Inhibition of Notch activity suppresses hyperglycemia-augmented polarization of macrophages to the M1 phenotype and alleviates acute pancreatitis

Acute pancreatitis (AP), characterized by acinar cell death and local or systemic inflammation, is the most common pancreatic disease [1]. The global incidence of AP is currently 33.74/100000 person-years and has been increasing worldwide [2,3]. It is critical to prevent AP from developing into severe disease, which is associated with multiple organ dysfunction and increased mortality [4]. In spite of progress in the development of AP treatment strategies over the past few decades, there are still no effective therapeutic medications to mitigate disease progression [3,5,6].

In clinical practice, most AP patients have elevated and fluctuating blood glucose levels, which may be due to stress response, β-cell injury, and nutritional treatment [7–10]. A previous study showed that diabetes is associated with an increased risk of severe AP [11]. However, whether hyperglycemic conditions and fluctuating blood glucose observed in the early phase of AP exacerbate pancreatic injury remains unclear.

During tissue injury and infection, monocytes migrate to tissues and differentiate into macrophages [12]. This cell type has two phenotypes: M1 macrophages represent the proinflammatory state and are characterized by iNOS expression and production of proinflammatory cytokines like interleukin (IL)-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), while M2 macrophages represent the anti-inflammatory state and are characterized by the production of IL-10 [13]. M1 macrophages dominate the proinflammatory phase of AP, while M2 macrophages dominate the phase of pancreatic repair and regeneration [14]. Diabetes is associated with tissue injury and inflammation in multiple organs [15,16], and a recent study showed that hyperglycemia during type 1 diabetes can aggravate acute liver injury by promoting the activation of liver-resident macrophages [17]. However, whether a hyperglycemic condition that follows AP would affect the polarization of pancreatic macrophages remains unknown.

The Notch signaling pathway is relatively conserved, primarily regulating embryonic development, multiple cell lineage differentiation, and adult tissue homeostasis [18]. The Notch receptors, Notch 1–4, are produced in the endoplasmic reticulum (ER) and interact with the Notch ligands, Delta, Jagged, and Serrate, on the surface of adjacent cells [19]. When the Notch pathway is activated, the Notch intracellular domain (NICD) becomes liberated and translocates into the cell nucleus, where it binds to the DNA-binding protein, Rbpj [20,21]. This interaction results in the transcription of Notch downstream targets, such as Hes and Hey family genes [22]. Notch-Rbpj signaling plays an important role in macrophage development, activation, and polarization [23,24]. In a diabetic retinopathy model, the Notch1 ligands, Jagged1 and Delta Like-4, are up-regulated during hyperglycemia and activate Notch signaling [25]. However, it remains unknown whether hyperglycemia uses Notch signaling to regulate macrophage polarization and pancreatic injury during AP.

Animal experiments were approved by the Institutional Review Board of Renmin Hospital of Wuhan University (WDRM 20190108). FVB/N mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and housed in pathogen-free conditions with free access to standard rodent chow and water. All animal experiments were performed in the Animal Experiment Center of Renmin Hospital of Wuhan University in accordance with standard guidelines. The AP model was established in 8–12-week FVB/N male mice using hourly intraperitoneal injections of cerulein (100 μg/kg body weight, C9026, Sigma–Aldrich, U.S.A.) six-times a day for 4 days [14,26]. Control mice (CON) were intraperitoneally injected with 0.9% saline. The AP with hyperglycemia group (APG) was induced after establishing the AP model using repeated intraperitoneal injections of 50% glucose (4 g/kg) 3 h apart, four-times per day for 1 week. The control with hyperglycemia group (CONG) was established using intraperitoneal injections of 50% glucose in control mice. Blood glucose levels were measured 1, 3, and 7 days after intraperitoneal injection of 50% glucose (4 g/kg) or saline. In detail, blood glucose levels were measured 0, 15, 30, 60, 90, and 120 min following glucose or saline injection with a glucometer (OneTouch UltraVue, Johnson & Johnson, U.S.A.) through the tail vein. Animals were killed at 1, 3, and 7 days after establishing the AP model. The mice were anesthetized using isoflurane inhalation before sample collection. Blood was obtained through the inferior vena cava, after which the mice died spontaneously, and their pancreases were immediately removed. Killing was confirmed using cervical dislocation. Pancreatic tissues were fixed in 4% paraformaldehyde or stored at −80°C for further histological and biochemical analyses.

