OBJECTIVE: To investigate the role of alveolar macrophages Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling in ventilator-induced lung injury (VILI) in rat model.
METHODS: Thirty adult male Sprague-Dawley rats were ventilated with high tidal volume (HTV) (40 ml/kg) for 240 minutes to establish VILI model after oral intubation. Then 4DegreesCelsius PBS was infused through the endotracheal tube and alveolar macrophages (AMs) were purified from the bronchoalveolar lavage fluid (BALF). The AMs were randomly divided into 3 groups (n=8 each): PBS stimulating group (group CON), TNF-α stimulating combined with PBS blocking group (STI group), and TNF-α stimulating combined with TLR4 monoclonal antibodies (TLR4 mAb) blocking group (ANT group). CON group was cultured for 16 hours after blocked by PBS for 2 hours. STI group was cultured with TNF-α (20 ng/mL) for 16 hours after blocked by PBS for 2 hours. ANT group was cultured with TNF-α (20 ng/mL) for 16 hours after blocked by PBS and TLR4 mAb for 2 hours. The cell culture supernatants were collected for determination of the expressions of TNF-α, IL-1β and IL-6 with ELISA. The expressions of TLR4, TLR9, MyD88 and nuclear factor κB (NF-κB) at both mRNA and protein levels were detected by reverse transcription PCR and Western blotting, respectively.
RESULTS: Compared with CON group, concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants increased significantly in STI group and ANT; the mRNA and protein levels of TLR4, MyD88 and NF-κB in AMs rose significantly in STI group, but there was no significant difference in the mRNA and protein levels of TLR4, MyD88 and NF-κB in ANT group. Compared with STI group, concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants, the mRNA and protein levels of TLR4, MyD88 and NF-κB in AMs decreased significantly in ANT group. There was no significant difference in TLR9 mRNA and protein levels among the three groups.
CONCLUSION: The stimulation of inflammatory cytokines can up-regulate the secretion of TLR4, MyD88 and NF-κB in AMs. TLR4-MyD88 signaling plays an important role in the development of ventilator-induced lung injury in rats.
