Dopamine D1 Receptor Agonist Alleviates Acute Lung Injury Via Modulating Inflammatory Responses In Macrophages And Barrier Function In Airway Epithelial Cells

Objective To explore the regulatory effects and underlying mechanisms of SKF38393(SKF),a dopamine D1 receptor agonist,on macrophage inflammatory response,oxidative stress,and airway epithelial barrier function in the lipopolysaccharide(LPS)-induced acute lung injury(ALI)mouse model.MethodsTHP-1 cells(differentiated into macrophage-like cells)and Beas-2B cells were cultured in vitro,and cells were stimulated with LPS with or without SKF/ML385.Cytokines(CCL2,IL-6,IL-1β)expression was assessed by real-time quantitative PCR(q PCR)and enzyme-linked immunosorbent assay(ELISA).The level of mitochondrial reactive oxygen species(ROS)was measured by a confocal microscope and fluorescent plate reader.Immunoblotting assay was used to detect the activation of related signaling pathways.Transepithelial electrical resistance(TEER)of Beas-2B cell monolayer to observe cell barrier function.Male C57BL/6 mice(6-8 weeks old)were randomly divided into PBS control group,SKF treatment group,LPS model group,LPS+SKF treatment group and LPS+SKF+ML385 inhibitor group.Subsequently,bronchoalveolar lavage fluid(BALF)and lung tissues were collected.The expression of IL-6,KC,G-CSF and TNF-αin BALF were determined by ELISA.The severity of lung injury,the secretion level of D1-like dopamine receptors(DRD1,DRD5)in macrophages and airway epithelial cells and the expression of tight junction protein(ZO-1,occludin)in airway epithelial cells were detected by histopathological,immunohistochemical(IHC)and immunofluorescence(IF)respectively.Immunoblotting assay was used to detect the activation of related inflammatory pathways.ResultsBy using THP-1 cell-derived macrophages and Beas-2B cells,the dopamine D1-like receptor agonist,SKF,effectively inhibited the expression of pro-inflammatory cytokines stimulated by LPS and mitochondrial ROS production and increased the level of TEER in airway epithelial cell monolayer and the expression of tight junction protein(ZO-1,occluding)in Beas-2B cells downregulated by LPS.In addition,SKF also exhibited extensive inhibitory effects on multiple inflammatory signaling pathways,including NF-κB,MAPK,and NLRP3/Caspase-1 pathways.In vivo experiments showed that dopamine D1-like receptors D1(DRD1)and D5(DRD5)were mainly distributed in pulmonary macrophages and alveolar epithelial cells.SKF pretreatment significantly restored the expression of DRD5,antioxidant factor NQO1,and tight junction protein ZO-1 and occludin in airway epithelial cells decreased by LPS,reduced the numbers of total cells,neutrophils as well as lymphocytes within the BALF.SKF could suppress total protein leakage and production of cytokines KC,IL-6,G-CSF in the mouse BALF.Additionally,SKF could inhibit the activation of NF-κB/NLRP3/Caspase-1 pro-inflammatory pathways.The protective effects of SKF were partially counteracted by ML385,an inhibitor of Nrf2,both in vivo and in vitro.ConclusionsHerein,we found the decreased expression of D1-like receptors in macrophages and airway epithelial cells within LPS-induced ALI lungs and determined the protective effects of SKF in these cell types,with the underlying mechanism linking to the activation of Nrf2 antioxidative system.Besides,SKF also exerted notable efficacy in alleviating pulmonary inflammatory injury in the ALI mouse model.