Effect of paeoniflorin on TLR2/4 pathwayin AGEs-induced RAW264.7 macrophages / 中国药理学通报

ABSTRACT

Aim To investigate the effect of paeoniflorin(PF)on TLR2/4 pathway in AGEs-induced RAW264.7 macrophages.Methods RAW264.7 macrophages were incubated at different time points in AGEs stimulation,as well as different concentrations of PF,to optimize experimental conditions.RAW264.7 macrophages were randomly divided into five groups control group(DMEM),bull serum albumin(BSA)group(200 mg·L-1 BSA),AGEs group(200 mg·L-1 AGEs),paeoniflorin group(200 mg·L-1 AGEs+10-5 mol·L-1 PF)and TLR2/4 inhibitor group(200 mg·L-1 AGEs+30 mg·L-1 OxPAPC).The expression of Toll-like receptor 2(TLR2),Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),p-IRAK1,TIR-domain containing adaptor protein-inducing IFN-β(TRIF),interferon regulatory factor 3(IRF3),p-IRF3,NF-κB p-p65,NF-κB p65,inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),interleukin-l β(IL-1β)and monocyte chemotactic protein-1(MCP-1)were measured by Western blot.Real-time PCR was used to detect the expression of TLR2 and TLR4 mRNA,while TNF-α,IL-1β and MCP-1 levels in cell supernatant were measured by ELISA.Results Compared with control group,AGEs significantly increased the expression of TLR2,TLR4,MyD88,p-IRAK1,TRIF,IRF3,p-IRF3,NF-κB p-p65,NF-κB p65,iNOS,TNF-α,IL-1β and MCP-1 proteins(P<0.01),as well as TLR2 and TLR4 mRNA(P<0.01).TNF-α,IL-1β and MCP-1 contents were also elevated in cell supernatant(P<0.01).The effects induced by AGEs were decreased significantly in PF and TLR2/4 inhibitor group(P<0.01).Conclusion PF plays an anti-inflammatory effect via inhibiting TLR2/4 pathway on macrophages,which may provide a new theoretical basis for the treatment of diabetic nephropathy.