The pancreatic human duct cell line, HPDE6-C7, was purchased from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone) supplemented with high glucose and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific) at 37°C in a 5% CO₂ incubator.

Cell viability assay using the MTT method was carried out to determine the most suitable damaging concentration of ATP (Solarbio) in vitro. In brief, HPDE6-C7 cells (1×10⁵ cells/ml) were treated with various concentrations of ATP (40, 20, 10, 5, 2.5 and 1.25 mM) for 16 h after 24 h of culture in 96-well plates. Subsequently, 10 µl MTT solution (5 mg/ml) were added into each well followed by 4 h of incubation at 37°C. Finally, the formazan crystals were dissolved with 100 µl dimethyl sulfoxide (DMSO) and measured with a microplate reader (BioTek) at a wavelength of 490 nm.

The HPDE6-C7 cells (1×10⁵ cells/ml) were treated with gradient concentrations of emodin (180, 90, 45, 22.5, 11.25 and 5.625 µM) (Solarbio) for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. Emodin was dissolved in DMSO to a final concentration of <0.1%. Finally, the MTT method was used to evaluate cell viability as described above.

To evaluate the effects of emodin on ATP-induced HPDE6-C7 cell injury, the cells were divided 5 five groups as follows: i) The control group; ii) ATP group; and iii) low-, iv) medium- and v) high-dose emodin groups. The cells in the control group were cultured under normal conditions without being subjected to any other treatment. The cells in the ATP group were pre-treated with 40 mM ATP for 16 h, while the cells in the emodin groups were respectively first treated with 11.25, 22.5 and 45 µM emodin for 24 h prior to a 16 h-incubation with ATP (40 mM). Cell viability was then assessed using the aforementioned MTT method. To observe the effects of emodin on the morphology of the HPDE6-C7 cells, the cells were grown in 6-well plates and divided into 3 groups as follows: i) The control group; ii) ATP group; and iii) high-dose emodin (45 µM) group. The cells were pre-treated as described above, and then imaged using a phase contrast microscope (Olympus Corp.).

Following culture in 6-well plates for 24 h at 37°C, the HPDE6-C7 cells were subjected to different treatments. The cells were then fixed in 4% paraformaldehyde and incubated with diluted anti-P2X7 antibody (1:100; cat. no. ab48871; Abcam) at 4°C overnight. The cells were then incubated with FITC-conjugated goat anti-rabbit IgG (1:50; cat. no. SA00003-2; ProteinTech Group) for 1 h and re-stained with DAPI (1 µg/ml; ProteinTech Group) for 5 min in the dark at 37°C. Immunohistochemical images (×100 magnification) were obtained using an Olympus BX63 fluorescence microscope (Olympus Corp.).

The HPDE6-C7 cells (1×10⁵ cells/ml) were seeded into 6-well plates for 24 h, and then transfected with a P2X7 overexpression plasmid using Lipofectamine 2000 according to the manufacturer's instructions (Thermo Fisher Scientific). The P2X7 overexpression plasmid, also termed pEX-3-P2X7 cDNA (cat. no. Y5251), was purchased from GenePharma Co., Ltd. Briefly, pEX-3-P2X7 cDNA (2 µg) was diluted in 200 µl of DMEM without serum, and then Lipofectamine 2000 (4 µl) was diluted in 200 µl of DMEM. Following a 5-min incubation at room temperature, the diluted DNA was combined with the diluted Lipofectamine 2000. This was followed by gentle mixing and incubation for 20 min at room temperature to allow the DNA-Lipofectamine 2000 complexes to form. Finally, 400 µl of the complexes were added to each well, and the wells were replenished with 1.6 ml of DMEM. Following 24 h of transfection, the cells were treated with emodin (45 µM) and ATP (40 mM). Finally, the P2X7, NLRP3, ASC and caspase-1 protein expression levels, and the contents of IL-1β and IL-18 in the cell supernatant were measured by western blot analysis and ELISA, respectively.

According to the manufacturer's instructions, the IL-1β and IL-18 contents in the cell supernatant were evaluated using commercially available human ELISA kits (Boster Bio). The OD values in each well were measured using a multifunctional microplate reader (BioTek) at a wavelength of 450 nm.