FVB/N mice were killed by cervical dislocation and sterilized by immersion in 75% ethanol. The femurs and tibias were prepared under sterile conditions. Bone marrow was flushed out of the bones using sterile Dulbecco’s modified Eagle’s medium (DMEM) (G4520, Servicebio, China), the cells were incubated in sterile erythrocyte lysate for 10 min to remove red blood cells, passed through a cell strainer (70 μm), and collected in a 50-ml tube. The cells were then centrifuged for 10 min at 2000 rpm (5804R, Eppendorf, Germany), washed with sterile phosphate-buffered saline (PBS), and resuspended in low-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (10999141, Gibco, Australia), 1% Penicillin–Streptomycin solution (P/S) (BL505A, BioSharp, China), and 20 ng/ml macrophage colony-stimulating factor (M-CSF) (315-02, PeproTech, U.S.A.), and maintained in a six-well culture plate. The cells were used for experiments 7 days after M-CSF stimulation [27].

Fresh pancreatic tissues were obtained from FVB/N mice and digested in type 1 collagenase (1.6 mg/ml, G0130, Sigma–Aldrich, U.S.A.) dissolved in 1× HBSS (BL561A, BioSharp, China) in a 37°C water bath for 20 min with gentle shaking. The enzymatic reaction was stopped with the addition of 1 mM CaCl₂. The digested tissue was centrifuged at 290×g at 4°C for 2 min and washed twice with 1× HBSS. The pellet was resuspended and filtered through a 70-μm cell strainer to obtain dispersed acinar cells. Cells were cultured in low-glucose DMEM supplemented with 10% FBS and 1% P/S in 5% CO₂ at 37°C.

The acinar cells were washed with sterile PBS and maintained in six-well plates in fresh DMEM. Prior to cocultivation, acinar cells were stimulated with 100 nM cerulein for 12 h, washed with sterile PBS, and seeded on to cell culture inserts (0.4-μm pore size, 3412, Corning, U.S.A.). The inserts were transferred into the wells containing bone marrow-derived macrophages (BMDMs), and co-incubation was maintained for 4 days [28]. The cells were then incubated in DMEM supplemented with 5.5, 25, or 50 mM glucose. The γ-secretase inhibitor, DAPT (S2215, Selleck, U.S.A.), was added to the medium to inhibit Notch signaling. BMDM morphology was assessed using an inverted microscope.

Mouse macrophages from the RAW264.7 cell line (Procell Life Science & Technology Co., Ltd., China) were cultured in low-glucose DMEM containing 10% FBS and 1% P/S in 5% CO₂ at 37°C.

Lentivirus against Notch1 was obtained from Shanghai GeneChem Co., Ltd., China. Prior to transfection with lentivirus, RAW264.7 cells were seeded in 12-well plates at a 1 × 10⁵ density per well, and fresh medium with lentivirus and HiTransG-P were added. After a 48-h transfection, 2 μg/ml puromycin (BS111, BioSharp, China) was added for the selection. After continuous selection with puromycin, the Notch1 knockdown RAW264.7 cell line was examined by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The lentivirus containing empty vector was used as a negative control. CON RAW264.7 cells and Notch1 knockdown RAW264.7 cells were stimulated with lipopolysaccharide (LPS) and cultured in DMEM with 5.5 or 50 mM glucose for 24 h. The cells were harvested for Western blotting and qRT-PCR analyses.

Pancreatic sections were stained with Hematoxylin and Eosin (H&E) and the severity of pancreatitis was analyzed by pathologic scoring. Injury indicators including edema, acinar necrosis, inflammation, perivascular infiltration, hemorrhage, and fat necrosis were analyzed by two researchers independently as described previously [29]. Edema, hemorrhage, and fat necrosis were assessed on whole tissue sections, while inflammatory infiltration, perivascular inflammation, and acinar necrosis were scored on ten 400× fields per slide.

