Dynasore Alleviates LPS-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis

Abstract

Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders without available pharmacological therapies. Dynasore is a cell-permeable molecule that inhibits GTPase activity and exerts protective effects in several disease models. However, whether dynasore can alleviate lipopolysaccharide (LPS)-induced ALI is unknown. This study investigated the effect of dynasore on macrophage activation and explored its potential mechanisms in LPS-induced ALI in vitro and in vivo.

Methods: Bone marrow-derived macrophages (BMDMs) were activated classically with LPS or subjected to NLRP3 inflammasome activation with LPS+ATP. A mouse ALI model was established by the intratracheal instillation (i.t.) of LPS. The expression of PYD domains-containing protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) protein was detected by Western blots. Inflammatory mediators were analyzed in the cell supernatant, in serum and bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assays. Morphological changes in lung tissues were evaluated by hematoxylin and eosin staining. F4/80, Caspase-1 and GSDMD distribution in lung tissue was detected by immunofluorescence.

Results: Dynasore downregulated nuclear factor (NF)-κB signaling and reduced proinflammatory cytokine production in vitro and inhibited the production and release of interleukin (IL)-1β, NLRP3 inflammasome activation, and macrophage pyroptosis through the Drp1/ROS/NLRP3 axis. Dynasore significantly reduced lung injury scores and proinflammatory cytokine levels in both BALF and serum in vivo, including IL-1β and IL-6. Dynasore also downregulated the co-expression of F4/80, caspase-1 and GSDMD in lung tissue.

Conclusion: Collectively, these findings demonstrated that dynasore could alleviate LPS-induced ALI by regulating macrophage pyroptosis, which might provide a new therapeutic strategy for ALI/ARDS.

Keywords: NLRP3 inflammasome; acute lung injury; acute respiratory distress syndrome; dynasore; inflammation; pyroptosis.

Figure 1

Dynasore inhibits the classical activation of macrophages by LPS. (A) Cell viability was tested in BMDMs stimulated with LPS (100 ng/mL) for 24 h by the XTT assay. (B and C) Relative mRNA expression of IL-6 and IL-12 in LPS-activated BMDMs (LPS 100 ng/mL, 4 h). (D–E) IL-6 and IL-12 secreted in untreated BMDMs or cells treated with dynasore (dyn 5, 10, 25, and 50 μM) and stimulated with LPS (100 ng/mL, 24 h). (F) Relative mRNA expression of pro-IL-1β in LPS-activated BMDMs (100 ng/mL, 4 h). (G) Pro-IL-1β protein detection in cell lysates of untreated or dynasore-pretreated BMDMs. Cells were primed with 100 ng/mL of LPS for 3 h. (H) Viability of cells incubated with the indicated concentrations of dynasore for 1 h and then stimulated with LPS at 100 ng/mL for 3 h before the addition of ATP. (I) Mature IL-1β secreted by untreated or dynasore-pretreated BMDMs, then stimulated with LPS (500 ng/mL, 3 h) and ATP (5 mM, 30 min). Data are shown as the mean ± SD (n = 4). *p < 0.05; **p < 0.01.

Figure 2

Dynasore inhibits NLRP3 inflammasome activation and pyroptosis in macrophages. (A) NLRP3, caspase-1 p10, and GSDMD-NT protein detection in the cell lysates of untreated or dynasore-pretreated BMDMs. Cells were primed with LPS (500 ng/mL) for 3 h, followed by treatment with ATP (5 mM, 30 min). (B) Cell death was quantified by measuring the percentage of LDH release. Cells were primed with LPS (500 ng/mL) for 3 h, followed by treatment with ATP (5 mM, 30 min). (C–E) Relative mRNA expression of NLRP3, caspase-1, and GSDMD in LPS-activated BMDMs (LPS 100 ng/mL, 4 h). Data (C–E) are shown as the mean ± SD (n = 3). *p < 0.05; **p < 0.01.

Figure 3

Dynasore downregulates NF-κB signaling and Drp1/ROS/NLRP3 inflammatory pathways. (A) NF-κB p65 and NF-κB p-p65 protein detection in cell lysates of untreated or dynasore-pretreated BMDMs. Cells were primed with 100 ng/mL of LPS for 1 h. (B) Images of NF-κB p65 nuclear translocation visualized by immunofluorescence in untreated BMDMs and BMDMs treated with LPS (100 ng/mL, 30 min) and dynasore (5, 10, 25, and 50 μM). (C) Quantification of mean fluorescence intensity of NF-κB p65 nuclear translocation analyzed in (B). (D) Drp1 and p-Drp1 protein detection in untreated or dynasore-pretreated BMDM cell lysates. Cells were primed with 100 ng/mL of LPS for 12 h. (E) Flow cytometric analysis of mROS expression detected using MitoSox dye in untreated or BMDMs pretreated with dynasore and activated with LPS (100 ng/mL, 3 h). (F) Fold-change in the mean mROS fluorescence intensity analyzed in (E). Data (C and F) are shown as the mean ± SD (n = 3). *p < 0.05; **p < 0.01.

Figure 4

Different concentrations of dynasore reduced key proinflammatory cytokine production in vivo. (A and B) The effects of dynasore (10 mg/kg, 30 mg/kg, or 50 mg/kg) pretreatment were assessed 24 h after the intratracheal instillation of LPS (10 mg/kg). IL-1β and IL-6 cytokines were assayed in BALF of 8 groups of mice. (C and D) Assay of IL-1β and IL-6 cytokines in the serum of 8 groups of mice (control group, dynasore 10 mg group, dynasore 30 mg group, dynasore 50 mg group, LPS group, dynasore 10 mg + LPS group, dynasore 30 mg + LPS group, and dynasore 50 mg + LPS group, n = 6). Data (A–D) are shown as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Dynasore alleviates lipopolysaccharide-induced acute lung injury in vivo. (A) The effects of dynasore (30 mg/kg) pretreatment on lung injury were assessed by H&E staining (representative images at ×200 and ×100) 24 h after the intratracheal instillation of LPS (10 mg/kg). Scale bars, 100 μm/200 μm. (B) Lung injury scores were calculated according to the severity of lung injury. (C–G) Assays of IL-1β, IL-6, and IL-12 cytokines in BALF and serum in 4 groups of mice: control group and dynasore 30 mg group, LPS group, and dynasore 30 mg + LPS group, n = 6. The experiments were independently repeated 3 times. Data (B–G) are shown as the mean ± SD (n = 3). *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 6

Dynasore decreases the co-expression of F4/80, caspase-1 and GSDMD in vivo. The distribution and protein co-expression of F4/80, caspase-1 and GSDMD were examined by immunofluorescence. The numbers of positive cells (F4/80 red; caspase-1, Orange; or GSDMD, green) and the total number of cells (DAPI, blue) in the 4 mice groups (control group, dynasore 30 mg group, LPS group, and dynasore 30 mg + LPS group, n = 6; scale bar, 100 μm) were counted using the counting function of Image J software. Fluorescence intensity of the co-expression of F4/80, caspase-1 and GSDMD was quantified using Image J software, and all values were normalized to the control group. The experiments were independently repeated 3 times. ***p < 0.001; ****p < 0.0001.

Figure 7

The signaling mechanism diagram of dynasore alleviates inflammation in macrophages and acute lung injury.