Baicalein Reduces Pyroptosis of Acinar Cells in Hyperlipidemic Acute Pancreatitis by Inhibiting M1 Polarization of Macrophages via the HMGB1/TLR4/NLRP3 Pathway

Abstract

Previous studies have shown that baicalein (BAI) can reduce pyroptosis of pancreatic acinar cells (PACs) in hyperlipidemic acute pancreatitis (HAP). This study aimed to elucidate the potential molecular mechanism of PAC pyroptosis mediated by BAI in HAP. A HAP rat model was established via a high-fat diet supplemented with 5% sodium taurocholate. Macrophages were treated with palmitic acid (PA). The rats and cells were treated with BAI. Molecular docking and DARTS assay were used to analyze BAI binding to HMGB1. Co-IP revealed that HMGB1 interacted with TLR4 and NLRP3 and that TLR4 interacted with NLRP3. The interaction between PA-induced macrophages and PACs was evaluated by cell coculture. BAI treatment improved pancreatic lesions, reduced iNOS expression, and decreased the number of M1 macrophages in HAP rats. BAI decreased CD86, HMGB1, NLRP3, ASC, cleaved caspase-1, and GSDMD-N expression in pancreatic tissue and serum IL-1β and IL-18 levels in HAP rats. Molecular docking results and DARTS assays revealed that BAI combined with HMGB1. Co-IP verified that HMGB1 interacted with TLR4 and NLRP3 and that TLR4 interacted with NLRP3. BAI and the HMGB1 inhibitor EP inhibited HMGB1, TLR4, and NLRP3 levels in PA-induced macrophages, increased cell viability, reduced pyroptosis, and ROS release, and inhibited M1 polarization. BAI and EP inhibited PA-induced M1 macrophage polarization and reduced PAC pyroptosis. HMGB1 overexpression partially reversed the effects of BAI on PA-treated macrophages and PACs. Under EP treatment, BAI had no significant effect on the above functions in PA-induced macrophages and PACs. BAI inhibited PA-induced macrophage M1 polarization through the HMGB1/TLR4/NLRP3 pathway, further inhibiting PAC pyroptosis. Our findings provide a theoretical and experimental basis for the molecular mechanism underlying BAI in the treatment of HAP.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02254-z.

Introduction

Acute pancreatitis (AP) is characterized by inflammation of the pancreas, which can lead to self-digestion of the pancreatic tissue, in which premature activation and retention of intracellular proteases can result in damage to and death of pancreatic acinar cells (PACs) [1]. Hyperlipidemic acute pancreatitis (HAP), also known as hypertriglyceridemic pancreatitis, is one of the major types of AP, accounting for approximately 10% of all AP cases [2]. Early persistent multiple organ failure and late infectious necrosis are associated with poor prognosis and mortality in HAP patients [3]. Further insights into the molecular regulation of HAP are essential to guide effective targeted therapy in the clinic.

Baicalein (BAI), a natural flavonoid isolated from the roots of Baicalensis, a traditional Chinese medicine, is believed to have a variety of biological actions, including antioxidation, inflammation, and neuroprotection [4]. The combination of emotion and BAI could significantly reduce tumor necrosis factor-alpha (TNF-α), interleukin (IL)−6, and myeloperoxidase expression in the pancreas of AP rats [5]. BAI protects against pancreatic injury in severe AP rats by inhibiting IL-6 and TNF-α expression [6]. BAI alleviates pancreatic inflammatory injury by regulating the activation of MAPK, STAT3, and nuclear factor kappa B (NF-κB) [7]. However, the molecular mechanism by which BAI affects PAC pyroptosis in HAP remains to be elucidated.

Macrophages are key cells involved in immune function in the human body [8]. In AP, M1 polarization of macrophages is significantly activated, accompanied by an increase in the secretion of inflammatory factors and the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a key proinflammatory factor [9]. Another study showed that peritoneal puncture and drainage treatment could improve AP by increasing the number of M2 macrophages and decreasing the number of M1 macrophages in animals [10]. However, whether BAI can regulate macrophage polarization in HAP remains unknown.