The Elivision Super HRP IHC Kit (KIT99-22, MXB, China) was used for immunohistochemistry according to the manufacturer’s instructions. Pancreatic sections were deparaffinized and rehydrated, and citrate buffer (pH 6.0, G1202, Servicebio, China) was used for antigen retrieval. Peroxidase blocking was performed in PBS with 3% H₂O₂ for 10 min, and permeabilization was conducted using 0.2% Triton X-100 (93443, Sigma–Aldrich, U.S.A.) for 45 min at room temperature. The slides were incubated with primary antibodies against IL-1β (1:200, GB11113, Servicebio), nuclear factor κ-B p65 (NF-κB p65) (1:200, GB11042, Servicebio), and Rbpj (1:200, 5313T, Cell Signaling Technology) overnight at 4°C. The slides were incubated with a response enhancer at 37°C for 20 min HRP-conjugated secondary antibody at 37°C for 20 min. AEC (ZLI-0936, ZSGB-Bio, China) or DAB (G1212, Servicebio, China) substrates were used for color development. Five sections in each group and ten 400× images per section were evaluated by calculating the number of positive cells/total number of cells.

Pancreatic sections were deparaffinized and rehydrated. Tris-EDTA (pH 9.0, G1203, Servicebio, China) was used for antigen retrieval, 0.2% Triton X-100 was used for permeabilization, and 10% donkey serum was used for blocking. The sections were incubated with primary antibodies overnight at 4°C and incubated with secondary antibodies for 1 h at 37°C. For immunofluorescent staining, primary macrophages were fixed in 4% paraformaldehyde for 20 min and blocked in 10% donkey serum. Incubation with primary antibodies was performed overnight at 4°C. Secondary antibody incubation was performed for 1 h at 37°C and DAPI (ab104139, Abcam, U.S.A.) was used to label the nuclei. The primary antibodies included CD68 (1:50, Sc-20060, Santa Cruz), iNOS (1:200, 18985-1-AP, Proteintech), CD206 (1:200, 18704-1-AP, Proteintech), IL-6 (1:200, GB11117, Servicebio), and Hes1 (1:200, ab108937, Abcam). The secondary antibodies included donkey anti-rabbit IgG H&L (Alexa Fluor® 488) (1:200, ab150073, Abcam), and donkey anti-mouse IgG H&L (Alexa Fluor® 594) (1:200, ab150108, Abcam). Five sections in each group were examined and ten 400× images per section were evaluated. iNOS⁺CD68⁺ cells represented M1 macrophages, CD206⁺CD68⁺ cells represented M2 macrophages, and Hes1⁺CD68⁺ cells represented Hes1-positive macrophages.

TUNEL staining was performed using the Click-iT Plus TUNEL Assay kit (C10618, Invitrogen, U.S.A.) with slight modifications in the manufacturer’s instructions. Deparaffinized sections were immersed in 4% paraformaldehyde for 15 min at 37°C and washed twice with PBS. The slides were permeabilized with proteinase K for 15 min at 37°C. After incubation in 4% paraformaldehyde for 5 min, the sections were incubated with terminal deoxynucleotidyl transferase (TdT) reaction buffer for 10 min at 37°C. TdT reaction mixture (TdT reaction buffer, EdUTP, TdT enzyme) was added to the slides and incubated at 37°C for 1 h. The slides were rinsed with 3% BSA and 0.1% Triton X-100 in PBS and incubated in 1× Click-iT Plus TUNEL SuperMix for 30 min at 37°C. DAPI (ab104139, Abcam, U.S.A.) with mounting medium was used to label the nuclei. Five sections were examined in each group, and ten 400× images per section were evaluated to calculate TUNEL-positive cells/total cells.

Pancreatic tissue and cells were lysed in RIPA buffer (P0013B, Beyotime, China) containing proteinase, phosphatase inhibitor cocktail (04693132001 and 0490684500, Roche, Switzerland), and PMSF (G2008, Servicebio, China). The lysates were centrifuged at 12000 rpm (5424R, Eppendorf, Germany) and 4°C for 10 min to remove debris, and the supernatants were collected and used for electrophoresis. Ten percent silk milk was used for blocking. The primary antibodies included Hes1 (1:1000, 11988s, Cell Signaling Technology), Rbpj (1:1000, 5313T, Cell Signaling Technology), cleaved caspase-3 (1:1000, 9661T, Cell Signaling Technology), TNF-α (1:1000, GB11188, Servicebio), IL-6 (1:1000, 12912T, Cell Signaling Technology), iNOS (1:1000, 18985-1-AP, Proteintech), and GAPDH (1:1000, GB11002, Servicebio). Antigen–antibody complexes were probed with HRP-conjugated secondary antibody for 1 h at room temperature. Immunoproducts were detected using the Ultra-sensitive ECL Chemiluminescence Kit (BL523A, BioSharp, China).

