Tumor necrosis factor-α promotes abnormal glucose metabolism after acute pancreatitis by inducing islet β-cell apoptosis via Bax/Bcl-2/caspase-3 signaling pathway

Abstract

BACKGROUND

Tumor necrosis factor-α (TNF-α) has been implicated in the development of diabetes following chronic pancreatitis. However, its role in abnormal glucose metabolism (AGM) after acute pancreatitis (AP) and post-pancreatitis diabetes mellitus remains unclear.

AIM

To investigate the role of TNF-α in AP-associated AGM and its effects on islet β-cell apoptosis, focusing on the underlying molecular mechanisms.

METHODS

Clinical data were collected to assess AGM’s incidence and identify the characteristics in 369 AP patients. In vitro, AP models were established using lipopolysaccharide in 266-6 acinar cells and MIN-6 β-cells. Cell proliferation, apoptosis, and protein expression were analyzed using the Cell Counting Kit-8 assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and western blotting. The TNF-α and insulin concentration in co-culture medium was measured by enzyme-linked immunosorbent assay. In vivo, an AP mouse model was induced using sodium taurocholate, and pancreatic tissues were analyzed through hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and western blotting. TNF-α levels were assessed by enzyme-linked immunosorbent assay. A TNF-α inhibitor was applied to the AP cell model to reassess apoptosis and protein expression.

RESULTS

AGM occurred in 40.38% of AP patients. Body mass index, severity grade, recurrence frequency, and lung injury were significantly associated with AGM. AP models in 266-6 and MIN-6 cells showed reduced β-cell proliferation, insulin secretion, and increased apoptosis, which correlated with inflammation severity. Similar findings of β-cell apoptosis were confirmed in the mouse model. TNF-α levels were significantly elevated in AP models, with higher levels in severe inflammation. Increased Bax and caspase-3 expression and decreased Bcl-2 expression were observed in both in vitro and in vivo models. These changes intensified with increasing inflammation. TNF-α inhibition reduced apoptosis and altered protein expression patterns, decreasing Bax and caspase-3, while increasing Bcl-2 in MIN-6 cells.

CONCLUSION

TNF-α contributes to β-cell apoptosis and AGM in AP through the Bax/Bcl-2/caspase-3 signaling pathway, suggesting TNF-α as a potential therapeutic target for preventing AP-associated AGM.

Key Words: Tumor necrosis factor-α; Abnormal glucose metabolism; Acute pancreatitis; Apoptosis; Bax; Bcl-2; Caspase-3

Core Tip: Acute pancreatitis (AP) often leads to abnormal glucose metabolism (AGM). This study investigated the role of tumor necrosis factor-α (TNF-α) in AP-associated AGM, and our results showed elevated TNF-α promoted β-cell apoptosis via the Bax/Bcl-2/caspase-3 signaling pathway, contributing to AGM. Inhibiting TNF-α reduced apoptosis and improved AGM, indicating its potential as a therapeutic target for preventing AGM after AP.

INTRODUCTION

Acute pancreatitis (AP) is a common gastrointestinal disorder and the third leading cause of hospitalization for gastrointestinal diseases[1]. It is characterized by pancreatic inflammation, local tissue damage, a systemic inflammatory response, and, in severe cases, multi-organ failure[2]. Emerging evidence suggests that metabolic disorders, particularly newly diagnosed prediabetes or diabetes mellitus (DM), often develop after an episode of AP[3,4]. Approximately 40% of patients with AP are diagnosed with prediabetes or DM upon discharge from the hospital[5]. Among the mechanisms involved, impaired insulin secretion is considered a key pathological process in pancreatitis-associated diabetes[6].