Pyroptosis is a newly identified mode of programmed cell death accompanied by an inflammatory response. NLRP3-mediated activation of caspase-1 leads to the release of proinflammatory factors and ultimately induces an inflammatory response and lytic inflammatory cell death [11]. Excessive fatty acids can induce pyroptosis in PACs by activating M1 polarization of macrophages [12], indicating that the interaction between macrophage polarization and pyroptosis is closely related to HAP pathogenesis. High mobility group box 1 (HMGB1) induces the release of inflammatory cytokines by specifically binding to Toll-like receptor 4 (TLR4) [13, 14]. TLR4 promotes the inflammatory cascade [15]. HMGB1 can activate NLRP3 in various disease processes, promote its formation and aggregation, and promote the inflammatory response and pyroptosis [16, 17]. Inhibiting the formation of the NLRP3 inflammasome improves the inflammatory response in AP [18]. HMGB1 promotes the polarization of M1 macrophages to alleviate inflammation in AP [19]. A previous study by our research group revealed that baicalin inhibits the NLRP3/caspase-1 pathway to alleviate PAC pyroptosis and inflammation in HAP [20].

This study investigated whether the interaction between HMGB1 and TLR4 mediated the influence of baicalin on macrophage polarization and pyroptosis in PACs in HAP.

Materials and Methods

Animal Model

Healthy Sprague–Dawley (SD) rats (weighing 150–200 g) were purchased from the Hunan Slack Jingda Laboratory Animal Co., Ltd. All the animal experiments in this study followed the ARRIVE guidelines and were approved by the Hunan Provincial People's Hospital Ethics Committee Ethics Department 2022 (Provincial No. 05). All the rats were housed under a 12-h light and dark cycle at a suitable temperature of 23 °C, a relative humidity of 50%–60%, and provided free access to drinking water and food. The rats were randomly divided into the sham, HAP, and BAI groups. As mentioned above, a HAP rat model was constructed [20]. In brief, the rats were fed a high-fat diet (77% normal diet, 20% animal fat, and 3% cholesterol) for two weeks. A pancreatitis model was subsequently established. The rats were then injected intraperitoneally with 50 mg/kg pentobarbital sodium. The abdomen was opened. The rats were then retrogradely injected with 1 mL/kg 5% sodium taurocholate (Sigma‒Aldrich, USA) into the common bile duct at a rate of 0.1 mL/min. Then, the injection site was pressed for 3 min, and the area of abdominal delamination was closed. BAI group rats were injected with 20 mg/kg BAI through the tail vein. The rats in the sham and HAP groups were injected with the same amount of normal saline. The rats were anesthetized with 50 mg/kg pentobarbital sodium 12 h after the operation. After the rats were deeply anesthetized, they were sacrificed via cervical dislocation. During the dislocation, the animals ceased spontaneous breathing for 2–3 min and lost their blink reflex. After the rats were sacrificed, the pancreatic tissue and serum were collected for further analysis and detection.

Primary Cell Isolation and Extraction

The production of bone marrow-derived macrophages is induced by bone marrow cells [20] In short, after the isolation of bone marrow cells, red blood cells were eliminated via red blood cell lysis buffer. The macrophages were cultured in complete medium supplemented with 15 ng/mL M-CSF for 7 days. The macrophage surface marker CD68 was detected via flow cytometry (MA5-28,262, eBioscience, USA). The pancreatic tissue was processed by using collagenase at 37 °C to separate the cells. After digestion, the tissue was further dispersed by passing through a nylon cloth to remove any remaining cellular debris. The isolated PACs were cultured in Dulbecco’s modified Eagle’s medium/Ham F-12 medium (DMIM-F12, HyClone, USA) supplemented with 10% fetal bovine serum (FBS, 10099141, Gibco, USA).

Cell Culture and Transfection

To investigate the potential toxicity of BAI on normal macrophages and its effect on PA-induced macrophage viability, the macrophages were treated with various concentrations of BAI (5, 10, 20, 40, 80, and 100 μmol/L) in a gradient manner. The macrophages in the control group were subsequently cultured normally without any treatment. The macrophages were treated with 0.1 mmol/L palmitic acid (PA) for 24 h [21]. Macrophages were treated with 5 mM HMGB1 inhibitor ethyd pyrrvate (EP) [22, 23] and/or 80 μmol/L BAI for 24 h, respectively. Macrophages were transfected with oe-NC and oe-HMGB1 plasmids via Lipofectamine 2000 (Invitrogen, CA) following the manufacturer's instructions. After transfection, the macrophages were treated with 80 μmol/L BAI or 0.1 mmol/L PA for 24 h.

Cell Coculture

To investigate the effects of PA-induced macrophages on PAC pyroptosis, the cells were divided into a NC group (normal culture of primary PACs), a control group (coculture of primary PACs with macrophages for 24 h), and a PA group (coculture of primary PACs with macrophages treated with 0.1 mmol/L PA for 24 h).

