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

Abstract

BACKGROUND

Acute pancreatitis (AP), a common acute gastrointestinal inflammation with rising incidence, imposes a substantial disease burden. However, targeted therapies for this condition remain elusive.

AIM

To investigate the role of aldehyde dehydrogenase 2 (ALDH2) in AP.

METHODS

Transcriptomic analysis of pancreatic tissue from murine AP models was conducted to identify dysregulated pathways. The porcine AP model was used as a preclinical model to evaluate ALDH2, nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, and autophagy-related markers. Pancreatic acinar cell-specific ALDH2 knockout (Aldh2^fl/flCela1-Cre) mice and ALDH2-specific siRNA transfected MPC-83 cells were created to interfere with ALDH2 expression. Rapamycin, ALDH2 agonist Alda-1, and chloroquine were used to investigate the role of autophagy in ALDH2-mediated regulation of the NLRP3 inflammasome.

RESULTS

We demonstrated that genes differentially expressed during AP were enriched in inflammatory immune and autophagy-related pathways. In porcine AP models, the severity of AP was associated with increase of ALDH2, NLRP3, apoptosis associated speck-like protein (ASC), and caspase-1 expression, along with reduced ALDH2 enzymatic activity and accumulation of autophagic substrates, as evidenced by elevated P62 and microtubule-associated protein light chain 3B-II levels and the presence of aggregated autophagic vesicles. Using in vivo murine and in vitro cellular AP models, we found that ALDH2 deficiency in pancreatic acinar cells further exacerbated AP, enhanced NLRP3 inflammasome activation, and aggravated autophagic substrate accumulation. Moreover, in cellular AP models, ALDH2 deficiency attenuated rapamycin-induced NLRP3 inflammasome inhibition. Conversely, Alda-1 reduced NLRP3 inflammasome activation, and this effect was reversed by chloroquine co-treatment, indicating that this reduction is autophagy-dependent.

CONCLUSION

In summary, ALDH2 deficiency in pancreatic acinar cells aggravates AP by exacerbating autophagic substrate accumulation and NLRP3 inflammasome activation, whereas ALDH2 activation reduces inflammasome components in an autophagy-dependent manner, suggesting this pathway as a potential therapeutic direction in AP.

Key Words: Acute pancreatitis; Aldehyde dehydrogenase 2; Autophagy; Nod-like receptor family pyrin domain-containing 3 inflammasome; Pancreatic acinar cell

Core Tip: Using caerulein-induced mouse and endoscope-based porcine acute pancreatitis (AP) models, this study found aldehyde dehydrogenase 2 (ALDH2) expression in pancreatic acinar cells increased while activity decreased, accompanied by autophagic substrate accumulation and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation. During AP, ALDH2 deficiency in pancreatic acinar cells exacerbated autophagic substrate accumulation and NLRP3 inflammasome activation in vivo and in vitro. ALDH2 deficiency attenuated rapamycin's inhibition of NLRP3 inflammasome activation, while ALDH2 agonist Alda-1 reduced inflammasome components in an autophagy-dependent manner, as evidenced by chloroquine reversal. These findings link ALDH2, autophagy, and NLRP3 inflammasome regulation in pancreatic acinar cells.

INTRODUCTION

Acute pancreatitis (AP) is a prevalent digestive disorder with an annual incidence of 15 to 83.7 cases per 100000 population in Western countries[1]. Gallstone diseases and alcohol consumption are considered the most common etiologies of AP[2]. The pathogenesis of AP, irrespective of the specific etiology, centers on intrinsic acinar cell damage, resulting in the inappropriate release and activation of trypsinogen to trypsin within pancreatic acini[3]. The presence of active trypsin activates a cascade of digestive enzymes, leading to the autodigestive injury of pancreatic tissue[4]. Acinar damage resulting from autodigestion triggers an inflammatory response, characterized by the infiltration of inflammatory cells and the release of inflammatory cytokines, eliciting local inflammation in the pancreas and peripancreatic tissues[4]. Furthermore, the systemic release of these cytokines can initiate an inflammatory cascade, culminating in systemic inflammatory response syndrome (SIRS) and potentially progressing to multiple organ dysfunction syndrome[5]. Therefore, preserving pancreatic acinar cell function and inhibiting localized inflammatory immune injury to acinar cells, as well as the subsequent development of SIRS represent crucial therapeutic strategies for AP.

Given the pivotal role of inflammatory cascades in AP progression, the NLR pyrin domain-containing protein 3 (NLRP3) inflammasome has emerged as a critical molecular mediator during this pathological process. Upon activation, the NLRP3 inflammasome exerts its biological function by facilitating the conversion of the precursor forms of interleukin (IL)-1β and IL-18 into their active forms. In addition, these cytokines exhibit a positive correlation with the severity of inflammation[6]. Researchers have found that in severe AP, the levels of free apoptosis-associated speck-like protein (ASC), an activation marker of the NLRP3 inflammasome, and IL-18 are significantly elevated[7]. Furthermore, experimental evidence from murine models has demonstrated the capability of NLRP3 inhibition to mitigate the severity of AP and associated organ injury, as supported by a systematic review and meta-analysis[8].

