FIG. 2.

H₂S inhibits hepcidin expression by decreasing IL-6 secretion in the pretreatment model in vitro. (A) A flowchart for the pretreatment in the CM model. NaHS was first applied to THP-1-derived macrophages 1 h before LPS (1 μg/ml) stimulation to produce different CMs. After 24 h, Huh7 cells were treated with these CMs for 6 h, and RNA was isolated for quantitative reverse transcription polymerase chain reaction. (B) NaHS suppressed HAMP expression in Huh7 cells induced by LPS-CM (n=5). (C) A similar effect was demonstrated with l-cysteine, the substrate for endogenous H₂S production (n=3). (D) NaHS significantly attenuated IL-6 concentration invoked by LPS in CM (n=3). (E) HAMP induction by LPS-CM was suppressed and completely abolished by anti-human neutralizing antibody (αIL6, 0.05, and 0.5 μg/ml) at a higher concentration (n=3). (F, G) NaHS inhibited LPS-CM-induced JAK2/STAT3 activation (F) and p-STAT3 translocation (G) in Huh7 cells. Representative immunoblots and immunofluorescence images are presented with densitometry analysis in Supplementary Data. Values are presented as mean±SEM from at least three independent experiments. ^##p<0.01 and ^###p<0.001 compared with the control group; *p<0.05 and **p<0.01 compared with the LPS-CM or IgG group. CM, conditioned medium. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars