Figure 7

Suppression of Toll-like receptor 4 (TLR4) with siRNA reduced the mRNA, protein expression and immunoreactivity of TNF-α, IL-1β and inducible nitric oxide synthase (iNOS) induced by hypoxic stress in BV-2 cells. (A) Reverse transcriptase (RT)-PCR analysis of TNF-α, IL-1β and iNOS gene expression in BV-2 cells transfected with control siRNA, transfected with control siRNA + hypoxia, transfected with TLR4 siRNA and TLR4 siRNA + hypoxia. Note that TNF-α, IL-1β and iNOS mRNA expression is increased significantly by different amountsrespectively after hypoxic exposure in control siRNA-transfected BV-2 cells (TNF-α, 5-fold; IL-1β, 6-fold; iNOS, 9-fold) (normalized with β-actin), but the increase was significantly suppressed in hypoxic TLR4 siRNA-transfected BV-2 cells compared with control siRNA-transfected BV-2 cells exposed to hypoxic stress. Notably, iNOS mRNA expression is decreased by 55% of the control. (B) Western blot analysis of TNF-α, IL-1β and iNOS protein expression in different groups of BV-2 cells given different treatments. The upper panel shows specific bands of TNF-α (25.6 kDa), IL-1β (17 kDa), iNOS (130 kDa) and β-actin (43 kDa). The lower panel of bar graphs shows significant changes in the optical density following hypoxic exposure (given as fold change of control siRNA transfected group). TNF-α, IL-1β and iNOS protein expressions are significantly increased after hypoxic exposure in control siRNA-transfected BV-2 cells and this increase is significantly decreased after hypoxic exposure in TLR4-transfected groups. (C) Immunofluorescence images show that TNF-α, IL-1β and iNOS immunoreactivity is markedly reduced in TLR4 siRNA-transfected BV-2 cells subjected to hypoxic exposure compared to cells transfected with control siRNA. *P <0.05 and **P <0.01. The values represent the mean ± SD in triplicate. Scale bar in C = 20 μm.