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that plays a pivotal role in detoxifying reactive aldehydes generated during the process of oxidative stress and ethanol metabolism[9]. ALDH2 confers a protective effect against blood-brain barrier injury induced by cerebral ischemia/reperfusion (I/R), septic cardiomyopathy, and acute gouty arthritis by attenuating oxidative stress and inhibiting inflammatory signaling pathways[10-12]. Within the gastrointestinal context, ALDH2 also exhibits a protective effect in pathological conditions such as alcohol-induced gastric ulceration and oxidative stress-induced gastric mucosal injury, primarily through its detoxification and anti-inflammatory mechanisms[13,14]. Specifically, a previous study investigated the role of ALDH2 in AP and demonstrated its protective role on pancreatic tissue by mitigating apoptosis through the attenuation of lipid peroxidation[15]. However, that study did not conduct an in-depth exploration of the inflammatory phenotype in AP nor the critical role of inflammatory immune responses in this context. Moreover, the impact of acinar cell-specific ALDH2 deficiency on AP, including its downstream effects on autophagy and NLRP3 inflammasome activation, has not been investigated. Further investigations are therefore warranted to explore the potential of ALDH2 as a preventive or therapeutic agent for AP.

As an important intracellular physiological process, autophagy can eliminate damaged proteins and organelles, achieve metabolic and energy renewal, thereby maintaining cellular homeostasis[16]. Accumulating evidence has indicated that efficient autophagy exerts anti-inflammatory and protective effects in various diseases, including inflammatory bowel disease and atherosclerosis[17,18]. In the specific setting of AP, the accumulation of autophagic vacuoles in pancreatic acinar cells serves as a hallmark feature in both experimental models and human disease[19]. Deficiencies in key proteins involved in the efficient autophagy pathway, such as autophagy-related protein 7 and lysosome associated membrane protein 2, have been shown to contribute to the onset and progression of pancreatitis, indicating an anti-inflammatory protective role of autophagy in AP[20,21]. Therefore, maintaining stable autophagic flux in acinar cells has been proposed as a potential protective mechanism against AP.

Notably, a functional interplay between ALDH2, autophagy, and the NLRP3 inflammasome has been implicated in other disease models. ALDH2 has been reported to promote autophagic flux in contexts such as hepatic I/R injury and acute kidney injury[22,23]. Furthermore, autophagy has been shown to suppress organelle stress-induced activation of the NLRP3 inflammasome[24]. However, the mechanistic relationship among ALDH2, autophagy, and NLRP3 inflammasome in AP pathogenesis remains largely unexplored. Therefore, in the present study, using in vivo murine models, a clinically relevant porcine model, together with in vitro studies, we hypothesized that deficiency of ALDH2 in acinar cells further exacerbated AP by aggravating autophagic substrate accumulation and enhancing NLRP3 inflammasome activation. We sought to elucidate the mechanism underlying the cyto-protective role of ALDH2 in pancreatic acinar cells, with a focus on its interplay with autophagy and the inflammatory immune response, aiming to provide a novel therapeutic rationale for AP.

MATERIALS AND METHODS

Animal care and use

Bama miniature pigs (3 in AP group and 3 in control group) weighing approximately 25-30 kg were used in this study. All pigs were healthy and maintained under standard laboratory conditions. C57BL/6 mice (6-8 weeks old, male) were obtained from SPF (Beijing) Biotechnology Co., Ltd (Beijing, China). Pancreatic acinar cell-specific ALDH2 knockout (Aldh2^fl/flCela1-Cre) mice and the wild-type (Aldh2^+/+) mice were acquired from Cyagen Biosciences Inc. (Suzhou, Jiangsu Province, China) using the Cre-Lox system. All mice were housed under standard laboratory conditions (12-hour light/dark cycle, 22 ± 2 °C, 50% ± 10% humidity) with free access to water and standard rodent chow. All animal experiments were approved by the Ethics Committee for Animal Experimentation of the Chinese PLA General Hospital and were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

Induction of AP experimental model

The murine AP model was accomplished via seven consecutive intraperitoneal injections of caerulein (Sigma, St. Louis, MO, United States), each at a dose of 50 μg/kg, with a one-hour interval between injections. Tissue harvesting was conducted one hour after the final injection (Supplementary Figure 1A). For the porcine AP model, pigs were fasted for 24 hours and deprived of water for 6 hours prior to the procedure. General anesthesia was induced by intravenous sodium thiamylal (20 mg/kg) and mechanical ventilation was maintained throughout the procedure. The pigs were positioned in the left lateral decubitus position, and an endoscope was inserted into the duodenum and advanced to the region of the pancreatic duct orifice. Under guidewire assistance, selective cannulation of the pancreatic duct was achieved, followed by slow infusion of iohexol (10 mL, Omnipaque) via the catheter. In the control group, cannulation of the pancreatic duct was not attempted.

Serum assay

Venous blood samples were obtained from pigs at baseline and 24 hours post-modeling. Blood samples were collected by removing the eyeballs from the sacrificed mice. Then the blood samples were centrifuged (3000 g, 10 minutes, 4 °C) to obtain serum. Serum amylase and lipase activities were quantified using commercial assay kits (Beijing Beijian & Xin Chuangyuan Biotechnology Co., Ltd., Beijing, China) following the manufacturer's protocols. Absorbance was measured at specific wavelength (405 nm for amylase, 550 nm for lipase) using a microplate reader. Enzyme activities were expressed as U/L.

Histological analysis

The pancreatic tissue was fixed in 4% paraformaldehyde for 24 hours, followed by paraffin embedding and sectioning for hematoxylin and eosin (HE) staining. The severity of pancreatic inflammation was scored based on previously reported criteria in the literature, which primarily included four aspects: Edema, inflammatory cell infiltration, vacuolization, and necrosis, each graded on a scale of 0 to 4[25].

Measurement of ALDH2 enzymatic activity

Tissue ALDH2 enzymatic activity was measured using a commercial assay kit (#BC5510, Solarbio) according to the manufacturer’s protocol. The assay quantifies ALDH2 activity by detecting the increase in absorbance at 340 nm, which reflects the rate of NAD⁺ reduction to NADH during the ALDH2-catalyzed oxidation of acetaldehyde to acetate.

RNA sequencing

Total RNA was extracted from pancreatic tissues of C57BL/6 mice (3 in AP group and 3 in control group) using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s protocol. The sequencing was carried out on an Illumina sequencing platform. Differential expression analysis was conducted using DESeq2. Genes with |log2 fold change| (|log2FC|) ≥ 0.585 and adjusted P value < 0.05 were considered as differentially expressed genes (DEGs) and were visualized using a volcano plot. Gene Ontology (GO) enrichment analysis was performed to identify significantly enriched biological processes, molecular functions, and cellular components among DEGs using the GOseq R package. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted to explore enriched signaling pathways using clusterProfiler R package. Gene Set Enrichment Analysis (GSEA) was carried out using the GSEA software (v4.2.3). |Normalized enrichment score| (|NES|) ≥ 1, nominal P value < 0.05, and FDR Q value < 0.25 were applied as significance thresholds.

Cell siRNA transfection and treatment

The murine pancreatic acinar cell line MPC-83 (Shanghai Biowing Biotechnology Application Co., Ltd, Shanghai, China) was cultured in Minimum Essential Medium (Biosharp, Beijing, China) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, United States) and 1% penicillin-streptomycin at 37 °C in a 100% humidified atmosphere containing 5% CO₂. MPC-83 cells were transfected with ALDH2-small interfering RNA and NC-siRNA employing Lipofectamine RNAiMAX transfection reagent (Invitrogen) following the manufacturer’s protocol. Briefly, cells at 60%-70% confluency were transfected with 30 nM siRNA complexed with Lipofectamine in Opti-MEM (Invitrogen) for 6 hours. After replacing with complete medium, cells were cultured for an additional 48 hours. The sequences of the siALDH2 are listed as follows: Sense, 5’-CCUGAAAUGUCUCCGCUAUUATT-3’ and anti-sense, 5’-UAAUAGCGGAGACAUUUCAGGTT-3’. The in vitro AP model was established by treating the cells with 100 nM caerulein. To investigate the role of autophagy in ALDH2-mediated NLRP3 inflammasome regulation, transfected cells were pretreated with the autophagy inducer rapamycin (MedChemExpress, Monmouth Junction, NJ, United States, 100 nM) for 1 hour, followed by the administration of caerulein (100 nM) to establish the AP model. A reverse validation experiment was performed using the ALDH2 agonist Alda-1 (MedChemExpress, Monmouth Junction, NJ, United States) and the autophagy inhibitor chloroquine (CQ, MedChemExpress, Monmouth Junction, NJ, United States). Cells were pretreated with Alda-1 (20 μM) for 4 hours prior to caerulein stimulation (100 nM), and CQ (25 μM) was added during the final 2 hours of caerulein stimulation.

