The Mechanism Of Chrysin-thiazolidine Derivative Xu Induced Macrophage Polarization Via TLR4

Macrophages as most plasticity in hematopoietic system,exsit in all tissues and show diversity fuctions.playing a great role in the development,homestasis,tissue repair and immunity.Two well-established polarized phenotypes are often referred to as classically activated macrophages(M1 macrophages)and alternatively activated macrophages(M2 macrophages).In response to IFN? or LPS stimulations,macrophages show a classically activation phenotype with increased production of reactive oxygen species and NO,releasing proinflammatory cytokine such as TNF?,IL-6 through activation of NF-?B and STAT1 activitiy.They mainly display antimicrobial,antitumor activity.Other stimuli such as IL4,IL-13 promote an alternative activation phenotype of macrophages,in which macrophages function in tissue repair,homestasis rebuild and immune regulation.Studies have shown that macrophage plays an important role in the pathogenesis of disease.Discovery of drug or small molecular targeting macrophage polarization is potential strategies in developing therapy of diseases.Chrysin is a very active flavonoid exerting a vast number of pharmacological properties such as anti-inflammatory,anti-oxidation and anti-tumor.However,its poor bioavailability prevents the futher application in therapy.So synthesis of derivatives/analogues to to further increase its biological effects is very important in developing new therapic drugs.We synthesize a small molecular using chrysin as a skeleton with a thiazolyl group,named as Xu.Based on previous work we focus on its impact on macrophage polarization.Our work shows as following:1.Xu promotes macrophage polarization.In vitro,we chose murine macrophage cell line ANA-1 as model cell.After Xu with IL-4 co-stimulated macrophage 24h,we analyzed expression of M2 marker(CD206/MGL1/2)by flow cytometry,arginase activity by arginase assay,expression of M2 specific gene Yml/Fizzl by PCR,these dates showed Xu promoted macrophage alternative activation induced by IL-4.To test M1 marker(CCR7)by flow cytometry,expression of proinflammatory cytokines(TNFa/IL-1?)by Q-PCR after Xu with LPS co-stimulating,we found that Xu enhanced the ability of LPS inducing macrophage polarization.The results indicate that Xu promotes macrophage polarization.2.Xu induces resting macrophage to Ml polarization.Compared with LPS,Xu also induced a pro-inflammatory phenotype in macrophages that includes M1 marker(CD80/CCR7)increased expression,pro-inflammatory cytokines(TNFa/IL-6)release,production of antimicrobial effectors such as nitric oxide.We then analysised expression of MHCII,phagocytic activity by flow cytometry.These dates indicates that Xu induces resting macrophage to M1 polarization.3.Xu promotes glycolysis of macrophage.To analyze the glucose consumption and production of lactic acid,we found increased glucose consumption and lactic acid release.These dates shows that Xu promotes glycolysis of macrophage.4.Xu induces the production of IFN? in macrophage.We tested the expression of IFN? by Q-PCR and ELISA,finding that Xu induced IFN? production.5.Mechanism of Xu mediated macrophage classical activation.Owing to Xu inducing classical activation of macrophage like LPS,we tested the expression of TLR4 by flow cytometry and western bloting,thus finding that Xu induces the increased expression of TLR4.Cell signaling analysis showed that NF-?B passway and STAT1 passway was activated.Using CRISPR/Cas9 technology to construct TLR4 knockout cell line,we found that Xu cannot induce macrophage to M1 polarization.In summary,we found a new small molecular Xu had the ability to induce macrophage activation that chrysin didn't have.To further investigate the molecular mechanism of Xu mediated macrophage classical activation,we found that Xu activated NF-?B/STAT1 passway via TLR4 to promote macrophage classical activation.