ALDH2 deficiency in pancreatic acinar cells aggravates acute pancreatitis by promoting NLRP3 inflammasome activation through autophagy

Figure 1 Aldehyde dehydrogenase 2 is upregulated with reduced enzymatic activity, alongside dysregulated inflammatory immune responses and autophagy in acute pancreatitis. Acute pancreatitis (AP) was induced in C57BL/6 mice by seven intraperitoneal injections of caerulein (50 µg/kg). A: The expression of aldehyde dehydrogenase 2 (ALDH2) in pancreatic tissues for caerulein group and control group by immunoblotting (n = 3 per group); B: Immunofluorescence analysis of ALDH2 in acinar cells of pancreatic tissues from caerulein group and control group (n = 3 per group). Scale bar: 20 μm, magnification, 10 μm. MIST-1 is used as a marker of acinar cells; C: ALDH2 enzyme activity in pancreatic tissues of caerulein group and control group (n = 3 per group); D: Quantitative analysis of relative ALDH2 protein expression; E: Quantitative analysis of relative ALDH2 fluorescence intensity; F: Volcano plot of differentially expressed genes (DEGs) in pancreatic tissue between the caerulein group and control group, with red indicating upregulation and blue indicating downregulation (n = 3 per group). Thresholds: P adjusted values < 0.05 and |log2 fold change| ≥ 0.585; G: Gene Ontology enrichment analysis for the DEGs in pancreatic tissue between the caerulein group and control group (n = 3 per group). The results presented the top five significant outcomes for each section; H: Kyoto Encyclopedia of Genes and Genomes pathway analysis for the DEGs in pancreatic tissue between the caerulein group and control group (n = 3 per group); I: Gene Set Enrichment Analysis of up-regulated cellular components (Immunoglobulin complex and T cell receptor complex) in the caerulein group (n = 3 per group). ^aP < 0.05. ALDH2: Aldehyde dehydrogenase 2; NES: Normalized enrichment score.

Figure 2 Acute pancreatitis induction promoted aldehyde dehydrogenase 2 enhancement with reduced enzymatic activity, nod-like receptor family pyrin domain-containing 3 inflammasome activation, and autophagic substrate accumulation in porcine models. Porcine acute pancreatitis (AP) was induced by endoscopic cannulation of the pancreatic duct followed by injection of contrast material (10 mL, Omnipaque). Pancreatic tissues were harvested at 24 hours after model induction. A: Representative hematoxylin and eosin staining images of porcine pancreas and corresponding pathological scores in the AP and control groups (n = 3 per group). Orange arrows denote inflammatory cells. Black arrows indicate vacuolization. Scale bar: 50 μm, magnification, 20 μm; B and C: Serum amylase, lipase, interleukin (IL)-1β, IL-6 and IL-18 levels before modeling and 24 hours after modeling in the porcine AP group (n = 3); D: Aldehyde dehydrogenase 2 (ALDH2) enzyme activity (biochemical assay) and protein expression (immunoblotting) in pancreatic tissues of AP group and control group (n = 3 per group); E: Immunofluorescence analysis of ALDH2 in pancreatic tissues of AP group and control group (n = 3 per group). Scale bar: 20 μm; F: The expression of nod-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis associated speck-like protein (ASC) and caspase-1 for AP group and control group in porcine pancreatic tissues by immunoblotting (n = 3 per group); G: Immunofluorescence analysis of NLRP3 (red) - ASC (green) and NLRP3 (red) - caspase-1 (green) in pancreatic tissues of AP group and control group. Scale bar: 20 μm, magnification, 10 μm; H: The expression of P62 and microtubule-associated protein light chain 3B (LC3B) for AP group and control group in porcine pancreatic tissues by immunoblotting (n = 3 per group); I and J: Immunofluorescence analysis of P62 and LC3B in pancreatic tissues of AP group and control group (n = 3 per group). Scale bar: 20 μm; K: Representative transmission electron microscopy images and quantitative analysis in pancreatic tissue of AP group and control group. Arrows denote autophagic vesicles. Scale bar: 5 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. AMY: Amylase; LPS: Lipase; LC3B: Light chain 3B; ALDH2: Aldehyde dehydrogenase 2; AP: Acute pancreatitis; IL: Interleukin; NLRP3: Nod-like receptor family pyrin domain-containing 3; ASC: Associated speck-like protein.

Figure 3 Aldehyde dehydrogenase 2 deficiency in pancreatic acinar cells exacerbated acute pancreatitis severity and nod-like receptor family pyrin domain-containing 3 inflammasome activation in mice. Acute pancreatitis (AP) was induced in pancreatic acinar cell-specific aldehyde dehydrogenase 2 (ALDH2) knockout mice (Aldh2^fl/flCela1-Cre) and the wild-type mice (Aldh2^+/+) by seven intraperitoneal injections of caerulein (50 µg/kg). A: The expression of ALDH2 in pancreatic tissues of Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice by immunoblotting (n = 3 per group); B: Representative hematoxylin and eosin staining images of pancreas and corresponding pathological scores in Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice (n = 3 per group). Orange arrows denote inflammatory cells. Black arrows indicate vacuolization. Scale bar: 50 μm, magnification, 20 μm; C: Serum amylase, lipase, interleukin (IL)-1β, IL-6 and IL-18 levels of AP model in Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice (n = 3 per group); D: Immunoblot analysis of nod-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis associated speck-like protein (ASC) and caspase-1 expression in pancreatic tissues from Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice with AP (n = 3 per group); E: Immunofluorescence analysis of NLRP3 (red) - ASC (green) and NLRP3 (red) - caspase-1 (green) in pancreatic tissues from Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice with AP. Scale bar: 20 μm, magnification, 10 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. AMY: Amylase; LPS: Lipase; ALDH2: Aldehyde dehydrogenase 2; IL: Interleukin; NLRP3: Nod-like receptor family pyrin domain-containing 3; ASC: Associated speck-like protein.

