Effect Of Berberine On Polarization Of Mouse RAW264.7 Macrophage Cells

Berberine,as heat of poison and antimicrobials has been used extensively in traditional Chinese medicine to treat enteritis.Recent studies suggested that berberine has many effects on diabetes,CVD(cardiovascular disease)tomour.Macrophage proliferation participate in the pathogenesis of acute and chronic inflammation.M1 macrophages secretion pro-inflammatory cytokines,resistance to pathoge,n invasion whereas lead to injury to the organism,M2 macrophages secretion anti-inflammatory cytokines,promote tissue repair,promot tumor formation.The immunopharmacology studies about berberine have revealed that berberine has anti-inflammatory and immune inhibitory effect,but the underlying mechanisms are not fully understood,neither the studiesits about its immunopharmacology mechanisms from the point of Macrophage proliferation.ObjectiveThis research through the experimental in vitro analysis the influence of berberine on macrophage polarization,from macrophage polarization Angle to explore the immune modulatory effect of berberine,to clarify berberine immune pharmacological mechanism.Methods1.Cell cultureMacrophage line by mouse origin RAW264.7 cell,the cell line from Abelson leukemia virus caused by tumor in mice.Batches of cells,frozen storage and recovery conditions are optimized,and determined by MTT method is used to test 72 h RAW264.7 cell growth curve and the effect of berberine on macrophage proliferation,establishment and stability of the experimental study of cell culture conditions.2.The effect of berberine on RAW264.7 cells polarization to M1 by LPS RAW264.7 cell concentration to 1×105mL-1 vaccination in 24 orifice,LPS stimulation 8 h,to medicine group with berberine preconditioning 2 h,reoccupy LPS stimulation after 8 h,after the training,respectively,for the following tests:ELISA to detect cell supernatant of TNF alpha,the secretion of IL-10;FCM detecting CD86 cell proportion;Fluorescence quantitative PCR detection of iNOS,SOCS3 mRNA expression;RT-PCR detection P2X7,both A2A mRNA expression3.The effect of berberine on RAW264.7 cells polarization to M2 by IL-4RAW264.7 cells with concentration of 1×108mL-1 vaccination in 24 orifice,IL-4 stimulus 24 h,to medicine group with berberine preconditioning 2 h,24 h after reoccupy IL-4 stimulation,training end respectively the following tests:ELISA to detect cell supernatant of TNF alpha,the secretion of IL-10;FCM detecting CD86 cell proportion;Fluorescence quantitative PCR detection Arg-1,SOCS2 mRNA expression.Results1.The effect of berberine on RAW264.7 cells proliferation was on dose-dependent.The IC50 of Berberine to RAW264.7 cells is 400?mol·L-1?800?mol·L-1.2.The effect of berberine on RAW264.7 cells polarization to M1 by LPSBerberine decrease the TNF-a secretion?CD86 cell proportion?iNOS?SOCS3?P2X7 mRNA expression level induced by LPS;and Berberine increased the A2A mRNA expression level induced by LPS.These rusults indicate that Berberine has inhibitory effect on macrophages polarization to M1 in vitro.3.The effect of berberine on RAW264.7 cells polarization to M2 by IL-4 Berberine decrease IL-10 secretion and Arg-1 mRNA expression level induced by IL-4,and increase TNF-a secretion and CD86 cell proportion,but SOCS2 mRNA expression level was not affected by berberine.Berberine has inhibitory effect on macrophages polarization to M2 in vitro.Conclusion1.RAW264.7 cell can be induced to M1 phenotypes by LPS;and RAW264.7 cell can be induced to M2 phenotypes by IL-4.2.Berberine has inhibitory effect on macrophages polarization to M1 and M2 in vitro,berberine possible plays a regulatory role on maintain the dynamic balance of M1/M2.