To explore the potential mechanism of BAI in PA-induced macrophage-mediated PAC pyroptosis, the cells were divided into a control group (primary PACs cocultured with macrophages for 24 h), a PA group (primary PACs cocultured with macrophages treated with 0.1 mmol/L PA for 24 h), a PA + BAI group (primary PACs cocultured with macrophages treated with 80 μmol/L BAI and 0.1 mmol/L PA for 24 h), a EP group (primary PACs cocultured with macrophages treated with 5 mM EP for 24 h), a PA + EP group (primary PACs cocultured with macrophages treated with 5 mM EP and 0.1 mmol/L PA for 24 h), a PA + BAI + EP group (primary PACs cocultured with macrophages treated with 80 μmol/L BAI, 5 mM EP, and 0.1 mmol/L PA for 24 h), a PA + BAI + oe-NC group (primary PACs cocultured with macrophages treated with 80 μmol/L BAI and 0.1 mmol/L PA and transfected with the oe-NC plasmid for 24 h), and a PA + BAI + oe-HMGB1 group (primary PACs cocultured with macrophages treated with 80 μmol/L BAI and 0.1 mmol/L PA and transfected with the oe-HMGB1 plasmid for 24 h). After coculture for 24 h, PACs were collected for molecular detection.

Hematoxylin and Eosin (H&E) Staining

Pancreatic tissue was fixed with 4% paraformaldehyde for 24 h. The sample was cut into 3 μm slices. The slices were subjected to baking at 62 °C for 8 h to remove any moisture and ensure proper fixation. To remove the residual paraffin, the slices were placed in xylene for 20 min. Next, the slices were sequentially immersed in a series of ethanol solutions with different concentrations to gradually rehydrate the tissue. Next, hematoxylin and eosin were used for 10 min. The staining was observed under an optical microscope (BA210T, MOTIC, Singapore).

Immunohistochemistry (IHC)

The H&E staining method was used to obtain 3 μm slices. The slices were immersed in 0.01 mol/L citrate buffer (pH of 6.0), heated to repair antigens, and placed in a periodate solution to inactivate endogenous enzymes. The slices were incubated overnight with iNOS (Rabbit, AWA45151, Abiowell, China) at 4 °C. The slices were incubated with HRP polyclonal goat anti-rabbit IgG (AWS0005, Abiowell, China). The slices were observed and analyzed under a microscope (MOTIC).

Immunofluorescence (IF)

The IHC staining method was used to identify the antigen. The slices were placed in 5% bovine serum albumin (BSA) and incubated for 60 min. The slices were incubated with primary antibodies against CD68 (mouse, ab955, Abcam, UK), HMGB1 (rabbit, ab79823, Abcam, UK), and NLRP3 (rabbit, 19,771–1-AP, Proteintech, USA) overnight at 4 °C. The slices were incubated with secondary antibodies, including CoraLite594-conjugated AffiniPure goat anti-rabbit IgG (H + L) (1:200, SA00013-4, Proteintech, USA) and CoraLite488-conjugated goat anti-mouse IgG (H + L) (1:200, SA00013-1, Proteintech, USA), at 37 °C for 60 min. The slices were preserved in buffer glycerin and observed under a microscope (MOTIC).

Cell Counting Kit-8 (CCK-8) Assay

A total of 5 × 10³ cells were added to 96-well culture plates and incubated at 37 °C. After adhesion, 10 μL of CCK-8 solution was added to each well. After the incubation period, the absorbance of each sample was measured via a spectrophotometer at a wavelength of 450 nm.

Enzyme-Linked Immunosorbent Assay (ELISA)

In accordance with the manufacturer's instructions, an IL-1β ELISA kit (KE20005, Proteintech, USA) and an IL-18 ELISA kit (CSB-E04610r, CUSABIO, China) were used to determine the IL-1β and IL-18 levels, respectively, in both the serum samples and the cell supernatants.

Flow Cytometry

The cells were washed with precooled buffer. The cells were incubated with CD86-FITC (11–0860-82, eBioscience, USA) at 4 °C for 40 min. The cells were subsequently centrifuged at 1,500 × g for 5 min and resuspended in PBS. Flow cytometry (A00-1–1102, Beckman, USA) was used to detect macrophage polarization. A Pyroptosis/Caspase-1 Assay Green Kit (ab219935, Abcam, UK) was used to evaluate pyroptosis via flow cytometry according to the manufacturer’s instructions. The degree of pyroptosis was evaluated by calculating the percentage of cells that were double positive for two markers, propidium iodide (PI) and caspase-1. In addition, to measure the release of reactive oxygen species (ROS), an ROS assay kit (S0033S, Beyotime, China) was used. The ROS levels in the treated cells were analyzed via flow cytometry (Beckman).

