Cerulein, curcumin and trigonelline were purchased from MilliporeSigma. The monoclonal antibodies against nuclear factor erythroid 2-related factor 2 (Nrf2; cat. no. sc-365949) and α-smooth muscle actin (SMA; cat. no. sc-32251) were purchased from Santa Cruz Biotechnology, Inc. The antibody against collagen I (ab34710)was purchased from Abcam. The antibody against heme oxygenase (HO)-1 (70081S) was purchased from Cell Signaling Technology, Inc. The TRIzol reagent and High-Capacity RNA-to-cDNA™ Kit were purchased from Thermo Fisher Scientific, Inc.

All experiments were performed according to the protocols of the Animal Care Committee of Wonkwang University and approved by the Institutional Animal Care and Use Committee Certification of Wonkwang University, South Korea (approval no. WKU 25-4). C57BL/6 female mice (age, 6–8 weeks; weight, 15–20 g; n=63 mice) were purchased from ORIENT BIO, INC. All animals were bred and housed in standard shoebox cages in a climate-controlled room with an ambient temperature of 23±2°C, humidity of 50±5%, and a 12/12-h light-dark cycle. The animals were fed standard laboratory chow, allowed water ad libitum and randomly assigned to either the control or experimental group (n=9/group). CP was induced by intraperitoneal injection of a supramaximal concentration of the stable cholecystokinin analog cerulein (50 µg/kg), administered six times/day at 1-h intervals, and repeated four times/week for a total of 3 weeks. The animals in the control group were administered saline instead of cerulein under the same conditions. Curcumin (10 or 20 mg/kg) or DMSO (control) was administered intraperitoneally 1 h before the first cerulein injection to the experimental and control groups, respectively. Mice were sacrificed 24 h after the last cerulein injection. Isoflurane (induction, 4.5; maintenance; 1.5%) in 95% O₂ and 5% CO₂ was used for anesthesia. CO₂ inhalation was used for euthanasia with a flow rate that displaced 50% of the cage vol/min and cervical dislocation was also performed to ensure death following CO₂ asphyxiation. The pancreatic samples were immediately collected for further examination.

For histological examination and scoring, the pancreas tissue was fixed in 4% formalin solution for overnight at room temperature, embedded in paraffin, cut into 4-µm sections, stained with hematoxylin-eosin (H&E) and examined under a light microscope. H&E staining was performed as follows: Hematoxylin for 8 min and eosin for 2 min at room temperature. The pancreases were analyzed in a blinded manner and graded using a semi-quantitative scoring system for edema, loss of acini and inflammatory cell infiltration. The samples were scored on a scale from 0 to 3 based on the presence of glandular atrophy and inflammation (0, normal, no glandular atrophy and inflammation; 1, mild, found in less than 25% of the pancreas. 2=moderate, found in less than 25 to 75% of the pancreas. 3=found in more than 75% of the pancreas). For Sirius Red staining, paraffin-embedded pancreatic sections (4-µm thick) were stained with 0.1% Sirius Red F3B in saturated picric acid for 1 h at room temperature (Sigma-Aldrich; Merck KGaA).

For immunofluorescence staining, tissues were sectioned at 9 µm thickness. The slides were blocked with blocked with serum (1% BSA; BSAS0.1; Bovogen Biologicals) at RT for 1 h and stained overnight with primary antibodies against α-SMA (1:1,000) and collagen I (1:1,000) at 4°C, followed by treatment with Alexa Fluor^®594-labeled secondary antibody (1:2,000; A11012, A11005; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Nuclei were counterstained with DAPI for 5 min at room temperature. Stained sections were visualized under a confocal laser microscope (Olympus Corporation).

mRNA transcripts in pancreatic tissue were analyzed using RT-qPCR. Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions. Total RNA (1 µg) was converted to cDNA using a High-Capacity RNA-to-cDNA™ kit (4387406; Applied Biosystems; Thermo Fisher Scientific, Inc.) at 37°C for 60 min and 95°C for 5 min. RT-qPCR was carried out using TaqMan™ Universal Master Mix II, no UNG (Applied Biosystems; Thermo Fisher Scientific, Inc.) on ABI StepOnePlus detection system according to the manufacturer's instructions (Applied Biosystems; Thermo Fisher Scientific, Inc.). The conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 10 sec and 60°C for 30 sec. The expression of the genes of interest were analyzed in triplicate and included a control reaction in which reverse transcriptase was not added to the reaction mixture. Relative gene expression (target gene expression normalized to that of the endogenous control gene) was calculated using the comparative Cq method (26). The results were normalized to those of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt). Forward, reverse and probe oligonucleotide primers were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.; actin α2 (Acta2), cat. no. Mm01546133_m1; fibronectin 1 (Fn1), cat. no. Mm01256734_m1; collagen, type I, alpha 1 (Col1a1), cat. no. Mm00801666_g1; collagen, type IV, alpha 1 (Col4a1), cat. no. Mm01210125_m1, transforming growth factor beta (Tgfb), cat. no. Mm00441726 _m1 and Hprt, cat. no. Mm03024075_m1). The sequences of the primers are not commercially available.