AP triggers a strong inflammatory response, leading to the release of various pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), as well as oxygen free radicals. TNF-α, in particular, plays a critical role in the pathogenesis of AP by amplifying systemic inflammation and contributing to organ dysfunction in severe cases[7]. It also regulates apoptotic signaling through the Bax/Bcl-2/caspase-3 pathway, where Bax promotes apoptosis, Bcl-2 inhibits apoptosis, and caspase-3 serves as a key effector of programmed cell death[8,9]. Dysregulation of this pathway is associated with pancreatic acinar cell apoptosis and systemic metabolic disturbances, including impaired glucose regulation[10]. Although previous studies have linked TNF-α to diabetes development following chronic pancreatitis (CP), its role in abnormal glucose metabolism (AGM) after AP remains poorly understood. This is particularly important, as AP-related AGM is a common long-term complication that can progress to post-pancreatitis DM (PPDM).

This study aims to determine whether TNF-α contributes to AGM following AP by modulating the Bax/Bcl-2/caspase-3 apoptotic signaling pathway. By elucidating the underlying molecular mechanisms, we seek to identify potential therapeutic targets to mitigate metabolic complications associated with AP, ultimately improving patient prognosis and reducing the burden of long-term glucose dysregulation. This research has both theoretical and clinical significance for the management of post-AP metabolic sequelae.

MATERIALS AND METHODS

Cells and reagents

The mouse pancreatic acinar cell line 266-6 and islet β-cell line MIN-6 were supplied by the National Collection of Authenticated Cell Cultures (Shanghai, China). Lipopolysaccharide (LPS) (Cat. No: ST1470), Cell Counting Kit-8 (CCK-8) (Cat. No: C0038), proteinase K (Cat. No: ST532), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay kit (Cat. No: C1098), RIPA lysis buffer (Cat. No: P0013C), pomalidomide (Cat. No: SC4345), and hematoxylin and eosin (HE) staining kit (Cat. No: C0105S) were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). Cell culture reagents, including Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS), were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and Serana (Germany), respectively. Sodium taurocholate (Cat. No: T4009) was obtained from Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). The mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Cat. No: KE10002) was obtained from Proteintech (IL, United States). The mouse insulin ELISA kit (Cat. No: ZEY00576MA), amylase detection kit (Cat. No: ZEY018-W96), and lipase detection kit (Cat. No: ZEY158-W96) were obtained from Zhuoeryou Biotechnology Co., Ltd. (Shanghai, China).

Study population and clinical data

A retrospective chart review was conducted from 2020 to 2023, involving patients diagnosed with AP who underwent glucose-related testing during hospitalization. The data collected included fasting blood glucose (FBG), hemoglobin A1c (HbA1c), and relevant clinical characteristics such as age, sex, body mass index (BMI), chronic conditions, AP etiology, severity grade, recurrence frequency, and the presence of lung injury. The overall FBG/HbA1c detection rate was 73.33% (451/615) in AP patients, confirming that inclusion was not biased toward specific subgroups.

Definition of AGM and severe AP

AGM was defined based on the criteria established by the American Diabetes Association. Specifically, AGM was diagnosed when FBG levels were between 5.6 mmol/L and 6.9 mmol/L and/or HbA1c levels were between 5.7% and 6.5%[11]. The diagnosis and severity classification of severe AP (SAP) were based on the revised Atlanta criteria[12].

Inclusion criteria for AP

Patients were eligible for inclusion if they were aged between 18 years and 80 years and met at least two of the following diagnostic criteria for AP: (1) Acute-onset epigastric pain, nausea, or vomiting occurring within 72 hours prior to hospital admission; (2) Serum amylase or lipase levels elevated to at least three times the upper limit of normal; and (3) Radiologic evidence consistent with AP on computed tomography, magnetic resonance imaging, or ultrasonography. In addition, patients were required to have a confirmed diagnosis according to the revised Atlanta classification or compatible diagnostic criteria, no evidence of other identifiable causes of abdominal pain (e.g., perforated ulcer or intestinal obstruction), and documented FBG and/or HbA1c results. Written informed consent for study participation was mandatory.

