A total of 90 male Sprague-Dawley rats, weighing 230–270 g, were randomly divided into 2 groups: an ANP group (n=60) and a control group (n=30). The animals were anesthetized with 10 mg/ml ketamine at a dose of 10 mg/100 g body weight injected intraperitoneally. ANP was induced by the retrograde injection of 3% sodium taurocholate into the pancreatic duct. The abdominal cavity was then entered through a midline incision. After identifying the duodenum and pancreas, the common bile duct was ligated at the position of the hepatic hilum. A duodenotomy was performed ~1 cm distal to the opening of the biliopancreatic duct into the duodenum and a polyethylene catheter was gently cannulated into the pancreatic duct via the duodenotomy incision. Sodium taurocholate solution (3%) was slowly injected at a constant rate of 0.5 ml/min. The catheter was removed after being kept in place for 30 min. The rats in the control group underwent sham surgery (open laparotomy with immediate closure). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of the Southeast University.

Fresh tissue was collected and fixed in 10% formaldehyde for 12 h, dehydrated in a conventional manner, embedded in paraffin, cut into slices and stained with hematoxylin and eosin (H&E). The morphological observation and histological assessment were conducted by an experienced pathologist, who was unaware of the sample identity, using previously described criteria (11,12).

A total of 20 animals were sacrificed at each time point (12, 24 and 36 h after the induction of ANP). Ten animals per group (sham operated) were used as the controls. Samples of the pancreas, lung and terminal ileum were harvested and a blood sample was collected from the hepatic portal vein of each animal at the time of pancreatectomy.

To determine the lung wet/dry weight ratio, the whole left lung, lobes or segments of the peripheral lung were weighed after initial removal and after drying in an oven at a constant temperature of 160°C for 48 h.

Prior to the closure of the midline incision, 3.7 MBq technetium-99m diethylenetriaminepentacetic acid (^99mTc-DTPA) was slowly injected into the apical portion of the jejunum of the rats in the ANP group. Urine was collected 12, 24 and 36 h after the ingestion of the tracer. The radioactivity of the tracer was detected and the urinary excretion rate of ^99mTc-DTPA was calculated according to the following equation: urinary excretion rate of ^99mTc-DTPA = [(urinary count-background) × urinary volume/(standard count-background) × 1000] × 100 (13).

Total RNA was extracted from the frozen tissue using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions and the integrity of the RNA was confirmed by 1% formaldehyde-agarose gel electrophoresis. The synthesis of cDNA was conducted using an RT-PCR kit (Takara Bio, Inc., Shiga, Japan) following the recommendations of the manufacturer. The primers were designed using Primer premier software. The primer sequences for the Reg I gene were: upstream: 5′-GCCAGGAGGCTGAAGAAG-3′ and downstream: 5′-CCAGTGTCCCAGGATTTG-3′. Moreover, the primer sequences for endogenous control GAPDH were: upstream: 5′-GTTCAACGGCACAGTCAA-3′ and downstream: 5′-CCTCAGTGTAGCCCAGGAT-3′. All primers were synthesized by Invitrogen Life Technologies. PCR was carried out in a 25-μl reaction mixture with 3 μl cDNA reaction product as the template mixed with 22 μl PCR mixture (including 25 mM MgCl₂, 5 U/μl Taq polymerase and 1.5 μl of each primer). The thermal cycling process began with an initial denaturing step at 94°C for 2 min followed by a denaturing step of 30 sec, and annealing at 54°C for another 30 sec. The synthesis of new DNA was initiated with an extension step lasting 1 min as the reaction temperature was raised to 72°C. The next cycle began with a return to 95°C for denaturation. The process ran for 32 cycles and involved a final step at 72°C for 7 min. After amplification, the RT-PCR products were submitted to electrophoresis on 1.5% agarose gel (100 V, 40 min). All PCR assays were conducted in triplicate and the results were semi-quantitated as the ratio amplicon/GAPDH and presented as the mean ± SD.

The histopathological score was evaluated based on the severity of injury in the pancreas, lung and intestine and used grading scale criteria. In the pancreas, according to the degree of edema, acinar necrosis, fat necrosis and perivascular infiltrate, the score varied from 0 to 4 and then the total score was obtained. Six parameters including hyaline membranes, microthrombi, edema, interstitial infiltrates, atelectasis and hemorrhage were taken into account in the microscopic observation of lung tissue. A score of 0 represents normal tissue, 1 shows discrete or small foci, 2 indicates moderate or large foci and 3 indicates severe or diffuse lesions. In the intestine, grade 0 represents normal mucosa and grades 1 to 5 indicate increasing damage of villi. Grades 6 to 8 represent crypt layer infarction to transmural infarction. A low score indicates a mild lesion, and a high score depicts serious damage. The mean was calculated from the sum of the respective scores.

Statistical analysis was achieved using SPSS software and comparisons between groups were made using an unpaired Student’s t-test. The correlation between Reg I and each histological score was assessed using the Pearson correlation test. Data are expressed as the mean ± SD and P<0.05 was considered to indicate a statistically significant result.