Enzyme-linked immunosorbent assay

Serum IL-1β (#JL18442 and #JL21874, Jianglai Biology, Shanghai, China), IL-6 (#88-7064, Thermo Scientific, Pittsburgh, PA, United States; #JL21880, Jianglai Biology, Shanghai, China), IL-18 (#JL20253 and #JL19261, Jianglai Biology, Shanghai, China; #RX501071P, Ruixin Biotech, Quanzhou, Fujian Province, China), NLRP3 (#JL10272, Jianglai Biology, Shanghai, China) and ALDH2 (#OKEH01438, Aviva Systems Biology, San Diego, CA, United States) were detected using the enzyme-linked immunosorbent assay (ELISA) kits. The levels of IL-1β (#88-7013, Thermo Scientific, Pittsburgh, PA, United States), IL-6 (#88-7064, Thermo Scientific, Pittsburgh, PA, United States) and IL-18 (#EK218, Lianke Bio, Hangzhou, Zhejiang Province, China) in the culture medium of MPC-83 cells were detected using the ELISA kits. The detection process was carried out in accordance with the manufacturer’s protocols.

Immunofluorescence staining

Paraffin-embedded sections were deparaffinized, rehydrated, and subjected to antigen repair. Cells grown on coverslips were fixed with 4% paraformaldehyde fix solution and permeabilize. After blocking with 5% bovine serum albumin, they were incubated overnight at 4 °C with primary anti-ALDH2 (#ab227021, Abcam, Cambridge, MA, United States, 1:1000; #15310-1-AP, Proteintech, Chicago, IL, United States, 1:500), anti-P62 (#ab211324, Abcam, Cambridge, MA, United States, 1:100; #ab232816-1001, Abcam, Cambridge, MA, United States, 10 μg/mL), anti-LC3B (#ab63817, Abcam, Cambridge, MA, United States, 1 μg/mL; #GTX127375, GeneTex, Irvine, CA, United States, 1:100), anti-NLRP3 (#WL02635, Wanleibio, Shenyang, Liaoning Province, China, 1:200; #19771-1-AP, Proteintech, Chicago, IL, United States, 1:100), anti-ASC (#GB113966, Servicebio, Wuhan, Hubei Province, China, 1:2000; #10500-1-AP, Proteintech, Chicago, IL, United States, 1:50), anti-Caspase-1 (#WLH4550, Wanleibio, Shenyang, Liaoning Province, China, 1:100; #22915-1-AP, Proteintech, Chicago, IL, United States, 1:100), anti-MIST1 (#GB114483, Servicebio, Wuhan, Hubei Province, China, 1:1000) antibodies. Appropriate secondary antibodies were applied for 1 hour at room temperature. Nuclei were counterstained with DAPI (Servicebio, Wuhan, Hubei Province, China). Images were acquired using a fluorescence microscope with consistent settings.

Western blotting

Tissue or cell was lysed in RIPA buffer with protease/phosphatase inhibitors (Beyotime Biotechnology, Shanghai, China), and protein concentrations were determined by bicinchoninic acid protein assay kit (Servicebio, Wuhan, Hubei Province, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels), transferred to PVDF membranes (Millipore, Billerica, MA, United States), blocked with 5% non-fat milk, and probed overnight at 4 °C with primary antibodies against ALDH2 (#ab227021, Abcam, Cambridge, MA, United States, 1:1000; #15310-1-AP, Proteintech, Chicago, IL, United States, 1:2000), P62 (#ab211324, Abcam, Cambridge, MA, United States, 1:1000; #ab232816-1001, Abcam, Cambridge, MA, United States, 0.2 μg/mL), LC3B (#ab63817, Abcam, Cambridge, MA, United States, 1:1000; #GTX127375, GeneTex, Irvine, CA, United States, 1:1000), NLRP3 (#F3229, Selleckchem, Houston, TX, United States, 1:1000; #19771-1-AP, Proteintech, Chicago, IL, United States, 1:1000), ASC (#GB113966, Servicebio, Wuhan, Hubei Province, China, 1:1000; #10500-1-AP, Proteintech, Chicago, IL, United States, 1:1000), pro-Caspase-1 (#ab179515, Abcam, Cambridge, MA, United States, 1:1000; #22915-1-AP, Proteintech, Chicago, IL, United States, 1:2000) and β-actin (#GB15003, Servicebio Wuhan, Hubei Province, China, 1:1000), followed by HRP-conjugated secondary antibodies. Images were acquired using the Amersham Imager 600 (Cytiva, Marlborough, MA, United States), and band intensities were quantified with Image J software.

Transmission electron microscopy

Samples were immediately fixed in cold Transmission electron microscopy (TEM) fixative (Servicebio Wuhan, Hubei Province, China), rinsed in 0.1 M phosphate buffer (pH 7.4), and post-fixed in 1% osmium tetroxide (2 hours, RT, protected from light). After dehydration in a graded ethanol series and acetone, samples were infiltrated with polybed 812 resin (SPI Supplies, West Chester, PA, United States) and polymerized at 60 °C for 48 hours. Ultrathin sections were prepared using a Leica EM UC7 ultramicrotome, double-stained with uranyl acetate and lead citrate, and subsequently examined under a HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, San Diego, CA, United States). Data are presented as the mean ± SD. Between-group comparisons were assessed by two-tailed Student’s t-test, with a P value < 0.05 considered statistically significant. Paired Student’s t-test was performed on serological data obtained from porcine models pre- and post-modeling.

