Male Sprague-Dawley (SD) rats (n=30; age, 8 weeks; weight, 230–270 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats were housed at a constant room temperature and humidity (21±2°C; 45–55%) under a 12-h light/dark cycle. The rats had ad libitum access to food and water. Animal experiments were approved by the Animal Care and Use Committee of Dalian Medical University (Dalian, China) and were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (22).

The rats were randomly assigned to the following five groups (n=6 rats/group): Control group, SAP group, emodin group, FK866 group and dexamethasone (DEX) group. To induce SAP, the rats were anesthetized with chloral hydrate (10%, 3.5 ml/kg bodyweight) and sodium taurocholate solution (5%, 1 ml/kg bodyweight) was retrogradely injected into the biliary-pancreatic duct. The control rats were anesthetized in the same manner however, without the sodium taurocholate solution injection. A total of 3 h after SAP induction, rats in the SAP, emodin, FK866 and DEX groups were intraperitoneally injected with a single dose of dimethyl sulfoxide, emodin (10 mg/kg bodyweight; both Aladdin, Shanghai, China), FK866 (10 mg/kg bodyweight; MedChem Express, Monmouth Junction, NJ, USA) or DEX (1 mg/kg bodyweight; Aladdin), respectively. After 24 h, peripheral blood was obtained, and lung and pancreatic tissues were excised. The rats were anesthetized with 10% chloral hydrate, and then sacrificed by exsanguination prior to tissue excision. Half of the tissues were immediately subjected to edema examination and the remaining tissues were fixed at room temperature for 48 h in 10% formaldehyde for hematoxylin and eosin (HE), and TUNEL staining.

A total of 24 h after treatment, the rats in each group were sacrificed and peripheral blood was obtained. PMNs were isolated from the peripheral blood using Histopaque-1083 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol and were immediately used for flow cytometry experiments.

RNA was extracted from PMNs using the RNApure Total RNA Rapid Extraction kit (BioTeke Corporation, Beijing, China) according to the manufacturer's protocol. RNA was then reverse-transcribed into cDNA with the following reaction system: 1 µg RNA, 1.2 µl RT primer (Tiangen Biotech Co., Ltd., Beijing, China), 0.75 µl dNTP (BioTeke Corporation), 4 µl 5X buffer, 0.25 µl RNasin, 1 µl super M-MLV reverse transcriptase (BioTeke Corporation) and sufficient ddH₂O to produce a final reaction volume of 20 µl. The following temperature protocol was applied for the RT reaction: 25°C for 10 min, 42°C for 50 min and 80°C for 5 min. qPCR analysis was performed using the Exicycler™ 96 Real-Time qPCR system (Bioneer Corporation, Daejeon, Korea) and the following reaction system: 1 µl cDNA, 0.5 µl forward primer, 0.5 µl reverse primer (Sangon Biotech Co., Ltd., Shanghai, China), 10 µl SYBR GREEN MasterMix (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and sufficient ddH₂O to produce a final reaction volume of 20 µl. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. The primer sequences were as follows: PBEF, forward 5′-GATGCCAAATCCCGAAGG−3′, reverse 5′-CCCACTCCAGCAGTTCATT-3′; and β-actin, forward 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′ and reverse 5′-GGCCGGACTCATCGTACTCCTGCTT-3′. Relative expression was determined using the 2^−ΔΔCq method (23).

The PMNs were lysed in kit lysis buffer (Whole Cell Lysis Assay kit; Wanleibio, Shenyang, China) and centrifuged at 10,005 × g for 10 min at 4°C. Total protein concentration was determined using a bicinchoninic acid kit (Wanleibio). Equal amounts of protein (40 µg) were separated by 10 or 13% SDS-PAGE and were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with nonfat milk, followed by incubation with primary antibodies against PBEF (cat. no. ab45890; 1:500; Abcam, Cambridge, UK), B-cell lymphoma (Bcl)-extra-large (xL; cat. no. BA3368; 1:400; Wuhan Boster Biological Technology Ltd., Wuhan, China), Bcl-2-associated X protein (Bax; cat. no. BA0315; 1:400, Wuhan Boster Biological Technology Ltd.), cleaved caspase-3 (cat. no. ab2302; 1:1,000; Abcam), Fas (cat. no. ab82419; 1:1,000; Abcam) and Fas ligand (L; cat. no. ab15285; 1:1,000; Abcam) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. WLA023 and WLA024; Wanleibio) at 37°C for 45 min. The bands were visualized by enhanced chemiluminescence (Wanleibio) and the blots were semi-quantified using Gel-Pro Analyzer (version 4.0; Media Cybernetics, Inc., Rockville, MD, USA) (24).

The lung and pancreatic tissues that were fixed in 10% formaldehyde at room temperature for 48 h were embedded in paraffin and cut into 5-µm sections. Subsequently, the sections were deparaffinized and rehydrated prior to staining with HE at 37°C (hematoxylin for 5 min and eosin for 3 min). The stained sections were examined, and images were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Blood was harvested and serum was separated by centrifugation at room temperature and 1,111 × g for 10 min. Subsequently, serum was diluted at a ratio of 1:10 in normal saline and incubated with pre-heated substrate buffer containing soluble starch (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at 37°C for 7.5 min, followed by incubation with 5 mM iodine solution (Sangon Biotech). Absorbance was measured at 660 nm.

