Adult male SPF Sprague-Dawley rats (weight, 200–250 g) were obtained from the Center for Animal Experiments of Wuhan University (Wuhan, China). All rats were housed in an animal care facility with a 12 h light/dark cycle and fed with standard commercial diets and water ad libitum. Experimental procedures were approved by the ethics committee of Wuhan University and performed in accordance with the principles of the 1983 Declaration of Helsinki.

Rats were fasted for 12 h prior to the experiment, but had free access to water. All rats were subjected to the induction of anesthesia in a closed chamber with 4% isoflurane (Abbott Laboratories, Shanghai, China) in 2 l/min oxygen, and were maintained under anesthesia for surgery with 2% isoflurane in 2 l/min oxygen. The SAP rat model was elicited by a standardized retrograde infusion of 1 ml/kg body weight 5% sodium taurocholate (STC) solution (Sigma-Aldrich, St. Louis, MO, USA) into the bile-pancreatic duct. Saline (20 ml/kg) was injected into the back subcutaneously to compensate for the fluid loss following the surgery.

The rats were randomly divided into three groups as follows: i) STC + thymosin β4 group (Tβ4 group, n=24). Thymosin β4 (GL Biochem Ltd., Shanghai, China; 30 mg/kg) was dissolved in saline at a concentration of 3 mg/ml and was administered intraperitoneally 30 min prior to STC infusion. ii) STC + saline (SAP group, n=24). Prior to the induction of SAP, rats in this group received saline by intraperitoneal injection. iii) Sham-operated group (SO group, n=24). In this group, rats were treated with sham surgery (the pancreas and duodenum were flipped a number of times) instead of STC infusion. Similar doses of thymosin β4 have been demonstrated to exert neuroprotective and neurorestorative effects in experimental models of traumatic brain injury (22).

Rats in the three groups were sacrificed at 3, 6 and 12 h after treatment (n=8 per group at each time-point). Blood samples were collected by intracardiac puncture at the respective time-points in the three groups. Samples were centrifuged at 3,000 × g for 15 min, and the serum was stored at −20℃ for subsequent biochemical and cytokine measurements. Following sacrifice, tissue from the head of the pancreas was harvested and fixed in 4% phosphate-buffered formaldehyde for histopathological observation. The remaining part of the pancreas was snap frozen in liquid nitrogen and stored at −80℃ for assay.

For histological analysis, paraffin-embedded pancreatic tissues were sliced to a thickness of 4 µm and stained with hematoxylin and eosin (H&E). The morphology of the pancreatic tissues was evaluated and documented by two experienced pathologists who were blinded to the experiment. Pancreatic histological assessment was scored for the severity of pancreatitis based on edema, inflammatory cell infiltrate and hemorrhage, and acinar necrosis according to the scale described by Schmidt et al (23).

Serum AMY and LIP levels were measured using standard techniques with a fully automatic chemistry analyzer (Olympus AU2700 Chemistry-Immuno Analyzer; Olympus Inc., Tokyo, Japan).

The serum concentrations of TNF-α, IL-1β and IL-6 were measured in units of pg/ml by the use of commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBioscience Ltd., Vienna, Austria). The absorbance was read by an automated microplate ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and concentrations were calculated by the standard curve run on each assay plate. All samples were measured in duplicate.

Neutrophil infiltration in the pancreas was quantitated by MPO activity. MPO activity was measured photometrically with 3,3′,5,5′-tetramethylbenzidine as a substrate, and the reaction was initiated by adding hydrogen peroxide to the medium. The MPO activity assay was performed with a commercial kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Pancreatic tissue sections were obtained from paraffin-embedded tissues. Sections of 4 µm thickness were deparaffinized with xylene, and 0.3% hydrogen peroxide was used to inactivate endogenous peroxidase activity. Sections were incubated in 5% normal goat serum diluted in phosphate-buffered saline (PBS). Endogenous biotin and avidin binding sites were blocked by avidin and biotin, respectively. The sections were incubated overnight with rabbit polyclonal anti-rat NF-κB p65 antibody (1:100; C22B4; Cell Signaling Technology, Inc., Danvers, MA, USA) in a humidity chamber at 4°C, then counterstained with hematoxylin. Negative control experiments were performed in which PBS was used instead of primary antibody.

