Effects of luteolin on the secretion of inflammatory cytokines from activated RAW264.7 macrophages / 医学研究生学报

ABSTRACT

Objective Inflammation is a defensive reaction of body , but excessive inflammatory response can lead to physi-cal injury.The aim of this study was to explore the effects of luteolin on the secretion of inflammatory cytokines from lipopolysaccharide (LPS) and interferon-g(IFN-γ) activated RAW264.7 cells. Methods RAW264.7 cells were divided into 5 groups control group (without any medicine), M1 group (polarized M1 cells activated by final concentration of 10 ng/mL LPS+20 ng/mL IFN-γ), M1+5L group (simultaneous activation of LPS and IFN-γplus final concentration of 5μmol/L luteolin), M1+10L group(simultaneous activa-tion of LPS and IFN-γplus 10μmol/L luteolin), M1+20L group(simultaneous activation of LPS and IFN-γplus 20μmol/L luteolin). The cell morphological transformation was observed by laser confocal microscope ;the mRNA levels of iNOS , IL-1βand IL-6 were test-ed by real-time quantitative PCR respectively;the secretion levels of TNF-αand IL-6 in culture supernatant were detected by ELISA;the changes of p-STAT3 (ser727) protein pathways were examined by western blot. Results Cellular morphology of activated RAW 264.7 cells changed obviously .Compared with the control group , the mRNA levels of iNOS, IL-1βand IL-6 decreased significantly in the other 4 groups(P<0.05).The iNOS level in M1+20L group significantly de-creased compared with M1 group[(29.52±3.07) vs (98.91±10.65), P<0.01].As to IL-1βlevel, it decreased significantly in M1+10L group(78.38±8.65) and M1+20L group(41.59±6.80) compared with M1 group(110.69±4.12)(P<0.05).While the IL-6 levels decreased significantly in M1+5L group(177.51±19.28), M1+10L group (106.14±5.63), M1+20L group(27.15±1.26), compared with M1 group(394.10±33.47)(P<0.05).LPS+IFN-γcould induce in-creased p-STAT3 (ser727) expression in M1 phenotype of RAW264.7 cells which was proved by its significant increase in M 1 group, M1+5L group and M1+10L group compared with control group (P<0.05).In comparison to M1 group, p-STAT3-ser expression in M1 phenotype downregulated in M1+5L group, M1+10L group, M1+20L group(P<0.05), along with dose-dependent characteristic.Com-pared with control group, the levels of IL-6 and TNF-αincreased significantly in M1 group, M1+5L group and M1+10L group.Com-pared with M1 group, the levels of IL-6 and TNF-αdecreased significantly in M1+5L group, M1+10L group and M1+20L group(P<0.05) , in which IL-6 showed concentration independence and TNF-αshowed no concentration independence . Conclusion Luteolin inhibits the secretion of pro-inflammatory cytokines through the down-regulation of p-STAT3 so as to exert anti-inflammatory effects .