All experiments were strictly in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA; NIH Publication No. 85-23, revised in 1996). The present study was approved by the Animal Ethics Committee of Jiangsu University (Zhenjiang, China).

Tricarbonyldichlororuthenium (II) dimer (CORM-2), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). CORM-2 was dissolved in DMSO to acquire a 40 mM stock solution. Inactive (i)CORM-2 was used as the negative control, and it was prepared as previously described (28). The primary antibodies of nuclear factor (NF)-κB, phosphorylated inhibitor of NF-κB subunit α (p-IκB-α), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The nuclear protein extraction buffer kit was purchased from Vazyme (Piscataway, NJ, USA). Tumor necrosis factor-α (TNF-α; JER-06), interleukin-6 (IL-6; JER-04) and IL-1β (JER-01) ELISA kits were obtained from Joyee Biotechnics Co., Ltd. (Shanghai, China). Caerulein (purity ≥97% by high-performance liquid chromatography) was obtained from Sigma-Aldrich (Merck KGaA).

C57BL/6 mice (n=180; male, 6–8 weeks old, 20±2 g) were obtained from the Experimental Animal Center of Jiangsu University, Zhenjiang, Jiangsu, China. The mice were housed in standard wire-topped cages and in temperature-controlled units (18–23°C with 40–60% humidity and 12-h light/dark cycle). Food and water were supplied ad libitum.

Caerulein was used to induce AP in the mice. The mice were randomly divided into four groups (n=30 mice/group): i) The control group, in which mice were treated hourly (×10) with normal saline (0.9% NaCl) (equal in volume to caerulein) intraperitoneally (i.p.); ii) the AP group, in which mice were treated hourly (×10) with caerulein (50 µg/kg, suspended in normal saline, i.p.); iii) the AP+CORM-2 group, in which mice received CORM-2 [8 mg/kg, intravenously (i.v.)] treatment 30 min after the induction of pancreatitis (first caerulein injection), and thereafter received hourly caerulein i.p. (×10); and iv) the AP+iCORM-2 group, in which mice received iCORM-2 (8 mg/kg, i.v.) treatment 30 min after the induction of pancreatitis (first caerulein injection), and thereafter received hourly caerulein i.p. (×10). The dosage of CORM-2 used in the present study was based on our previous studies (29,30).

In another set of experiments, mice were randomly divided into four groups (n=15 mice/group), and were monitored for 5 days to observe their survival rate.

A total of 12 h after the induction of pancreatitis, the mice were sacrificed by overdose of anesthesia. Blood samples were collected by cardiac puncture and serum was immediately obtained by centrifugation 3,000 × g for 5 min at 4°C. The separated serum was used for the subsequent determination of lipase, amylases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Meanwhile, pancreas, lung and liver tissues were harvested and immediately frozen in liquid nitrogen or fixed with 10% formalin at room temperature overnight for following analysis.

The activity of serum amylase and lipase was detected to assess pancreatic damage using a commercial kit (SNM144-BOU, Biolebo Technology Co., Ltd., Beijing, China), according to the manufacturers' protocol. Liver injury was assessed by measuring the enzymatic activities of ALT and AST in serum samples using a set of commercial kits (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's protocol.

Samples of 10% formalin-fixed pancreas, lung and liver tissues were embedded in paraffin and segmented to 4 µm for routine histology. The fixed tissues were stained with hematoxylin and eosin (H&E), and examined by two experienced morphologists who were unaware of the sample identity. A total of 10 randomly selected microscope fields (magnification, ×200) were tested for each pancreas tissue sample. The severity of pancreatic injury was scored in accordance with the previously described 0–4 (normal to severe) scale (31).

A terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) kit (Roche Diagnostics, Indianapolis, IN, USA) was used to measure apoptosis in pancreatic cells, according to the manufacturers' protocol. Tissue was fixed in 10% neutral formalin overnight, dehydrated and embedded in paraffin wax. Paraffin-embedded pancreas sections (4 µm) were incubated for 20 min at 60°C prior to being deparaffinized and rehydrated. Following digestion with 20 µg/ml proteinase K at room temperature for 20 min, the sections were washed with PBS three times, and incubated in 25 mM cobalt chloride according to the manufacturer's protocol. Tissue sections were mounted using Aqua-Poly/Mount mounting medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Finally, apoptotic cells were observed under a microscope, and 10 microscope fields (magnification, ×200) were randomly selected for analysis in each viewed sample. Finally, apoptotic cells were observed under a microscope, and 10 microscope fields (magnification, ×200) were randomly selected for analysis in each viewed sample.

A previous study included a detailed description of the measurement of whole lung wet/dry ratio, an index of lung edema (32). Lung tissues were dissected from the heart and large blood vessels, the trachea was separated at the carina, external liquid was removed by blotting, and the lungs were placed on a pre-weighed pan to obtain the wet weight. Subsequently, the lungs were incubated overnight in a dry atmosphere at 85°C and were re-weighed to obtain the dry weight.

Immunohistochemical staining. Pancreatic tissues were fixed in 10% formalin and 3–4 µm slices were prepared from paraffin-embedded tissues. Following paraffin removal and rehydration, slices were subjected to heat-mediated antigen repair in sodium citrate buffer (10 mM sodium citrate, pH 6.0) and blocked in 10% normal goat serum (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The samples were subsequently incubated with the primary antibodies against ICAM-1 (1:200; sc-1511; Santa Cruz Biotechnology, Inc.) and VCAM-1 (1:200; sc1504; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following washing three times with PBS, samples were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:100; sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 20 min. Subsequently, when the samples had again been washed three times with TBS, they were stained with freshly prepared diaminobenzidine chromogen (brown) for 3–5 min at room temperature, washed with distilled water, and counterstained with hematoxylin for 10 sec at room temperature. The samples were dehydrated with a gradient of ethanol. Finally, the slides were mounted with mounting medium, labelled and viewed under the microscope (IX51-A12PH; Olympus Corporation, Tokyo, Japan). Among the pancreas samples, 10 microscope fields (magnification, ×200) were randomly selected for analysis in each tissue sample. The average optical density of ICAM-1 and VCAM-1 was evaluated using Image Pro-Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

MPO activity was determined in tissues from the pancreas, lung and liver, according to a previous study (33). Tissue samples were homogenized in 50 mM potassium phosphate buffer (PB, pH 6.0) and centrifuged at 10,000 × g for 10 min at room temperature. The precipitate was collected and the pellet was suspended in 50 mM PB containing 0.5% hexadecyltrimethylammonium bromide. The samples were sonicated (30 times, and each time was 5–10 s at room temperature at 20 kHz and 200 W) and centrifuged again at 10,000 × g for 10 min at room temperature. Aliquots of 0.3 ml were added to 2.3 ml reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H₂O₂ solution. One unit of enzyme activity was regarded as the MPO content which resulted in a change in absorbance detection at 460 nm for 3 min. MPO activity is expressed as U/g tissue.

The MDA levels in pancreatic, lung and liver tissue samples were also assessed. The tissue samples were homogenized with a 1.15% KCl solution. An aliquot (100 µl) of the homogenate was added to a reaction mixture, which included 200 µl 8.1% SDS, 1,500 µl 20% acetic acid (pH 3.5), 1,500 µl 0.8% thiobarbituric acid and 700 µl distilled water. The samples were boiled for 1 h at 95°C and centrifuged for 10 min at 3,000 × g at room temperature. The absorbance at 650 nm was determined by spectrophotometry.

To determine the levels of TNF-α, IL-1β and IL-6 in the serum and tissue homogenates of the pancreas, lung and liver, ELISA kits were used, according to the manufacturer's instructions of each kit.