RESULTS

ALDH2 is upregulated with reduced enzymatic activity during AP, alongside dysregulated inflammatory immune responses and autophagy

Through the intraperitoneal injection of caerulein in C57BL/6 mice, the AP model was successfully established, as confirmed by histopathological and serological analyses (Supplementary Figure 1B and C, Supplementary Table 1). Following caerulein treatment, ALDH2 expression was mildly increased, as demonstrated by both western blotting and immunofluorescence staining (Figure 1A and B). However, its enzymatic activity was significantly reduced (P < 0.05, Figure 1C). Statistical analysis confirmed that the increase in ALDH2 expression was significant (P < 0.05, Figure 1D and E). To further explore the pathways enriched with DEGs during AP occurrence, transcriptome sequencing analysis was therefore conducted. A total of 6979 DEGs were identified, including 3522 upregulated and 3457 downregulated genes (Figure 1F). In the GO analysis, the results presented the top five significantly enriched terms for each section (Figure 1G). The AP mice showed significant enrichment in biological processes such as ribonucleoprotein complex biogenesis and mRNA processing. For cellular components, significant enrichment was observed in the actin cytoskeleton and microtubule, while molecular functions were notably enriched in transcription coregulator activity and guanyl nucleotide binding. KEGG analysis indicated that AP was enriched in autophagy pathways and inflammation-related pathways, such as NF-kappa B signaling pathway, TNF signaling pathway, mTOR signaling pathway and MAPK signaling pathway (Figure 1H). GSEA further demonstrated the up-regulation of genes associated with inflammatory immune responses, such as immunoglobulin complex and the T cell receptor complex (Figure 1I). We further assessed the in situ interaction among ALDH2, autophagy, and inflammatory immunity. Triple immunofluorescence co-staining of pancreatic tissue sections demonstrated a marked increase in the co-localization of ALDH2, P62, and NLRP3 in murine AP model compared to the control group (Supplementary Figure 1D). Taken together, these results suggest that despite a reduction in its enzymatic activity, the expression of ALDH2 in pancreatic acinar cells is elevated during AP, and that autophagy and inflammatory immunity also play crucial roles during the disease process.

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.

Occurrence of AP induced ALDH2 upregulation, reduced enzymatic activity, NLRP3 inflammasome activation, and autophagic substrate accumulation in porcine models

To closely mimic the clinical scenario of AP, we utilized a large animal model of AP in pigs. Inflammatory cell infiltration and cellular vacuolization were observed in the pancreatic tissue by HE staining (Figure 2A). The pathological score of the AP group was significantly higher than that of the control group (P < 0.05, Figure 2A, Supplementary Table 2). Concurrently, serum amylase and lipase levels were elevated, along with increased levels of the inflammatory cytokines IL-1β, IL-6, and IL-18 (Figure 2B and C). Consistent with the observations in murine AP models, a comparable upregulation of ALDH2 expression was confirmed in the porcine model of AP with reduced enzymatic activity (P < 0.05, Figure 2D and E). Furthermore, we investigated the expression of the NLRP3 inflammasome in the porcine AP model. The results demonstrated that the induction of AP promoted the activation of the NLRP3 inflammasome compared with the control group (Figure 2F and G). Regarding the status of autophagy during AP, the results revealed that compared with the control group, the expressions of P62 and LC3B-II were significantly increased in the AP group (P < 0.05, Figure 2H-J). To further investigate, TEM was employed to examine the intracellular ultrastructure, and the results showed a marked accumulation of autophagic vesicles in the AP group compared to the control group (Figure 2K). Taken together, these data suggest that the induction of AP in porcine models results in an upregulation of ALDH2 along with a relative decrease in its enzymatic activity, the activation of the NLRP3 inflammasome, and the accumulation of autophagic substrates.

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.

Pancreatic acinar cell-specific ALDH2 knockout aggravated the severity of AP and NLRP3 inflammasome activation in murine AP models

To investigate the cell-specific role of ALDH2 in pancreatic acinar cells during AP, we generated pancreatic acinar cell-specific ALDH2 knockout (Aldh2^fl/flCela1-Cre) mice. A significant reduction in ALDH2 expression was observed in Aldh2^fl/flCela1-Cre mice compared to their wild-type group (P < 0.05, Figure 3A). AP models were successfully established by intraperitoneal caerulein injection in both Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice. Histological HE staining results indicated that ALDH2 deficiency in pancreatic acinar cells exacerbated the inflammatory responses (Figure 3B, Supplementary Table 3). Serum analysis yielded consistent findings, demonstrating elevated serum amylase and lipase levels, as well as increased levels of serum IL-1β, IL-6 and IL-18 (Figure 3C). Subsequently, we examined the impact of ALDH2 deficiency on the NLRP3 inflammasome within pancreatic tissues. We found that following AP induction, the NLRP3 inflammasome was further activated in Aldh2^fl/flCela1-Cre mice compared to Aldh2^+/+ mice (Figure 3D and E). Taken together, these data suggest that ALDH2 deficiency in pancreatic acinar cells is associated with aggravated pancreatitis severity and enhanced NLRP3 inflammasome activation in murine AP models.

