Effect Of Surfactin On Lipopolysaccharide-Induced Production Of Proinflammatory Cytokines In Macrophages

Traditional anti-inflammatory drugs have side effects on organs and tissues. Many studies have shown that surfactin have lower body injury, lower drug resistance and can improve immunity. The objective of this study is to investigate the anti-inflammatory activity of surfactin on mouse peritoneal macrophages through the inhibition of proinflammatory cytokines via related pathways.The proliferation index of macrophages were measured by MTT assay. Phagocytosis of macrophages was determined by neutral red dye uptake method. NO production was detected by NO kit. The morphology, nucleic acid metabolism and glycogen metabolism of macrophages were measured by electron microscopy, AO staining and glycogen staining. TNF-a, IFN-y, IL-6, IL-12, iNOS and COX-2 genes expression was detected by RT-PCR. TLR4 protein expression, NF-κB and MAPK pathways activation were detected by Western blot.The proliferation index of macrophages were 108.5% and 113.0% after cells were induced by 60,80 μg/mL surfactin for 24 h. Proliferation index was 137.0% after induced by 5 p.g/mL LPS for 24 h. Compared with LPS group, proliferation index of macrophages were 150.8% induced by surfactin (80 μg/mL)+LPS. This proved that there were no cytotoxicity in the concentration range of surfactin and LPS. Phagocytosis activity and NO production of macrophages induced by LPS can be significantly inhibited by surfactin, and 60 μg/mL surfactin had the best inhibition effect. The phagocytosis activity was 47.6% (p<0.01) less than LPS group and NO production reached to 18.7 μmol/L from 59.6 μmol/L (p<0.01). The change of morphology and high level of nucleic acid metabolism and glycogen metabolism induced by LPS were inhibited by surfactin. The expression level of TLR4 was 38.4%(p<0.01) less than LPS group after treated by surfactin (60 μg/mL)+LPS. RT-PCR results demonstrated that the genes expression of TNF-a, IFN-y, IL-6, IL-12, iNOS and COX-2 in macrophages were 83.3%,80.4%,55.4%,55.8%,63.9% and 66.3% (p<0.01) less than that in LPS group after induced by surfactin (60 μg/mL)+LPS. Western blot analysis showed that the degradation of IκB-α and NF-κB p65 nuclear translocation in macrophages induced by LPS were inhibited by surfactin; and also showed that the phosphorylation of ERK1/2, p38 MAPK and JNK induced by LPS were suppressed by surfactin.LPS induced phagocytosis activity, NO production and TLR4 protein expression in macrophages were significantly inhibited by surfactin. Surfactin has effective inhibitory effects on proinflammatory cytokines, iNOS and COX-2 via inhibition of NF-κB and MAPK pathways.