A nuclear protein extraction buffer kit (Vazyme) was used for nucleic protein extraction. BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to evaluate the protein concentrations. Samples (10 µg of protein) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1.5 h and subsequently incubated with anti-mouse NF-κB-specific polyclonal antibody (1:1,000; cat. no. sc-514451) or anti-mouse p-IκB-α-specific polyclonal antibody (1:1,000; cat. no. sc-7977) at 4°C overnight and secondary HRP-conjugated goat anti-mouse IgG antibody at the correct concentration (1:10,000; cat. no. sc-2031; all from Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Enhanced chemiluminescence was used to visualize the bands using FluorChem FC3 (ProteinSimple, San Jose, CA, USA), and AlphaView 3.4.0 software (ProteinSimple) was used for quantitative analysis.

GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used for the statistical analyses. All experiments were repeated at least three times and the data are presented as the mean ± standard deviation. Comparisons between groups were analyzed using one-way factorial analysis of variance followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

To confirm the therapeutic effects of CORM-2, caerulein hyperstimulation animal models of AP were used. The survival rate was calculated to be 100% (15/15) in the control group, 20% (3/15) in the AP group, 73.3% (11/15) in the CORM-2 group, and 20% (3/15) in the iCORM-2 group at 5 days (Fig. 1A). This suggested that the administration of CORM-2 led to significantly lower mortality compared with the AP group and protected against the lethality of AP, and that treatment with iCORM-2 did not serve a protective role against mortality (Fig. 1A). The levels of serum amylase and lipase were examined, as these reflect the degree of pancreatic injury. The levels of serum amylase and lipase were increased at 12 h post-AP induction, and were reduced by the administration of CORM-2 (Fig. 1B and C). The AP animals treated with iCORM-2 exhibited unaltered serum amylase and lipase activity compared with the AP group (Fig. 1B and C).

The severity of AP was also assessed by histological examination (Fig. 2). The pancreatic architecture was normal in control group mice (Fig. 2Aa), whereas the pancreatic tissues in AP group mice exhibited severe pathological damage at 12 h post-AP induction (Fig. 2Ab). Treatment with CORM-2 improved the pancreatic damage induced by caerulein (Fig. 2Ac), while iCORM-2 treatment had no marked alleviating effect on injury in the pancreas (Fig. 2Ad). Histopathological scoring of the pancreatic injury indicated that caerulein induction induced edema, inflammatory cell infiltration, and necrosis of the acinar cells compared with the control group (Fig. 2C). This damage was markedly alleviated by treating the AP mice with CORM-2 (Fig. 2C). However, iCORM-2 treatment failed to obviously improve the histological score (Fig. 2C). Previous clinical and experimental studies have reported that apoptosis is observed during the course of AP (34,35). In order to further investigate the effect of CORM-2 on pancreatic cell apoptosis in caerulein-induced AP, a TUNEL assay was performed to detect pancreatic cell apoptosis. As presented in Fig. 2B, a mass of apoptotic cells (including pancreatic acinar cells, intercalated ducts cells and certain types of islet cells) with brown nuclei was observed in the pancreases from mice of the AP group (Fig. 2Bb), although not in mice of the control group (Fig. 2Ba). The number of apoptotic cells with brown nuclei was significantly reduced by treatment with CORM-2 (Fig. 2Bc). No significant alterations in staining were seen in the iCORM-2 treatment group compared with the AP group (Fig. 2Bd).

In addition to pancreas injury, caerulein injections may also induce injury to the lung and liver in wild-type mice, as previously reported (2,12,25,26). The severity of lung and liver injury was evaluated by histological analysis in the current study (Fig. 3). H&E staining demonstrated the normal structure of the sections of lung and liver in the control group (Fig. 3A and E). In caerulein-induced AP mice, increased numbers of alveolar epithelial cells and red blood cells with inflammatory cell infiltration were observed in the lung (Fig. 3B). Furthermore, marked degeneration and necrosis of liver tissues, hyperemia in the central veins of the liver and infiltration of the liver by inflammatory cells was also observed (Fig. 3F). These morphological alterations caused systemic inflammation and organ damage in the lung and liver, and were also observed in the iCORM-2-treated AP group (Fig. 3D and H). Following in vivo CORM-2 treatment, the pathological alterations in the lung and liver were decreased (Fig. 3C and G), indicating that CORM-2 had a protective effect on vital organs in AP.