Exclusion criteria for AP

The exclusion criteria were as follows: (1) Current or prior diagnosis of DM; (2) Known history of CP; and (3) Lack of available FBG or HbA1c measurements during clinical evaluation. Patients who declined or were unable to provide informed consent were also excluded.

Cell culture

The mouse pancreatic acinar cell line 266-6 and islet β-cell line MIN-6 were cultured in DMEM supplemented with 10% FBS, 50 mmol/L β-mercaptoethanol (Cat. No: M6250, Sigma, MO, United States), and 100 U/mL penicillin along with 100 μg/mL streptomycin (P/S). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

CCK-8

MIN-6 cells in the logarithmic growth phase were seeded into 96-well plates and cultured in DMEM containing 10% FBS, 50 mmol/L β-mercaptoethanol, and P/S at 37 °C with 5% CO₂. After 24 hours of incubation, the cells were divided into five groups: (1) Control group; (2) LPS group (5 μg/mL); (3) LPS group (10 μg/mL); (4) AP group (conditioned medium collected from 266-6 cells treated with 5 μg/mL LPS for 24 hours); and (5) AP group (conditioned medium collected from 266-6 cells treated with 10 μg/mL LPS for 24 hours). After exposure to the respective conditioned media for 24 hours, cell incubation was performed for 1 hour at 37 °C, supplemented with 20 μL of CCK-8 solution. Cell viability was assessed by the measurement of absorbance at the wavelength of 490 nm using a microplate reader.

Mouse model of AP

Male BALB/c mice (n = 8 per group) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and randomly assigned to three groups: Control group, mild AP group, and SAP group. The AP model was established via retrograde injection of sodium taurocholate into the biliopancreatic duct, as previously described[13]. To minimize potential confounding effects of sex hormones on experimental outcomes, only male mice were used, as recommended in prior experimental pancreatitis studies[14]. Mice in the mild AP group received 10 μL of 5% sodium taurocholate, while those in the SAP group received 10 μL of 10% sodium taurocholate, both at an infusion rate of 5 μL/minute. Control mice were injected with 10 μL of 0.9% sodium chloride solution. Blood samples were collected from the retrobulbar venous plexus on days 1, 3, and 7 post-induction to measure serum TNF-α levels. Serum amylase and lipase activity were measured on day 1 post-induction. These data were included in the Supplementary Figure 1. On day 7, the mice were anesthetized with intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and sacrificed. Pancreatic tissues were harvested to evaluate islet β-cell apoptosis.

AP cell model

An in vitro AP model was established by inducing inflammation in the murine pancreatic acinar cell line 266-6 using LPS[15]. Cells were cultured in DMEM supplemented with 10% FBS, 50 mmol/L β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL P/S for 24 hours, then seeded into culture dishes at the logarithmic growth phase. The cells were treated for 24 hours with varying concentrations of LPS and 10 mmol/L pomalidomide (a TNF-α inhibitor). The concentrations were selected based on preliminary experiments, which indicated that 10 mg/L LPS significantly enhanced both TNF-α secretion and apoptosis in pancreatic acinar cells compared to 5 mg/L LPS. In contrast, 10 mmol/L pomalidomide markedly attenuated LPS-induced TNF-α secretion and apoptosis. The conditioned medium was then collected. MIN-6 cells were maintained in fresh medium for 24 hours and exposed to the conditioned medium for an additional 24 hours. Cell responses were assessed through several assays: (1) Cell viability was evaluated by the CCK-8 assay; (2) Apoptosis index was quantified using the TUNEL assay; and (3) Expression of apoptosis-related proteins was analyzed via western blotting.

Measurement of amylase and lipase activity

Serum amylase and lipase levels in the AP mouse model were measured using commercial kits according to the manufacturer’s protocols.