Primary macrophages or RAW264.7 cells were homogenized in TRIzol reagent (G3013, Servicebio, China) for total RNA extraction. After incubation in TRIzol for 30 min at room temperature, the cells were scraped off the culture dish. Chloroform was added and mixed thoroughly, and the lysates were incubated at room temperature for 15 min and centrifuged for 10 min at 4°C. The supernatants were aspirated into fresh 1.5-ml EP tubes and precooled isopropanol was added. After incubation at 4°C for 15 min, the lysates were centrifuged at 12000 rpm (5424R, Eppendorf, Germany) for 10 min to obtain RNA products. The RNA products were washed three times with 75% ethanol and dissolved in 20 μl DEPC water (BL510A, BioSharp, China). The concentration of RNA was determined using Nanodrop 2000. RNA products were reverse transcribed into cDNAs using a GoScript reverse transcription system (K1691, Thermo Fisher, U.S.A.). The qRT-PCR was performed with SYBR Green reagent (G3320-01, Servicebio, China). Primer sequences for qRT-PCR are listed in Supplementary Table S1. Gene expression was normalized to GAPDH and analyzed using the 2^−ΔΔC_T method.

Data are presented as mean ± SEM, and analyzed by SPSS 26.0 (IBM, U.S.A.) and Prism 8.0 software (GraphPad, U.S.A.). An unpaired Student’s t test was used to evaluate the differences between two groups. P<0.05 was considered statistically significant.

A murine AP model was established through intraperitoneal injections of cerulein six-times a day for 4 days. After establishing the AP model, the hyperglycemic condition was created through intraperitoneal administration of 50% glucose (4 g/kg) for the indicated time period (Figure 1A). Significantly elevated blood glucose levels were observed following glucose injections 1, 3, and 7 days after AP (Figures 1B–D). The pancreases of non-AP mice that received glucose injections (CONG) were not different from those of control mice (CON) receiving saline (Figure 1E), indicating that the hyperglycemic condition did not injure the pancreas in the absence of pancreatitis. Compared with the normal pancreas structure observed in the CON group, obvious edema, acinar necrosis, and inflammatory cells infiltration were observed in pancreases from the AP group (Figure 1F). APG mice exhibited more severe pancreatic injury than AP mice at each indicated time point (Figure 1F,G). The detailed score for each pathological parameter in each group is listed in the Supplementary Table. These findings indicate that hyperglycemic conditions could aggravate pancreatic injury caused by AP.

To evaluate pancreatic inflammation in each group, IL-1β, NF-κB p65, and IL-6 expression was assessed by immunostaining and found to be significantly higher in the AP groups than in the CON groups. APG mice had higher levels of pancreatic inflammation than AP mice as shown by increased staining of IL-1β, NF-κB, and IL-6 at each time point (Figure 2A–F). Cleaved caspase-3 protein levels were also higher in pancreases from the AP group than those from the CON group. High-concentration glucose treatment resulted in a further increase in cleaved caspase-3, indicating that hyperglycemic conditions exacerbated apoptosis (Figure 2G,H). TUNEL staining also showed an increased number of apoptotic cells in APG mice than AP mice (Figure 2I,J). These findings suggest that hyperglycemia exacerbated pancreatic inflammation and increased cellular apoptosis during AP.

