Inhibition of PTP1B Through NADH Dehydrogenase Activity Reduces Acute Pancreatic Acinar Cell Pyroptosis by Inhibiting the ROS/TXNIP/NLRP3 Pathway.

Following CAE treatment, the protein expression of PTP1B was significantly elevated in MPC83 cells (Fig.  1 F). CAE treatment significantly decreased the proliferative activity of MPC83 cells (Fig.  1 A), and notably elevated the levels of IL-1β and IL-18 cytokines (Fig.  1 B–C). PI staining revealed a significant increase in the number of positive cells in the CAE group (Fig.  1 D). GSDMD immunofluorescence (Fig.  1 E) and Western blot analysis showed upregulation of AP-1, ASC, cleaved-Caspase-4, cleaved-Caspase-5, cleaved-Caspase-11, cleaved-caspase-1, and NLRP3 in the CAE-treated AP model group (Fig.  1 F). These results suggest that CAE induces PTP1B expression and promotes inflammation and pyroptosis in MPC83 cells. Fig. 1 CAE caused increased levels of inflammation and pyroptosis in MPC 83 cells. A The proliferative activity of MPC 88 cells was measured by CCK-8; B , C levels of cytokines IL-18 ( B ) and IL-1β ( C ) were measured by ELISA; D , E PI staining of cells by fluorescence microscope (Scale = 10㎛) ( D ) and PI Staining Quantification ( E ); F fluorescence intensity and cell distribution of GSDMD by immunofluorescence (Scale = 10㎛); G–O : Level of PTP 1 B and pyroptosis-related proteins were measured by Western blot. ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001 CAE caused increased levels of inflammation and pyroptosis in MPC 83 cells. A The proliferative activity of MPC 88 cells was measured by CCK-8; B , C levels of cytokines IL-18 ( B ) and IL-1β ( C ) were measured by ELISA; D , E PI staining of cells by fluorescence microscope (Scale = 10㎛) ( D ) and PI Staining Quantification ( E ); F fluorescence intensity and cell distribution of GSDMD by immunofluorescence (Scale = 10㎛); G–O : Level of PTP 1 B and pyroptosis-related proteins were measured by Western blot. ** P  < 0.01; *** P  < 0.001; **** P  < 0.0001 Treatment with PTP1B inhibitors reduced IL-1 β and IL-18 levels in AP cells (Fig.  2 A, B), and fluorescence microscopy revealed a decrease in PI-positive cells (Fig.  2 C, D). Immunofluorescence analysis showed reduced fluorescence intensity of GSDMD distribution in the cytoplasm compared to the AP model group (Fig.  2 E). Western blot analysis demonstrated a significant decrease in the expression and activation of pyroptosis-related proteins, including AP-1, ASC, cleaved-caspase-4, cleaved-caspase-5, cleaved-caspase-11, cleaved-caspase-1, and NLRP3 (Fig.  2 F–N). The results indicate that inhibiting PTP1B activity can effectively mitigates CAE-induced pyroptosis in AP cells. Fig. 2 Inhibition of PTP1B reduces inflammation and necrosis of pancreatic acinar cells. A , B ELISA detection of the cytokine IL-1β ( A ) and the level of IL-18 ( B ); C , D , PI staining of cells under a fluorescence microscope (Scale = 10㎛) ( C ) and PI Staining Quantification ( D ); E Immunofluorescence detection of the fluorescence intensity and cell distribution of GSDMD (Scale = 10㎛); F–N Level of PTP1B and pyroptosis-related proteins were measured by Western blot. ns: No statistical difference; * P  < 0.05; ** P  < 0.01; *** P  < 0.001 Inhibition of PTP1B reduces inflammation and necrosis of pancreatic acinar cells. A , B ELISA detection of the cytokine IL-1β ( A ) and the level of IL-18 ( B ); C , D , PI staining of cells under a fluorescence microscope (Scale = 10㎛) ( C ) and PI Staining Quantification ( D ); E Immunofluorescence detection of the fluorescence intensity and cell distribution of GSDMD (Scale = 10㎛); F–N Level of PTP1B and pyroptosis-related proteins were measured by Western blot. ns: No statistical difference; * P  < 0.05; ** P  < 0.01; *** P  < 0.001 To explore the role of PTP1B in modulating ROS production via NADH dehydrogenase activity, we treated CAE-induced AP cells with a series of inhibitors. Cells were treated with the PTP1B inhibitor MSI-1436, the mitochondrial complex I inhibitor Rotenone, and the ROS scavenger N-Acetyl- l -cysteine (NAC). CAE induction significantly suppressed mitochondrial complex I activity, as evidenced by experimental results. Notably, MSI-1436 and NAC treatments significantly enhanced mitochondrial complex I activity compared to the CAE group. Conversely, co-treatment with MSI-1436 and Rotenone significantly reduced mitochondrial complex I activity compared to MSI-1436 treatment alone (Fig.  3 A). Further analysis revealed that CAE induction increased ROS levels within the cells. Treatment with MSI-1436 and NAC significantly reduced ROS levels, while co-incubation with MSI-1436 and Rotenone increased ROS levels compared to the MSI-1436 alone (Fig.  3 B). Mitochondrial membrane potential assays showed that MSI-1436 and NAC treatments prevented the red-to-green shift in mitochondrial fluorescence, suggesting mitigation of CAE-induced mitochondrial damage (Fig.  3 C). Collectively, these findings suggest that PTP1B inhibition promotes ROS production by enhancing NADH dehydrogenase (mitochondrial complex I) activity, identifying PTP1B as a potential therapeutic target for regulating oxidative stress and pyroptosis in pancreatic cells. Fig. 3 Inhibition of PTP1B inhibits ROS generation and AP cell apoptosis by restoring mitochondrial complex I activity. A MitoCheck Complex I Activity Assay Kit for detecting the activity of mitochondrial complex I; B ROS assay kit for detecting cellular ROS levels; C JC-1 assay kit for detecting mitochondrial membrane potential (Scale = 10㎛); D , E ELISA detection of the levels of IL-18 ( D ) and IL-1β ( E ); F , G , PI staining under a fluorescence microscope (Scale = 10㎛) ( F ) and PI Staining Quantification ( G ); H Immunofluorescence detection of the fluorescence intensity and cell distribution of GSDMD (Scale = 10㎛); I–Q , Level of PTP 1 B and pyroptosis-related proteins were measured by Western blot. ns: No statistical difference; * P  < 0.05; ** P  < 0.01; *** P  < 0.001 Inhibition of PTP1B inhibits ROS generation and AP cell apoptosis by restoring mitochondrial complex I activity. A MitoCheck Complex I Activity Assay Kit for detecting the activity of mitochondrial complex I; B ROS assay kit for detecting cellular ROS levels; C JC-1 assay kit for detecting mitochondrial membrane potential (Scale = 10㎛); D , E ELISA detection of the levels of IL-18 ( D ) and IL-1β ( E ); F , G , PI staining under a fluorescence microscope (Scale = 10㎛) ( F ) and PI Staining Quantification ( G ); H Immunofluorescence detection of the fluorescence intensity and cell distribution of GSDMD (Scale = 10㎛); I–Q , Level of PTP 1 B and pyroptosis-related proteins were measured by Western blot. ns: No statistical difference; * P  < 0.05; ** P  < 0.01; *** P  < 0.001 Further investigation of cellular pyroptosis revealed that treatment with MSI-1436, Rotenone, and NAC significantly inhibited the CAE-induced upregulation of IL-18 and IL-1β expression (Fig.  3 D, E). Additionally, PI staining showed that MSI-1436 and NAC treatments reduced apoptosis levels compared to the CAE group, while Rotenone treatment increased apoptosis relative to the MSI-1436 group (Fig.  3 F, G). Immunofluorescence and Western blot analysis of GSDMD showed trends consistent with PI staining. Expression levels of pyroptosis-related proteins—including GSDMD, AP-1, ASC, cleaved-caspase-4/5/11, and cleaved-caspase-1, and NLRP3—were significantly reduced following MSI-1436 and NAC treatment compared to the CAE group. Conversely, co-treatment with MSI-1436 and Rotenone significantly increased pyroptosis-related protein expression compared to MSI-1436 treatment alone (Fig.  3 H–Q). Notably, the ROS scavenger NAC exerted the strongest inhibitory effect on CAE-induced pyroptosis among all treatment groups, suggesting that PTP1B promotes ROS generation by inhibiting mitochondrial complex I activity, thereby contributing to pyroptosis in AP. To further elucidate the role of the TXNIP/NLRP3 pathway as a downstream mediator of ROS-induced pyroptosis in AP cells, the TXNIP inhibitor Ruscogenin was employed. Experimental results showed that CAE treatment significantly reduced mitochondrial complex I activity compared to controls, whereas Ruscogenin treatment restored mitochondrial complex I activity (Fig.  4 A). Moreover, Ruscogenin significantly reduced CAE-induced ROS accumulation (Fig.  4 B) and mitigated mitochondrial damage (Fig.  4 C). Importantly, Ruscogenin treatment markedly decreased CAE-induced pyroptosis. Specifically, Ruscogenin treatment significantly reduced in IL-1β and IL-18 levels (Fig.  4 D, E), decreased the number of PI-positive cells, and downregulated GSDMD expression (Fig.  4 F–H). Furthermore, Ruscogenin significantly downregulated the expression of pyroptosis-related proteins in AP cells, while PTP1B expression remained unchanged (Fig.  4 I–Q). These results suggest that PTP1B promotes ROS accumulation by inhibiting mitochondrial complex I activity, thereby activating the TXNIP/NLRP3 pathway and inducing pyroptosis in pancreatic acinar cells. Fig. 4 Inhibition of PTP1B inhibits ROS activation by restoring mitochondrial complex I activity and inhibits pancreatic acinar cell pyroptosis through the TXNIP/NLRP3 pathway. A MitoCheck Complex I Activity Assay Kit for detecting the activity of mitochondrial complex I; B ROS assay kit for detecting cellular ROS levels (Scale = 10㎛); C JC-1 assay kit for detecting mitochondrial membrane potential (Scale = 10 ㎛); D , E ELISA detection of the levels of IL-18 ( D ) and IL-1β ( E ); F , G PI staining under a fluorescence microscope (Scale = 10㎛) ( F ) and PI Staining Quantification ( G ); H Immunofluorescence detection of the fluorescence intensity and cell distribution of GSDMD (Scale = 10㎛); I–Q Level of PTP 1 B and pyroptosis-related proteins were measured by Western blot. ns: No statistical difference; * P  < 0.05; ** P  < 0.01; *** P  < 0.001 Inhibition of PTP1B inhibits ROS activation by restoring mitochondrial complex I activity and inhibits pancreatic acinar cell pyroptosis through the TXNIP/NLRP3 pathway. A MitoCheck Complex I Activity Assay Kit for detecting the activity of mitochondrial complex I; B ROS assay kit for detecting cellular ROS levels (Scale = 10㎛); C JC-1 assay kit for detecting mitochondrial membrane potential (Scale = 10 ㎛); D , E ELISA detection of the levels of IL-18 ( D ) and IL-1β ( E ); F , G PI staining under a fluorescence microscope (Scale = 10㎛) ( F ) and PI Staining Quantification ( G ); H Immunofluorescence detection of the fluorescence intensity and cell distribution of GSDMD (Scale = 10㎛); I–Q Level of PTP 1 B and pyroptosis-related proteins were measured by Western blot. ns: No statistical difference; * P  < 0.05; ** P  < 0.01; *** P  < 0.001

The pancreatic acinar cells MPC83 cells (BNCC340245) was used to construct an AP model by treating with 10 μmol/L CAE for 12 h. Then process according to groupings, using an inhibitor of PTP1B (MSI-1436, 1 μmol/L, purchased from AbMole), an inhibitor of mitochondrial complex I (Rotenone, 10 nmol/L, purchased from Selleck), an inhibitor of TXNIP (Ruscogenin, 10 μmol/L, purchased from Selleck), and a ROS scavenger (N-Acetyl- l -cysteine, 5 mmol/L, purchased from Solarbio) for treatment over 48 h. Then, the cell proliferation ability was tested, and ELISA was used to detect IL-18 and IL-1β in the supernatant of each group of cells. Cell death was detected by propidium iodide (PI) staining, and the expression and distribution of gasdermin (GSDMD) were detected by immunofluorescence. Mitochondrial membrane potential and ROS were detected, and fluorescence microscopy was used for observation and photography. Extract total cell proteins for Western blot detection of pyroptosis-related protein levels. Each group of samples was repeated 3 times. Cell Counting Kit-8 (Beyotime) was used to detect cell proliferation activity. Digest each group of cells with trypsin, resuspend cells in a culture medium containing 10% FBS, and inoculate in 96 well plate × 10 5 cells/well. Incubate the 96 well plate in a 37℃ constant temperature incubator containing 5% CO 2 for 0, 24, 48, 72 h. The 10 μL CCK-8 reagent was added at the corresponding time, continue to cultivate for 2 h, and then use a Microplate reader to detect the absorbance at 450 nm. Inoculate logarithmic growth phase cells into a culture dish, wait for the cells to adhere to the wall, and perform different treatments according to the experimental groups. After 48 h of drug intervention, cells were digested and collected, total protein was extracted, and protein concentration was measured by BCA method. The expression level of cytokines IL-18 and IL-1β in cell supernatant was detected by according to manufacturer's instructions for IL-1 beta Mouse ELISA Kit and IL-18 Mouse ELISA Kit (Thermo Fisher). Collect cells was wash twice in PBS. Fix the cells in 70% ethanol pre-cooled at 4 ℃ for 1 h; Wash twice with PBS, centrifuge at a speed of 850 g in a centrifuge, and discard the supernatant; Add 50 µL RNase storage solution (100 µg/mL), 200 µL of PI working solution (50 µg/mL storage solution, Solarbio) and incubate at room temperature for 15 min; Seal the film after rinsing. Fluorescence microscopy was used to observe DNA at excitation wavelength of 488 nm and emission wavelength of 630 nm. The DNA showed red fluorescence and was photographed for preservation. Take out the cell slides and place them in a used 60 mm cell culture dish, then wash three times with PBS. Remove the supernatant from the plate, add 4% cold paraformaldehyde and fix for 20 min, then wash with PBS three times. Add 0.2% Triton X-100 and react for 10 min. Block the same host serum as the secondary antibody for 30 min. Wash the cells in PBS three times, then incubate overnight with antibodies against GSDMD and PTP1B (Abcam) at 4 °C. After washing the cells with PBS for the three time, incubate the cells in the presence of 4′,6-diamino-2-phenylindole (DAPI) in the dark for 10 min. Observe and save images using confocal laser scanning microscopy, wash off PBS with distilled water, and seal with glycerol for preservation. Protein extraction kit (Thermo Fisher) extracts total cell protein, BCA kit (Thermo Fisher) measures protein concentration, 50 μg/well, 10% SDS-PAGE gel was separated for 1 h, transferred to PVDF membrane, sealed with PBS buffer containing 5% skimmed milk powder for 1 h, added rabbit anti human primary antibody and internal reference β-actin antibody of each target protein, respectively, stayed overnight at 4 °C, rinsed three times with PBST buffer, 5 min each time, added secondary antibody (goat anti rabbit), incubated at 37 °C for 2 h. PBST buffer solution was rinsed three times, ECL was developed, and protein gel imaging system was used for imaging. Image J analyzes the grayscale value of the target protein. Target protein antibodies PTP1B, pro-Caspase-1, cleaved-Caspase-1, pro-Caspase-11/4/5, cleaved Caspase-11/4/5, AP-1, ASC, NLRP3 (Abcam). Remove the culture medium in a 6-well plate of MPC83 cells and add 1 mL of fresh culture medium. Then, evaluate the changes in MMP using the JC-1 assay kit (Beyotime), according to the manufacturer's instructions. Immediately analyze the sample using a fluorescence microscope after the operation is completed. Measure the fluorescence intensity of JC-1 aggregates after excitation at 525 nm and emission at 590 nm (red), as well as the fluorescence intensity of JC-1 monomers after excitation/emission at 490/530 nm (green). The Cell Mitochondria Separation Kit (Beyotime) were isolated Mitochondria from 5 × 10 6 MPC83 samples. The activity of mitochondrial complex I was measured using a trace mitochondrial respiratory chain complex I activity assay kit (Solarbio, China) according to the manufacturer's instructions. Add mitochondrial homogenate to the corresponding reaction buffer. Transfer the reaction mixture to a preheated (30 °C) quartz colorimetric dish and immediately place it in a spectrophotometer. Measure the absorbance of the reaction mixture at 340 nm. Cell ROS levels were detected using the Mito Tracker Red CMXRos assay kit (Beyotime). Collect cells from cell culture and wash them twice with PBS. Afterward, strictly follow the instructions of the reagent kit for operation. Observe and take photos using a fluorescence microscope. All experiments were performed in triplicate and repeated independently three times. Data are presented as mean ± standard error. Statistical analyses were conducted using GraphPad Prism 9.0. Student’s t -test was used for comparisons between two groups, and one-way ANOVA followed by Tukey’s post hoc test was applied for multiple group comparisons. A P  < 0.05 was considered statistically significant differences. Fluorescence data were quantified using ImageJ software.

In summary, our results demonstrate that PTP1B inhibition in pancreatic acinar cells suppresses the ROS/TXNIP/NLRP3 pathway by enhancing NADH dehydrogenase activity, thereby alleviating CAE-induced pyroptosis.

AP is a prevalent and destructive gastrointestinal disorder. AP initially presents as local inflammation, which can progress to systemic inflammatory responses. PTP1B, localized to the endoplasmic reticulum, acts as a critical regulator of signaling pathways involving the epidermal growth factor receptor (EGFR), integrin, and insulin receptors. PTP1B is implicated in multiple inflammatory signaling pathways and is associated with inflammatory mediators, including interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), extracellular signal-regulated kinase (ERK), protein kinase B (PKB/AKT), human epidermal growth factor receptor 2 (HER2), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Studies have shown that inflammation induces PTP1B overexpression, and its alleviates inflammation in various disease models [ 18 ]. PTP1B knockdown prevents ox-LDL-induced inflammatory injury and cellular dysfunction in HUVECs via regulation of the AMPK/SIRT1 signaling pathway [ 19 ]. Moreover, PTP1B deficiency ameliorates experimental colitis in mice by promoting the expansion of myeloid-derived suppressor cells (MDSCs) [ 20 ]. Importantly, PTP1B is also closely linked to pancreatic function, as its deficiency has been associated with glucose intolerance and severe diabetes in murine models. Global PTP1B deficiency has been shown to mitigate severe diabetes caused by the deletion of insulin receptor substrate 2 [ 4 ]. Additionally, PTP1B expression is significantly upregulated during the early stages of AP [ 21 ]. These findings collectively suggest a strong association between PTP1B and AP pathogenesis. In this study, we investigated the role of pancreatic PTP1B in AP using a CAE-induced AP cellular model. Our results demonstrated that PTP1B expression is closely associated with inflammation and focal cell death in AP. Notably, PTP1B inhibition significantly alleviated inflammation and cell death in AP cells. Furthermore, we found that PTP1B-induced cell death was associated with ROS accumulation. ROS accumulation was driven by the inhibition of mitochondrial complex I activity, which subsequently activated the TXNIP/NLRP3 inflammasome pathway and triggered pyroptosis. This study provides preliminary mechanistic insights into the role of PTP1B in the pathophysiology of acute pancreatitis. Mitochondrial damage is a critical pathogenic process in AP [ 22 ]. Mitochondrial dysfunction disrupts energy (ATP) metabolism, enhances ROS production, and triggers oxidative stress. Oxidative stress damages mitochondrial DNA and membranes, leading to impaired mitochondrial function. Notably, ROS accumulation induces pyroptosis by through NLRP3 activation [ 23 ]. Liu et al. demonstrated that CIRP induces excessive ROS accumulation by disrupting mitochondrial function and mitophagy, thereby activating pyroptosis in l -arginine-induced AP [ 22 ]. Moreover, rotenone induces apoptosis by increasing mitochondrial ROS production [ 24 ]. Therefore, AP progression can be mitigated by protecting mitochondrial function to reduce ROS accumulation. The mitochondrial electron transport chain is the primary source of ROS production. Mitochondrial complex I’s NADH dehydrogenase (mitochondrial complex I) is a candidate site for ROS generation. In response to damage, mutations, or an accumulation of NADH due to low ATP demand, mitochondrial respiration is