HE staining and TUNEL assay

Pancreatic tissues were fixed in 4% paraformaldehyde for 48 hours, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned. Sections were deparaffinized in xylene, rehydrated through descending ethanol concentrations, and then stained with HE and subjected to the TUNEL assay. Histological changes were examined under an Olympus BX-51 optical microscope. Pancreatic injury was scored on a scale from 0 to 3 (0 = normal, 3 = severe) based on the extent of necrosis, edema, and inflammation[16]. Pathologists evaluating the sections were blinded to the experimental groups.

ELISA

TNF-α levels were measured at days 1 and 3 post-induction based on established protocols from previous studies[17]. An additional measurement was performed on day 7, when mice were euthanized and pancreatic tissue samples were collected, as apoptotic changes were relatively obvious by this point. Serum samples from mice and culture supernatants from MIN-6 cells were collected for analysis. TNF-α levels were quantified using an ELISA kit according to the manufacturer’s instructions. Briefly, standard solutions were prepared, and each sample was diluted 1:1 with diluent (50 μL sample + 50 μL diluent). A total of 100 μL of each standard or sample was added to the appropriate wells and incubated at room temperature for 2.5 hours. The wells were then washed 3 times, followed by the addition of 100 μL/well of biotinylated TNF-α detection antibody per well and incubated for 1 hour. Next, 100 μL of streptavidin-horseradish peroxidase (HRP) was added per well and incubated for 45 minutes, with washing steps in between. Subsequently, 100 μL of 3,3’,5,5’-tetramethylbenzidine substrate was added to each well, and incubation continued for 30 minutes. The reaction was stopped by adding 50 μL of stop solution for 5 minutes. Absorbance was measured at 450 nm using a Victor^™ X3 microplate reader. Analyte concentrations were determined from the standard curve provided with the ELISA kit. Simultaneously, serum insulin levels in mice were measured using commercial ELISA kits according to the manufacturer’s protocols.

Glucose-stimulated insulin secretion analysis

MIN-6 cells were subjected to glucose-stimulated insulin secretion assays under various conditions. Briefly, cells were washed twice with modified Krebs-Ringer bicarbonate buffer (KRBB) and preincubated for 30 minutes at 37 °C in KRBB containing 1.67 mmol/L glucose. The cells were then stimulated with 1.67 mmol/L or 16.7 mmol/L glucose in KRBB for 1 hour at 37 °C. Supernatants were collected for insulin quantification.

Western blotting analysis

Protein expression levels of caspase-3, Bax, and Bcl-2 in MIN-6 cells and mouse pancreatic tissues were assessed by western blotting. Total protein was extracted using RIPA lysis buffer, and protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk at room temperature for 2 hours and then incubated overnight at 4 °C with the following primary antibodies: Anti-caspase-3 (1:2000, Cat. No: 82202-1-RR, Proteintech, IL, United States), anti-Bax (1:2000, Cat. No: Ab32503, Abcam, United Kingdom), and anti-Bcl-2 (1:2000, Cat. No: Ab182858, Abcam, United Kingdom). After washing, membranes were incubated with HRP-conjugated secondary antibody (Goat Anti-Mouse IgG HRP, 1:10000, Cat. No: SA00001-1, Proteintech, IL, United States) in PBST at 37 °C for 1 hour. β-actin (1:10000, Cat. No: 20536-1-AP, Beyotime Biotechnology, China) was used as the internal control. Protein bands were visualized using an enhanced chemiluminescence detection kit (Cat. No: P0018S, Beyotime Biotechnology, China). Data were obtained from three independent replicates and presented as mean ± SD.

Statistical analysis

All statistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, United States) and GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, United States). Continuous variables were expressed as mean ± SD and compared using independent-samples t-test or one-way analysis of variance (ANOVA). Categorical variables were described as percentages or counts and compared using the χ² test or Fisher’s exact test. A two-tailed P-value of < 0.05 was considered statistically significant.