Western Blotting

PACs and fresh pancreatic tissue were added to RIPA lysis buffer (P0013B; Beyotime, China) for lysis. Total protein was extracted, and bicinchoninic acid (BCA) kits were used for determination. The same protein mass was separated via 10% and 15% SDS polyacrylamide gels (Bio-Rad, USA) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk at room temperature for 45 min. The membrane was then incubated with primary antibodies, including anti-ASC (rabbit, 10500–1-AP, Proteintech, USA), anti-cleaved caspase-1 (rabbit, #4199, Cell Signaling Technology, Inc., USA), anti-GSDMD-N (rabbit, ab215203, Abcam, UK), anti-GSDMD (rabbit, 20770–1-AP, Proteintech, USA), anti-HMGB1 (rabbit, ab79823, Abcam, UK), anti-TLR4 (rabbit, ab13556, Abcam, UK), anti-NLRP3 (rabbit, 19771–1-AP, Proteintech, USA), and the negative control β-actin (mouse, 66009–1-Ig, Proteintech, USA), overnight at 4 °C. The membrane was incubated with an HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (AWS0001, Abiowell, China) and an HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (AWS0002, Abiowell, China) at room temperature for 1 h. The ChemiScope6100 system (CLiNX) was used to capture the chemiluminescence signal from the membrane. ImageJ was used for grayscale analysis. The original images can be found in Fig. S3-4. The results are expressed as the expression of the target protein relative to β-actin.

Coimmunoprecipitation (Co-IP)

HEK-293 T cells (AW-CNH086, Abiowell, China) were lysed with RIPA buffer. The cell lysate was immunoprecipitated with anti-HMGB1 (rabbit, ab79823, Abcam, UK), anti-TLR4 (rabbit, ab13556, Abcam, UK), and goat anti-rabbit IgG (B900610, Proteintech, USA) at 4 °C overnight. Then, 20 μL of protein A/G agarose beads was mixed with 200 μL of IP lysate and centrifuged at 3000 rpm for 3 min. Next, the cell lysate was incubated with anti-HMGB1, anti-TLR4, and anti-NLRP3 (rabbit, 19,771–1-AP, Proteintech, USA) antibodies at 4 °C overnight. The mixtures were incubated with protein A/G beads for 3 h at 4 °C. The beads were washed at 4 °C with PBS containing a protease inhibitor mixture, centrifuged five times, resolved with 10% SDS buffer, and then analyzed via western blotting. The original images can be found in Fig. S3-4.

Molecular Docking Verification

The 3D structure of the protein was obtained from the PDB database (http://www.rcsb.org/). Using PyMOL software, ligands and water molecules were removed from the protein structure. The 3D structure of the compound was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). AutoDock Tools version 1.5.6 software was used to prepare the protein and compound structures for molecular docking. This involved adding atomic charges and assigning atomic types to the molecules. All necessary parameters and settings for the molecular docking simulation were set up in AutoDock Vina. The molecular docking patterns were visualized via PyMOL (version 2.4).

Drug Affinity Responsive Target Stability (DARTS)

DARTS was conducted as described previously [24, 25]. Briefly, recombinant HMGB1 protein (0.5 μg/mL) was incubated with BAI (0, 20, 40, 80, or 100 μmol/L) at room temperature for 1 h, followed by the addition of pronase for 20 min. The reactions were terminated by the addition of loading buffer and analyzed by Western blot analysis. The original images can be found in Fig. S3.

Statistical Analysis

All the measured values are reported as the means ± standard deviations (SDs), and the data were analyzed and statistically evaluated via GraphPad Prism 8.0 statistical software. Statistical significance was assessed via one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

Results

BAI Treatment Reduced the Number of M1 Macrophages in the Pancreatic Tissue of HAP Rats

To investigate the effect of BAI on the polarization of macrophages in HAP rats, a HAP model was induced with high-fat and high-sodium taurine cholate. H&E staining revealed that the morphology of the pancreas in the sham group was normal, with a clear structure and no obvious acinar abnormalities. The pancreatic tissue of the HAP group exhibited interstitial edema, necrosis, and inflammatory cell infiltration. BAI treatment improved pancreatic tissue lesions in HAP rats (Fig. 1A). IHC revealed greater iNOS expression in the pancreatic tissue of the HAP group than in that of the sham group. BAI decreased iNOS levels in the pancreatic tissues of HAP rats (Fig. 1B). Compared with that in sham rats, the proportion of CD86⁺ cells in the pancreatic tissues of HAP rats was greater. BAI reduced the proportion of CD86⁺ cells (Fig. 1C). Our results showed that BAI treatment reduced the number of M1 macrophages in the pancreatic tissue of HAP rats.