impaired, resulting in an increased NADH/NAD ratio and subsequent ROS formation [ 25 ]. Interfering with NADH dehydrogenase is a critical strategy to mitigate ROS accumulation. In this study, we demonstrated that the PTP1B inhibitor MSI-1436 effectively slowed cellular AP progression, significantly reducing the levels of inflammatory cytokines IL-1β and IL-18, as well as pancreatic acinar cell pyroptosis. Furthermore, MSI-1436 restored NADH dehydrogenase (mitochondrial complex I) activity, thereby reducing mitochondrial ROS production. Further investigations confirmed that PTP1B inhibition alleviated ROS accumulation and reduced cell death by restoring NADH dehydrogenase activity. Our findings confirm the work of Yang et al., demonstrating that knocking down PTP1B reduces reactive oxygen species (ROS) accumulation and consequently alleviates BMSC senescence. This protective effect is achieved through the activation of AMPK-mediated mitochondrial autophagy [ 26 ]. PTP1B inhibition also enhanced mitochondrial function by upregulating SIRT1 expression and decreasing p65 NF-κB phosphorylation, which reduced ROS generation, JNK activation, and apoptosis [ 27 ]. Julia et al. demonstrated that PTP1B localizes to the outer mitochondrial membrane with a peritail anchor, interacting with the mitochondrial EGFR under external stimulation. This interaction may induce mitochondrial dysfunction by modulating oxygen consumption, electron transport chain activity, glucose uptake, lactate production, and the ATP/ADP ratio [ 28 ]. Therefore, PTP1B inhibition in AP may alleviate cell death by suppressing ROS production. Pancreatic acinar cell pyroptosis is a crucial pathological process in acute pancreatitis, though its precise regulatory mechanisms remain unclear [ 29 ]. The NLRP3 inflammasome plays a pivotal role in activating GSDMD and interleukin (IL)−1β, thereby driving the pathological progression of various diseases [ 22 ]. Previous studies have shown that TXNIP mediates NLRP3 activation and contributes to pancreatic acinar cell injury in AP. During AP onset, endoplasmic reticulum stress induces upregulation of TXNIP expression in cells [ 30 ]. This upregulation further activates NLRP3, exacerbating cellular damage. Furthermore, cellular injury during AP progression leads to excessive mitochondrial ROS production, enhancing the TXNIP-NLRP3 interaction and exacerbating inflammation and pyroptosis in AP [ 31 ]. In this study, we observed compromised mitochondrial membrane potential in pancreatic acinar cells following CAE treatment, accompanied by increased ROS accumulation. Simultaneously, NLRP3 and GSDMD expression levels were significantly elevated. In contrast, treatment with Ruscogenin reduced ROS levels and significantly decreased the expression of ASC, cleaved-caspase-1, GSDMD, and other pyroptosis-related proteins. These findings further support the role of ROS accumulation in regulating pyroptotic cell death in AP through the TXNIP/NLRP3 pathway. This study provides preliminary insights into the molecular mechanisms through which inhibition of PTP1B alleviates pyroptosis in pancreatic acinar cells during AP. Specifically, the findings highlight enhanced NADH dehydrogenase activity and the suppression of the ROS/TXNIP/NLRP3 signaling pathway. However, several limitations should be acknowledged. Firstly, the lack of in vivo data limits our understanding the effects of PTP1B inhibition in AP animal models. Furthermore, despite rigorous experimental design, the possibility that pharmacological inhibition of PTP1B may affect additional downstream pathways—such as ROS accumulation and TXNIP/NLRP3 activity—cannot be completely ruled out. In addition, although our results align with previous reports indicating that PTP1B inhibition enhances NADH dehydrogenase activity [ 32 ], direct evidence confirming that PTP1B directly modulates NADH dehydrogenase remains lacking. Therefore, further studies are warranted to elucidate the precise mechanisms through which PTP1B and its pharmacological inhibition influence the onset and progression of AP.