Mouse PSCs were prepared from pancreatic tissue as previously described (7). The pancreas was immediately cut into small pieces with scissors and digested for 20 min at 37°C in a shaking water bath using Gey's balanced salt solution (GBSS) containing collagenase, filtered through a 100-µm nylon mesh and subjected to isopycnal separation with Nycodenz solution (D2158-100G; Sigma Aldrich). The PSCs were collected from the upper layer of the gradient, washed with GBSS and cultured in DMEM, high glucose, pyruvate (11995; Gibco™) containing 10% fetal bovine serum (cat. no. 16000-044; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin. The isolated PSCs were incubated at 37°C with 5% CO₂, and the cells from passages 3 to 5 were used for further experiments. PSCs were cultured in serum-free DMEM for 24 h before treatment with the experimental reagents at 37°C. To investigate the effect of curcumin on HO-1, PSCs were treated with curcumin (1, 5, 10 or 20 µM) for 6, 9, 12 or 24 h. PSCs were treated with 10 µM cobalt protoporphyrin (CoPP) for 6 h as a positive control for HO-1 expression. To investigate the effect of curcumin on the Nrf2/HO-1 pathway, PSCs were treated with 10 µM curcumin for 6 h in the presence or absence of trigonelline 10 µM pretreated and then treated with curcumin. To investigate whether curcumin-induced HO-1 was associated with the antifibrotic effect in PSCs, PSCs were treated with or without pretreatment with 10 µM tin protoporphryin-IX (SnPP) and curcumin to determine the expression of HO-1. Next, PSCs were divided into groups treated with 10 µM curcumin for 1 h and 0.5 ng/ml TGF-β1 for 24 h.

Mouse PSCs were lysed on ice with radioimmunoprecipitation assay lysis buffer (iNtRON Biotechnology). Then, the lysates were boiled in 62.5 mM Tris-HCl buffer, pH 6.8, containing 2% SDS, 20% glycerol and 10% 2-mercaptoethanol. Protein samples were quantified using BSA and were loaded into each well (20 µg) and separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were blocked with 5% skimmed milk in PBS-0.1% Tween-20 (PBST) for 2 h at room temperature and incubated overnight with antibodies against HO-1 (1:1,000) for overnight at 4°C. After washing three times with PBST, each blot was incubated with peroxidase-conjugated secondary antibody (1:5,000; SA002-500; GenDEPOT) for 1 h at room temperature. The proteins were visualized using an enhanced chemiluminescence detection system (GE Healthcare) according to the manufacturer's protocols. The bands were detected and quantified by using Quantity One software (version 4.5.2; Bio-Rad Laboratories, Inc.).

Mouse PSCs were plated at density of 1×10⁵ cells/well in a chamber slide and incubated with 10 µM curcumin for 6 h at 37°C. The cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed thrice with PBST. The cells were treated with 0.1% Triton X-100 for 15 min at room temperature. After washing with PBST, non-specific binding sites were blocked with serum (1% BSA) for 1 h at room temperature and incubated overnight with Nrf2 antibody at 4°C (1:1,000). The cells were washed with PBST and incubated with AlexaFluor^®594 secondary antibody (1:2,000; A11012; Invitrogen) for 2 h at room temperature in the dark. For nuclear staining, the cells were incubated with DAPI (5 mg/ml) for 5 min at room temperature. The slides were washed with PBST and mounted for examination under a confocal laser microscope (Olympus Corporation).

Data are expressed as mean ± standard error of the mean. Statistical significance was evaluated using one-way analysis of variance. Post-hoc analysis using the Duncan method for multiple comparisons among groups. P<0.05 was considered to indicate a statistically significant difference. All experiments were conducted in triplicate.

To evaluate the effect of curcumin on CP, mice were intraperitoneally administered either DMSO (control) or curcumin (10 or 20 mg/kg) 1 h before the first administration of cerulein (Fig. 1A). Following CP induction, H&E staining was conducted to investigate histological changes in the pancreas. The pancreas of CP mice was characterized by the loss of acinar cells, inflammatory cell infiltration and edema, which were suppressed by curcumin treatment (Fig. 1B). The curcumin treatment group showed a dose-dependent decrease in the edema, inflammation, and glandular atrophy compared to the CP group (Fig. 1C).