The activation of pancreatic macrophages and their subphenotypes were examined by CD68, iNOS, and CD206 immunostaining in the pancreases of mice from each group. CD68 and iNOS costaining represented the proinflammatory M1 phenotype (Figure 3A), while CD68 and CD206 costaining represented the M2 phenotype (Figure 3B). Minimal expression of CD68 in the CON and CONG groups indicated that macrophages were not activated in the absence of pancreatitis. The injured pancreases of AP mice showed massive infiltration of CD68 positive macrophages, and this number further increased in mice receiving glucose insults (APG) (Figure 3C), suggesting that hyperglycemia facilitated macrophage activation. In addition, the proportion of each macrophage subtype differed between the AP and APG groups (Figure 3D). While both the AP1d and APG1d groups had a high proportion of M1 macrophages, the APG group had significantly more M1 macrophages than the AP group at days 3 and 7. In contrast, while M2 macrophages were activated in both the AP1d and APG1d groups, a significant increase in M2 macrophages was observed in the AP3d and APG3d groups. The APG7d group had significantly less M2 macrophage infiltration than the AP7d group. In addition, the ratio of M1 to M2 macrophages was higher in the APG than in the AP group on days 3 and 7 (Figure 3E). These data suggest that the hyperglycemic condition promoted differentiation of pancreatic macrophages towards an M1 phenotype and inhibited M2 polarization.

The results described above indicate that hyperglycemia promotes macrophage polarization to the M1 phenotype, however, the associated mechanism remains unclear. To evaluate Notch activity in pancreases from each group, Rbpj immunostaining was performed. Higher Rbpj staining was observed in acinar-to-ductal metaplasia (ADM) structures and interstitial infiltrations in AP than CON pancreases (Figure 4A). In addition, pancreases from the APG3d and APG7d groups had significantly more Rbpj positive cells than pancreases from the AP3d and AP7d groups (Figure 4A,B), suggesting that hyperglycemic condition further elevated Notch activity after AP. Western blot analysis also showed significantly higher Rbpj and Hes1 levels in pancreatic tissues from APG versus AP mice (Figure 4C–E). To investigate the impact of Notch up-regulation on macrophage polarization, immunofluorescent costaining of Hes1 and CD68 was performed. CD68⁺Hes1⁺ macrophages were markedly higher in AP than CON pancreases, and the proportion of co-expressing cells was significantly higher in the APG groups than the corresponding AP groups (Figure 4F,G). These results show that the increased M1 polarization that occurs under hyperglycemic conditions may be associated with up-regulated Notch signaling.

To investigate the role of Notch signaling in macrophage polarization in vitro, BMDMs (Figure 5A) and pancreatic acinar cells were isolated from FVB/N mice, and CD68 immunostaining was used to identify isolated macrophages after a 7-day M-CSF stimulation. A co-incubation system using BMDMs and cerulein-stimulated acinar cells was established. In brief, pancreatic acini were treated with cerulein to simulate the pancreatitis model, and the cerulein-stimulated acini were transferred to the co-culture system to cultivate BMDMs (Figure 5B). While acini-stimulated BMDMs showed robust Notch activity, the administration of the Notch signaling inhibitor, DAPT, to the co-culture system significantly reduced macrophage Notch activity in a dose-dependent manner as shown by reduced Rbpj and Hes1 levels (Figure 5C–E).

To further explore the impact of hyperglycemia on macrophage polarization, different concentrations of glucose were added to the co-culture system. Notch1, Notch2, and Rbpj mRNA levels in the macrophages increased significantly after culture with cerulein-stimulated acini and rose even higher after hyperglycemia was induced. However, the addition of DAPT (40 μM) to the co-culture system successfully reduced Notch1, Notch2, and Rbpj mRNA levels (Figure 5F–H). Western blot analysis also showed significantly higher Rbpj and Hes1 protein levels in macrophages after high concentration glucose treatment, while blocking Notch signaling with DAPT reversed this phenomenon (Figure 5I–K). Levels of M1 macrophage cytokines, IL-1β, IL-6, and TNF-α, increased significantly in macrophages in the co-culture system, and cytokine mRNA levels further increased under hyperglycemic conditions, indicating that hyperglycemia increased M1 polarization. DAPT significantly reduced the expression of these cytokines, suggesting that polarization towards the M1 phenotype was inhibited by Notch suppression (Figure 5L–N).

Assessment of cellular morphology showed that some macrophages developed a fusiform shape after coculture with cerulein-stimulated acinar cells, suggesting that polarization had occurred. High concentration glucose insult further promoted changes to macrophage morphology, while DAPT treatment inhibited morphology changes (Figure 6A). Immunostaining of CD68 and either Hes1 or iNOS showed significant increase in macrophage Notch activity and increased M1 polarization in the co-culture system following high concentration glucose stimulation, while DAPT treatment reduced both phenomena (Figure 6B–E). These findings suggest that up-regulated macrophage Notch activity in hyperglycemic conditions promoted macrophage polarization towards an M1 phenotype.