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.

Pancreatic acinar cell-specific ALDH2 knockout exacerbated the accumulation of autophagic substrates in murine AP models

In the AP model of Aldh2^fl/flCela1-Cre mice and Aldh2^+/+ mice, we further investigated the intracellular autophagy. Compared to the Aldh2^+/+ mice, Aldh2^fl/flCela1-Cre mice exhibited further elevated levels of P62 and LC3B-II in acinar cells following AP induction (Figure 4A and B). TEM analysis also revealed a greater accumulation of autophagic vesicles in Aldh2^fl/flCela1-Cre mice compared to the control group (Figure 4C). Taken together, these data suggest that specific deletion of ALDH2 in pancreatic acinar cells worsens the accumulation of autophagic substrates in murine AP models.

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.

ALDH2 deficiency aggravated NLRP3 inflammasome activation in caerulein-stimulated pancreatic acinar cells

We then conducted in vitro investigation in pancreatic cells. Pancreatic acinar cell MPC-83 ALDH2 knockdown cell model was successfully constructed using siRNA (Figure 5A). After the stimulation with caerulein, cell supernatant culture medium ELISA results demonstrated that the levels of inflammatory cytokines IL-1β, IL-6 and IL-18 were significantly elevated, and the knockdown of ALDH2 further promoted the production of these inflammatory cytokines (P < 0.05, Figure 5B-D). Consistent with this trend, the onset of AP induced NLRP3 inflammasome activation, which was further potentiated by ALDH2 deficiency (Figure 5E-I). Taken together, these results indicate that the knockdown of ALDH2 leads to exacerbated NLRP3 inflammasome activation following caerulein stimulation in pancreatic acinar cells.

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.

ALDH2 deficiency exacerbated the accumulation of autophagic substrates in caerulein-stimulated pancreatic acinar cells

By knocking down ALDH2 and establishing in vitro cellular AP models, we further investigated the autophagy-related changes. During the onset of AP, the protein levels of P62 and LC3B-II were increased. ALDH2 knockdown further exacerbated the accumulation of these autophagic substrates (P < 0.05, Figure 6A-F). Furthermore, TEM analysis revealed that ALDH2 deficiency led to a greater accumulation of autophagic vesicles, which aligns with the aforementioned findings (Figure 6G and H). Taken together, these results indicate that the deficiency of ALDH2 leads to exacerbated accumulation of autophagic substrates following caerulein stimulation in pancreatic acinar cells.

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.

ALDH2 deficiency attenuated rapamycin-induced NLRP3 inflammasome inhibition in caerulein-stimulated pancreatic acinar cells

To further investigate whether ALDH2 modulates the NLRP3 inflammasome through autophagy and affects the severity of AP, we applied rapamycin while knocking down ALDH2 in the in vitro AP model. The ELISA analysis of the supernatant culture medium revealed that rapamycin reduced the secretion of inflammatory cytokines IL-1β, IL-6, and IL-18, whereas ALDH2 knockdown promoted their release during the onset of AP. Furthermore, ALDH2 knockdown partially attenuated the protective effects of rapamycin (P < 0.05, Figure 7A-C). We then investigate the expression of the NLRP3 inflammasome. The results demonstrated that in AP, the autophagy inducer rapamycin reduced the activation of the NLRP3 inflammasome. However, this suppressive effect could be attenuated by pancreatic acinar cell-specific ALDH2 knockdown (Figure 7D-H). Taken together, these results indicate that in caerulein-stimulated pancreatic acinar cells, ALDH2 knockdown impairs rapamycin-mediated inhibition of the NLRP3 inflammasome.

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.

Alda-1 reduced NLRP3 inflammasome components in an autophagy-dependent manner

In the in vitro AP model, we further performed a reverse validation experiment using the ALDH2 agonist Alda-1, combined with an intervention using the autophagy inhibitor CQ. In caerulein-stimulated pancreatic acinar cells, Alda-1 treatment reduced the protein levels of NLRP3, ASC, and caspase-1 compared with the control group. Furthermore, CQ co-treatment reversed the Alda-1-induced reductions in the expression levels of NLRP3 inflammasome components (P < 0.05, Figure 8A-D). In addition, we examined the protein levels of the autophagy markers LC3B-II and P62. Compared with the control group, Alda-1 treatment decreased both LC3B-II and P62 protein levels, whereas CQ co-treatment significantly increased the levels of both proteins (P < 0.05, Figure 8E-G). Taken together, these results indicate that in the in vitro AP model, pharmacological activation of ALDH2 by Alda-1 reduces NLRP3 inflammasome components in an autophagy-dependent manner, as evidenced by chloroquine reversal.