Fig. 1.

BAI reduced the number of M1 macrophages in the pancreatic tissue of HAP rats. (A) H&E staining was used to observe the histopathological morphology of the pancreas. (B) IHC analysis of iNOS expression in pancreatic tissues. (C) Flow cytometry was used to evaluate the proportion of CD86⁺ cells in pancreatic tissue. *P < 0.05 vs. the sham group; # P < 0.05 vs. the HAP group

BAI Treatment Inhibited Pyroptosis in HAP Rats

The effect of BAI on pyroptosis in HAP rats was further investigated. CD68, HMGB1, and NLRP3 expression in pancreatic tissues was greater in the HAP group than in the sham group. BAI treatment decreased CD68, HMGB1, and NLRP3 expression in the pancreatic tissue of HAP rats (Figs. 2A and B). Compared with those in the sham group, ASC, cleaved caspase-1, and GSDMD-N expression was increased in the HAP group. BAI treatment inhibited ASC, cleaved caspase-1, and GSDMD-N levels in HAP rats. GSDMD expression did not differ between the groups (Fig. 2C). The serum IL-1β and IL-18 levels in HAP rats were greater than those in the sham group, whereas BAI treatment reduced the serum IL-1β and IL-18 levels in HAP rats (Fig. 2D). Our findings suggested that BAI inhibited pyroptosis in HAP rats.

Fig. 2.

BAI decreased macrophage pyroptosis in HAP rats. (A and B) IF analysis of CD68 andHMGB1, CD68 and NLRP3 expression in pancreatic tissues. (C) Western blot analysis of ASC, cleaved caspase-1, GSDMD, and GSDMD-N expression. (D) IL-1β and IL-18 levels. * P < 0.05 vs. the sham group; # P < 0.05 vs. the HAP group

BAI Regulated the HMGB1/TLR4/NLRP3 Pathway

The potential molecular mechanism of BAI in HAP rats was further investigated. Compared with those in the sham group, the HMGB1, TLR4, and NLRP3 levels in the HAP group were increased. BAI treatment decreased HMGB1, TLR4, and NLRP3 expression levels in HAP rats (Fig. 3A). To verify the degree of binding between BAI small molecules and the HMGB1 protein, BAI was used as a ligand, and HMGB1 was used as a receptor. The binding between the receptor and ligand is more stable when the binding energy is lower [26, 27]. Molecular docking results revealed that the overall and local landscape of BAI interacted with HMGB1. The binding affinity of BAI for HMGB1 was −6.1 kcal/mol, which was less than −5.0 kcal/mol (Fig. 3B), indicating stable binding [28]. We also conducted a DARTS assay and found that HMGB1 exhibited increased resistance to pronase-induced proteolysis upon BAI treatment (Fig. 3C). The interactions between HMGB1 and TLR4, HMGB1 and NLRP3, and TLR4 and NLRP3 were subsequently verified via Co-IP (Fig. 3D). Our results showed that BAI inactivated the TLR4/NLRP3 pathway by binding to HMGB1.

Fig. 3.

BAI regulated the HMGB1/TLR4/NLRP3 pathway. (A) Western blot analysis of HMGB1, TLR4, and NLRP3 expression. (B) Docking patterns between the small-molecule BAI and HMGB1 protein. (C) DARTS confirmed the binding of BAI to HMGB1. (D) Co-IP detection of the interaction of HMGB1 with TLR4 and NLRP3 and the interaction of TLR4 with NLRP3. *P < 0.05 vs. the sham group; # P < 0.05 vs. the HAP group

BAI Inhibited Macrophage Pyroptosis and M1 Polarization Through the HMGB1/TLR4/NLRP3 Pathway

The potential mechanism of the effect of BAI in PA-induced macrophages was further explored at the cellular level. Figure 4A shows the morphology of the macrophages and the identification of the macrophage surface marker CD68 via flow cytometry, indicating the successful acquisition of primary macrophages. The results of the CCK-8 assay revealed that BAI had no effect on normal macrophage viability at concentrations lower than 80 μmol/L. Moreover, BAI increased PA-induced macrophage activity in a dose-dependent manner, and 80 μmol/L BAI had the most significant effect (Fig. 4B). Therefore, 80 μmol/L BAI was selected for subsequent studies.

Fig. 4.

