Abstract
BackgroundThe 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.MethodsFor 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.ResultsWe 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.ConclusionsOur 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.
Highlights
The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury
In vivo study of LPS-induced pulmonary edema and epithelial hyperpermeability We performed in vivo studies to determine effect of LPS in Acute lung injury (ALI), in pulmonary epithelial permeability
We found that LPS-injected mice presented higher Bronchoalveolar lavage Fluid (BALF)/Blood fluorescent intensity ratio (Fig. 1a), implicating the breakdown of pulmonary epithelial barrier
Summary
The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. We performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp signal pathway in LPS-induced ALI. It has been frequently implicated that exposure to LPS leads to activation of various signal pathways, Zhijie et al have performed an in-vivo study to determine the critical role of Toll-like receptor 4 (TLR4) in LPS-induced ALI [13]. The activation of p38 MAPK has been implicated as a critical step in the process of pulmonary barrier dysfunction induced by various stimulus, including pertussis toxin, LPS, and H2O2. Emerging evidences highlight role of p38 MAPK in pulmonary epithelial injury [15,16,17,18,19]. We seek to uncover the potential role of TLR4-p38MAPK-Hsp signal pathway in LPS-induced pulmonary epithelial hyperpermeability
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