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.

DISCUSSION

The effective management of AP is crucial, as it directly determines patient survival, influences complication rates, and affects long-term quality of life. Despite numerous studies on AP, there is still a lack of effective strategies for its prevention and treatment. In this study, we demonstrated that ALDH2 in pancreatic acinar cells exerted a significant protective effect against the onset and progression of AP. Through a series of in vivo and in vitro experiments, we found that ALDH2 deficiency in pancreatic acinar cells exacerbated the accumulation of autophagic substrates and the activation of NLRP3 inflammasome during AP. Using pharmacological induction of autophagy, we found that ALDH2 deficiency attenuated the suppressive effect of autophagy inducer on NLRP3 inflammasome activation. Conversely, pharmacological activation of ALDH2 reduced the protein levels of NLRP3 inflammasome components, and this effect was reversed by the autophagy inhibitor chloroquine, indicating that ALDH2 reduces these components in an autophagy-dependent manner. These findings further suggested that the presence of ALDH2 in pancreatic acinar cells mitigated inflammatory responses in AP by suppressing NLRP3 inflammasome activation through autophagy-related mechanisms.

ALDH2, a pivotal mitochondrial enzyme within the ALDH family, exists as a homotetrameric complex localized to the mitochondrial matrix. It primarily functions to catalyze the oxidation of toxic aldehydes into their less reactive carboxylic acid derivatives, thereby playing a critical role in cellular detoxification and metabolic homeostasis[26]. During the onset of AP, ALDH2 enzyme activity decreased, even though its protein levels increased relatively as a compensatory response. The observed impairment in enzyme activity was consistent with other conditions such as cerebral I/R injury and myocardial injury[10,11]. Regarding the clinical data, we conducted a preliminary exploratory study (Ethics approval No. S2025-046-01), and the results were presented in Supplementary Table 4. Serum levels of ALDH2, NLRP3, and IL-18 were measured before and after endoscopic retrograde cholangiopancreatography (ERCP) in patients who developed post-ERCP pancreatitis (PEP) and in matched controls without PEP. While the small sample size precluded definitive statistical conclusions, the results suggested a trend of increased serum ALDH2 levels in patients with PEP, whereas levels remained stable or slightly decreased in non-PEP controls. Similar trends were observed for NLRP3 and IL-18. These findings preliminarily indicate that ALDH2 may be further upregulated under inflammatory stress, consistent with our animal model results. This observation aligns with a previous transcriptomic study of AP patients, where ALDH2 was identified among a set of upregulated genes in peripheral blood[27].

A previous study has explored the therapeutic potential of Alda-1, a small-molecule activator of ALDH2, in the context of AP. The researchers established AP models in both C57BL/6J mice and rat pancreatic acinar cells using caerulein hyperstimulation. By examining markers including malondialdehyde, 4-hydroxynonenal, B-cell lymphoma-2, BCL2 associated x, and cleaved caspase-3, they demonstrated that Alda-1 exerted protective effects in AP by attenuating lipid peroxidation and reducing apoptosis[15]. Nevertheless, the study omitted the analysis of the inflammatory and immunological mechanisms central to AP pathogenesis, and failed to identify the specific cell types mediating the in vivo effects. In the present study, we utilized a murine AP model for transcriptome analysis of pancreatic tissue, which revealed significant engagement of inflammatory immune pathways during AP progression. These findings were further corroborated in porcine AP models, where activated NLRP3 inflammasome and concomitant increases in pro-inflammatory cytokines secretion were observed. In addition to using siRNA-mediated ALDH2 knockdown in pancreatic acinar cells, we generated pancreatic acinar cell-specific ALDH2 knockout mice. Both models demonstrated that ALDH2 deficiency in pancreatic acinar cells exacerbated NLRP3 inflammasome activation and amplified the inflammatory responses in AP.

