examine the m RNA and protein levels of CSE in peritoneal macrophages of C57BL/6 mice.In vitro experiments, Raw264.7 cells were treated with Hcy, GYY4137 or PAG. The methylene blue method was applied to examine H2 S level in the cell culture supernatants. ELISA was applied to determine TNF-α and IL-1β levels in the cell culture supernatants.Results: Dietary supplementation with 2% methionine in drinking water resulted in significant elevations in plasma Hcy and pro-inflammatory cytokine(TNF-α, IL-1β) levels, accompanied by the reduction in plasma H2 S level in C57BL/6 mice. Moreover, the CSE m RNA and protein expressions decreased approximately by 60% and 45%, respectively, in the peritoneal macrophages isolated from the methionine-treated mice compared to control dieted mice. GYY4137(50mg/kg/day, i.p.) co-treatment led to a marked decrease of pro-inflammatory cytokines TNF-α and IL-1β level in the plasma of methionine-treated mice, while PAG(37.5mg/kg/day, i.p.) aggravated the increases of these cytokines level. Neither GYY4137 nor PAG had influence on the plasma Hcy level induced by methionine diet.The observations were confirmed in raw264.7 cells. 100 μM Hcy reduced the H2 S production but enhanced TNF-α and IL-1β generation. H2S-releasing agent GYY4137 co-treatment attenuated the Hcy-induced elevations of TNF-α and IL-1β production in a dose-dependent manner. Pre-treatment with H2 S precursor cysteine(Cys, 1 m M) in raw264.7 cells also attenuated the increases of TNF-α and IL-1β generation, both of which could be reversed by the addition of PAG(1 m M). Co-treatment with PAG, in the absence of Cys, did not obviously affect the cytokines level in Hcy-treated cells.Conclusions: These results indicate the down-regulation of CSE-H2 S generation may be involved in Hcy-induced elevations of pro-inflammatory cytokines in both the plasma Part Ⅱ Effects of homocysteine on CSE/H2 S signaling in macrophagesand related mechanismsObjective: To investigate the influence of homocysteine on the CSE/H2 S signaling in macrophages and related mechanisms.Methods: Western blot and Quantitative PCR were applied to examine the protein and m RNA expressions of CSE in Hcy-treated Raw264.7 macrophages. The promoter activity of cse gene was measured by luciferase promoter activity analyses. The m RNA expression and activity of DNMTs were determined by Quantitative PCR and Epi Quik DNMT Activity/Inhition Assay Ultra Kit, respectively. The methylation status in cse gene promoter region was determined by bisulfite sequencing assay. DNMTs inhibitor AZA and si RNA were used to inhibit DNMTs activity. The knockdown efficiency of si RNA was determined by western blot assay.Results: Hcy inhibited CSE expression and promoter activity, accompanied by the increases of DNA methyltransferase(DNMT) expression and DNA hypermethylation in cse promoter region. DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages.Conclusions: These results indicate that DNA hypermethylation of Cp G rich region in cse promoter may contribute to the down-regulation of CSE transcription and thus the decrease in H2 S production in Hcy-treated Raw264.7 macrophages.