ICA, dimethyl sulfoxide (DMSO), cerulein, NaCl, hexadecyl-trimethyl-ammonium bromide (HTAB), and Tris-HCl were purchased from Sigma-Aldrich (Merck KGaA). ICA stock solutions were using DMSO. Easy-Blue™ total RNA extraction kit was purchased from iNtRON Biotechnology. Anti-phosphorylated extracellular signal-regulated kinase (ERK)1/2 (1:1,000; cat. no. 9101L), phosphorylated c-Jun N-terminal kinase (JNK 1/3; 1:1,000; cat. no. 9251L), and p38 (1:1,000; cat. no. 9211L) antibodies and SB203580 (p38 inhibitor) were purchased from Cell signaling Technology, Inc. Total MAPK antibodies against ERK 1/2 (1:1,000; sc-93), JNK 1/3 (1:1,000; sc-474) and p38 (1:1,000; sc-535), inhibitory κ-Bα (Iκ-Bα; 1:1,000; sc-371), and β-actin antibody (1:1,000; sc-1615) were obtained from Santa Cruz Biotechnology, Inc. In addition, horseradish peroxidase (HRP)-conjugated secondary antibodies, including chicken anti-rabbit IgG-HRP (1:5.000; sc-516087) and chicken anti-goat IgG-HRP (1:10.000; sc-516086) were purchased from Santa Cruz Biotechnology, Inc.

All experiments were performed in accordance with the animal care regulations of Wonkwang University set forth and approved by the Wonkwang University Animal Ethics Committee.

All experiments were performed according to the protocols approved by the Animal Care Committee of Wonkwang University. C57BL/6 mice (n=324, 6-8 weeks old, female, weighing 15-20 g), were purchased from Orient Bio. All animals were bred and housed in standard shoebox cages in a climate-controlled room with an ambient temperature of 23±2°C under a 12 h light/dark cycle for 7 days. Animals were fed standard laboratory chow, and water ad libitum. Mice were randomly assigned to control or experimental groups.

AP was induced via six intraperitoneal injections (i.p.) of cerulein (50 µg/kg) at intervals of 1 h. Control animals were administered DMSO under the same conditions. In the pre-treatment groups, ICA (5, 10 or 20 mg/kg, i.p.), SB203580 (1 mg/kg i.p., n=6) or DMSO (i.p.) were administered 1 h before the first cerulein injection, in the experimental (n=6) and the control group (n=6) respectively. Mice were sacrificed 6 h after the last cerulein injection. In the post-treatment groups, ICA (20 mg/kg, n=6) or DMSO (control group, n=6) were administered 1, 3 and 5 h after the first cerulein injection. Mice were sacrificed 6 h after the last cerulein injection. Blood (withdrawn from the heart), the pancreas, and the lung were rapidly removed and stored at −80°C for further analysis. For the detection of MAPKs and NF-κB, mice were pretreated with ICA (10 or 20 mg/kg) or DMSO 1 h before the intraperitoneal injection of cerulein (50 µg/kg), and sacrificed at 0, 15, 30 and 60 min after cerulein injection, respectively. For immunofluorescence, mice were pretreated with ICA (20 mg/kg) or DMSO 1 h before the intraperitoneal injection of cerulein (50 µg/kg), and sacrificed at 30 min after cerulein injection. Mice were sacrificed via CO₂ asphyxiation. The CO₂ flow rate displaced 20% of the cage volume per minute. To ensure death following CO₂ asphyxiation, cervical dislocation was performed. The pancreas was rapidly removed and stored at −80°C for further analysis.

The appearance of the entire pancreas and the semi-quantitative index based on edema and inflammation were examined from each treatment group. The pancreas and lung tissues were fixed in 4% formalin solution at room temperature overnight, embedded in paraffin, cut into 4 µm sections, stained with hematoxylin for 8 min and eosin for 2 min overnight. Samples were examined under a light microscope. Tissue sections from each sample, representing a minimum of 100 fields were examined and scored on a scale of 0-3 (0 corresponding to normal appearance and 3 corresponding to severe disease), based on the presence of interstitial edema and interstitial inflammation. In the assessment of the lung, the sections were examined for the presence of wall thickening and inflammation. The levels of edema, and pro-inflammatory cell infiltration were scored by pathologists in a blinded manner who were unware of the study design; scores were set based on a scale of 0 (normal) to 3 (severe) (20). In brief, a score of 0 presented the absence of edema and inflammatory cells, 1 indicated focal edema between lobules and 10 inflammatory cells; 2 suggested diffuse edema between lobules and 10-20 inflammatory cells, while a score of 3 reflected diffuse edema between acinar cells and >20 inflammatory cells.