RESULTS

Comparison of clinical characteristics of AP patients with AGM and normal glucose tolerance

To investigate whether AP contributes to AGM in a clinical setting, FBG and/or HbA1c levels were analyzed in AP patients. Correlation analyses were performed between AGM and various clinical parameters. The results revealed that BMI, severity grade, recurrence frequency, and the presence of lung injury were significantly associated with AGM. However, other clinicopathological factors, such as age, sex, etiology of AP, or chronic conditions were comparative between AGM and normal glucose tolerance groups (Table 1). These findings suggest that AP may play a key role in the development of AGM.

Table 1 Comparison of clinical characteristics of acute pancreatitis patients with abnormal glucose metabolism and normal glucose tolerance.

Characteristics	AGM group (n = 149)	NGT group (n = 220)	Statistical value	P value
Age, n			0.643	0.423
≥ 65	35	44
< 65	114	176
Sex, n			0.254	0.614
Male	102	156
Female	47	64
BMI, mean ± SD, kg/m2	26.701 ± 3.363	25.630 ± 3.904	2.730	0.007b
Chronic conditions1, n			0.357	0.550
Yes	55	88
No	94	132
Etiology, n			4.893	0.180
Biliary	50	99
Hyperlipidemia	45	57
Alcoholic	32	38
Other	22	26
Severity grade, n			4.772	0.029a
Mild	119	194
Severe	30	26
Frequency, n			25.195	0.001c
Non-recurrent AP	98	193
Recurrent AP	51	27
Lung injury, n			7.063	0.008b
Yes	67	69
No	82	151

¹Chronic conditions include hypertension, cerebral infarction, or coronary heart disease.

^aP < 0.05.

^bP < 0.01.

^cP < 0.001.

AGM: Abnormal glucose metabolism; NGT: Normal glucose tolerance; BMI: Body mass index; AP: Acute pancreatitis.

Impaired viability of islet β-cells in AP models

To explore the relationship between AP and AGM, both in vitro and in vivo AP models were established. The in vitro model, 266-6 pancreatic acinar cells were exposed to varying concentrations of LPS for 24 hours, after which the conditioned medium was transferred to MIN-6 islet β-cells to simulate the AP microenvironment. The CCK-8 assay revealed a significant reduction in the proliferation of MIN-6 cells compared to the control group, and this inhibitory effect was both dose- and time-dependent (Figure 1A). In addition, the TUNEL assay showed a significant increase in apoptosis, and ELISA analysis demonstrated a corresponding decrease in insulin levels in MIN-6 cells exposed to conditioned medium from LPS-treated 266-6 cells (Figure 1B-D). The in vivo model, AP was induced by sodium taurocholate. HE staining and TUNEL assays of pancreatic tissue revealed obvious apoptosis of islet β-cells in the AP mouse model, especially in the SAP group (Figure 1E). Quantitative analysis confirmed the increased apoptosis in these tissues (Figure 1F). These results collectively suggest that AP inhibits the proliferation and promotes the apoptosis of islet β-cells.

Figure 1 Impaired viability of islet β-cells in acute pancreatitis models. A-F: Cell Counting Kit-8 assay was used to assess the proliferation of islet β-cells (MIN-6) under control conditions, lipopolysaccharide stimulation (5 μg/mL or 10 μg/mL), and co-culture with media from lipopolysaccharide-treated 266-6 cells (A). Apoptosis in islet β-cells was evaluated in both acute pancreatitis cell and mouse models using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (B and E). Quantitative analysis of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive β-cells (C and F). Insulin secretion by islet β-cells in the acute pancreatitis model was quantified with an enzyme-linked immunosorbent assay kit (D). Representative results from three independent replicates are shown (n = 8). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. LPS: Lipopolysaccharide; AP: Acute pancreatitis; MAP: Mild acute pancreatitis; SAP: Severe acute pancreatitis; HE: Hematoxylin and eosin.

