The Mechanism Of TLR4/NF-κB Signaling Pathway In Palmitate-induced Inflammatory Cytokines Expression In Human Aortic Vascular Smooth Muscle Cells

Objectives:.To investigate IL-8、IL-6and MCP-1expression by palmitate in humanaortic vascular smooth muscle cells, and to investigate IL-8、IL-6and MCP-1expression bydifferent signaling pathway inhibitors in human aortic vascular smooth musclecells.Methods:Human aortic vascular smooth muscle cells were treated with different dosesof palmitate for different time (6h,12h,24h),every time point set control group (include2%BSA),and then the LPS were used to be positive control. Cell RNA was isolated and thenRT-PCR was used to check IL-8、IL-6and MCP-1mRNA., IL-8、IL-6and MCP-1proteinlevels were measured by ELISA..Protein kinase C(PKC)、phosphotylinosital3kinase(P13K)、ceramide、NF-κB and MAPK inhibitors were added to the serum-free media for30min followed by treatment with palmitic acid for additional6h,cell and culture medium werecollected,IL-8、IL-6and MCP-1mRNA were detected by quantitative real-time PCR., IL-8、IL-6and MCP-1protein levels were measured by ELISA..Results: Palmitate induced IL-8、IL-6and MCP-1mRNA and protein expression clearly and in a time and dose dependentmanner.NF-κ B inhibitor.parthenolide inhibited HA-VSMC IL-8mRNA and proteinexpression by palmitate clearly，PKC inhibitor calphostinCand helerythrine,but myriocin andwortmannin implicated no effects. parthenolide and PKC inhibitor chelerythrine downregulate IL-6mRNA and protein expression in HA-VSMC by palmitate,but MAPK inhibitorPD98059and PI3K inhibitor wortmannin can not.Parthenolide inhibited HA-VSMCMCP-1mRNA and protein expression by palmitate，but chelerythrine、wortmanninandmyriocin implicated no effects. Conclusions:①Palmitate induced IL-8、 IL-6andMCP-1expression by TLR4signal pathway in human aortic vascular smooth muscle cells,leading the inflammation of aortic vascular smooth muscle cells.②Different signaling pathway inhibitors inhibit IL-8、IL-6and MCP-1expression in human aortic vascular smoothmuscle cells.,and Palmitate induces IL-8and IL-6expression in HAVSMCs through NF-κBand PKC pathway. Palmitate induces MCP-1expression in HAVSMCs through NF-κBpathway. Objectives:To investigate the role of TLR-4/NF-κ B signaling pathway inpalmitate-induced inflammatory cytokines expression in human aortic vascular smoothmuscle cells Methods: The recombinant adenovirus Toll Like receptor4(TLR4)shRNA vector(pGSadeno-TLR4)was constructed and transfected into hunamn aortic vascular smoothmuscle cells(HA-VSMC). Cell total protein was isolated and then Western blot was used tocheck TLR4protein expression TLR4mRNAwere detected by quantitative real-time PCR. Toinvestigate palmitate induced IL-8、 IL-6and MCP-1mRNA expression and proteinlevels.NF-κB activation were studied by NF-κB luciferase reporter assay and ELISA.Results:pGSadeno-TLR4was constructed and transfected into hunamn aortic vascular smooth musclecells, it inhibited NF-κB activity and IL-8、IL-6and MCP-1mRNA and protein expression.This results show that Palmitate induces IL-8、IL-6and MCP-1expression in HAVSMCsthrough TLR4/NF-κB pathway.Conclusions: Palmitate induces IL-8、IL-6and MCP-1expression in HAVSMCs through TLR4/NF-κB pathway.