Glibenclamide Alleviates LPS-Induced Acute Lung Injury through NLRP3 Inflammasome Signaling Pathway

Abstract

Glibenclamide displays an anti-inflammatory response in various pulmonary diseases, but its exact role in lipopolysaccharide- (LPS-) induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) remains unknown. Herein, we aimed to explore the effect of glibenclamide in vivo and in vitro on the development of LPS-induced ALI in a mouse model. LPS stimulation resulted in increases in lung injury score, wet/dry ratio, and capillary permeability in lungs, as well as in total protein concentration, inflammatory cells, and inflammatory cytokines including IL-1β, IL-18 in bronchoalveolar lavage fluid (BALF), and lung tissues, whereas glibenclamide treatment reduced these changes. Meanwhile, the increased proteins of NLRP3 and Caspase-1/p20 after LPS instillation in lungs were downregulated by glibenclamide. Similarly, in vitro experiments also found that glibenclamide administration inhibited the LPS-induced upregulations in cytokine secretions of IL-1β and IL-18, as well as in the expression of components in NLRP3 inflammasome in mouse peritoneal macrophages. Of note, glibenclamide had no effect on the secretion of TNF-α in vivo nor in vitro, implicating that its anti-inflammatory effect is relatively specific to NLRP3 inflammasome. In conclusion, glibenclamide alleviates the development of LPS-induced ALI in a mouse model via inhibiting the NLRP3/Caspase-1/IL-1β signaling pathway, which might provide a new strategy for the treatment of LPS-induced ALI.

Figure 1

The effect of glibenclamide on the pathological injury in LPS-induced ALI. (a) The pathological alternations in lung tissues were evaluated with HE staining (n = 4). (b) Lung injury scores in four groups. (c) Wet/dry ratio in four groups (n = 4). (d) Alveolar-capillary leakage in four groups (n = 3). (e) The total protein concentration in BALF (n = 3). Scale bars, 100 μm. Data are representative of three independent experiments (mean and SD). ns: not significant. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (unpaired Student's t-test).

Figure 2

The effect of glibenclamide on the LPS-stimulated inflammatory response in lungs. (a) The total cell number counts in BALF (n = 3). (b, c) The percentage of neutrophils and macrophages is presented (n = 3). (d–i) The content of IL-1β, IL-18, and TNF-α in (d–f) BALF (n = 3) and (g–i) lung tissues (n = 3) was measured by ELISA. Data are representative of three independent experiments (mean and SD). ns: not significant. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 (unpaired Student's t-test).

Figure 3

The inhibition role of glibenclamide in the NLRP3/Caspase-1/IL-1β signaling pathway induced by LPS. (a) Relative NLRP3 mRNA expression in lungs was measured by real-time PCR (n = 3). (b, c) Representative images of Western blot of NLRP3 and Caspase-1/p20 in the lungs and quantitative analysis (n = 3). (d, e) Representative images of immunohistochemical staining of NLRP3 in the lungs and quantitative analysis (n = 3). Scale bars, 100 μm. Data are representative of three independent experiments (mean and SD). ns: not significant. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 (unpaired Student's t-test).

Figure 4

A protective role of glibenclamide in vitro inflammatory model. (a) CCK-8 assay was used to determine glibenclamide cytotoxicity (n = 3). (b) Relative NLRP3 mRNA expression in PMs was measured by real-time PCR (n = 3). (c, d) Representative images of Western blot of NLRP3 and Caspase-1/p20 in PMs and quantitative analysis (n = 3). (e) Representative immunofluorescence staining of NLRP3 in PMs. (f) Calculated percentage of NLRP3-positive nuclei (n = 3). (g–i) The level of IL-1β, IL-18, and TNF-α in cell supernatant was measured by ELISA (n = 3). Data are representative of three independent experiments (mean and SD). Scale bars, 20 μm. ns: not significant. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 (unpaired Student's t-test).

Figure 5

Schematic diagram of the role of glibenclamide on NLRP3 inflammasome in the pathogenesis of LPS-induced ALI. Glibenclamide blocked the activation of NLRP3 inflammasome induced by LPS and thus reduced the release of proinflammatory cytokines and then attenuated the lung injury consequently.