Role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced pulmonary epithelial hyperpermeability

Abstract

Background: The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. Actin cytoskeleton has been implicated to be critical in regulation of epithelial barrier. Here, we performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced ALI.

Methods: For in vivo studies, 6-8-week-old C57 mice were used, Bronchoalveolar lavage Fluid /Blood fluorescent ratio, wet-to-dry lung weight ratio, as well as protein concentrations and neutrophil cell counts in BALF were detected as either directly or indirectly indicators of pulmonary alveolar barrier dysfunction. And hematoxylin and eosin staining was performed to estimate pulmonary injury. The in vitro explorations of transepithelial permeability were achieved through transepithelial electrical resistance measurement and testing of FITC-Dextran transepithelial flux in A549. In addition, cytoskeletal rearrangement was tested through F-actin immunostaining. And SB203580 was used to inhibit p38 MAPK activation, while siRNA was administered to genetically knockdown specific protein.

Results: We showed that LPS triggered activation of p38 MAPK, rearrangement of cytoskeleton which resulted in severe epithelial hyperpermeability and lung edema. A549 pretreated with TLR4 siRNA、p38 MAPK siRNA and its inhibitor SB203580 displayed a lower permeability and fewer stress fibers formation after LPS stimulation, accompanied with lower phosphorylation level of p38 MAPK and Hsp27, which verified the involvement of TLR4-p38 MAPK-Hsp27 in LPS-evoked alveolar epithelial injury. Inhibition of p38 MAPK activity with SB203580 in vivo attenuated pulmonary edema formation and hyperpermeability in response to LPS.

Conclusions: Our study demonstrated that LPS increased alveolar epithelial permeability both in vitro and in vivo and that TLR4- p38 MAPK- Hsp27 signal pathway dependent actin remolding was involved in this process.

Keywords: ALI; Alveolar barrier dysfunction; Cytoskeletal rearrangement; Hsp27; LPS; P38 MAPK; TLR4.

Fig. 1

LPS induce pulmonary edema and alveolar epithelial hyperpermeability in vivo. C57 mice were intraperitoneal injected with PBS or PBS-diluted LPS (15 mg/kg) for 8 h. (a) BALF/blood fluorescence intensity ratio and (b) Wet-to-dry lung weight ratio were measured; (c) protein concentration and (d) neutrophil cell counts in BALF were obtained; n = 3, *P < 0.05 versus control. (e) Lung Hematoxylin and eosin staining were obtained and lung injury score was calculated. n = 5, *P < 0.05

Fig. 2

LPS induce A549 hyperpermeability and cytoskeleton rearrangement. A549 cells were stimulated by LPS in (a, c) time- or (b, d) dose-dependent manner, and (a, b) TER value and (c, d) permeability coefficient for dextran (Pd) in dextran trans-epithelial flux were evaluated. (e) A549 were treated with or without 100 ng/mL LPS and the image of rhodamine-phalloidin stained F-actin was obtained through a laser confocal microscopy; (f) Fluorescence intensity of F-actin was qualified. n = 3, *P < 0.05 versus control

Fig. 3

Effect of LPS on p38 MAPK phosphorylation. A549 cells were stimulated by LPS in (a) time- or (b) dose-dependent manner. p-p38 was detected by western blotting. n = 3, *P < 0.05 versus control

Fig. 4

Role of TLR4 in LPS-induced A549 hyperpermeability. A549 was pretreated with TLR4 siRNA followed by LPS exposure. (a) TER value and (b) permeability coefficient for dextran (Pd) in dextran trans-epithelial flux were measured; (c) P-p38 and P-Hsp27 were detected by western blotting; (d) The image of rhodamine-phalloidin stained F-actin was obtained through a laser confocal microscopy; (e) Fluorescence intensity of F-actin was qualified. n = 3, *P < 0.05 versus control, #P < 0.05 versus LPS

Fig. 5

Effect of p38 siRNA in LPS-induced A549 hyperpermeability. A549 was pretransfected with p38 siRNA followed by LPS stimulation. (a) TER value and (b) permeability coefficient for dextran (Pd) in dextran trans-epithelial flux were measured; (c) P-p38 and P-Hsp27 were detected by western blotting; (d) The image of rhodamine-phalloidin stained F-actin was obtained through a laser confocal microscopy; (e) Fluorescence intensity of F-actin was qualified. n = 3, *P < 0.05 versus control, #P < 0.05 versus LPS

Fig. 6

Role of p38 inhibitor SB203580 in LPS-induced A549 hyperpermeability. A549 was pretreated with SB203580 followed by LPS exposure. (a) TER value and (b) permeability coefficient for dextran (Pd) in dextran trans-epithelial flux were measured; (c) P-p38 and P-Hsp27 were detected by western blotting; (d) The image of rhodamine-phalloidin stained F-actin was obtained through a laser confocal microscopy; (e) Fluorescence intensity of F-actin was qualified; (f) The image of rhodamine-phalloidin stained F-actin was obtained through a laser confocal microscopy by using primary mice pulmonary epithelial cells.  n = 3, *P < 0.05 versus control, #P < 0.05 versus LPS

Fig. 7

In vivo study of effect of p38 MAPK in LPS-induced ALI. C57 mice were pretreated with SB203580 before LPS (15 mg/kg) injection. (a) BALF/blood fluorescence intensity ratio and (b) Wet-to-dry lung weight ratio were measured; (c) protein concentration and (d) neutrophil cell counts in BALF were obtained; n = 3~ 4, *P < 0.05 versus control. (e) Lung Hematoxylin and eosin staining were obtained and lung injury score was calculated. n = 5, *P < 0.05