CP is typically accompanied by pancreatic fibrosis due to activation of PSCs, which express α-SMA and produce ECM components, such as collagen and fibronectin (6,7). Therefore, the activation of PSCs in the pancreas was evaluated using immunofluorescence staining of α-SMA. A marked increase of α-SMA-positive PSCs was displayed in tissue from cerulein-induced CP mice. However, curcumin treatment significantly decreased the α-SMA-positive area of pancreatic tissue (Fig. 2A and B). RT-qPCR was performed to examine the mRNA expression of α-SMA. The elevated mRNA expression levels of Acta2 (which encodes α-SMA) associated with CP was decreased by curcumin treatment (Fig. 2C).

Next, the effects of curcumin on ECM production in activated PSCs were investigated. Sirius Red staining was performed to evaluate the production and deposition of collagen, which is a representative ECM component (27). In the cerulein-induced CP group, collagen production and deposition increased significantly, whereas in the curcumin-treated group, collagen production and deposition decreased in a concentration-dependent manner (Fig. 3A and B). Collagen I levels were evaluated using immunofluorescence staining. The collagen I-positive area significantly increased in the cerulein-induced CP group but decreased in the curcumin-treated group (Fig. 3C and D). In addition, curcumin decreased the mRNA expression levels of ECM components, such as Col1a1, Col4a1 and Fn1, which were increased in cerulein-induced CP samples (Fig. 3E-G).

Curcumin affects inflammation, oxidative stress and fibrosis via the Nrf2/HO-1 pathway (19,28). Therefore, PSCs were isolated from the mouse pancreas to investigate the effects of curcumin. The changes in the mRNA and protein expression of HO-1 in PSCs following exposure to curcumin were examined. PSCs were treated with curcumin at different doses (1, 5, 10 or 20 µM) for 6, 9, 12 or 24 h. PSCs were treated with 10 µM CoPP for 6 h as a positive control for HO-1 expression. mRNA expression of HO-1 in PSCs increased following 6 h treatment with 5 and 10 µM curcumin and decreased at 20 µM curcumin (Fig. 4A). In addition, HO-1 mRNA expression significantly increase after 3 h treatment with 10 µM curcumin, reached its highest level after 6 h and decreased after 9 and 24 h. (Fig. 4B).

To investigate changes in the protein expression levels of HO-1, PSCs were treated with 10 µM curcumin for 1, 2, 3, 6, 9 or 24 h. HO-1 protein expression began to increase from 3 h after curcumin treatment, reached the highest level at 9 h, and showed a tendency to slightly decrease after 24 h. (Fig. 4C). As curcumin is known to produce HO-1 via the Nrf2/HO-1 pathway (19,28), changes in Nrf2 expression in PSCs were investigated. Nrf2 exists in the cytoplasm; however, when activated, it translocates to the nucleus, binds antioxidant-responsive elements (ARE) and transcribes related factors such as HO-1, NAD(P)H quinone oxidoreductase 1 and superoxide dismutase (29). To determine whether curcumin increased expression of HO-1 via the Nrf2/HO-1 pathway, PSCs were pretreated with 10 µM trigonelline, an Nrf2 inhibitor, for 6 h and curcumin (10 µM for 6 h). In the DMSO-treated control group, Nrf2 was present in the cytosol; however, in the curcumin-treated group, it was activated and translocated to the nucleus (Fig. 4D). Additionally, pretreatment with trigonelline decreased the mRNA expression levels of HO-1, which were increased by curcumin (Fig. 4E).

Whether curcumin affects PSC activation and ECM production in PSCs by inducing HO-1 production was investigated. The effect of curcumin on HO-1 expression was investigated after treatment with SnPP, a HO-1 inhibitor. PSCs were pretreated in the presence or absence of SnPP (10 µM) for 1 h and treated with curcumin (10 µM). After 24 h, HO-1 expression was evaluated at the protein level. When the PSCs were treated with curcumin (10 µM) alone, HO-1 expression increased; however, in the group pretreated with SnPP, the increase in HO-1 caused by curcumin was suppressed (Fig. 5A). This suggested that SnPP treatment effectively suppresses the effect of curcumin on HO-1 expression. Next, whether curcumin-induced HO-1 affected PSC fibrosis was investigated. PSCs were treated with TGF-β1 (0.5 ng/ml for 24 h) for activation and the markers of PSC activation and ECM production (Acta2, Tgfb1, Col1a1 and Col4a1) were measured. Increased expression following TGF-β1 treatment and a significant decrease in expression of all factors following curcumin treatment were observed. However, in the SnPP-pre-treated group, the inhibitory effects of curcumin on PSC activation and ECM production were reversed (Figs. 5B-D).