Blood samples for the determination of serum amylase and lipase levels were obtained 6 h after the last injection of cerulein. Mice were sacrificed via CO₂ asphyxiation followed by cervical dislocation; blood samples were then withdrawn from the heart. Amylase and lipase activities were determined by an assay kit of Bio Assay Systems.

Sequestration of neutrophils within the pancreas and lung was evaluated by measuring the MPO activity within tissues. Briefly, tissue samples were weighed, homogenized with 20 mM phosphate buffer (pH 7.4), and centrifuged at 10,000 × g for 10 min, 4°C. The pellets were then re-suspended in 50 mM phosphate buffer (pH 6.0) containing 0.5 % HTAB. The samples were centrifuged at 10,000 × g for 5 min, 4°C, and mixed with 80 mM sodium phosphate buffer (pH 5.4) containing 1.6 mM TMB. The mixture was incubated at 37°C for 110 sec, and the reaction was terminated with 2 M H₂SO₄. Tissue MPO activity was determined by measuring the absorbance at 450 nm with a Versa Max microplate reader (Molecular Devices, LLC) and was expressed as U/mg protein.

mRNA transcripts of mouse pancreatic tissues were analyzed by RT-qPCR. Total RNA was isolated from the mouse pancreas using Easy-Blue™ and subjected to RT using ABI cDNA synthesis kit according to the manufacturer's protocols (Applied Biosystems; Thermo Fisher Scientific, Inc.). For each sample, triplicate test reactions and a control reaction lacking reverse transcriptase were analyzed for the expression of the gene of interest, and the results were normalized to those of the 'housekeeping' hypoxanthine phosphoribosyltransferase (HPRT) mRNA. qPCR was performed using a standard protocol from the TaqMan™ Universal Master Mix II, no UNG (Applied Biosystems; Thermo Fisher Scientific, Inc.) on StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed at 50°C for 2 min and 95°C for 10 min, followed by 60 cycles of amplification at 95°C for 10 sec and 60°C for 30 sec. Forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.). The forward, reverse and probe oligonucleotide primers for multiplex real-time TaqMan PCR were as follows: For mouse TNF-α forward, 5′-TCT CTT CAA GGG ACA AGG CTG-3′, reverse, 5′-ATA GCA AAT CGG CTG ACG GT-3′; mouse IL-1β forward, 5′-TTG ACG GAC CCC AAA AGA T-3′, reverse, 5′-GAA GCT GGA TGC TCT CAT CTG-3′; mouse IL-6 forward, 5′-TTC ATT CTC TTT GCT CTT GAA TTA GA-3′, reverse, 5′-GTC TGA CCT TTA GCT TCA AAT CCT-3′; mouse HPRT (forward, 5′-GAC CGG TCC CGT CAT GC-3′; reverse, 5′-CAT AAC CTG GTT CAT CAT CGC TAA-3′).

Pancreatic tissues were homogenized and 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. Proteins were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate-buffered saline with Tween-20 for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. After washing three times, the membrane was incubated with secondary antibodies 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.).

Immunofluorescence for the analysis of phosphorylated p38 was performed on pancreas tissue. The pancreas tissues were embedded in frozen section compound, cut into 9 µm sections, fixed in 100% methanol at −20°C for 5 min The tissues were incubated with the primary antibodies against phosphorylated p38 (1:250) at 4°C overnight followed by the fluorescence labeled secondary antibodies Alexa Fluor^® 488 goat anti rabbit (1:2,000; A27034; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. Nuclei were counter stained with DAPI (5 ng/ml) at room temperature for 5 min. Stained sections were visualized using a confocal laser microscope (Olympus Corporation).

