Chlorogenic Acid Mediates JAK-STAT1 And NF-κB Pathways To Promote M1 Phenotype Polarization In Mouse Bone Marrow-Derived Macrophages

Purpose: Chlorogenic acid(CGA)is a natural bioactive polyphenol with a variety of pharmacological activities such as antibacterial,immunomodulatory,inhibiting free radical oxidation,anti-tumourigenic and anti-viral activities,and is found in a variety of natural medicinal plants such as Lonicera.Macrophages are a key component of the body’s immune system,by fixing pathogens,performing phagocytosis and digestion,and activating lymphocytes to respond to pathogens.There are two different functional phenotypes of macrophages,M1 and M2 macrophages,with M1 macrophages having a strong anti-pathogen and anti-tumour effect.On the other hand,M2 macrophages have tumour-associated cellular properties under certain characteristic circumstances.It has been shown that chlorogenic acid regulates the polarization of both macrophage phenotypes,but the exact signalling mechanism remains unclear.The aim of this study was to investigate the mechanism of the polarization response of CGA on mouse bone marrow-derived macrophages(BMDMs)towards the M1 phenotype in an in vitro model and to verify whether JAK-STAT1 and NF-κB are involved in the CGA induced polarization response of M1 macrophages.Methods: M1-positive group and IL-4(20ng/m L)induced by using LPS(15ng/m L)+ IFN-γ(15ng/m L)and M2-positive group,CGA as a drug group;Flow cytometry detects the expression of F4/80,CD80,CD86 and MHC Ⅱ,CCR7,CD206 and FITC-Dextran on the surface of BMDMs.CCK-8 and LDH assays are perform to detect the cell viability of BMDMs after CGA treatment;RT-q PCR detects to the expression levels of TNF-α,IL-6,IL-12 and CD206,Chil3,IL-10,Arg1 m RNA in BMDMs;ELISA detects the concentrations of TNF-α,IL-6,IL-12 and CD206 in BMDMs;And Griess method detects the levels of NO in BMDMs;Western Blot analysis i NOS,NF-κB protein expression and JAK-STAT1 phosphorylation in BMDMs;LB agar plates treat with CGA were counted against Escherichia coli and Staphylococcus aureus.coli after co-incubation.Results: The purity of F4/80 in BMDMs was >90% as determined by flow cytometry.CGA significantly promoted the expression of MCHⅡ,CD80 and CD86 molecules on the surface of BMDMs,and it increase the expression of M1 macrophage marker CCR7(C-C chemokine receptor type7)and decreased the expression of M2 macrophage marker CD206(P < 0.05).After CGA treatment the expression of TNF-α,IL-6 and IL-12 m RNA was significantly increased in BMDMs,while the expression of CD206,Chil3,IL-10 and Arg1 m RNA was significantly decreased(P < 0.05).ELISA result shows that TNF-α,IL-6 and IL-12 in BMDMs increase significantly(P < 0.05);In an in vitro bacterial phagocytosis and killing assay,CGA increase the phagotrophy of FITC-Dextran by BMDMs and decrease the number of bacterial survival in Escherichia coli and Staphylococcus aureus;In addition,i NOS,NF-κBp65 and JAK-STAT1 protein expression levels are significantly increase in BMDMs(P <0.05).These data shows that CGA promote M1 phenotypic polarization and expression of associated cytokines is closely associate with activation of NF-κBp65 and JAK-STAT1.Conclusion: CGA induces polarization of BMDMs towards M1-type macrophages and inhibits the trend towards M2-type macrophages,which is regulate by mediating JAK-STAT1 and NF-κB signaling.