BAI inhibited macrophage pyroptosis and M1 polarization through the HMGB1/TLR4/NLRP3 pathway. (A) Primary macrophage identification. (B) Cell viability. * P < 0.05 vs. the control group. (C) Western blot analysis of HMGB1, TLR4, and NLRP3 expression in macrophages. (D) Cell viability was measured via a CCK-8 assay. (E) Pyroptosis of macrophages was detected via flow cytometry. (F) Western blot analysis of ASC, cleaved caspase-1, GSDMD, and GSDMD-N expression in macrophages. (G) ROS levels in macrophages were detected via flow cytometry. (H) ELISA analysis of IL-1β and IL-18 levels. (I) Flow cytometry analysis of the proportion of CD86⁺ cells. *P < 0.05 vs. the control group; # P < 0.05 vs. the PA group; & P < 0.05 vs. the PA + BAI + oe-NC group

PA increased the expression of HMGB1, TLR4, and NLRP3 in macrophages, whereas BAI and the HMGB1 inhibitor EP inhibited the PA-induced expression of HMGB1, TLR4, and NLRP3 in macrophages. Compared with those in the PA + BAI + oe-NC group, HMGB1, TLR4, and NLRP3 expression in macrophages in the PA + BAI + oe-HMGB1 group was increased. However, under EP treatment, BAI did not affect the expression of HMGB1, TLR4, or NLRP3 in PA-induced macrophages (Figs. 4C and S1A). The CCK-8 results revealed that PA-induced macrophage activity decreased, whereas BAI and EP increased PA-induced macrophage activity. Compared with that in the PA + BAI + oe-NC group, the decreased macrophage activity was observed in the PA + BAI + oe-HMGB1 group. However, BAI did not affect PA-induced macrophage activity under EP treatment (Figs. 4D and S1B).

Flow cytometry revealed that PA induced macrophage pyroptosis, whereas BAI and EP inhibited PA-induced macrophage pyroptosis. Compared with that in the PA + BAI + oe-NC group, macrophage apoptosis was increased in the PA + BAI + oe-HMGB1 group. Under EP treatment, BAI did not affect PA-induced macrophage pyroptosis (Figs. 4E and S1C). Compared with those in the control group, ASC, cleaved caspase-1, and GSDMD-N expression in the PA group was increased, whereas BAI and EP reduced ASC, cleaved caspase-1, and GSDMD-N expression in PA-induced macrophages. Compared with that in the PA + BAI + oe-NC group, ASC, cleaved caspase-1, and GSDMD-N expression in macrophages in the PA + BAI + oe-HMGB1 group was increased. BAI did not affect ASC, cleaved caspase-1, or GSDMD-N expression in PA-induced macrophages under EP treatment. No significant differences in GSDMD expression were observed among the groups (Figs. 4F and S1D). ROS release was increased in PA-induced macrophages, whereas BAI and EP inhibited ROS release in PA-induced macrophages. Compared with that in the PA + BAI + oe-NC group, ROS release was greater in the PA + BAI + oe-HMGB1 group. BAI did not affect ROS release in PA-induced macrophages under EP treatment (Figs. 4G and S1E). IL-1β and IL-18 levels were increased in the supernatant of PA-induced macrophages. In contrast, BAI and EP decreased the IL-1β and IL-18 levels in the supernatant of PA-induced macrophages. oe-HMGB1 increased the IL-1β and IL-18 levels in the PA + BAI + oe-NC group. BAI did not affect IL-1β or IL-18 levels in PA-induced macrophages under EP treatment (Figs. 4H and S1F). In addition, PA increased the number of CD86⁺ macrophages, whereas BAI and EP decreased the number of CD86⁺ macrophages. oe-HMGB1 increased the number of CD86⁺ macrophages in the PA + BAI + oe-NC group. Meanwhile, BAI did not affect the number of CD86⁺ macrophages among PA-induced macrophages under EP treatment (Figs. 4I and S1G). Our results showed that BAI reduced macrophage pyroptosis and M1 polarization by inhibiting the HMGB1TLR4/NLRP3 pathway.

Macrophage Polarization Promoted Pyroptosis in PACs in Pancreatitis

The effect of PA-induced macrophages on PACs was investigated via a cell coculture technique. The results revealed that PA-induced macrophages inhibited the activity of PACs (Fig. 5A) and promoted pyroptosis (Fig. 5B). ASC, cleaved caspase-1, and GSDMD-N levels were greater in the PA group than in the control group, while GSDMD expression did not differ significantly between the groups (Fig. 5C). Compared with that in the control group, ROS release in PACs was increased in the PA group (Fig. 5D). Compared with those in the control group, the IL-1β and IL-18 levels in the PA group were increased (Fig. 5E). Our results suggested that PA-induced macrophages promoted pyroptosis in PACs.