The presence of ANP in the taurocholate-induced animals was confirmed by serum amylase activity and histology (Table I and Fig. 1). As demonstrated in Table I, the levels of circulating amylase in the ANP group were significantly higher than those in the sham-operated animals 12, 24 and 36 h after surgery (P<0.05). Bloody ascites in the abdominal cavity, pancreatic bleeding spots and necrosis were observed when harvesting the samples from the ANP animals. The histological worsening of the pancreatitis in the pancreatic parenchyma following sodium taurocholate treatment was evidenced by hemorrhage (Fig. 1B and C) and necrosis (Fig. 1D) as compared with the control (Fig. 1A). The histological scoring of the pancreas was conducted according to the criteria previously described by Schmidt et al(14). As shown in Table II, the histological scores for the pancreas 12, 24 and 36 h after injection in the ANP group were significantly higher than those in the controls (8.2±1.40, 10.1±1.80, 11.3±1.81 vs. 0.5±0.53, 0.4±0.52, 0.6±0.7, respectively, P<0.01).

As shown in Fig. 2, the pulmonary injury induced by ANP was characterized by thickening of the alveolar wall, pulmonary edema (Fig. 2B), infiltration of neutrophils and hemorrhage (Fig. 2C and D) compared with the controls (Fig. 2A). Disruption of the pulmonary alveolus followed by consolidation and interstitial vasodilation was also observed 36 h after injection (Fig. 2D). The histological scores for lung injury were calculated according to previously described criteria (11). As demonstrated in Table III, the histological scores for pulmonary injury 12, 24 and 36 h after injection in the ANP group were significantly higher than those in the controls (8.95±1.88, 12.05±2.11, 13.65±1.81 vs. 0.5±0.71, 0.3±0.48, 0.3±0.67, respectively, P<0.01). The ratios of rat lung wet/dry weights were calculated and are shown in Table IV. The ratios increased significantly in the ANP animals 12, 24 and 36 h after injection compared with those in the controls (5.28±0.13, 5.39±0.21, 5.47±0.21 vs. 4.47±0.16, 4.48±0.17, 4.47±0.17, respectively, P<0.05.

Pancreatitis-induced intestinal injury was mainly manifested by mild hyperemia and edema in the intestinal mucosa proper layer 12 h after surgery (Fig. 3B) and accelerated edema as well as focal necrosis were observed 24 h after surgery (Fig. 3C). Massive inflammatory cell infiltration and exfoliation of the mucosal epidermis microvilli were found 36 h after surgery (Fig. 3D) compared with the controls (Fig. 3A). The histological scores for intestinal injury were calculated according to previously described criteria (12). As demonstrated in Table V, the histological scores for pancreatitis-associated intestinal injury 12, 24 and 36 h after the induction of ANP were significantly higher than those in the controls (1.8±0.89, 3.3±1.17, 4.2±0.95 vs. 0.2±0.42, 0.3±0.48, 0.3±0.48, respectively, P<0.01). The intestinal permeability was assessed by measuring the 12-, 24- and 36-h urinary excretion rates of ingested ^99mTc-DTPA. As demonstrated in Table VI, the urinary excretion rate of ^99mTc-DTPA was significantly elevated at each time-point following the induction of ANP as compared with the controls (34.70±4.03, 54.63±6.94, 66.83±7.56 vs. 4.62±1.17, 6.14±1.42, 7.48±0.92, respectively, P<0.01). The excretion rate of ^99mTc-DTPA tended to increase as the time post-surgery increased, indicating increased intestinal permeability.

To determine whether Reg I mRNA is expressed in rat lung and intestinal tissue, the total RNA was extracted and RT-PCR was performed as described in Materials and methods. As shown in Fig. 4, a PCR product of the expected size (316 bp) was amplified with specific Reg I primers as well as the endogenous control GAPDH with an expected size of 671 bp. There was a trend towards an increased expression of Reg I mRNA in the lung or intestinal tissue as compared with the controls, indicating that the overproduction of Reg I mRNA correlated with the severity of the disease, while the expression of endogenous control GAPDH was basically unchanged. All RT-PCR trials were performed in triplicate and the intensity ratio of amplicon/GAPDH was analyzed in a semi-quantitative manner.

To determine whether the expression levels of Reg I mRNA were correlated with the severity of the pancreatitis-induced pulmonary and intestinal injury, correlation analysis was performed between the Reg I mRNA expression levels and histological scores, ratios of lung wet/dry weights and intestinal permeability indices, respectively. A strong positive correlation between the expression levels of Reg I mRNA in the lung tissue and the histological scores of pulmonary injury was found, with a correlation coefficient of 0.5212 (Fig. 5A, r=0.5212, P<0.01), implying that the upregulation of Reg I mRNA in lung tissue is correlated with the severity of the pancreatitis-induced pulmonary injury. Similarly, a positive correlation between the expression levels of Reg I mRNA and the ratio of lung wet/dry weights was also observed (Fig. 5B, r=0.4797, P<0.01). In addition, we also identified a positive correlation between the Reg I mRNA expression levels in intestinal tissue and the histological scores of intestinal injury (Fig. 5C, r=0.6728, P<0.01) as well as with the intestinal permeability index (Fig. 5D, r=0.7092, P<0.01), implying that the overproduction of Reg I mRNA in intestinal tissue is correlated with the severity of the pancreatitis-induced intestinal injury.