TNF-α levels were elevated in the AP model

To explore the molecular mechanism underlying AP-induced inhibition of islet β-cell growth, TNF-α levels were measured in both cell culture supernatants and mouse serum. ELISA results revealed that TNF-α levels in the culture medium of LPS-treated 266-6 cells were significantly elevated compared to the control group (Figure 2A). Similarly, TNF-α levels in the serum of AP mice were significantly higher than those in the control mice (Figure 2B). These findings suggest that TNF-α may play a role in mediating the inhibitory effects of AP on islet β-cell growth.

Figure 2 Tumor necrosis factor-α levels were elevated in acute pancreatitis models. A: Tumor necrosis factor-α levels in conditioned media from MIN-6 cells were quantified by enzyme-linked immunosorbent assay under control conditions, lipopolysaccharide stimulation (5 μg/mL or 10 μg/mL), and co-culture with media from lipopolysaccharide-treated 266-6 cells; B: Serum tumor necrosis factor-α levels in acute pancreatitis mouse models were quantified using the same methodology. Representative results from three independent replicates are shown. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. AP: Acute pancreatitis; TNF-α: Tumor necrosis factor-α; LPS: Lipopolysaccharide; MAP: Mild acute pancreatitis; SAP: Severe acute pancreatitis.

AP regulated TNF-α related apoptotic signaling in islet β-cells

To investigate whether TNF-α mediates AP-induced apoptosis in islet β-cells, the expression of key apoptosis-related proteins, including Bax, Bcl-2, and caspase-3, was examined. Western blotting analysis revealed that co-culture with conditioned medium from LPS-treated 266-6 cells significantly upregulated Bax and caspase-3 expression in MIN-6 cells while downregulating Bcl-2, compared to the control group (Figure 3A-D). Consistent with these in vitro findings, in vivo analysis in the AP mouse model also showed increased Bax and caspase-3 levels and reduced Bcl-2 expression compared to control mice (Figure 3A and E-G).

Figure 3 Acute pancreatitis induces islet β-cell apoptosis through the tumor necrosis factor-α-dependent Bax/Bcl-2/caspase-3 apoptotic signaling pathway. A: Western blot analysis was performed to detect the expression of caspase-3, Bcl-2, and Bax in MIN-6 cells under control conditions, lipopolysaccharide stimulation, and co-culture with media from lipopolysaccharide-treated 266-6 cells, and in acute pancreatitis mouse tissues; B-D: Quantitative analysis of Bcl-2, Bax and caspase-3 relative protein expression in vitro acute pancreatitis model; E-G: Quantitative analysis of Bcl-2, Bax and caspase-3 relative protein expression in vivo acute pancreatitis model. Representative results from three independent replicate assays are shown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Data are presented as the mean ± SD. AP: Acute pancreatitis; LPS: Lipopolysaccharide; MAP: Mild acute pancreatitis; SAP: Severe acute pancreatitis.

To confirm the involvement of TNF-α in AP-induced apoptosis, the AP cell model was treated with the TNF-α inhibitor pomalidomide. ELISA confirmed that pomalidomide effectively suppressed TNF-α levels (Figure 4A). The TUNEL assay revealed that inhibition of TNF-α significantly reduced the number of apoptotic MIN-6 cells, compared to the untreated AP group (Figure 4B and C). Additionally, ELISA indicated that as TNF-α levels decreased, insulin secretion increased correspondingly (Figure 4D). Furthermore, western blotting analysis showed that pomalidomide reversed the AP-induced upregulation of Bax and caspase-3 and restored Bcl-2 expression to normal levels (Figure 5). These findings suggest that AP-induced islet β-cell apoptosis is mediated, at least in part, by TNF-α-dependent apoptotic signaling pathways.

