| Objective: Cordycepin exerts anti-inflammatory effects by inhibiting the production of inflammatory cytokines and chemokines during inflammation.Our previous research found that cordycepin can inhibit the phosphorylation of inhibitory protein(Inhibitor of nuclear factor kappa-B,IκB)and block IκB Kinase γ(IκB Kinase γ)in HEK-293 T cells by reducing the transcription activity of p65,IKKγ)to inhibit tumor necrosis factor(Tumor necrosis factor,TNF-α)-induced activation of nuclear factor-κB(NF-κB),we also found that cordycepin can completely inhibit T cell receptor(TCR)signaling cascade in a mouse paw inflammation model induced by Complete Freund’s Adjuvant(CFA),thereby inhibiting T cell activation.Therefore,we hypothesized that the mechanism which cordycepin inhibits inflammation in the soles of mice is related to its inhibition of macrophage inflammation to produce inflammatory cytokines and chemokines.In this experiment,a mouse foot inflammation model was established in vivo and cell inflammation model of macrophages was established in vitro.Different doses of cordycepin were given before inflammation.The aim was to explore the mechanism of cordycepin on small cells by studying the mechanism of cordycepin on macrophages and anti-inflammatory effect in inflammation of rat paw.(1)Female KM mice were randomly divided into 6 groups(10 in each group): control group(Control),saline(Saline)+ CFA treatment group,dexamethasone(Dexamethasone,Dexa)(2 mg/kg)+ CFA treatment group,high cordycepin(H-Cor: 20 mg/kg)dose + CFA treatment group,cordycepin(M-Cor:10 mg/kg)dose + CFA treatment group,low cordycepin(L-Cor: 5 mg/kg)dose+ CFA treatment group.Using immunohistochemistry to detect cytokines TNF-α,interleukin-6(Interleukin-6,IL-6),interferon-inducible protein-10(IP-10),Expression of interferon-induced mononuclear factor(Monokine induced by IFN-γ,Mig)and γ-interferon(Interferon-γ,IFN-γ).Enzyme-linked immunosorbent assay(ELISA)to detect the expression of chemokines IP-10 and Mig in mouse serum.(2)Mouse macrophages RAW264.7 with good growth conditions were plated at a certain density,and culture 24 hours,they were randomly divided into seven groups,namely: normal control group,Lippolysaccharide(LPS)treatment group,Dexa(2 μg/m L)+ LPS treatment group,Cordycepin(25 μM,50 μM,100 μM,200 μM)+ LPS treatment group.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)technology was used to detect the expression levels of pro-inflammatory cytokines TNF-α and IL-6m RNA.ELISA was used to detect the expression levels of TNF-α and IL-6pro-inflammatory cytokines in the supernatant of mouse macrophages RAW264.7.Western Blot detecte the expression levels of interleukin-1 receptor associated kinas(IRAK-1)and transforming growth factor kinase 1(TGF-beta-activated kinase 1,TAK1)phosphorylated protein in mouse macrophages RAW264.7.(3)Mouse macrophages RAW264.7 with good growth state were plated at a certain density and culture 24 hours,the mouse macrophages RAW264.7 were Methods:randomly divided into a normal control group and IFN-γ treatment group,cordycepin(25 μg/m L,50 μg/m L,100 μg/m L)+ IFN-γ treatment group.Western Blot detected the expression level of phosphorylated protein of Signal transducers and activators of transcription-1(STAT1)in RAW264.7 mouse macrophages,ELISA detected the expression levels of chemokines IP-10 and Mig in the supernatant.Results:1.Immunohistochemical detected the expression levels of proinflammatory cytokines TNF-α and IL-6 increased significantly in the inflammatory tissues of the soles of mice in the Saline + CFA treatment group.The expression of pro-inflammatory cytokines TNF-α and IL-6 in inflammatory tissues was significantly reduced in the soles of mice pretreated with cordycepin.2.Immunohistochemical detected the expression levels of chemokines IP-10 and Mig were significantly increased in the inflammatory tissues of the soles of mice in the Saline + CFA treatment group,The expression levels of chemotaxis IP-10 and Mig were significantly reduced in the inflammatory tissues of the soles of mice pretreated with cordycepin.At the same time,it was found by ELISA that the expression levels of chemokines IP-10 and Mig in Saline + CFA treatment group increased significantly in mice serum,while the expression levels of chemokines IP-10 and Mig in Cordycepin pretreatment group decreased..3.Immunohistochemical detected the expression of cytokine IFN-γ was significantly increased in the inflammatory tissues of the soles of mice in the Saline + CFA treatment group,The amount of expression of cytokine IFN-γ in the inflammatory tissues of the soles of mice treated with cordycepin was significantly reduced.4.RT-qPCR technology found that the expression of proinflammatory cytokines TNF-α and IL-6 m RNA in macrophages stimulated by LPS was significantly increased,and the proinflammatory cytokines TNF-α and IL-6 in the cordycepin pretreatment group IL-6 m RNA expression decreased;meanwhile,ELISA detected the expression of proinflammatory cytokines TNF-α and IL-6 in the LPS treatment group of macrophages supernatant was significantly increased.The expression levels of cytokines TNF-α and IL-6decreased in a concentration-dependent manner in the cordycepin pretreatment group.5.Western Blot detected signal pathway protein the expression of phosphorylated protein of IRAK-1 and TAK1 increased significantly in the LPS treatment group.The expression of phosphorylated protein of IRAK-1 and TAK1 gradually decreases in macrophage pretreated with cordycepin.6.Western Blot detected the expression level of STAT1 phosphorylated protein in the IFN-γ treated group increased significantly and the expression level of STAT1 phosphorylated protein in macrophage pretreated with cordycepin gradually decreased.7.ELISA method showed that the expression levels of chemokines IP-10 and Mig in the IFN-γ treatment group of the macrophage supernatant increased significantly,and the expression levels of the chemokines IP-10 and Mig showed concentration dependence is reduced in the cordycepin pretreatment group.Conclusion:1.Cordycepin can inhibit LPS-induced phosphorylation of IRAK-1 protein in mouse macrophages RAW264.7,inhibit IRAK-1 mediated phosphorylation of TAK1 protein,and inhibit the expression and secretion of TNF-α and IL-6,reduce in the inflammatory tissue of the paw,thereby reducing the inflammatory response.2.Cordycepin can inhibit IFN-γ-induced phosphorylation of STAT1 protein in mouse macrophages RAW264.7,down-regulate the expression and secretion of chemokines IP-10 and Mig,and reduce the expression of IP-10 and Mig in inflammatory tissues of the paw,Thereby reducing macrophage chemotaxis. |
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