The Role Of Tlr2/4 In Responed Of Inflammation Under Hyperoxia-induced Acute Lung Injury

Part one The role of expression of Toll-like receptor 2/4 in hyperoxia-induced lung injury in ratObjectiveTo investigate the role of Toll-like receptor -2/4(TLR-2/4) in pathogenesis of hyperoxia-induced acute lung injury(HALI).MethodsThirty-two Sprague-Dawley(SD)rats were randomly divided into normal control group, hyperoxia for 24 hours group, hyperoxia for 48 hours group, and hyperoxia for 72 hours group, with 8 rats in each group. At corresponding exposure time points the animals were killed, the left lung was removed and measured the wet/dry weight ratio, the severity of lung injury was assessed by lung histopathology scores; the gene expression of TLR-2/4 in lung tissue were assessed by real-time polymerase chain reaction(Real-time PCR); the expression of TLR-2/4 protein in lung tissue were measured by Western Blotting ;the amount of IL-6 protein in lung tissue was examined by enzyme-linked immunosorbent assay (ELISA).ResultsAfter hyperoxia exposure ,the increase in lung wet/dry weight ratio as compared with the normal control group .Lung injury scores in hyperoxia for 48,72 hours group was significantly higher than that of the normal group(P<0.01). Real-time PCR results showed that hyperoxia exposure group of TLR2/4 gene expression in lung tissue were significantly higher than the air control group, and reached the peak at 24h(P < 0.05). Western Blotting study showed increased protein expression of TLR-2/4 in lung tissues compared with the control group , Respectively,peaked at 48h and 72h; ELISA assay also demonstrated upregulation of IL-6 levels in lung tissue compared with the normal control group,and at 48h reached the peak (P <0.05).ConclusionThe prolonged exposure to hyperoxia may causes acute lung injury in rat, and TLR2/4 plays an important role in the development of hyperoxia-induced acute lung injury in rat. Part two expression and function of Toll-like receptor 2/4 in Alveolar epithelial cells under hyperoxia-induced oxidative stressObjectiveTo observe the relation of intracellar ROS and TLR2/4 gene and protein and function of TLR2/4 pathway in alveolar epithelial cells under hyperoxia-induced oxidative stress for elucidating the possible mechanisms of responed inflammation in hyperoxia-induced acute lung injury.MethodsThe A549-human alveolar epithelia cell line was treated by hyeroxia >90% FIO2 for various times (1 h, 2 h, 6 h ,12h,24h and 48h)(1)intracellular ROS levels were measured by flow cytometry.(2) TLR2/4 gene expression were observed by real-time PCR.(3)TLR2/4 protein expression were observed by flow cytometry.(4) The activity of NF-κBin A549 cells was measured with EMSA.(5)The IL-6 and IL-8 concentrations in the supernatant of cultured A549 cells were tested with ELISA .(6) In some experiment , with NAC pretreated A549 cells, then exposed hyperoxia for 6h , The conditioned medium was collected at the time indicated for the assay of IL-6 and IL-8 protein concentration and the cells were washed with ice-cold phosphate buffered saline(PBS) and then used for the TLR2/4mRNA and protein assay and measurement of intracellular ROS and activation of NF-κB.ResultUsing reverse transcripition-PCR , we show that A549 cells express TLR1 through TLR10 mRNA under basal conditions, the result was confirmed by the use of human mononuclear cells as a positive control. flow cytometric demonstrate that A549 cells express TLR2 and TLR4 mainly intracellularly. Stimulation with hyperoxia for various times , the level of intracellular ROS increase with the prolonged exposure to hyperoxia, and the expression of TLR2 and TLR4 significantly increased . the activity of NF-κB and the levels of IL-6 and IL-8 also increased remarkably. Under pre-treatment of NAC, significantly lower levels of intracellular ROS, the expression of TLR2 and TLR4 also decreased in A549 cells, the NF-κB nuclear transcription activity and the levels of IL-6 and IL-8 in supernatant level compared with air control group no significant difference, but significantly lower than the group of high oxygen exposure.ConclusionA549 cells exposed to hyperoxia, the intracellular ROS can activate TLR2 / 4 expression in human alveolar epithelial cell, leading to pro-inflammatory cytokines IL-6 and IL-8 in the large release. This process may be through the signal transduction pathway of TLR2/4-NF-κB- inflammatory mediators . Part three TLR2 siRNA and TLR4 siRNA regulates the function of alveolar epithelial cell under hyperoxia-induced oxidative stressObjectiveAfter the TLR2 small interfering RNAs (siRNA) and TLR4 siRNA were transferred into cultured A549 cells with Lipofectamine 2000, to observe the TLR2/4 gene and protein and function of TLR2/4 pathway in alveolar epithelial cells under hyperoxia-induced oxidative stress to demonstrate that intracellular ROS can activate the signal transduction pathway of TLR2/TLR4-NF-κB- inflammatory mediators , it trigged the responed inflammation in hyperoxia-induced acute lung injury.MethodAfter the TLR2 siRNA and TLR4 siRNA were transferred into cultured A549 cells with Lipofectamine 2000 were exposed to 90% fractional inspired oxygen(FIO2) for 6 hrs. (1) the inhibition effect of TLR2/4 gene expression were observed by real-time PCR.(2) the inhibition effect of TLR2/4 protein expression were observed by flow cytometry.(4) The activity of NF-κB in A549 cells was measured with EMSA.(5) The IL-6 and IL-8 concentrations in the supernatant of cultured A549 cells were tested with ELISA .Result Levels of TLR2 and TLR4 Protein and mRNA were significantly reduced in TLR2-siRNA and TLR4-siRNA treated A549 cells compared to cells trallsfected with control siRNA. Transfection of A549 cells with TLR2 - siRNA and TLR4siRNA resulted in down regulation of hyperoxia– induced the activity of NF-κB and the levels of IL-6 and IL-8 compared with the group of high oxygen exposure, but with air control group no significant difference.ConclusionUnder hyperoxia condition, intracellular ROS can activate the signal transduction pathway of TLR2/4-NF-κB-inflammatory mediators , Leading to the production of pro-inflammtion IL-6 and IL-8.