Figure 4 Inhibition of tumor necrosis factor-α expression attenuated the impairment of islet β-cell in acute pancreatitis models. A: Tumor necrosis factor-α levels in MIN-6 conditioned media were measured by enzyme-linked immunosorbent assay under control conditions, lipopolysaccharide stimulation (10 μg/mL), and co-culture with media from lipopolysaccharide-treated 266-6 cells, with or without treatment with a tumor necrosis factor-α inhibitor; B: Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay; C: Quantitative analysis of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive β-cells; D: Insulin secretion by islet β-cells in the acute pancreatitis model was quantified using an enzyme-linked immunosorbent assay kit. Representative results from three independent replicate assays are shown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. AP: Acute pancreatitis; TNF-α: Tumor necrosis factor-α; LPS: Lipopolysaccharide.

Figure 5 Inhibition of tumor necrosis factor-α expression reversed the alterations in the Bax/Bcl-2/caspase-3 apoptotic signaling pathway in acute pancreatitis models. A: Western blot analysis of caspase-3, Bcl-2, and Bax expression in MIN-6 cells under control conditions, lipopolysaccharide stimulation (10 μg/mL), and co-culture with media from 266-6 cells, with or without tumor necrosis factor-α inhibitor treatment; B-D: Quantitative analysis of Bcl-2, Bax and caspase-3 relative protein expression in vitro acute pancreatitis model. Representative results from three independent replicate assays are shown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Data are presented as the mean ± SD. LPS: Lipopolysaccharide.

DISCUSSION

DM is a common metabolic complication of pancreatic diseases and is a heterogeneous disorder caused by multiple mechanisms. Diabetes resulting from exocrine pancreatic diseases is classified as type 3c DM (T3cDM), which accounts for approximately 9.2% of all diabetes cases[18]. The pathogenesis of T3cDM primarily involves a reduced number and decreased function of islet cells due to pancreatic autoinflammation, fibrosis, and sclerosis, leading to impaired insulin secretion. This distinguishes T3cDM from T1DM and T2DM. T3cDM encompasses PPDM, pancreatic cancer-associated diabetes, and other forms[19]. Although T3cDM is linked to various benign and malignant exocrine pancreatic diseases, CP is the most common cause, accounting for 78.5% of T3cDM cases[20]. Moreover, 80% of CP patients develop T3cDM, with 33% progressing to PPDM within five years[21,22].

Although T3cDM related to CP has been widely studied, research on its association with AP is still limited but increasingly recognized. Lee et al[23] studied 3187 patients with AP and found that their risk of developing new-onset diabetes was 2.1 times higher than that of healthy individuals. Similarly, Shen et al[24] reported in a cohort of 2966 patients with AP that the incidence of new diabetes was 2.5 times greater in those with AP compared to those without. Patients experiencing two or more AP recurrences showed significant reductions in total pancreatic volume and β-cell mass, leading to a substantially increased risk of PPDM, whereas those with one or no recurrence did not show significant volume loss or increased risk[10,25,26]. These studies collectively highlight that AP is lined to AGM and an increased risk of T3cDM. In our analysis, BMI, severity grade, recurrence frequency, and lung injury were significantly associated with AGM occurrence, supporting the view that AP contributes to AGM and aligning with previous evidence identifying AP as a risk factor for T3cDM.

García-Compeán et al[27] proposed that the pathophysiology of PPDM is multifactorial, involving several mechanisms that affect different levels of glucose metabolism regulation. One or more of these mechanisms may predominate in the different diabetes phenotypes, including pancreatic necrosis[28], local and systemic inflammation[29], autoimmunity against β cells and other components of Langerhans islets[30], metabolic dysregulation[31,32], and disturbances in the gut-pancreas axis[33]. In particular, local and systemic inflammation in patients with AP, especially SAP, is reported to be more intense than in CP, with significantly higher concentrations of various inflammatory factors[34-36]. These cytokines contribute to the progression of AP from localized inflammation to systemic inflammatory response syndrome[37]. Key inflammatory cytokines, including TNF-α, IL-1, and IL-6, play crucial roles in the pathogenesis of AP. Among these, TNF-α is considered the first cytokine released and a major mediator of the immune response, playing a critical role by directly damaging acinar cells, leading to necrosis, inflammation, and edema[38,39]. Additionally, gastrin-releasing peptide levels are elevated in patients with AGM following AP and correlate with TNF-α concentrations[40]. Although the biological functions of TNF-α have been extensively studied, its role in predicting pancreatitis severity remains controversial[41]. Our results demonstrate elevated TNF-α levels both in vivo and in vitro. TNF-α concentrations in the culture medium significantly increased and correlated with inflammatory severity in our established AP cell model. Similarly, serum TNF-α was elevated in AP mice and increased with disease severity. In addition, our results revealed an inverse correlation between TNF-α concentration and insulin secretion, indicating that reduced TNF-α levels promote insulin secretion.

