Study Of Rutin On The Effect Of Anti-inflammatory And Mechanism In Vitro

Inflammation is an immune defense response in living tissues with vascular system against a variety of inflammatory factors and the caused damage.It is an adaptive defense response,which can eliminate various inflammatory factors and promote the repair of injury.However,many damages are often associated with the occurrence of the disease,including blood circulation disorder,the release of inflammatory mediators and tissue degeneration and necrosis.At present,the main anti-inflammatory drugs are steroids and non-steroidal anti-inflammatory drugs in clinic.Although they exhibit better therapeutic effect,they also have many side effects.Therefore,the research and development of safe,effective and new anti-inflammatory drugs has gradually become the attention focus.Rutin,also known as the rutinoside,is a kind of natural flavonoids compound,which widely exists in many natural plants with rich content.It has been reported that rutin has better anti-inflammation effect.In this study,the anti-inflammatory effect and mechanism of rutin were evaluated with LPS induced RAW264.7 cells.The content of cytokines,such as tumor necrosis factor-?(TNF-?),interleukin-1 ?(IL-1?),interleukin-6(IL-6)and interleukin-10(IL-10)in LPS induced RAW264.7 cell was detected by enzyme-linked immunosorbent assay(enzyme linked immunosorbent assay,ELISA)for evaluation the effect of rutin on the cytokinecontent.The effect of rutin on cell morphology was observed by light microscope.The effect of rutin on inflammatory mediator was evaluated by assay the content of NO with Griess method.The phagocytosis activity of rutin on LPS induced RAW264.7 cell was measured by neutral red phagocytosis experiment.Real-time fluorescence quantitative PCR(Quantitative Real-time PCR,q PCR)method was used to measure the m RNA expression of i NOS gene and other related gene in TLR4-My D88-TRAF6-NF-?B signaling pathway.Immunoblotting assay(Western Blot)method was used for detection of the effect of rutin on the protein expression of i NOS and other related proteins in TLR4-My D88-TRAF6-NF-?B signaling pathway.These results indicated that rutin could significantly decrease the content of TNF-?,IL-1 ? and IL-6 in LPS induced RAW264.7 cell,and increase the content of IL-10,and improve the morphological changes.Rutin could also effectively inhibit cell differentiation,and decrease the content of NO and the phagocytic activity.Meanwhile,rutin significantly inhibited the upregulation of i NOS,TLR4,My D88,TRAF6,p65 gene induced by LPS,and enhanced the m RNA expression level of I?B gene.In addition,rutin significantly decreased the expression of i NOS,TLR4,My D88,TRAF6 and p65 proteins,and increased the protein expression of I?B,and decreased the phosphorylation of p65 and I?B protein.In conclusion,rutin can play an anti-inflammatory role by regulation the production of the cytokines and inflammatory mediators through TLR4-My D88-TRAF6-NF-?B signaling pathway.