Okra Polysaccharide-2 Plays A Vital Role On The Activation Of RAW264.7 Cells By TLR2/4-mediated Signal Transduction Pathways

Okra(Abelmoschus esculentus L.)is a common vegetable,which contains a lot of polysaccharides in our daily life.RPS-2 is one of the three polysaccharides(RPS-1,RPS-2,RPS-3)isolated from okra in our previous study,which has good immunomodulatory activities both in vivo and in vitro.The immune regulation mechanism of RPS-2 on RAW264.7 cells is still unclear.This article aims to investigate whether RPS-2 activates RAW264.7 cells through TLR2/4-mediated NF-κB and MAPKs signal transduction pathways,which can reveals tthe immune regulation mechanism.The research can provide a theoretical basis for the application of RPS-2,and has certain academic and application prospects.Firstly,the phagocytic activity of RPS-2 on mouse macrophages RAW264.7 was determined by flow cytometry.Results showed that in the concentration range of8～200μg/m L,RPS-2 significantly enhanced the phagocytic activity of RAW264.7macrophages in a concentration-dependent manner,and when the concentration of RPS-2was 200μg/m L,the phagocytic activity is the strongest.Secondly,anti-TLR2 antibody and anti-TLR4 antibody were used for antibody neutralization to explore the effect of TLR2/4 on the production of NO,IL-10 and TNF-αin the process of RPS-2 activation of RAW264.7 macrophages.The results showed that anti-TLR2 antibody and anti-TLR4 antibody almost blocked the production of NO,IL-10 and TNF-α in RAW264.7 cells induced by RPS-2,which indicates that RPS-2 can activate RAW264.7 cells through TLR2/4 receptors.Thirdly,in order to explore whether RPS-2 can activate macrophages through TLR2/4-mediated NF-κB and MAPK signaling pathways,in this experiment,Western blot was used to detect the expression and phosphorylation levels of NF-κB and MAPKs signaling pathway-related proteins.The results showed that RPS-2 can significantly enhance the protein phosphorylation level of NF-κB and IκBα.After adding PDTC,a specific inhibitor of NF-κB signaling pathway,the phosphorylation level of NF-κB and IκBα was significantly reduced;RPS-2 significantly enhanced the expression of p38,ERK1/2 and JNK phosphorylated proteins in the MAPKs signaling pathway.MAPKs specific inhibitors(SB203580,PD98059 and SP600125)significantly inhibited the levels of p38,ERK1/2 and JNK phosphorylation induced by RPS-2.In summary,RPS-2 can activate macrophages through TLR2/4-mediated NF-κB and MAPKs signaling pathways.Finally,in order to verify the above conclusions,the experiment tested the effects of specific inhibitors of NF-κB and MAPKs signaling pathways on the production of NO,IL-10 and TNF-α in macrophages induced by RPS-2.The results showed that specific inhibitors of NF-κB pathway and MAPKs pathway(SB203580,PD98059 and SP600125)significantly inhibited the production of NO,IL-10 and TNF-α in macrophages induced by RPS-2.In summary,the okra polysaccharide-2 can activate RAW264.7 macrophages through TLR2/4 receptor-mediated NF-κB and MAPKs signaling pathways.