Acute Pancreatitis (AP) is an inflammatory disorder of the pancreas characterized by a high risk of mortality. It represents one of the most complex and clinically challenging abdominal conditions. Despite advances, the pathogenesis of AP remains incompletely understood, and no specific pharmacological therapy is currently available to halt its progression [ 1 ]. Therefore, elucidating the etiology and identifying regulatory targets are crucial for improving prognosis and developing targeted therapies. Protein Tyrosine Phosphatase 1B (PTP1B) is a ubiquitously expressed non-receptor phosphatase and a key regulator of signaling pathways involving epidermal growth factor receptor, integrins, and insulin receptors. Emerging evidence suggests a functional association between PTP1B and pancreatic physiology. In pancreatic tumors, elevated PTP1B expression is positively correlated with tumor progression and distant metastasis. Furthermore, inhibiting of PTP1B has been shown to enhance pancreatic cell proliferation and confer protection against cellular injury, thereby delaying diabetes progression [ 2 , 3 ]. Notably, PTP1B expression is significantly upregulated during the early stages of CAE-induced AP [ 4 ]. In addition, inhibition of pancreatic T-cell PTP expression alleviates CAE-induced AP in murine models [ 5 ]. These findings implicate PTP1B as a key player in pancreatic function; however, its precise role in AP pathogenesis remains unclear. Oxidative stress-induced pyroptosis is a major contributing factor in AP. During AP progression, elevated levels of inflammation and ROS activate GSDMD and NLRP3, triggering pyroptosis events that exacerbate disease severity [ 6 ]. Excessive ROS has been shown to activate NLRP3/IL-1β axis via the thioredoxin (TRX)/TXNIP pathway, thereby promoting inflammation and pyroptosis [ 7 , 8 ]. Significant ROS accumulation has been observed during AP [ 9 ], and its reduction via inhibition of NADH dehydrogenase activity has been shown to prevent pancreatic cell pyroptosis in AP mouse models [ 10 , 11 ]. Moreover, ROS accumulation also serves as a major inducer of endoplasmic reticulum (ER) stress and GSDMD-mediated pyroptosis. Multiple studies have have demonstrated a strong association between elevated GSDMD expression and AP [ 12 ]. GSDMD-mediated pyroptosis promotes the release of inflammatory cytokines, triggering systemic inflammatory responses and intestinal mucosal barrier disruption in murine models of severe acute pancreatitis (SAP) [ 13 ]. Inhibition of GSDMD mitigates AP progression by reducing ER stress-induced pancreatic necrosis and systemic inflammation [ 14 ]. These findings underscore ROS accumulation as a central driver of inflammation and pyroptosis, highlighting ROS inhibition as a promising therapeutic strategy for AP. Importantly, PTP1B has been implicated in ROS production under oxidative stress conditions. Selective PTP1B inhibitors have been shown to reduce ROS and NO accumulation in adipocyte-derived progenitor cells, thereby attenuating apoptosis and oxidative stress [ 15 ]. Moreover, targeting the PTP1B/RNF213/CYCLD/SPATA axis effectively controls ROS-induced pyroptosis in hypoxic brain tumors models [ 16 ]. These findings suggest a close interplay between PTP1B-mediated ROS regulation and cell death pathways. However, limited evidence exists regarding the role of PTP1B in regulating ROS production and clearance during AP pathogenesis [ 17 ]. Therefore, this study aims to investigate the mechanistic role of PTP1B in AP pathogenesis and to identify potential therapeutic targets for clinical intervention.