A total of 48 healthy male Sprague Dawley rats (weight, 350±30 g; age, 12–14 weeks), were provided by the Experimental Animal Center of the Third Xiangya Hospital (Changsha, China). Sodium taurocholate was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Amylase polyclonal antibody (cat. no. YT5164) and enzyme-linked immunosorbent assay (ELISA) kit for HMGB1 (Total HMG-1 Cell-Based Colorimetric ELISA kit) were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA). Rabbit anti-HMGB1 (cat. no. ab79823) and anti-Beclin 1 (cat. no. ab55878) primary antibodies were from Abcam (Cambridge, MA, USA). Goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA; cat. no. 111-005-144). The color pre-stained protein marker was purchased from Fermentas (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Bradford protein concentration assay kit was obtained from Beyotime Institute of Biotechnology (Haimen, China). TRIzol^® reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The SYBR Green quantitative polymerase chain reaction (qPCR) Mix was purchased from Toyobo Co., Ltd. (Osaka, Japan).

The present study was approved by the ethics committee of the Experimental Animal Center of the Third Xiangya Hospital. The rats were housed at a constant temperature of 22°C under a 12-h light/dark cycle. The rats had access to standard food and water ad libitum. The ANP rat model was created according to the method described by Aho et al (19). The rats were fasted for 24 h, with ad libitum access to water. Subsequently, the rats were anesthetized by intraperitoneal injection with 10% chloral hydrate (0.3 ml/100 g). An incision was made into the ventral midline for entry into the abdominal cavity, in order to expose the common bile duct and the pancreatic duct. The biliopancreatic duct was occluded at the distal duodenum using a vascular clip. For the experimental group, the canal was infused slowly with 5% sodium taurocholate (0.1 ml/100 g) using a 1-ml syringe at the proximal end. For the control group, the canal was infused with an equal quantity of normal saline. After injection, the needle remained motionless for 10 min to prevent drug reflux. After modeling, the skin was disinfected, the peritoneum was closed with a continuous single-layer suture, and the skin was sutured with a full-layer simple-interval suture. After the abdomen had been closed, the rats were injected with 5 ml saline at the inside position of the lower limb muscle to supplement water lost during the operation.

Experimental (ANP) rats were grouped according to the following time points after modeling: 0 h (n=4), 3 h (n=8), 6 h (n=8), 12 h (n=8) and 24 h (n=8). The control rats were grouped according to the following time points: 3 h (n=3), 6 h (n=3), 12 h (n=3) and 24 h (n=3). Rats were sacrificed, at the aforementioned time points, by cardiac puncture following anesthetization by intraperiotneal injection with 10% chloral hydrate (0.3 ml/100 g body weight).

Rats were anesthetized and sacrificed. Blood samples (~5 ml) were collected from the heart, which were maintained at 4°C for 30 min and centrifuged at 1,000 × g for 15 min at normal temperature. The supernatant was then collected and preserved at −80°C, avoiding repeated freezing and thawing. For electron microscopy, pancreatic specimens were maintained in 4% paraformaldehyde for 24 h at 4°C, and were then transferred to tissue preservation solution (0.2% sodium azide) for preservation. Other pancreatic tissues were collected, placed in cryogenic tubes and maintained in liquid nitrogen.

Serum amylase and HMGB1 levels were used to determine if ANP modeling was successful. Serum amylase and HMGB1 levels were assessed using ELISA, according to the manufacturer's protocol with the amylase antibody used at a dilution of 1:20,000.

The pancreatic tissues were trimmed to 1×1×1 mm3, fixed with 2.5% pentanediol for 24 h, and post-fixed with 2% osmium tetroxide for 2 h; dehydrated with a gradient of 50%, 70%, 90%, and 100% dehydrated acetone, in each solution three times with each time 10 min. The samples were then placed in an epoxy resin and pure acetone mixture (1:1) at 37°C for 24 h, prior to embedding in a mixture of Epon812 resin, methyl nadic anhydride, dodecenylsuccinic anhydride, and dimethylaminomethyt phenol, at 60°C for 24 h. The blocks were trimmed with semi-thin glasscutter, stained with toluidine blue, and observed under light microscopy to select areas with pancreatic acinar structure. Ultrathin sections with a thickness of 500 Å were obtained with an ultrathin microtome and stained with uranyl acetate and lead nitrate. The sections were observed under transmission electron microscopy (Nissan HT7700; Nissan Corporation, Tokyo, Japan) to examine changes of the pancreatic acinar autophagosomes.

Sections (4 µm) of paraffin-embedded pancreatic tissues were dewaxed and hydrated, and were washed three times with phosphate-buffered saline (PBS; pH 7.4; 5 min/wash). Subsequently, the samples were boiled under high-pressure in 10 mM citric acid buffer (pH 6.0) for 30 min for antigen retrieval, cooled to room temperature, and washed with distilled water and PBS (2×5 min). Sections were incubated with 3% hydrogen peroxide at room temperature for 1 min, and were washed three times with PBS (5 min/wash). Sections were then blocked with goat antiserum (Abcam) for 7 min at 37°C, and were washed with PBS (5 min). Subsequently, the samples were incubated with the following primary antibodies at 4°C overnight: Anti-HMGB1 or anti-Beclin 1 (1:200). Sections were then rinsed three times with PBS (5 min/wash), and a biotinylated secondary antibody (1:100) was then added to the sections, which were incubated at room temperature for 1 h and rinsed in a humidified chamber. Subsequently, enzyme-labeled streptavidin (50 µl; Wuhan Boster Biological Technology, Co., Ltd., Wuhan, China) was added, incubated at room temperature for 30 min and rinsed in a humidified chamber. Sections were stained with DAB (Sigma-Aldrich; Merck Millipore), observed under a BX53 microscope (Olympus Corporation, Tokyo, Japan), and when the color was suitable, the staining reaction was terminated by rinsing. Sections were then counterstained with hematoxylin, rinsed with water, dehydrated in gradient alcohol, cleared with xylene, and finally sealed with neutral resin. Images were captured using a camera system (Motic B5; Motic, Xiamen, China). Yellow-brown staining of the nuclei or cytoplasm was recognized as positive staining. Densitometric quantitative analysis of the expression levels was carried out using the Image-Pro Plus 6.0 image system (Media Cybernetics, Inc., Rockville, MD, USA).

Tissue samples were washed with saline to remove blood. Subsequently, 400 µl solution A [200 µl KCl (10 mM), 2 ml HEPES (10 mM, pH 7.9), 20 µl EDTA (0.1 mM), 1.2 ml MgCl₂ (1.5 mM), 200 µl DTT (1 mM), 100 µl PMSF (0.5 mM) and 16.28 ml of dH₂O] was added. Tissue samples were homogenized and placed on ice for 15 min. Samples were then centrifuged at 15,000 × g for 1 min at 4°C. The supernatants (cytoplasmic proteins) were transferred to a new tube, and the pellet (nuclei) was resuspended in 400 µl solution B [5 ml glycerol (25%), 4 ml HEPES (20 mM; pH 7.9), 4 ml NaCl (400 mM), 200 µl EDTA (1 mM), 200 µl DTT (1 mM), 200 µl PMSF (1 mM) and 6.4 ml dH₂O). Samples were placed on ice for 20 min, and were then centrifuged at 15,000 × g for 10 min at 4°C. The supernatants (nuclear proteins) were transferred to new tubes and were stored at −80°C until further use.

