ABSTRACT
Objective To observe the expression of toll like receptor 4(TLR4) Signaling and the release of inflammation factors in rat tubular epithelial cell(NRK-52E) under high glucose condition after TLR4-siRNA transfection.Methods Three TLR4-siRNA sequences were designed and synthesized.The transfection efficiency was observed by fluorescence microscope after transfection,and the expression of TLR4 mRNA was detected by real time PCR.The most effective siRNA was selected to be used for forward experiments.After transfection for 24 h,cells were stimulated with 25 mmol/L glucose and/or 10-7 mmol/L Angiotension Ⅱ (Ang Ⅱ) for 12 h,24 h; cells without stimulation were as normal control.Real-time PCR was used to analyze TLR4 and myeloid differentiation factor 88 (MyD88) mRNA expression; Western blot was used to observe TLR4/MyD88 and NF-κB protein expression.ELISA assay was used to detect the concentration of monocyte chemoattractant protein-1 (MCP-1),interleukin-6(IL-6) in cell supernatant after cells were stimulated for 24 h.Results TLR4/ MyD88 mRNA and TLR4/MyD88/NF-κB protein were highly expressed under high glucose or Ang Ⅱ co -incubated NRK-52E(P ＜ 0.01),the MCP-1 and IL-6 levels were also increased markedly compared with normal control group (P ＜ 0.01).TLR4/MyD88 mRNA and TLR4/MyD88/NF-kB protein expressions were obviously inhibited in cells that were transfected with TLR4-siRNA compared with high glucose group(P ＜ 0.01),MCP-1 and IL-6 production decreased remarkably compared with high glucose or Ang Ⅱ co-stimulated group(P ＜ 0.01).Conclusions High glucose can lead to the activation of TLR4/ MyD88/NF-kB signaling and the secretion of inflammation factors in NRK-52E,Ang Ⅱ further augments these effects.The effect can be blocked efficiently by specific siRNA gene silence.TLR4 signaling plays a pivotal role in the innate-immune inflammatory reaction in NRK-52E.