Lentivirus transfection was used to knockdown Notch1 in RAW264.7 cells in order to further verify the role of Notch signaling in macrophage polarization. Fluorescent images 48 h after lentivirus transfection are shown in Figure 7A. Notch1 mRNA levels, and Rbpj and Hes1 protein levels decreased significantly after Notch1 knockdown (Figure 7B–E), suggesting that Notch activity was effectively inhibited. To mimic inflammation in RAW264.7 cells, a series of exogenous LPS concentrations were administered. After LPS stimulation for 24 h, Notch activity and TNF-α production in RAW264.7 cells increased in a dose-dependent manner (Figure 7F,G). Thus, 1 μmol/ml LPS was selected for the subsequent experiment. The hyperglycemic condition was established using 50 mM glucose stimulation. For the CON-RAW264.7 cells, LPS stimulation significantly increased Notch1, Hes1, and Rbpj mRNA, and Hes1 and Rbpj protein levels, and expression was further increased under hyperglycemic conditions. Notch1, Hes1, and Rbpj mRNA, and Hes1 and Rbpj protein levels were significantly lower in Notch1 knockdown RAW264.7 cells than CON-RAW264.7 cells (Figure 8A–F). IL-1β, IL-6, and TNF-α mRNA, and IL-6 and TNF-α protein levels were significantly higher after LPS and high-concentration glucose treatment of CON-RAW264.7 cells than Notch1 knockdown RAW264.7 cells (Figure 8D,G–K). To quantify the number of M1 macrophages, iNOS protein levels were measured in each group. LPS treatment significantly increased iNOS expression in CON-RAW264.7 cells, and hyperglycemia further elevated iNOS. However, iNOS protein levels were lower in Notch1 knockdown RAW264.7 cells than CON-RAW264.7 cells in response to the indicated treatment (Figure 8L,M). These findings suggest that inhibition of Notch activity via Notch1 knockdown in RAW264.7 cells prevented LPS and hyperglycemia-induced M1 polarization of macrophages.

In the present study, a cerulein-induced AP model with hyperglycemia was established. Animal experiments demonstrated that hyperglycemic conditions aggravated the pancreatic injury, promoted M1 polarization, and elevated Notch signaling following AP. In vitro experiments confirmed that hyperglycemia facilitated M1 polarization by amplifying Notch signaling in macrophages. Inhibition of Notch signaling of macrophages by DAPT or Notch1 knockdown prevented M1 polarization and decreased the production of proinflammatory factors. These data suggest that hyperglycemia conditions can aggravate AP and controlling blood glucose or inhibiting Notch signaling during early AP may prevent progression to severe disease.

Although studies have assessed the impact of diabetes on the progression of AP, the role of hyperglycemia in AP patients remains poorly understood. In clinical practice, hyperglycemia is observed in most patients with AP, potentially resulting from an acute stress response that activates the neuroendocrine system and causes the release of glucagon [30,31]. In addition, AP-induced β-cell injury reduces insulin secretion [32], and the complex interplay between multiple hormones and cytokines causes insulin resistance, which further aggravates hyperglycemia [31]. Enteral and parenteral nutrition treatment during AP can also induce transient hyperglycemia [9].

Previous studies have shown that diabetes aggravates AP and AP diabetic mice receiving insulin treatment have less cell death and increased expansion of acinar cells [33–35]. The current study focused on the impact of hyperglycemia on pancreatitis during early AP, rather than diabetes. Acute hyperglycemia was shown to aggravate pancreatic injury and inflammatory infiltration, and increase acinar cell apoptosis following AP. Interestingly, hyperglycemia increased the number of macrophages that infiltrated the pancreatic parenchyma, promoted M1 polarization, inhibited M2 polarization, and increased the release of proinflammatory factors. Macrophages are important innate immune cells in mice and humans, and the balance of M1 and M2 macrophages plays a critical role in regulating the immune response [36]. Hyperglycemia is shown to exacerbate hepatic ischemia/reperfusion injury by promoting M1 polarization and inhibiting M2 polarization, further supporting the findings from the current study [37].