Following extraction of the cytoplasmic and nuclear proteins, 5% SDS-polyacrylamide gel electrophoresis was used to separate the proteins (10 µg samples), which were then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk at room temperature for 2 h, the PVDF membranes were agitated overnight at 4°C with anti-HMGB1 or anti-Beclin 1 (1:1,000). Subsequently, the membranes were agitated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1,000) at room temperature for 1 h. The blots were visualized following treatment with enhanced chemiluminescence solution in a dark room for light exposure. The PVDF membrane was then cleaned with eluent to remove antibodies, and was hybridized with rabbit polyclonal anti-β-actin antibodies (1:1,000; Abcam; cat. no. ab8227) or rabbit anti-histone H3 (1:1,000; RayBiotech, Inc., Norcross, GA, USA; cat. no. 168-10463) and a goat anti-rabbit horseradish-peroxidase secondary antibody (1:1,000; Abcam; cat. no. ab6721). The films were scanned, the optical density values of HMGB1, Beclin 1 and β-actin (internal control) bands were measured using an image analysis system (Quantity One software version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the ratios were obtained for semi-quantitative analysis.

Total RNA was extracted from the pancreatic tissues using TRIzol^® at various time points (0, 3, 6, 12 and 24 h) following ANP modeling. For each sample, 2.0 µg total RNA was reverse transcribed to cDNA using 1 µl oligo (dT) (0.5 µg/µl) primer according to the protocol of the First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). PCR primers were designed according to the gene sequences downloaded from National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genbank/) using Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA). The PCR primer sequences were sequenced by Sangon Biotech., Co., Ltd. (Shanghai, China) as follows: HMGB1, sense 5′-GCTCAGAGAGGTGGAAGAC-3′, antisense 5′-CCAATGGATAAGCCAGGAT-3′; Beclin 1, sense 5′-TGTGGAATGGAATGAAATCAA-3′, antisense 5′-CCCCCAGAACAGTACAACGGC-3′; and β-actin (internal reference), sense 5′-CCCATCTATGAGGGTTACGC-3′ and antisense 5′-TTTAATGTCACGCACGATTTC-3′. The PCR cycling conditions were as follows: Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 sec, and annealing and extension at 58°C (HMGB1 and β-actin) or 60°C (Beclin 1) for 30 sec, for a total of 35 cycles. The PCR reaction was performed in a total of 20 ml, including 10 µl 2X SYBR Green qPCR mix, 1 µl primers (10 µM), 1 µl cDNA and 8 µl DEPC-water, the reaction was conducted in an Eppendorf MasterCycler Realplex qPCR machine. Fluorescence was read at each extension phase. The Cq values were obtained and calculated by 2-ΔΔCq formula (20) relative to the mean of 0 h values, to get the relative expression values.

Data are presented as the mean ± standard deviation, and were analyzed using Student's t-test and one-way analysis of variance followed by the Student-Newman-Keuls post-hoc test. SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

As presented in Table I, amylase levels were increased 3 h after modeling. No rats succumbed within the 24 h experimental period. This is consistent with our previous modeling approach (21).

As presented in Fig. 1, phagocytic vesicles appeared in some pancreatic cells at 3 h. The number of phagocytic vesicles increased in a time-dependent manner. These vesicles exhibited typical autophagy-like morphology (Fig. 1A-E).

The protein expression levels of HMGB1 and Beclin 1 were detected by western blotting (Fig. 2). As presented in Fig. 2A and B, the protein expression levels of HMGB1 began to increase at 12 h; this finding was supported by the results of a qPCR (Fig. 3A). However, unlike HMGB1, the translation (Fig. 2A and C) and transcription levels (Fig. 3B) of Beclin 1 began to increase at 3 h. Similar expression patterns of HMGB1 and Beclin 1 were observed using immunohistochemistry (Figs. 4 and 5, Table II).

Based on the TEM results, autophagy appeared at 3 h, which corresponded with the increase in amylase levels at 3 h. However, HMGB1 levels did not increase until 12 h. Since HMGB1 is a nuclear factor, it may promote autophagy from the nucleus (22). Therefore, the nuclei were isolated in the present study. Notably, HMGB1 was shown to be accumulated in the nuclei at the early stage of ANP (Fig. 6).

It has previously been reported that HMGB1 may be secreted outside of cells, where is has a role as an inflammatory cytokine (23). ELISA was used to evaluate the presence of HMGB1 in the serum at various time points after ANP modeling. As presented in Fig. 7, compared with the control group, HMGB1 levels in the serum began to increase at 3 h, and were highest at 24 h after modeling.