ABSTRACT
Objective To observe the regulation of Toll-like receptor 4 (TLR4) signal and the release of inflammation factors after angiotensin Ⅱ (Ang Ⅱ) stimulation in rat mesangial cells under high glucose condition,revealing the innate immune-related mechanism of injury by Ang Ⅱ on mesangial cells under high glucose.Methods After synchronization,cells incubated with Ang Ⅱ (10-7 mmo/L) and/or high glucose (25 mmol/L) were used as the stimulation group,cells without stimulation were as normal control (5.6 mmol/L glucose).To determine the role of TLR4 and the adaptor myeloid differentiation factor 88 (MyD88),equal number of HBZY-1 cells were added with 10-5 mmol/L irbesartan and/or TLR4 blocker (10 mg/L) for 1 h and then incubated with Ang Ⅱ (10-7 mmo/L) and/or high glucose (25 mmol/L) for 12 h or 24 h respectively.Real-time PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression after 12 h.Immunofluorescence was used to observe TLR4 protein expression after 24 h; Western blotting was used to observe TLR4,MyD88 and nuclear factor κB (NF-κB) protein; ELISA was used to detect the concentration of MCP-1,IL-6 in cell supernatant respectively.Results Compared with normal control group,TLR4 mRNA and MyD88 mRNA were highly expressed in high glucose or Ang Ⅱ-induced HBZY-1 cells (P ＜ 0.01),TLR4,MyD88 and NF-κB protein as well as MCP-1,IL-6 were also up-regulated significantly (P ＜ 0.01).Compared with high glucose or Ang Ⅱ group,MyD88 and NF-κB protein as well as MCP-1,IL-6 were further up-regulated markedly in Ang Ⅱ and high glucose costimulated group (P ＜ 0.01).In HBZY-1 cells that were preincubated with irbesartan and/or TLR4 blocker,TLR4 and MyD88 protein expression were obviously inhibited,IL-6 and MCP-1 production were also decreased remarkably compared with high glucose and/or Ang Ⅱ group (P ＜ 0.01).Conclusions High glucose and Ang Ⅱ stimulate the release of proinflammatory factors in rat glomerular mesangial cells via TLR4-MyD88 pathway.This process is inhibited by irbesartan or TLR4 blocker via modulation of the signal.Ang Ⅱ has the positive-regulation potential on the release of inflammation factors via TLR4 signal in rat mesangial cells under high glucose condition.