Notch signaling is pivotal to the development and differentiation of progenitor cells in the embryonic pancreas [38], and plays a decisive role in the differentiation of multiple cell lineages including dendritic cells and macrophages [39]. Prior studies have demonstrated a role for Notch signaling in the regulation of inflammation and apoptosis [40,41]. Available data confirm that Notch signaling is activated in the cerulein-induced AP model, and previous research has shown that Notch activity is elevated in the injured pancreases of AP patients and essential to the regeneration of impaired pancreatic acinar cells during AP [26,42,43]. However, excessively activated Notch signaling is harmful and is shown to aggravate renal podocyte injury in mouse models of diabetic and adriamycin-induced nephropathy [44]. Other studies have demonstrated that Notch signaling can aggravate hypoxia/reoxygenation-induced cardiomyocyte injury [45]. These studies suggest that excessively activated Notch signaling can aggravate organ or tissue injury in multiple disease models. It also has been shown that the Notch-Rbpj pathway selectively regulates M1 macrophage-associated gene expression in LPS-induced BMDMs injury [46]. In a bacterial infection model, up-regulation of macrophage Notch signaling promotes macrophage polarization towards an M1 phenotype, resulting in increased production of inflammatory factors and aggravating tissue damage [47]. In the AP model described in the current study, Notch signaling was activated in pancreatic macrophages, and the hyperglycemic condition further increased Notch activity and promoted macrophage polarization towards an M1 phenotype. DAPT treatment or Notch1 knockdown in primary macrophages and macrophage cell lines depressed M1 polarization and decreased the production of proinflammatory factors. These data suggest that Notch signaling is a potential target for the prevention and treatment of AP.

The present study demonstrates that hyperglycemia promotes macrophage polarization towards an M1 phenotype, increases the production of proinflammatory factors, and aggravates pancreatic injury during AP, all of which are associated with higher Notch signaling. These results provide evidence that controlling blood glucose is critical for the effective treatment of AP. The present study reveals a novel mechanism for Notch signaling in AP and provides a molecular basis for AP treatment.

• Most patients with AP suffer from hyperglycemia. Previous research indicates that Notch signaling is increased in the injured pancreases of AP patients but it remains unclear whether hyperglycemia impacts pancreatic Notch signaling and disease severity after AP.

• Findings from the present study suggest that hyperglycemia promotes pancreatic macrophage polarization to the M1 phenotype during AP. Inhibiting Notch activity prevents M1 polarization and mitigates pancreatic injury after AP.

• The present study shows that reduced Notch activity and well-controlled blood glucose are necessary to prevent AP disease progression, and Notch signaling may be a potential target for the treatment of AP. Further studies are required to establish clinical evidence.

Data presented in this manuscript are available upon reasonable request from the corresponding authors.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the National Natural Science Foundation of China [grant numbers 81800574, 82170651 (to Qiao Shi) and 81870442 (to Weixing Wang)].

Ning Hu: Data curation, Investigation, Methodology, Writing—original draft. Xiaoyi Zhang: Data curation, Investigation, Methodology, Writing—original draft. Xuanzhe Zhang: Data curation, Investigation, Methodology. Yongjun Guan: Investigation, Methodology. Ruyuan He: Investigation. Enfu Xue: Investigation. Xiaoyi Zhang: Investigation. Wenhong Deng: Investigation. Jia Yu: Investigation. Weixing Wang: Conceptualization, Funding acquisition, Writing—review & editing. Qiao Shi: Conceptualization, Funding acquisition, Data curation, Investigation, Writing—review & editing.

AP

acute pancreatitis

BMDM

bone marrow-derived macrophage

CON

control

DAB

diaminobezidin

DAPT

N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HRP

horseradish peroxidase

IL

interleukin

iNOS

inducible nitric oxide synthase

LPS

lipopolysaccharide

M-CSF

macrophage colony-stimulating factor

NF-κB p65

nuclear factor κ-B p65

P/S

Penicillin–Streptomycin

PBS

phosphate-buffered saline

qRT-PCR

quantitative real-time polymerase chain reaction

Rbpj

Recombination signal binding protein for immunoglobulin kappa J region

TNF-α

tumor necrosis factor-α

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling