Healthy, adult Sprague Dawley rats weighing between 250 and 280 g were purchased from Dashuo Laboratories (Chengdu, China). All animals were individually housed in plastic cages containing wood shavings, and maintained in a temperature-controlled environment with a 12-h light/dark cycle and free access to food and water. All animals were allowed to acclimate to these conditions for 1 week prior to experimental treatment. All experimental animal procedures were approved by the Ethics Committee for Animal Experiments of the General Hospital of Chengdu Military Command (Chengdu, China), and all animal experiments were performed according to the National Animal Welfare Law of China.

Thirty-six Sprague Dawley rats were randomly divided into sham-operated, SAP and SAP plus berberine groups (n=12/group), which were referred to as the Sham, SAP and SAP+ber groups, respectively. Berberine (Sigma-Aldrich, St. Louis, MO, USA; 50 mg/kg dissolved in 1 ml normal saline/200 g body weight) or normal saline (1 ml/200 g body weight) were administered intragastrically once a day for 5 days, prior to the induction of pancreatitis.

After 12 h of fasting, rats were anesthetized with an intraperitoneal injection of 50 mg/kg phenobarbital and a midline laparotomy was performed. In the Sham group, upon opening the abdominal cavity, the pancreas and duodenum were only moved prior to the closure of the abdominal wall, using a double layer of sutures, and the return of the rats to their cages. In the SAP and SAP+ber groups, subsequent to opening the abdominal cavity, pancreatitis was induced using retrograde injection of 3% Na taurocholate (Sigma-Aldrich; 1 ml/kg body weight) into the pancreatic duct over a period of 2 min. Following the surgery, rats in all three groups were injected subcutaneously with 5 ml 0.9% NaCl solution twice a day to supplement the blood volume. Twenty-four hours after the surgery, rats were re-anesthetized and laparotomies were performed. Samples of blood and tissue were obtained immediately.

Ileal and pancreatic specimens were fixed in a 4% paraformaldehyde solution immediately following isolation. Tissues were then fixed, dehydrated, paraffin-embedded and sectioned, according to standard methods. The 4-μm-thick sections were stained with hematoxylin and eosin (H&E), and the pathological changes in the small intestine and pancreas were examined using an optical microscope (Olympus, Tokyo, Japan). A pathologist who was blinded to the grouping scored the ileal specimens according to the method described by Chiu et al (15,16). The mucosal damage was graded from zero (normal) to five (severely damaged).

Serum DAO activity and endotoxin levels are used as indices of small intestinal mucosal mass and integrity. Rat blood samples were collected as described above, measured and then centrifuged at 1,509 × g for 10 min at 4°C. The supernatant was transferred to sterile labeled tubes and stored at −80°C until use. Serum endotoxin levels were assessed using a Chromogenic Limulus Amebocyte Lysate assay kit (Shanghai Med & Chem Institute, Shanghai, China). DAO activity was examined using a commercial kit (Nanjing Jianchen Co. Ltd., Nanjing, China) according to the manufacturer’s instructions. In brief, DAO catalyzes the oxidation of the substrate putrescine, and the product is quantitatively oxidized by peroxidase in proportion to the quantity of hydrogen peroxide produced, resulting in the production of o-dianisidine, which has an absorption maximum at 440 nm.

MLNs were harvested under sterile conditions, prior to being homogenized, incubated at 37°C in an agitated water bath for 18 h, plated on MacConkey II agar (Oxoid Ltd., Basingstoke, UK) and incubated aerobically at 37°C for 24 h. A blinded visual inspection was then performed. Results were recorded as positive for growth, without quantification, or no growth. The culture was considered positive if bacterial growth was observed, and the incidence of bacterial translocation was calculated by determining the number of rats with a positive bacterial culture divided by the total number of rats studied.

The rats were sacrificed by overdose of anesthesia and segments of the ileum were isolated, flushed and immediately placed in RNAiso Plus buffer (Takara Biotechnology Co. Ltd., Dalian, China). Total RNA was extracted according to the manufacturer’s instructions. cDNA was synthesized by reverse transcription using the PrimeScript RT Reagent kit (Takara Biotechnology Co. Ltd.) with an oligo (dT) primer. qPCR analysis was performed using SYBR^® Premix EX Taq™ II (Takara Biotechnology Co. Ltd.) in a Bio-Rad IQ5 system (Bio-Rad, Hercules, CA, USA). Primers for zonula occludens (ZO)-1, occludin and GAPDH were synthesized by Takara Biotechnology Co. and had the following sequences: ZO-1, 5′-GCTCCTCCCACCTCG CACGT-3′ (forward) and 5′-GACCTGCTGGAGCATAGG GCTG-3′ (reverse); occludin 5′-TGGAGTTGCGGGAGAGCG ATC-3′ (forward) and 5′-GGGCAGTCGGGTTGACTCCCA-3′ (reverse); GAPDH, 5′-TCCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-AGATCCACAACGGATACATT-3′ (reverse). The reaction conditions were as follows: Initial denaturation at 95°C for 1 min, template denaturation at 95°C for 20 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min (for a total of 40 cycles) and a final extension at 72°C for 10 min. The cycle threshold (CT) was determined using automatic baseline calculations. A CT value of >30 was considered unacceptable. The relative gene expression was calculated using the 2^−ΔΔCT method. GAPDH was used as an internal control.