Fig. 5.

Polarization of macrophages promotes pyroptosis of PACs in pancreatitis. (A) Cell viability was measured via a CCK-8 assay. (B) Pyroptosis of PACs was detected by flow cytometry. (C) Western blot detection of ASC, cleaved caspase-1, GSDMD, and GSDMD-N expression in PACs. (D) ROS levels in PACs. (E) IL-1β and IL-18 levels in the cell supernatant. *P < 0.05 vs. the control group

BAI Mediated M1 Macrophage Polarization to Inhibit Pyroptosis in PACs Through the HMGB1/TLR4/NLRP3 Pathway

The potential molecular mechanism of PA-induced macrophage pyroptosis in PACs was investigated via a cell coculture technique. The results showed that PA-induced macrophages inhibited the activity of PACs. BAI and EP increased PAC activity in PA-induced macrophages. Compared with that in the PA + BAI + oe-NC group, PAC activity was inhibited in the PA + BAI + oe-HMGB1 group (Figs. 6A and S2A). BAI and EP inhibited pyroptosis in PACs, while oe-HMGB1 promoted pyroptosis in PACs (Figs. 6B and S2B). Compared with that in the PA group, ASC, cleaved caspase-1, and GSDMD-N expression in the PA + BAI group and PA + EP group was lower. oe-HMGB1 promoted ASC, cleaved caspase-1, and GSDMD-N expression in PACs. GSDMD expression did not differ between the groups (Figs. 6C and S2C). Compared with that in the PA group, ROS release in the PA + BAI group and PA + EP group was decreased. oe-HMGB1 promoted ROS release in PACs (Figs. 6D and S2D). IL-1β and IL-18 expression in the PA + BAI and PA + EP groups was lower than that in the PA group. oe-HMGB1 increased IL-1β and IL-18 expression in PACs (Figs. 6E and S2E). Our results suggested that BAI inhibited pyroptosis in PACs via macrophage M1 polarization through the HMGB1/TLR4/NLRP3 pathway.

Fig. 6.

BAI mediated M1 macrophage polarization to inhibit pyroptosis in PACs through the HMGB1/TLR4/NLRP3 pathway. (A) Cell viability. (B) Pyroptosis of PACs. (C) Western blot detection of ASC, cleaved caspase-1, GSDMD, and GSDMD-N expression in PACs. (D) Flow cytometry analysis of ROS levels in PACs. (E) IL-1β and IL-18 levels in the cell supernatant. *P < 0.05 vs. the control group; # P < 0.05 vs. the PA group; & P < 0.05 vs. the PA + BAI + oe-NC group

Discussion

BAI reduced the degree of pancreatic lesions in HAP rats; decreased iNOS expression and the number of M1 macrophages; downregulated CD68, HMGB1, NLRP3, ASC, cleaved-caspase1, and GSDMD-N expression; and decreased serum IL-1β and IL-18 levels. Molecular docking and DARTS results revealed that the BAI small molecule was bound to the HMGB1 protein. Co-IP results revealed that HMGB1 interacted with TLR4 and NLRP3 and that TLR4 interacted with NLRP3. As indicated by in vitro experiments, BAI and EP decreased HMGB1, TLR4, and NLRP3 levels induced by PA in macrophages, improved viability, and inhibited pyroptosis, ROS release, and M1 polarization. In addition, BAI and EP inhibited the ability of PA-treated macrophages to promote pyroptosis in PACs. The overexpression of HMGB1 partially reversed the effect of BAI on PA-induced macrophages and PACs.

Macrophages play a central role in various inflammatory diseases [29]. Previous studies have shown that CO combined with hemoglobin vesicle therapy can polarize macrophage lines toward the M2-like phenotype, inhibit HMGB1 expression, and improve AP in mice [30]. MALAT1 promotes macrophage M1 polarization to induce AP via the HMGB1/TLR4 axis [19]. In this work, BAI inhibited HMGB1 expression in rat pancreatic tissue, reduced the number of M1 macrophages, and improved HAP. The in vitro results revealed that BAI reduced HMGB1 levels in PA-induced macrophages and inhibited M1 polarization. In addition, the molecular docking and DARTS results revealed that the small molecule BAI was stably bound to the HMGB1 protein, indicating that BAI inhibited M1 macrophage polarization by binding to HMGB1, thereby alleviating HAP.