At 24 h post-treatment, lung and pancreatic samples were immediately excised and weighed. The samples were then dried for 72 h, until the weight remained unchanged, and were then weighed again. The wet/dry (W/D) weight ratio was subsequently calculated.

DNA fragmentation was measured by TUNEL assay using an In Situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland) (25). Briefly, 5-µm tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% H₂O₂. The sections were washed with PBS, followed by incubation with TdT reaction mixture at 37°C for 1 h. Subsequently, the sections were incubated with converter-POD for 30 min and then with DAB substrate (Beijing Solarbio Science & Technology). Images of the stained cells were captured under a fluorescence microscope (Olympus Corporation).

PMNs were isolated from the peripheral blood of rats in the control group (n=6) and were cultured in vitro in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a 5% CO₂ incubator. PMNs were then treated with emodin (30 µg/ml), FK866 (10 nM) or DEX (100 nM) for 16 h in the presence of lipopolysaccharide (LPS; 50 ng/ml; Aladdin). Untreated PMNs were used as controls.

Cell apoptosis was analyzed using an Apoptosis Detection kit (Wanleibio). PMNs were harvested after the indicated treatments, and were resuspended in binding buffer. Subsequently, Annexin V-FITC and PI were added to stain the cells. Following a 15-min incubation at room temperature in the dark, the cells were analyzed by flow cytometry (Accuri™ C6; BD Biosciences, San Jose, CA, USA) and the system software (BD Accuri C6 Software; BD Biosciences, USA).

Data are presented as the mean ± standard deviation of three independent repeated experiments. GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA) was used to analyze statistical differences. The results were analyzed using one-way analysis of variance with Bonferroni post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Following treatment with the indicated reagents; PMNs were isolated from the peripheral blood of rats in each group. Subsequently, PBEF expression was detected in PMNs using RT-qPCR and western blotting. The results indicated that the mRNA (Fig. 1A) and protein expression levels of PBEF (Fig. 1B) were significantly higher in the SAP group compared with in the control group. Conversely, PBEF expression in the emodin, FK866 and DEX groups was markedly decreased compared with in the SAP group.

The results of histological examination with HE staining indicated that the lung and pancreatic tissue structures of the control rats were normal. Following the induction of SAP, a thickened alveolar septum, edema, hemorrhage and infiltration of inflammatory cells were all observed in the lung tissues (Fig. 2A). In the SAP group, HE staining of the pancreatic tissues identified pancreatic edema, hemorrhage, acinar cell necrosis and inflammatory cell infiltration (Fig. 2B). Conversely, these histopathological alterations were alleviated in SAP rats treated with emodin, FK866 and DEX.

The severity of SAP-associated ALI was confirmed by measuring serum amylase activity. As presented in Fig. 3A, serum amylase activity was significantly increased in rats in the SAP group compared with in the control group. However, following treatment with emodin, FK866 and DEX, SAP rats exhibited reduced serum amylase activity compared with in the untreated SAP rats.

The degrees of pulmonary and pancreatic edema were examined by measuring water content. The results demonstrated that the W/D weight ratios of the lung (Fig. 3B) and pancreatic tissues (Fig. 3C) in the SAP group were higher than those in the control group. However, the W/D weight ratios were markedly decreased following treatment with emodin, FK866 and DEX compared with in the SAP group.

The present study detected cell apoptosis in the lung and pancreatic tissues using TUNEL staining. The results indicated that the number of apoptotic cells in the lung (Fig. 4A) and pancreatic tissue sections (Fig. 4B) were markedly increased following the induction of SAP compared with in the control group. However, treatment with emodin, FK866 and DEX markedly decreased the number of apoptotic cells in the lung and pancreatic tissues compared with in the SAP group.

The present study also examined PMN apoptosis using flow cytometry. The results demonstrated that rats in the SAP group (10.28±0.77%) exhibited reduced apoptosis of PMNs compared with in the control rats (49.01±2.62%) (Fig. 5A). However, emodin (25.48±4.02%), FK866 (43.2±3.65%) and DEX treatment (23.72±2.13%) significantly increased cell apoptosis of PMNs, compared with in the SAP group.

The protein expression levels of cleaved caspase-3, Bax, Bcl-xL, Fas and FasL were measured by western blotting. The results demonstrated that the expression levels of Bax, cleaved caspase-3, Fas and FasL in the SAP group were significantly decreased compared with in the control group, whereas Bcl-xL expression was increased in the SAP group (Fig. 5B). Following treatment of the SAP rats with emodin, FK866 and DEX, the expression levels of Bax, cleaved caspase-3, Fas and FasL were markedly increased, whereas Bcl-xL expression was decreased compared with the untreated SAP rats.

Flow cytometric analysis indicated that LPS significantly inhibited PMN apoptosis compared with the control group (Fig. 6A). Compared with the LPS group, incubation with emodin, FK866 and DEX significantly induced PMN apoptosis and attenuated the anti-apoptotic effects of LPS on PMNs.

The expression levels of apoptosis-associated proteins were detected using western blotting. The results demonstrated that, following LPS stimulation, the expression levels of Bax, cleaved caspase-3, Fas and FasL were markedly decreased, whereas Bcl-xL was increased (Fig. 6B). However, treatment with emodin, FK866 and DEX markedly elevated Bax, cleaved caspase-3, Fas and FasL expression in LPS-stimulated PMNs, and reduced Bcl-xL expression.