Figure 4 Aldehyde dehydrogenase 2 deficiency in pancreatic acinar cells exacerbated accumulation of autophagic substrates in murine acute pancreatitis models. Acute pancreatitis (AP) was induced in pancreatic acinar cell-specific aldehyde dehydrogenase 2 knockout mice (Aldh2^fl/flCela1-Cre) and the wild-type mice (Aldh2^+/+) by seven intraperitoneal injections of caerulein (50 µg/kg). A: The expression of P62 and microtubule-associated protein light chain 3B (LC3B) in pancreatic tissues of Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice by immunoblotting (n = 3 per group); B: Immunofluorescence analysis of P62 and LC3B in acinar cells of pancreatic tissues from Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice with AP (n = 3 per group). Scale bar: 20 μm, magnification, 10 μm. MIST-1 is used as a marker of acinar cells; C: Representative transmission electron microscopy images and quantitative analysis in pancreatic tissue from Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice with AP. Arrows denote autophagic vesicles. Scale bar: 5 μm. ^aP < 0.05, ^bP < 0.01. Aldh2^fl/flCela1-Cre: Aldehyde dehydrogenase 2 knockout mice.

Figure 5 Aldehyde dehydrogenase 2 deficiency aggravated nod-like receptor family pyrin domain-containing 3 inflammasome activation in caerulein-stimulated pancreatic acinar cells. MPC-83 cells were transfected with aldehyde dehydrogenase 2 (ALDH2)-siRNA and NC-siRNA. The induction of acute pancreatitis (AP) was established by caerulein (100 nM) treatment. A: The expression of ALDH2 in ALDH2-siRNA treated cells and their corresponding controls by immunoblotting (n = 3 per group); B-D: Supernatant cell culture medium interleukin (IL)-1β, IL-6 and IL-18 Levels in each group (n = 3 per group); E-H: Immunoblot analysis of nod-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis associated speck-like protein (ASC) and caspase-1 for cells in each group (n = 3 per group); I: Immunofluorescence analysis of NLRP3 (red) - ASC (green) and NLRP3 (red) - caspase-1 (green) for cells in each group. Scale bar: 20 μm, magnification, 10 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. NS: Not significant; CER: Caerulein; IL: Interleukin; ALDH2: Aldehyde dehydrogenase 2; NLRP3: Nod-like receptor family pyrin domain-containing 3; ASC: Associated speck-like protein.

Figure 6 Aldehyde dehydrogenase 2 deficiency exacerbated the accumulation of autophagic substrates in caerulein-stimulated pancreatic acinar cells. MPC-83 cells were transfected with aldehyde dehydrogenase 2 (ALDH2)-siRNA and NC-siRNA. The induction of acute pancreatitis (AP) was established by caerulein (100 nM) treatment. A and B: Immunoblot analysis of P62 and microtubule-associated protein light chain 3B (LC3B) for cells in each group (n = 3 per group); C-F: Immunofluorescence analysis of P62 and LC3B for cells in each group (n = 3 per group). Scale bar: 20 μm, magnification, 10 μm; G and H: Representative transmission electron microscopy images and quantitative analysis of cells in each group. Arrows denote autophagic vesicles. Scale bar: 1 μm. ^aP < 0.05, ^bP < 0.01. NS: Not significant; LC3B: Light chain 3B; ALDH2: Aldehyde dehydrogenase 2; CER: Caerulein.

Figure 7 Aldehyde dehydrogenase 2 deficiency attenuated rapamycin-induced nod-like receptor family pyrin domain-containing 3 inflammasome inhibition in caerulein-stimulated pancreatic acinar cells. MPC-83 cells were transfected with aldehyde dehydrogenase 2 (ALDH2)-siRNA and NC-siRNA. Cells were then pre-treated with the autophagy inducer rapamycin (100 nM) for 1 hour, then stimulated with caerulein (100 nM) for 24 hours before sample collection. A-C: Supernatant cell culture medium interleukin (IL)-1β, IL-6 and IL-18 levels in each group (n = 3 per group); D-G: Immunoblot analysis of nod-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis associated speck-like protein (ASC) and caspase-1 for cells in each group (n = 3 per group); H: Immunofluorescence analysis of NLRP3 (red) - ASC (green) and NLRP3 (red) - caspase-1 (green) for cells in each group. Scale bar: 20 μm, magnification, 10 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. CER: Caerulein; Rapa: Rapamycin; IL: Interleukin; ALDH2: Aldehyde dehydrogenase 2; NLRP3: Nod-like receptor family pyrin domain-containing 3; ASC: Associated speck-like protein.

Figure 8 Alda-1 reduced nod-like receptor family pyrin domain-containing 3 inflammasome components in an autophagy-dependent manner. Cells were pretreated with Alda-1 (20 μM) for 4 hours prior to caerulein stimulation (100 nM), and chloroquine (25 μM) was added during the final 2 hours of caerulein stimulation. A-D: Immunoblot analysis of nod-like receptor family pyrin domain-containing 3, apoptosis associated speck-like protein and caspase-1 for cells in each group (n = 3 per group); E-G: Immunoblot analysis of P62 and microtubule-associated protein light chain 3B for cells in each group (n = 3 per group). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. NS: Not significant; CER: Caerulein; LC3B: Light chain 3B; CQ: Chloroquine; NLRP3: Nod-like receptor family pyrin domain-containing 3; ASC: Associated speck-like protein.