Studies have shown that protocatechuic acid prevents L-arginine-induced AP in rats via the HMGB1/TLR4 pathway [31]. The inhibition of miRNA-340-5p mediated by CCCTC binding factors aggravates myocardial injury in AP rats by activating the HMGB1/TLR4 axis [32]. Chaiqin Chengqi decoction reduces the severity of AP by inhibiting TLR4 and the NLRP3 inflammasome [33]. Combined antibiotic treatment inhibits intestinal bacterial translocation to the pancreas and inhibits NLRP3, cleaved caspase-1, and IL-1β levels in colon and pancreatic tissue, thereby preventing experimental AP [34]. Paeonol inhibits macrophage ROS release and M1 polarization through the NLRP3 inflammasome pathway, thereby preventing AP [18]. Proanthocyanidins inhibit the macrophage NLRP3 inflammasome, ROS levels, and M1 polarization and improve cell viability to alleviate AP in mice [35]. In this study, our results revealed that BAI and EP reduced the levels of NLRP3, ASC, cleaved caspase-1, GSDMD-N, IL-1β, and IL-18 in the pancreatic tissue of HAP rats and in PA-induced macrophages. BAI and EP increased PA-induced macrophage activity and decreased apoptosis and ROS release. Our results are consistent with those of previous studies. In addition, our findings revealed that HMGB1 interacted with TLR4 and NLRP3, that TLR4 interacted with NLRP3, and that the overexpression of HMGB1 interrupted the effect of BAI on PA-induced macrophages. The overexpression of HMGB1 increased the levels of HMGB1, TLR4, NLRP3, ASC, cleaved caspase-1, GSDMD-N, IL-1β, and IL-18 in PA-induced macrophages; decreased cell viability; and promoted apoptosis, ROS release, and M1 polarization. Under EP treatment, BAI had no significant effect on the above functions in PA-induced macrophages and PACs. Our findings suggested that BAI binding to HMGB1 inhibited PA-induced macrophage M1 polarization and pyroptosis via the TLR4/NLRP3 pathway.

The active ingredient of Chaiqin Chengqi decoction alleviates pancreatic necrosis and inflammation in AP mice through inhibiting the pyroptosis of APCs by targeting GSDMD [36]. Studies have shown that loss of S100A9 in the pancreatic duct alleviates AP by targeting VN1-mediated ROS release in PACs to inhibit NLRP3 activation [37]. Excessive fatty acids induce pyroptosis in PACs through the polarization of M1 macrophages [12]. A previous study by our research group revealed that BAI inhibited the NLRP3/caspase-1 pathway, alleviating PAC pyroptosis and inflammation in hyperlipidemic pancreatitis [20]. In this work, our results further revealed that PA-induced macrophages inhibited PAC activity and promoted apoptosis, pyroptosis, and ROS release. BAI and EP prevented this effect, increased PAC activity, and inhibited apoptosis, pyroptosis, and ROS release, whereas HMGB1 overexpression decreased PAC activity and promoted apoptosis, pyroptosis, and ROS release. Our findings suggested that BAI binding to HMGB1 inhibited PA-induced macrophage M1 polarization through the TLR4/NLRP3 pathway, thereby reducing pyroptosis in PACs, as illustrated in Fig. S5. Our results are consistent with those of previous studies. In future work, we will further explore other molecular mechanisms of BAI in HAP. BAI is a prospective candidate bioactive dentin bonding agent [38]. However, BAI has not been applied in the clinic for HAP and is still being studied in a preclinical setting.

Conclusion

Our results suggested that BAI reduced PAC pyroptosis in HAP by inhibiting M1 polarization of macrophages via the HMGB1/TLR4/NLRP3 pathway. Our findings might provide new targets for the clinical treatment of HAP.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Xiangyang Wang, Peng Liu: conceptualization, methodology, project management, resources, validation, Formal analysis, visualization, Writing - original draft and Writing - review & editing.

Yaxiong Zhou, Yilei Liu, Tingting Mo: data curation, Formal analysis, investigation and Writing - review & editing.

Zhiyuan Chen, Yu Zhang: data curation.

Li Yang: Supervision; conceptualization, methodology, Funding acquisition, resources, validation, Formal analysis, visualization and Writing - review & editing.

Funding

The study was supported by funds from Natural Science Foundation of Hunan Province, China [grant number 2023JJ30351].

Data Availability

The dataset supporting the conclusions of this article is included within the article.

Declarations

Ethics Approval

All animal experiments in this study was followed the ARRIVE guideline and approved by Hunan Provincial People's Hospital Ethics Committee Ethics Department 2022 (Provincial No. 05).

Competing Interests

The authors declare no competing interests.