Fig 1.
Schematic diagram of experimental design.
(A) Experimental protocol in vivo. (B) Experimental protocol in vitro.
Fig 2.
Effects of sevoflurane on expiratory resistance (Re) and dynamic lung compliance (Cldyn) in mice after different concentrations of methacholine (Mch) after LPS challenge.
(A) Ratios of Re changed with different concentrations of Mch. (B) Ratios of Cldyn changed with different concentrations of Mch. Data are presented as means ± SEM (n = 8 per group). *P < 0.05 versus PN; #P < 0.05, versus PL.
Fig 3.
Effects of sevoflurane on histopathologic outcome and cell counts in bronchoalveolar lavage fluid (BALF) after LPS challenge in vivo.
(A) Representative photomicrographs of lung sections stained with hematoxylin-and-eosin from mice anesthetized with phenobarbital or sevoflurane in the absence or presence of LPS challenge. The mice in group PN and SN showed much less inflammatory cell infiltration, alveolar septal thickening, and pulmonary edema in the lung compared with the mice exposed to LPS (NL+SL). Sevoflurane administered after intraperitoneal sensitization could mitigate lung inflammation upon re-challenge with LPS. Original magnification 100×. (B) Total cell and neutrophil counts in BALF. Data are presented as means ± SEM (n = 8 per group).*P < 0.05 versus PN; #P < 0.05, versus PL.
Table 1.
Effects of sevoflurane on concentrations of cytokines in BALF of mice exposed to LPS (ng/L).
Fig 4.
Immunohistochemical staining of TLR4 in trachea and effects of sevoflurane on TLR4 protein and mRNA expression after LPS challenge in vivo.
(A) Representative micrographs of TLR4 in trachea of mice anesthetized with phenobarbital or sevoflurane in absence or presence of LPS challenge. The presence of TLR4 was observed only in the smooth muscle cell layer, whose cell membrane and cytoplasm stained tan as detected by horseradish peroxidase–labeled antibodies. Scale bars = 50 μm. (B) Histogram for quantification of TLR4 in smooth muscle cell layer. The mean optical density values (MOD) were calculated after normalizing against PN. (C) Representative western blot and quantitative analysis of TLR4 isolated from smooth muscle cell layer after LPS challenge. (D) Quantitative real-time PCR of TLR4 mRNA expressions in isolated smooth muscle cell layer. (E) Representative western blot and quantitative protein analysis of NF-κB in nuclear extracts from smooth muscle cell layer after LPS challenge. (F) Quantitative real-time PCR of NF-κB mRNA expressions in smooth muscle cell layer after LPS challenge. Sevoflurane prevented significant increases in protein and mRNA expressions of TLR4 and NF-κB in isolated smooth muscle cell layer from mice after LPS challenge. The relative integral density values (IDVs) were calculated after normalizing against GAPDH in each sample and presented as relative protein expression units. Data are presented as means ± SEM (n = 8 per group). *P < 0.05 versus PN; #P < 0.05, versus PL.
Fig 5.
Effects of sevoflurane on TLR4 protein expression in airway smooth muscle cells (ASMCs) after continuous LPS exposure for 1, 3, 5, 12, and 24 h in vitro.
(A) Representative western blot of TLR4 in cultured ASMCs under different protocols. (B-F) Quantitative protein analysis of TLR4 in cultured ASMCs under different protocols. The relative integral density values (IDVs) were calculated after normalizing against GAPDH in each sample and presented as relative protein expression units. Sevoflurane prevented TLR4 increases in ASMCs at 3, 5, 12, and 24 h after continuous LPS exposure. Data are presented as means ± SEM. * P < 0.05 versus Group Air; #P < 0.05 versus AL.
Fig 6.
Effects of sevoflurane on NF-κB protein expression in nuclear extracts of airway smooth muscle cells (ASMCs) after continuous LPS exposure for 1, 3, 5, 12, and 24 h in vitro.
(A) Representative western blot of NF-κB in cultured ASMCs under different protocols. (B-E) Quantitative protein analysis of NF-κB in cultured ASMCs under different protocols. The relative integral density values (IDVs) were calculated after normalizing against GAPDH in each sample and presented as relative protein expression units. Sevoflurane prevented NF-κB increases in ASMCs at 3, 5, 12, and 24h after continuous LPS exposure. Data are presented as means ± SEM. * P < 0.05 versus Group Air; #P < 0.05 versus AL; $P < 0.05 versus SL.