The mechanisms by which inflammatory factors induce AGM and potentially contribute to T3cDM in patients with AP remain unclear. Previous studies have shown that elevated IL-1β levels can trigger β-cell apoptosis via the p38 mitogen-activated protein kinases signaling pathway, playing a role in the pathogenesis of both T1DM and T2DM[42]. Similarly, TNF-α has been implicated in β-cell apoptosis via activation of caspase-3[43]. In our study, an AP mouse model exhibited significant islet β-cell apoptosis, which was exacerbated by inflammation. Correspondingly, in vitro AP models using 266-6 and MIN-6 cell lines showed decreased cell proliferation and increased apoptosis, which worsened with the intensity of the inflammatory response. Western blotting analysis indicated upregulated Bax and caspase-3 expression and downregulated Bcl-2 in both pancreatic tissues and MIN-6 cells, with these changes becoming more pronounced as inflammation progressed. Previous studies have demonstrated that inhibition of TNF-α, activin A, and nuclear factor-kappa B signaling pathways can effectively reduce the severity of AP[44]. In this study, treatment with a TNF-α inhibitor significantly attenuated apoptosis in MIN-6 cells, accompanied by decreased Bax and caspase-3 expression and increased Bcl-2 expression. These findings indicate that TNF-α inhibition can reverse the alterations in apoptosis-related proteins induced by AP in islet β-cells. The novelty of this study lies in revealing how TNF-α promotes islet β-cell apoptosis during AP, connecting inflammatory responses to the activation of key apoptotic signaling pathways. Similarly, the study by Tsai et al[45] demonstrated that inhibiting TNF-α secretion with pomalidomide attenuated pancreatic injury in a mouse model of AP. Our data further support these findings, showing that suppression of TNF-α led to decreased β-cell apoptosis and enhanced insulin secretion, underscoring the therapeutic potential of targeting TNF-α in AP-associated AGM.

Several limitations of this study should be acknowledged. Firstly, as a single-center, retrospective study, clinical bias cannot be ruled out. Future studies should incorporate additional functional metabolic assessments, beyond FBG and HbA1c in patients with AP. Secondly, the limited follow-up duration in this study restricts the long-term clinical implications of our findings, which could be addressed by longer follow-up periods in future. Thirdly, the small sample size and the exclusive use of male mice in the AP mouse model may limit the generalizability of these findings. Finally, while this study focused on the Bax/Bcl-2/caspase-3 signaling pathway, we did not investigate upstream or downstream networks of TNF-α, such as β-cell apoptosis mediated by TNF-α receptors (TNFR1/TNFR2), or the interplay between nuclear factor-kappa B/mitogen-activated protein kinases and apoptotic pathways. Further research is required to fully delineate these complex signaling cascades and their role in AP-associated AGM.

CONCLUSION

In conclusion, AGM was closely related to AP. Our findings demonstrated that elevated TNF-α promoted islet β-cell apoptosis via the Bax/Bcl-2/caspase-3 pathway, correlating with the severity of inflammation. Importantly, pomalidomide effectively attenuated TNF-α-induced apoptosis and reversed the expression of key apoptotic proteins, highlighting its therapeutic potential in AP-associated AGM.