Figures
Fig.1 Effects of BMP326 on the viability of RAW264.7 macrophages. (a) Chemical structure of BMP326. (b)RAW264.7 macrophages were treated with different concentrations of LPS(0-1000ng/ml) for 24 h, Griess Reagent Kit detected that 100ng/ml LPS is the optimal stimulate condition for following researches. (c) RAW264.7 macrophages were pretreated with BMP326 (0-20 μM) for 3 h and were stimulated with or without LPS (100 ng/ml) for another 24 h. The cells viability was determined by CCK-8 assays. #P < 0.05 vs. Control group; **P < 0.01 vs. LPS alone.
Fig.2 BMP326 inhibited LPS-induced iNOS gene expression and NO synthesis in RAW264.7 macrophages. RAW264.7 Macrophages were pretreated with or without various concentrations of BMP326(0-20 μM) for 3 h before LPS(100 ng/ml) stimulation for another 24 h.(a) The culture medium was used for measuring the level of NO by Griess reagents.(b,c,d) The mRNA levels of iNOS, COX-1 and COX-2 were measured by qRT-PCR.Values are referred as mean ± SD,#P < 0.05 vs. Control group,*P < 0.05 vs. LPS alone, **P < 0.01 vs. LPS alone.
Fig.3 BMP326 inhibited the inflammatory cytokines secretion and expression of mRNA in LPS-induced RAW264.7 macrophages. RAW264.7 macrophages were treated as described before (a,b,c). The mRNA levels of IL-6 , TNF-α and IL-1β were measured by qRT-PCR. (b) The effects of BMP326 on secretion of IL-6 , TNF-α and IL-1βwere measured by ELISA. Values are referred as mean ± SD, ##P < 0.01 vs. Control group,*P < 0.05 vs. LPS alone, **P < 0.01 vs. LPS alone.
Fig.4 Effects of BMP326 on the NF-κB and the MAPK signaling pathways in LPS-induced RAW264.7 macrophages. Cells were pretreated with the indicated concentrations(0-20 μM) of BMP326 for 3 h and then stimulated with LPS (100ng/mL) for another 30 min. Protein lysate were harvested and analyzed by western blot with specific antibodies.(a) Protein levels of phospho-p65 were measured.(b)Protein levels of phospho-IκBα were measured. Protein levels of phospho-ERK(c),phospho-JNK(d) and phospho-p38(e) were measured. GAPDH was used as an internal standard control and blot bands densitometry were analyzed with Image J software. Values are referred as mean ± SD, ##P < 0.01 vs. Control group,*P < 0.05 vs. LPS alone, **P < 0.01 vs. LPS alone.
Fig.5 The possible molecular mechanism underlying the anti-inflammatory effect of BMP326. BMP326 suppressed the production of inflammatory mediators and pro-inflammatory cytokines through inhibition of NF-κB and MAPKs signaling pathway in LPS-stimulated RAW264.7 cells.