Rosiglitazone was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA) and GW9662 was purchased from Enzo Life Sciences Inc. (Ann Arbor, MI, USA). Additionally, sodium taurocholate (TCA) and dimethyl sulfoxide were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Male specific pathogen-free Sprague Dawley rats (weight, 150–200 g) were obtained from the Experimental Animal Center of Hubei Academy of Medical Sciences (Wuhan, China). All animal procedures were approved by the ethics committee of Wuhan University (Wuhan, China) and performed in compliance with the EC regulations and the NIH standards (Guide for the Care and Use of Laboratory Animals, NIH publication, 85–23, revised 1996).

The experimental design is shown in Fig. 1. Sixty animals were randomly divided into two groups, 40 rats received intragastric administration of a high-fat diet (77% normal diet, 20% animal fat and 3% cholesterol) for 2 weeks, which induced experimental hyperlipidemia. The remaining rats received a normal diet and ate freely. Rats receiving the normal diet were divided into 2 groups; the sodium TCA group (SB group, n=10) and the control group (SA group, n=10). The SB group was subjected to sodium TCA-induced SAP and the SA group were injected with saline instead of sodium TCA. Rats with hyperlipidemia were randomly divided into 4 groups: the hyperlipidemia and sodium TCA group (SLA group, n=10); the hyperlipidemia, rosiglitazone and sodium TCA group (SR group, n=10); the hyperlipidemia, GW9662, rosiglitazone and sodium TCA group (SRI group, n=10); and the hyperlipidemia group (SL group, n=10). The SLA and SR groups were subjected to sodium TCA-induced AP and the SR group was additionally treated with 10 mg/kg rosiglitazone by intraperitoneal injection (IP) 1 h prior to sodium TCA. The SRI group were treated in the same way as the SR group with the additional administration of 0.3 mg/kg GW9662 by IP injection 30 min prior to rosiglitazone. The SL group was treated with saline only (Fig. 1).

Twelve hours prior to the start of the experiment, rats were deprived of food but allowed access to water ad libitum. The SAP model was induced by the method by Paszkowski et al (11), with improvements. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 ml/kg). SAP was induced by the retrograde infusion of 5% sodium TCA (1 ml/kg; Sigma-Aldrich Co.) into the ampulla of Vater, transduodenally, using an Angiocath with a standardized pressure-controlled infusion rate under laparotomy. Following infusion, the section of the bile-pancreatic duct entering the duodenum was clipped by a noninvasive Angiocath for 5 min. After checking for bile leakage, the opening in the duodenum lateral wall was sutured. The abdomen was closed, and the rats recovered from the anesthetic and were allowed access to water. All rats were sacrificed by exsanguination 12 h following the induction of pancreatitis, and blood samples were obtained by direct intra-cardiac puncture. The pancreas was removed immediately and the head of the pancreas was fixed in formaldehyde. Tissue sections were paraffin-embedded and continual sections were cut. Portions of this organ were frozen in liquid nitrogen and stored at −80°C until assayed.

Serum amylase (AMY) activity was measured using an automatic biochemistry analyzer (Olympus Optical Co., Ltd., Tokyo, Japan). Serum triglyceride (TG) and total cholesterol (TC) concentrations were measured in triplicate using commercially available colorimetric assay kits (Diagnosticum Rt, Budapest, Hungary) adapted to 96-well plates, as described previously (12). The accuracy of the assays was monitored using standard lipid controls (Sentinel, Milan, Italy).

Continuous sections of paraffin-embedded pancreatic tissue were stained with hematoxylin and eosin for pathological examination. Morphometric documentation for pancreatic sections using a light microscope (Olympus Optical Co., Ltd., Tokyo, Japan) were evaluated by two independent pathologists, each of whom was blinded to the other’s assessment and recorded their findings on an evaluation form for pancreatic injury. The evaluation of pancreatic injury, including the graded assessment of pancreatic edema, vascular edema, fat necrosis, acinar necrosis and calcification, was determined by the histological score of pancreatic injury (13).