Data were expressed as the mean ± standard error of the mean. Statistical significance of intergroup differences was evaluated using two-way analysis of variance, with time and dose as variables, followed by a Duncan's post-hoc test. All statistical analyses were performed using SPSS version 10.0 statistical analysis software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference. All experiments were conducted in triplicate.

To evaluate the prophylactic effects of ICA against cerulein-induced AP, histological examination of the pancreas and lung was carried out. In the DMSO-treated mice, histological features of the pancreas and lung showed typical normal architecture. In the DMSO-treated mice with AP, histological features of damaged pancreas tissue were observed, as characterized by interstitial edema and inflammatory cell infiltration. However, treatment with ICA ameliorated the pancreatic histological damage in a dose-dependent manner (Fig. 1B). In addition, histological features of the lung in cerulein-induced AP were observed; lung injury was characterized by alveolar wall thickening and inflammatory cell infiltration. Nevertheless, lung injury was significantly reduced by treatment with ICA compared with the control (Fig. 1D and E). In addition, to assess the effects of ICA on the severity of cerulein-induced AP, PW/BW ratio, serum amylase and lipase levels were measured. In the cerulein-induced AP mice, the PW/BW ratio was increased than in the control mice due to pancreatic edema; however, cerulein-induced increases in the PW/BW ratio was inhibited by ICA treatment in a dose-dependent manner (Fig. 1F). Furthermore, the levels of serum digestive enzymes, including amylase and lipase were significantly increased in the cerulein-induced AP mice than in the control mice; the levels of both enzymes were decreased by ICA treatment (Fig. 1G and H).

Neutrophil migration into target tissue is increased when inflammation occurs (21). Therefore, we examined MPO activity in the pancreas and lung after the induction of AP, in order to measure neutrophil infiltration. As shown in Fig. 2A, MPO activity was significantly higher in cerulein-induced AP mice than in control mice; however, the increase of MPO activity was inhibited by ICA treatment in the pancreas and the lung in a dose dependent manner. Of note, the production of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α were determined to increase during cerulein-induced AP (22). Thus, we examined the pancreatic mRNA levels of IL-1β, IL-6 and TNF-α. As shown in Fig. 2B, the expression levels of these pro-inflammatory cytokines significantly increased in cerulein-induced AP mice compared with the control; however, ICA treatment inhibited IL-1β, IL-6 and TNF-α expression in a dose-dependent manner.

To investigate the inhibitory mechanisms of ICA on AP, the activation of MAPKs and NF-κB was examined in the pancreas. Mice were administered ICA (20 mg/kg) or DMSO for 1 h and then stimulated with cerulein for 0, 15, 30 and 60 min. Cerulein treatment triggered the phosphorylation of MAPKs and the degradation of Iκ-Bα. However, administration of ICA inhibited the phosphorylation of p38, but not of ERK 1/2, JNK, and degradation of Iκ-Bα (Fig. 3A). Consistent with Fig. 3A, p38 phosphorylation was also inhibited by ICA as determined via immunofluorescence staining (Fig. 3C). To determine whether the inhibition of p38 activation was responsible for the effects of ICA, we administered ICA at different concentrations (10 or 20 mg/kg). As shown in Fig. 3D and E, the phosphorylation of p38 was inhibited by ICA in a dose-dependent manner.

To determine whether inhibition of p38 could affect the reduction of inflammatory responses, a p38 inhibitor (SB203580, 1 mg/kg) was employed, after which the severity of AP was evaluated. As presented in Fig. 4, inhibition of p38 by SB203580 suppressed the histological damage to the pancreas and lung, and reduced the PW/BW ratio, and the levels of amylase and lipase compared with AP mice (Fig. 4). These observations were similar to the effects of ICA (Fig. 1). Additionally, administration of SB203580 significantly reduced MPO activity, and the mRNA levels of IL-1β, IL-6 and TNF-α compared with in AP mice (Fig. 5).

To examine the therapeutic effects of ICA in cerulein-induced AP, we applied ICA after the onset of AP. Post-treatment of ICA at 1 h but not 3 and 5 h after first cerulein injection significantly reduced the PW/BW ratio, serum amylase and lipase activities, and histological injury of the pancreas and lung compared with the AP mice, which suggests that ICA could exhibit therapeutic properties in the early phase of AP (Fig. 6).