Total proteins from the HPDE6-C7 cells were extracted using a protein extraction kit (cat. no. KGP2100; KeyGen Biotech), and the protein concentrations were determined using a BCA protein assay kit (cat. no. KGP902; KeyGen Biotech). Protein samples were separated by SDS-PAGE (10–12%) and then transferred onto PVDF membranes (EMD Millipore). The membranes were then blocked and incubated with primary antibodies against P2X7 (cat. no. ab48871), NLRP3 (cat. no. ab214185), ASC (cat. no. ab155970) and caspase-1 (cat. no. ab1872) (all from Abcam and 1:800 dilution in TBST) overnight at 4°C. Following incubation with the secondary antibody [cat. no. SA00001-2; HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) ProteinTech Group] (1:2,000 dilution in TBST) at room temperature for 2 h, the protein expression in the membranes was visualized by enhanced ECL using Tanon-5200 Multi Gel Imaging System (Tanon Science and Technology). β-actin (cat. no. ab179467; Abcam) was used as the internal control. Quantitative analysis was performed using Gel-Pro analyzer 4.0 software (Media Cybernetics).

Data are expressed as the means ± standard deviation (SD) and analyzed using SPSS 17.0 software (SPSS, Inc.). Differences between multiple groups were analyzed using one-way analysis of variance with Tukey's post-hoc test. Comparisons between 2 groups were made using an unpaired Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

In order to establish a model of ATP-induced injury using normal human pancreatic ductal epithelial cells, we adopted various concentrations of ATP to stimulate the HPDE6-C7 cells. As shown in Fig. 1A, ATP at a concentration of 40 mM significantly decreased the viability of the HPDE6-C7 cells. Thus, 40 mM was selected as the stimulus dose for the in vitro model in the subsequent experiments. Furthermore, to identify the safe concentration of emodin, the cytotoxicity of emodin against the HPDE6-C7 cells was evaluated by MTT assay. As shown in Fig. 1B, the results indicated that treatment with 90 µM emodin exerted marked cellular cytotoxicity (P<0.001), which demonstrated that 45 µM was the maximum safe concentration of emodin. In addition, as shown in Fig. 1C, emodin (45 µM) notably increased cell viability compared with the ATP model group. Furthermore, the cell morphology images presented in Fig. 1D indicated that ATP significantly induced HPDE6-C7 cell death, and that this was attenuated by emodin (45 µM). Therefore, these results suggested that emodin exerted a potent protective effect against ATP-induced HPDE6-C7 cell injury.

In order to examine the effects of emodin on P2X7 protein expression, we performed a P2X7 immunofluorescence assay. As shown in Figs. 2 and S1, the number of P2X7-positive cells exhibiting green fluorescence was significantly increased in the ATP group; however, treatment with emodin (45 µM) markedly decreased this green fluorescence. Subsequently, we evaluated whether the P2X7/NLRP3 signaling pathway is involved in the protective effects of emodin against ATP-induced HPDE6-C7 cell injury. The results shown in Fig. 3 revealed that the expression of proteins associated with the P2X7/NLRP3 signaling pathway, including P2X7, NLRP3, ASC and caspase-1 in the ATP group was significantly increased compared with the control group. Treatment with emodin (45 µM) markedly inhibited the levels of these proteins. Therefore, emodin can attenuate HPDE6-C7 cell injury induced by ATP through the inhibition of the P2X7/NLRP3 signaling pathway.

The present study examined the effects of emodin on the conents of IL-1β and IL-18 in ATP-stimulated HPDE6-C7 cells. The results shown in Fig. 4 indicated that the contents of IL-1β and IL-18 in the cell supernatant were significantly increased in the model group, and that these effects were reversed by treatment with emodin (45 µM).

In order to explore the role of P2X7 on the protective effects of emodin against ATP-induced HPDE6-C7 cell injury, a P2X7-cDNA plasmid was used to overexpress the P2X7 gene. As shown in Fig. 5, the protein level of P2X7 was notably increased following transfection with the P2X7 plasmid compared with the negative control group. Furthermore, as shown in Figs. 6 and S2, the morphological damage and the number of P2X7-positive cells in the P2X7 overexpression group were significantly increased compared with the negative control group. However, these effects were not attenuated by treatment with emodin (45 µM). Therefore, P2X7 overexpression reversed the protective effects of emodin against ATP-induced HPDE6-C7 cell injury.

In addition, as shown in Fig. 7, following transfection with the P2X7 overexpression plasmid, regardless of the presence of emodin, the expression levels of P2X7, NLRP3, ASC and caspase-1 were all notably upregulated compared with the control group. In addition, as shown in Fig. 8, similar results were also observed regarding the release of IL-1β and IL-18 in the cell supernatant. Overall, these results suggested that P2X7 overexpression abrogated the regulation of the P2X7/NLRP3 signaling pathway mediated by emodin, which further demonstrated that the protective actions of emodin on ATP-induced pancreatic ductal cell injury were mainly due to the inhibition of the P2X7/NLRP3 signaling pathway.