Autophagy is an evolutionarily conserved, self-degradative cytoprotective mechanism that facilitates the delivery of dysfunctional cellular components, such as damaged organelles and protein aggregates, to lysosomes for degradation and subsequent recycling of macromolecules. This process plays a critical role in maintaining cellular homeostasis, mitigating stress, and preventing the accumulation of toxic substances[28,29]. Intact autophagic function confers essential protective effects against inflammatory disorders. For example, autophagy induction mitigates the activation of NLRP3 inflammasome and excessive in inflammation during intestinal I/R injury[30]. Consistently, promoting autophagy-mediated inactivation of the NLRP3 inflammasome also attenuates intestinal injury in experimental colitis[31]. This autophagy-mediated suppression of the NLRP3 inflammasome has also been observed in lipopolysaccharide-induced pneumonia and gouty arthritis[32,33]. As previously reviewed by our group, impaired autophagy plays a critical pathogenic role in the initiation and progression of AP[29]. Impaired autophagic flux could lead to a marked upregulation of NLRP3 inflammasome activation and IL-1β production within the pancreatic tissue[34]. Consistent with previous studies, in our study, transcriptomic analysis of the murine AP models indicated involvement of the autophagic pathway in AP pathogenesis. This was further corroborated in porcine AP models, which exhibited increased expression of P62 and LC3B-II, along with an accumulation of autophagic vesicles in pancreatic tissue, a pattern consistent with altered autophagic flux. Using both in vivo and in vitro models of acinar cell-specific ALDH2 knockdown, we found that ALDH2 deficiency was associated with more pronounced alterations in autophagic flux during AP. Moreover, pharmacological induction of autophagy attenuated NLRP3 inflammasome activation and mitigated pancreatic inflammation, which was consistent with the previous study[35]. However, this protective effect could be attenuated by ALDH2 deficiency in pancreatic acinar cells. Conversely, ALDH2 activation by Alda-1 decreased NLRP3 inflammasome components, an effect reversed by chloroquine, further supporting that this reduction is autophagy-dependent. These results indicate that intact autophagic function contributes to the anti-inflammatory protective mechanisms associated with ALDH2 in pancreatic acinar cells during AP.

In the present study, in addition to employing murine AP models to investigate fundamental mechanisms at the cellular and molecular level, we also utilized porcine models of AP that could mimic clinically relevant PEP[36]. The porcine AP model provided translational support, while detailed mechanistic conclusions were drawn from the murine and cellular models. Such dual-model strategy allowed us to bridge mechanistic insights from controlled rodent studies with pathophysiological features in a large-animal setting that closely resembled clinical conditions. Furthermore, we generated pancreatic acinar cell-specific ALDH2 knockout mice (Aldh2^fl/flCela1-Cre) to investigate the role of ALDH2 in a cell-type-targeted manner. We believe that the findings derived from these models may provide a foundation for future therapeutic exploration in AP.

The present study extends prior work on ALDH2 in AP in several respects. First, while earlier research has largely focused on its anti-apoptotic effects via lipid peroxidation, our findings suggest a previously unrecognized mechanism by which ALDH2 contributes to the regulation of NLRP3 inflammation through autophagy in AP. Second, our study employs acinar cell-specific ALDH2 deficiency, providing cell-type-specific mechanistic insights. Finally, the use of autophagy induction and inhibition experiments provides functional support for ALDH2-mediated reduction of inflammasome components in an autophagy-dependent manner.

Nevertheless, it should be noted that this study has several limitations. First, the sample size for the porcine AP model and some immunofluorescence analyses was relatively small, which may limit the generalizability of these findings. Future studies with larger sample sizes are warranted to further validate the translational relevance of ALDH2 and its related pathways in AP. Second, the clinical sample size is limited, and the relationship between ALDH2 gene polymorphisms and AP remains insufficiently understood, although a previous study suggested that the ALDH2*2 genotype may reduce the risk of alcohol-induced AP[37]. Future large-scale prospective studies are needed to integrate ALDH2 genotyping, longitudinal protein expression measurements, and inflammatory biomarker profiling, so as to clarify trends and associations among these factors in AP. Third, the in vitro experiments were conducted using an immortalized pancreatic acinar cell line, which may not fully recapitulate the behavior of primary acinar cells. In addition, the pharmacological experiments were performed only in vitro, and their in vivo relevance remains to be further validated. Nonetheless, the key mechanistic conclusions are supported by the in vivo genetic evidence from acinar cell-specific ALDH2 knockout mice. Given that pancreatic acinar cells represent the predominant cellular population in the pancreas, this study specifically centered on elucidating the role and mechanistic actions of ALDH2 in this cell type. The contribution of immune cell ALDH2 to the AP phenotype therefore remains to be determined. Fourth, while we utilized pancreatic acinar cell-specific ALDH2 knockout mice, financial and practical constraints precluded the utilization of ALDH2 overexpression models, which will be addressed in subsequent work. Finally, the role of ALDH2 in alcoholic pancreatitis, consistent with its metabolic function, remains an important unanswered question beyond the scope of the current caerulein-induced AP models. Overall, additional experiments are still required to address these limitations.

CONCLUSION

This study identified the essential protective role of ALDH2 in pancreatic acinar cells against AP in both in vivo and in vitro models. The protective effect of ALDH2 in pancreatic acinar cells is closely linked to its involvement in the regulation of autophagy, which contributes to the attenuation of NLRP3 inflammasome activation. We believe that these findings may provide a foundation for future investigation into the therapeutic relevance of ALDH2 in AP.

ACKNOWLEDGEMENTS

We thank our peer reviewers for the insightful and thorough suggestions on this manuscript.