Additionally, lung injury induced by caerulein (Fig. 4) was also characterized by marked pulmonary edema (Fig. 4F), sequestration of lung neutrophils (increase in MPO content; Fig. 4B), lipid peroxidation (increase in MDA content; Fig. 4C) and an increase in pro-inflammatory cytokine levels (TNF-α, IL-1β and IL-6; Fig. 4D). The manifestation of the liver injury induced by caerulein also included an increase in the serum concentrations of ALT and AST (Fig. 4A), while CORM-2 treatment effectively relieved the injury to the lung and liver, and this was not the case with iCORM-2 (Fig. 4).

The levels of MPO and MDA that were increased by caerulein injection were reduced by treatment with CORM-2. The results for MPO levels were also in accordance with the histological results. In the AP group, significant areas of inflammatory cell infiltration were evident (Fig. 5A) and the histological score of inflammatory cell infiltration was significantly increased (Fig. 2Cb). Treatment with CORM-2 reduced neutrophil infiltration and the extent of lipid peroxidation in the pancreas (Fig. 5A and B). There was no significant difference between the AP group and the AP+iCORM-2 group (Fig. 5A and B).

Increased adherence of neutrophils to endothelial cells leads to the accumulation of neutrophils in tissues, which is primarily mediated by adhesion molecules (36). Sequestered neutrophils in the pancreas augment the injury to the tissue (37). Therefore, the expression of the ICAM-1 and VCAM-1 adhesion molecules was assessed, as they facilitate the attachment of neutrophils to the endothelium. No positive staining for ICAM-1 and VCAM-1 was observed in the pancreatic tissue sections that were obtained from mice in the control group (Fig. 6A and B). Sections obtained from AP mice 12 h post-caerulein induction exhibited strong positive staining for ICAM-1 and VCAM-1 (Fig. 6A and B). The degree of pancreatic staining for ICAM-1 and VCAM-1 was reduced in tissue sections obtained from CORM-2-treated AP mice (Fig. 6A and B). No marked alterations in staining were observed in the iCORM-2-treated AP group compared with the AP group (Fig. 6A and B). Quantitative analysis of the average optical density for ICAM-1 and VCAM-1 is presented in Fig. 6C.

For the purpose of investigating the regulation of inflammation by CORM-2 treatment, ELISA was performed to detect the levels of the pro-inflammatory cytokines IL-6, IL-1β and TNF-α in the serum and pancreas of mice. The levels of IL-6, IL-1β and TNF-α in the serum and pancreas of the mice markedly increased at 12 h post-AP induction (Fig. 7A). In vivo administration of CORM-2 significantly decreased the caerulein-induced increase in the levels of IL-6, IL-1β and TNF-α (Fig. 7). However, treatment with iCORM-2 did not alter the levels of these cytokines compared with the AP group (Fig. 7).

To examine whether the NF-κB pathway was involved in the observed anti-inflammatory effects of CORM-2-derived CO, western blotting was used to detect the expression of NF-κB (p65) in the pancreas. The results revealed that AP mice exhibited a significant increase in NF-κB expression compared with control animals (Fig. 8A). This increase was prevented by treatment with CORM-2, although not by iCORM-2 (Fig. 8A). Furthermore, the phosphorylation of IκB-α (p-IκB-α), which is required for the initiation of NF-κB activation, was also examined by western blotting in the pancreas. There was a significant increase in the level of p-IκB-α in the pancreas of AP mice, which was suppressed by administration of CORM-2 (Fig. 8B). No significant difference in the p-IκB-α level in the pancreas was observed following treatment with iCORM-2 compared with the pancreas from AP mice (Fig. 8B).