The Impacts Of Regulating Toll-Like Receptor 2/Nuclear Factor-KB Signal Pathway On Rats With Ventilator-Induced Lung Injury

OBJECTIVE To evaluate the role of Toll-like receptor 2/nuclear factor-κ B (TLR2/NF-κ B) signal pathway pretreated with TLR2 mAb in rats modeling of ventilator-induced lung injury.METHOD Both in vitro and in vivo experimrnts were carried out. Sixty male Sprague-Dawley rats were randomly divided into 6 groups (n=10). In vivo, Group A:ventilated with a nomal VT of 8 ml/kg. Group B:rats were given concentration of 25 ug/ml TLR2 monoclonal antibodies (TLR2mAb,10 ug/kg) by slow instilled through tracheal catheter, and then ventilated with a high tidal volume (VT) of 40 ml/kg. Group C:rats were tracheally instilled saline, and then ventilated with a high VT of 40 ml/kg. After 4h ventilation, blood serum, bronchoalveolar lavage fluid (BALF) and lung tissue was collected. In vitro, rats in group D were ventilated with a normal VT of 8ml/kg, rats in group E and group F were ventilated with a high VT of 40 ml/kg. And then macrophages in three groups were extracted after 4h ventilation and culture. Group D:control group, and PBS as the intervented drug and stimulator. Group E:macrophages were pretreated with concentration of 25 ug/kg TLR2mAb, lh later TNF-a was given as a stimulator. Group F:macrophages were given PBS before stimulation of TNF-α. Sixteen hours later, cultural supernatants and macrophages were collected. The lung wet to dry ratio (W/D) was calculated, and the changes in pathology and ultrastructure in lung tissue were observed with microscope. Enzyme linked immunosoebent assay (ELISA) was performed to determine the concentration of interleukins (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) in serum, BALF and culture supernatants. Real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB) was used to assess the messenger RNA expressions and protein level of TLR2, NF-κ B and myeloid differentiation factor 88 (MyD88) in lung tissue, respectively.RESULTS No obvious pathological changes in lung were foud in group A and group B, and no obvious damages to ultra-microstructure were found in lung macrophages, type Ⅰ and type Ⅱ epithelial cells. However, in group C, pathological changes were observed, including pulmonary alveoli fusion, alveoli septum thickening, inflammatory cells infiltration, and damages to ultrastructure of lung macrophages, damamges to cell membrane of typr Ⅰ and type Ⅱ epithelial cells, cacuoles in cytoplasm, damage to organelle, and even pyknosis and perinuclear cistern thickening. Compared with group C, W/D ratio and mean concentration of inflammatory cytokines in serum and BALF showed a significant decrease in group A and group B; mRNA expression and protein level of TLR2, NF-κ B, MyD88 in lung tissue were significantly decrease in group A and group B. Compared with group F, concentration of IL-1β, IL-6 and TNF-α in culture supernantants, mRNA expression and protein level of TLR2, NF-κ B and MyD88 in macrophages were significantly decrease in group D and group E.CONCLUSION To some extent, pre-intervention withTLR2mAb to block the TLR2/NF-κB signal pathway can inhibit the release of pro-inflammatory factors, and regulate the ventilator-induced lung injury (VILI).