Abstract
The present study investigated the relationship between connexin 43 (Cx43) expression in alveolar type II epithelial cells (ATII) and alveolar air-blood barrier permeability, and the effect of microRNA-206 (miR-206) on the expression of Cx43 in sepsis-induced acute lung injury. For the in vivo study, rats were divided into the sham, caecum ligation and perforation (CLP), CLP+Cx43 inhibitors (Cx43-In) and CLP+miR-206 analogs (miR-206-Mi) groups. CLP method was used to prepare an acute lung injury model of sepsis. Following successful modeling, lung tissue was collected for hematoxylin and eosin (HE) staining, and the wet to dry weight ratio (W/D) and protein content in bronchoalveolar lavage fluid (BALF) were detected. Cx43 expression in lung tissue was determined by immunohistochemistry and western blot analysis. Additionally, miR-206 and Cx43 expression levels in lung tissue were detected by reverse transcription-quantitative polymerase chain reaction. Rat ATII cells were cultured in Transwells plates to form monolayers, then treated with Cx43 mRNA inhibitors or miR-206 analogs. The cell monolayers were then stimulated with lipopolysaccharide and their permeability was evaluated by detecting fluorescein-labeled dextran at the lower chamber of the Transwells. The dual luciferase reporter gene assay was used to investigate whether miR-206 targeted the 3' untranslated region of Cx43 mRNA to regulate Cx43 expression, thereby regulating the permeability of the alveolar air-blood barrier. Results demonstrated that the CLP method induced damage to the alveolar structure, thickened the alveolar wall, caused hyperemia and hemorrhage in the pulmonary interstitium and caused infiltration of inflammatory cells. Edema in the pulmonary interstitium and alveolar space, exudation of neutrophilic granulocyte and pink edema fluid in alveolar cavities were also observed. W/D ratio, the BALF protein content, and expression of Cx43mRNA and Cx43 were increased significantly, whilst miR-206 expression decreased compared with the control group. The lung tissue inflammatory response was attenuated, and the W/D ratio and BALF protein content decreased in the Cx43-In and miR-206-Mi groups compared with the CLP group. Moreover, Cx43 mRNA and protein expression were decreased significantly in the Cx43-In and miR-206-Mi groups. In addition, the dual luciferase reporter gene assay determined that the untranslated region of Cx43 mRNA had a complementary sequence to miR-206. Of note, Cx43 mRNA expression in the miR-206-Mi group was not significantly decreased in vitro. In conclusion, the increase in ATII cell Cx43 expression during sepsis-induced acute lung injury resulted in an increase in the permeability of the alveolar air-blood barrier. miR-206 targeted the Cx43 mRNA 3'untranslated region to downregulate Cx43 expression, which further improved the permeability of the alveolar air-blood barrier.