Table 1.
Comparison of PIP and arterial blood gases between two groups.
Figure 1.
Effect of MV and LPS on lung inflammatory injury.
Concentrations of total protein (A) and IgM (B) in BALF, wet/dry lung weight ratio (C), lung MPO activity (D), neutrophils (E) and total cell numbers (F) were measured from the four groups of rats exposed to sham maneuver, MV, LPS and MV+LPS. Sections from the left lung lobe were stained with hematoxylin and eosin. Representative photos are shown for each experimental group (original magnification × 400). Note that lung sections from sham group (G) showed a normal alveolar structure, MV group (H) and LPS group (I) showed mild inflammatory changes (I), MV+LPS group (J) showed pronounced leukocyte infiltration and increased septal thickening. Lung injury histological scores (K). Data shown are means±SD from 6 rats per group. *, p<0.05 vs sham group; #, p<0.05 vs LPS group.
Figure 2.
Effect of MV and LPS on HMGB1 expression and cytokines production.
Quantitative RT-PCR was performed with total RNA from each animal, and HMGB1 mRNA expression in the lung was shown as percentage over β-actin gene in the same sample (A). HMGB1 protein expression was determined by Western blotting in the lung and the intensity ratio of HMGB1 to β-actin for each band is shown (B). ELISA was performed for protein levels of IL-1β (C), IL-6 (D), TNF-α (E), MIP-2 (F) and HMGB1 (G) in BALF from each animal. Data shown are means±SD from 6 rats per group. *, p<0.05 vs sham group; #, p<0.05 vs LPS group.
Figure 3.
Involvement of p38 MAPK and NF-κB signaling in MV and LPS induced lung injury.
Sham and LPS-treated rats randomized to either spontaneous respiration or moderate tidal volume mechanical ventilation (10 ml/kg) for 4 h. The proteins from lung tissues were used for the detection of p38, ERK, JNK (A) or IκB-α and p65 subunit of NF-κB in the nucleus and cytoplasm (B). Human lung epithelial cells (A549) were pre-incubated with NF-κB inhibitor SN-50, p38 inhibitor SB203580, ERK1/2 inhibitor PD98059 or JNK inhibitor SP 600125 followed by cyclic stretch (CS) at 20% strain, 30 cycle/min for 4 hrs. Cells were then lysed and protein was extracted for Western blot analysis (C). Note remarkable inhibition of HMGB1 expression in animals with SN-50 or SB203580 treatment, but not PD98059 or SP600125. β-actin was used as the loading control. Representative blots of three experiments are shown. All data are expressed as mean±SD. *, p<0.05 vs control group; #, p<0.05 vs CS+LPS group.
Figure 4.
Effect of HMGB1 blocking antibody on ventilator-induced lung injury in rats.
Animals received anti-HMGB1 blocking antibody intratracheally before challenged by LPS and MV. BALF concentrations of total protein (A) and IgM (B), neutrophils (C), lung MPO activity (D), lung wet/dry weight ratio (E), IL-1β (F) and MIP-2 (G) concentrations, and histopathology (H, I) were measured. H: MV+LPS; I: MV+LPS+ HMGB1 antibody. Data are expressed as mean±SD. *, p<0.05 vs IgY group.
Figure 5.
Proposed mechanism of HMGB1 in sustaining inflammatory response in ventilator-induced lung injury.
Mechanical stretch is sensed by mechanosensor apparatus and activates p38 MAPK and NF-κB cascades, initiating HMGB1 genes transcription. Nuclear HMGB1 then translocates to the cytoplasm and subsequent being released into the extracellular milieu. The extracellular HMGB1 forms complexes with other molecules (e.g. LPS, IL-1β) and such complexes in turn activate the cells. This forms a positive autocrine/paracrine feed back loop, which prolongs and expands the process of VILI.