Pancreatic NF-κB p65, I-κBα and ICAM-1 levels at 12 h after STC infusion were determined by Western blot analysis. Cytoplasmic and nuclear proteins were extracted using the Nuclear-Cytosol Extraction kit (Applygen Technologies Inc., Beijing, China). Concentrations of protein in the samples were evaluated by the Bradford method with bovine serum albumin as a standard. Equal amounts of protein samples were electrophoresed using 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Non-specific binding was blocked with 5% skimmed milk in Tris-buffered saline and 0.1% Tween 20 (TBST) buffer at room temperature for 2 h. The cytoplasmic proteins were incubated with primary antibodies of rabbit polyclonal anti-rat inhibitor of κB (I-κB)α antibody (1:1,000; sc-371; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), actin antibody (1:2,000; sc-130656; Santa Cruz Biotechnology, Inc.) and ICAM-1 antibody (1:1,000; sc-7891; Santa Cruz Biotechnology, Inc.). In addition, the nuclear proteins were incubated with rabbit polyclonal anti-rat NF-κB p65 (1:1,000; C22B4; Cell Signaling Technology, Inc.) and histone H3 antibody (1:1,000; sc-10809; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following extensive rinsing with TBST, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature, and developed with the use of an electrochemiluminescent reagent (Immobilon Western HRP Substrate; EMD Millipore, Bedford, MA, USA) and images were captured on light-sensitive imaging film (Kodak, Rochester, NY, USA). The protein bands were quantified by densitometry using Quantity One 4.5.0 software (Bio-Rad Laboratories, Inc., Richmond, CA, USA).

Data was analyzed using SPSS statistical software, version 16.0 (SPSS Inc., Chicago, IL, USA). All data are expressed as the mean ± standard deviation. Data were compared between all groups by one-way analysis of variance (ANOVA). A two-tailed P-value of less than 0.05 was considered statistically significant.

As shown in Fig. 1, the SAP group had significant increased serum AMY and LIP levels from 3 h to 12 h compared with those in the SO group (P<0.05). Compared with the SAP group, treatment with thymosin β4 prior to STC infusion significantly reduced the serum levels of AMY and LIP at each time-point (P<0.05).

Representative pathological sections of the pancreatic tissue are shown in Fig. 2A–C. In the SAP group, massive areas of acinar necrosis accompanied by infiltration of inflammatory cells, vacuolization and hemorrhage were observed. Rats in the SO group showed little morphological evidence of pancreatic injury, with the exception of mild interstitial edema. As shown in Fig. 2D, the pancreatic histological score was significantly decreased in the Tβ4 group compared with the SAP group.

Serum proinflammatory cytokine levels were analyzed to determine the effect of thymosin β4 on the inflammatory process of SAP. As illustrated in Fig. 3A–C, serum concentrations of IL-1β, IL-6 and TNF-α increased continuously with time. However, the increases in the concentrations of these cytokines following SAP were markedly decreased by the administration of thymosin β4 (P<0.05). Neutrophil infiltration in the pancreas was evaluated by the measurement of MPO activity after SAP. The MPO activity of the SAP group increased significantly at each time-point compared with that in the SO group (P<0.05). When administered prophylactically, thymosin β4 reduced MPO activity in the pancreatic tissue at 6 to 12 h compared with that in the SAP group (P<0.05; Fig. 3D).

To investigate the localization of NF-κB p65 expression, an immunohistochemical assay was conducted. In the SO group, NF-κB p65 was expressed mainly in the cytoplasm in pancreatic tissues (Fig. 4A). NF-κB p65 immunoreactivity was highly expressed in the nucleus following the administration of STC (Fig. 4B). However, a marked reduction in NF-κB p65 staining was observed in the cell nuclei with thymosin β4 pretreatment, and the staining increased in the cytoplasm (Fig. 4C).

Fig. 5A and B shows that the expression of NF-κB p65 protein in the pancreas in the SAP group was higher than that in the SO group at 12 h. In the Tβ4 group, the NF-κB p65 protein was expressed at a much lower level than in the SAP group (P<0.05). Furthermore, the SAP group showed a significant reduction of pancreatic I-κBα protein expression (Fig. 5D). As shown in Fig. 5C, the expression of pancreatic ICAM-1 protein in the SAP group was much higher than that in the SO group. However, ICAM-1 protein expression was much lower in the Tβ4 group than in the SAP group (P<0.05).