Treatment of Severe Acute Pancreatitis and Related Lung Injury by Targeting Gasdermin D-Mediated Pyroptosis

Abstract

The functional relevance and effects of the pyroptosis executioner gasdermin D (GSDMD) on severe acute pancreatitis (SAP)-associated lung injury are unclear. We established caerulein-induced mouse models of SAP-associated lung injury, which showed that GSDMD-mediated pyroptosis was activated in both pancreatic and lung tissues. Compared with Gsdmd wild-type SAP mouse models, Gsdmd knockout (Gsdmd^-/- ) ameliorated SAP-induced pancreas and related lung injury. Additionally, we investigated the effects of disulfiram on the treatment of SAP. Disulfiram is a Food and Drug Administration (FDA)-approved anti-alcoholism drug, which is reported as an effective pyroptosis inhibitor by either directly covalently modifying GSDMD or indirectly inhibiting the cleavage of GSDMD via inactivating Nod-like receptor protein 3 inflammasome. We demonstrated that disulfiram inhibited the cleavage of GSDMD, alleviated caerulein-induced SAP and related lung injury, and decreased the expression levels of proinflammatory cytokines (IL-1β and IL-18). Collectively, these findings disclosed the role of GSDMD-mediated pyroptosis in SAP and the potential application of disulfiram in the treatment of SAP.

Keywords: GSDMD; acute lung injury; disulfiram; inflammation; programmed cell death; pyroptosis; severe acute pancreatitis.

FIGURE 1

GSDMD-mediated pyroptosis is activated in pancreatic tissues in severe acute pancreatitis mouse models. Mice were sacrificed 24 h after LPS injection, and samples were collected. (A,B) The pyroptosis-associated molecules GSDMD, IL-1β, and IL-18 were analyzed by Western blotting and IHC. Scale bar, ×200. (C) Serum IL-1β and IL-18 levels were detected by ELISA. *p < 0.05 vs. other groups; ^#p < 0.05 vs. Gsdmd ^–/– sham group. Experiments were repeated three times. Data are expressed as mean ± SD, n = 10 per WT group, n = 8 per Gsdmd ^–/– group. GSDMD, gasdermin D; LPS, lipopolysaccharide; IHC, immunohistochemistry; WT, wild type.

FIGURE 2

GSDMD-mediated pyroptosis is activated in lung tissues in severe acute pancreatitis mouse models. Mice were sacrificed 24 h after LPS injection, and samples were collected. (A,B) The pyroptosis-associated molecules GSDMD, IL-1β, and IL-18 were analyzed in the lung by Western blotting. Experiments were repeated three times. (C) Immunofluorescence labeling of IL-1β (green), IL-18 (red), and DAPI (blue) in the four groups, Scale bar, × 200. All the data are expressed as mean ± SD, n = 10 per WT group, n = 8 per Gsdmd ^–/– group. GSDMD, gasdermin D; LPS, lipopolysaccharide; WT, wild type.

FIGURE 3

Gsdmd deficiency attenuates the injury of the pancreas and lung. Mice were sacrificed 24 h after LPS injection, and samples were collected. (A,B) H&E staining of the pancreas and lung in four groups was performed, and the morphologies were photographed at ×200 enlargement. (C–F) The levels of serum lipase, amylase, TNF-α, and IL-6. Experiments were repeated three times. *p < 0.05 vs. other groups; ^#p < 0.05 vs. Gsdmd ^–/– sham group in the expression of lipase, amylase, TNF-α, and IL-6. (G) Lung tissue wet/dry ratio was measured. All the data are expressed as mean ± SD, n = 10 per WT group, n = 8 per Gsdmd ^–/– group. LPS, lipopolysaccharide; WT, wild type.

FIGURE 4

Inhibition of pyroptosis with DSF alleviates the inflammation of the pancreas and lung. WT mice were separately treated with DSF of 25, 50, and 100 mg/kg. At 24 h after LPS injection, all mice were sacrificed. (A,B) Representative H&E staining of pancreatic and lung tissue. Scale bars, × 200. (C–F) Levels of lipase, amylase, TNF-α, and IL-6 in the serum were tested using ELISA. Experiments were repeated three times. (G) Wet/dry ratio was measured in the lung tissue. Data are presented as mean ± SD (n = 10 per group). *p < 0.05 vs. other groups; ^#p < 0.05 vs. WT-SAP group, WT-SAP DSF (25 mg/kg); ^#p > 0.05 vs. sham, sham DSF (50 mg/kg), WT-SAP DSF (100 mg/kg). DSF, disulfiram; WT, wild type; LPS, lipopolysaccharide; SAP, severe acute pancreatitis.

FIGURE 5

DSF inhibits the cleavage of GSDMD and decreases the levels of IL-1β and IL-18 in pancreatic tissues. WT-sham and WT-SAP mouse models were treated with DSF (50 mg/kg). The mice were sacrificed 24 h after LPS injection. (A,B) Western blotting and IHC analysis of GSDMD, IL-1β, and IL-18 in pancreatic tissue. Scale bars, ×200. (C) Serum IL-1β and IL-18 were analyzed by ELISA. Experiments were repeated three times. Data are presented as mean ± SD (n = 10 per group). *p < 0.05 vs. other groups; ^#p < 0.05 vs. WT-SAP group, WT-SAP DSF (25 mg/kg); ^#p > 0.05 vs. sham + sham DSF (50 mg/kg), WT-SAP DSF (100 mg/kg). DSF, disulfiram; GSDMD, gasdermin D; WT, wild type; SAP, severe acute pancreatitis; LPS, lipopolysaccharide; IHC, immunohistochemistry.

FIGURE 6

DSF inhibits the cleavage of GSDMD and decreases the levels of IL-1β and IL-18 in lung tissues. WT-sham and WT-SAP mouse models were treated with DSF (50 mg/kg). The mice were sacrificed 24 h after LPS injection. (A) Western blotting analysis of GSDMD, IL-1β, and IL-18 in the lung. Scale bars, × 200. Experiments were repeated three times. (B) Immunofluorescence labeling of IL-1β (green), IL-18 (red), and DAPI (blue) in the four WT groups, Scale bar, × 200. Data are presented as mean ± SD (n = 10 per group). DSF, disulfiram; GSDMD, gasdermin D; WT, wild type; SAP, severe acute pancreatitis; LPS, lipopolysaccharide.