Frozen pancreatic tissue was mechanically homogenized in 1 ml ice-cold extraction buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM ethylene diamine tetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 0.1% sodium dodecylsulfate and 1 μg/ml each of aprotinin and leupeptin). Tissue samples were incubated on ice for 30 min, then centrifuged at 13,000 × g at 4°C for 30 min, and the supernatant was collected and stored at −80°C. The protein concentration of each sample was determined using the Bradford method with bovine serum albumin as the standard. Protein samples (40 μg) were electro-phoresed using sodium dodecyl sulfate-polyacrylamide gels at 100 V for 120 min. The separated proteins were transferred to a nitrocellulose membrane. Moreover, the membrane was blocked with blocking buffer [Tris-buffered saline (TBS) containing 5% non-fat dry milk and 0.1% Tween-20] for 120 min at room temperature, then washed three times for 10 min each in TBS with 0.1% Tween-20 and incubated with the primary antibodies. The primary antibodies used were goat polyclonal anti-rat ICAM-1 (1:600; Santa Cruz Biotechnology Inc., CA, USA) antibody, rabbit polyclonal anti-rat TNF-α antibody (1:1,000; Abcam, MA, USA) and rabbit polyclonal anti-rat β-actin antibody (1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), and were stored overnight at 4°C. Tissue samples were washed three times for 10 min each with TBS containing 0.05% Tween-20 (TBST) and the membranes were incubated with secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-goat immunoglobulin G (1:3,000, Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature. Following repeated washings with TBST, the antibody-antigen complex was detected with enhanced chemiluminescence reagent (Immobilon Western HRP Substrate; Millipore Corp., Bedford, MA, USA) and scanned for densitometric analysis using a bio-image analysis system (Bio-Rad Laboratories Inc., Baltimore, MD, USA) for quantification. The results from each experimental group are expressed as the relative integrated intensity of ICAM-1 and TNF-α compared with the β-actin band densities in the same batch.

Data are expressed as the mean ± standard deviation. Statistical analysis was performed with SPSS for Windows, version 17.0 (SPSS, Inc., Chicago, IL, USA). Means of the different groups were compared using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Following the 2-week high-fat diet period, the rats with hyperlipidemia were heavier than the rats that had received a normal diet, although not significantly so. However, the 2-week high-fat diet significantly increased the serum cholesterol and TG levels from 1.72±0.13 and 0.61±0.12 mmol/l to 10.86±1.47 and 1.24±0.28 mmol/l, respectively (P<0.05; Table I).

Representative histological sections of pancreatic tissue are shown in Fig. 2. The histological severity of pancreatitis was measured with a validated scale (pancreatitis score) based on the degree of edema, inflammatory cell infiltration, hemorrhage and necrosis. No pathological injury in the rats of the SA and SL groups was observed. The total pancreatitis score of the SLA group was significantly higher compared with that of the SB group (P<0.05). Pretreatment with rosiglitazone significantly reduced the inflammatory changes in the SA group compared with those of the SR group (P<0.05); however, the total pathological scores of the SLA and SRI groups were not significantly different (P>0.05; Fig. 3).

In a rat model of SAP similar to that used in the present study (14), the protein expression level of ICAM-1 in the pancreas peaked at 12 h. In the present study, western blot analysis of pancreatic tissues also identified ICAM-1 expression at 12 h following the induction of SAP (Fig. 4). The expression level of ICAM-1 in the pancreatic tissues was significantly higher in the SLA group than in the SL group (P<0.01), and higher in the SB group than in the SA group. The ICAM-1 protein expression level was greatly reduced in the SR group compared with that of the SLA group (P<0.01), and showed no significant difference when compared with those of the SA and SL groups (P>0.05). Furthermore, no significant differences were observed between the SLA and SRI groups with regard to ICAM-1 protein expression (P>0.05). However, the expression of ICAM-1 in the pancreatic tissues was significantly increased in the SLA group compared with that of the SB group (P<0.05; Fig. 4).

Western blot analysis of the pancreatic tissue (Fig. 5) identified that the protein expression level of TNF-α was significantly higher in the SLA group than in the SL group (P<0.01), and higher in the SB group than in the SA group. The TNF-α protein level was greatly reduced in the SR group compared with that in the SLA group (P<0.01), and showed no difference when compared with those of the SA and SL groups (P>0.05). Furthermore, no significant difference in TNF-α expression between the SLA and SRI groups was observed (P>0.05). However, TNF-α protein expression levels were significantly increased in the SLA group compared with those of the SB group (P<0.05; Fig. 5).