Ilea were immediately placed in cold radio-immunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) subsequent to flushing. Each sample was then sonicated on ice three times for 30 sec using a Sonic Dismembrator (Thermo Fisher Scientific, Waltham, MA USA) and centrifuged at 14,000 × g for 15 min. Supernatants were then collected and the protein concentrations were determined according to the Bradford method using bicinchoninic acid assay reagent (Beyotime Institute of Biotechnology). Samples containing 20 μg protein were loaded onto SDS-PAGE gels, electrophoresed using a Bio-Rad mini gel system (Bio-Rad) and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked using 5% bovine serum albumin in 50 mM Tris-HCl (pH 7.5), 140 mM NaCl and 0.1% Tween and incubated at 4°C overnight with primary antibodies against ZO-1 (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), occludin (1:1,000; Santa Cruz Biotechnology, Inc.), MLC (1:1,000; Epitomics Inc., Burlingame, CA, USA) or phosphorylated MLC (pMLC; 1:1,000; Abcam PLC, Cambridge, MA, USA). Membranes were then washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies. Membrane imaging was performed using an enhanced chemiluminesce detection system (Millipore) according to the manufacturer’s instructions.

Kruskal-Wallis and Mann-Whitney U tests were performed for comparisons among groups. The incidence of gram-negative bacterial translocation to the MLNs was assessed using a χ² test. A value of P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS 18.0 statistical software (SPSS Inc., Chicago, IL, USA).

To determine whether SAP rat models were successfully established, sections of pancreatic tissue were stained with H&E and examined using light microscopy (Fig. 1). Pancreatic tissues from the Sham group exhibited a normal macroscopic and histological appearance. By contrast, the H&E-stained sections from the SAP and SAP+ber groups revealed atypical pancreatic architectures, with marked interstitial edema, acinar cell necrosis, leukocyte infiltration and scattered hemorrhage. These findings suggest that SAP rat models were successfully established; however, berberine had no significant effect on the histological changes in the pancreas.

To identify the role of berberine in the maintenance of intestinal barrier function, the intestinal membrane permeability was evaluated by assaying serum DAO activity and endotoxin concentration (Fig. 2A and B). In the Sham group, the baseline levels of serum DAO activity and endotoxin concentration 24 h after the sham surgery were 0.235±0.053 kU/l and 0.218±0.086 EU/ml, respectively. Serum DAO activity and endotoxin levels were observed to be higher in the SAP group than those in the Sham group (P<0.05). Furthermore, berberine pretreatment was observed to significantly decrease serum DAO activity (33.0%) and endotoxin levels (16.7%) compared with levels in the SAP group (P<0.05).

To further assess the effect of berberine on intestinal barrier function, the incidence of bacterial translocation was evaluated (Fig. 2C). No bacterial translocation was observed in the MLNs from the Sham group; however, the incidence of bacterial translocation to the MLNs in the SAP group 24 h after the retrograde injection of 3% Na-taurocholate was observed to be 83.3%. Berberine treatment was found to reduce the rate of bacterial translocation to 58.3%, which was significantly lower than that observed in the SAP group (P<0.05). Serum DAO activity and endotoxin levels were also observed to decrease with berberine treatment.

These results indicate that berberine treatment may ameliorate the intestinal mucosal barrier damage associated with SAP induced by the retrograde injection of Na-taurocholate.

Animals were sacrificed 24 h after surgery. H&E-stained ileal sections were examined using an optical microscope and scored by a pathologist who was blinded to the grouping, according to the aforementioned method. The results of the pathological scoring are shown in Fig. 3. In the Sham group, the epithelial cells were observed to be closely arranged in a regular fashion. Twenty-four hours after the induction of SAP, the ileal mucosa was injured. Vacuolated epithelial cells, shortened villi and infiltrating lymphocytes were observed. The mean pathological score of the ilea in the SAP group increased to 1.833, compared with 0.333 in the Sham group (P<0.05). Compared with the rats in the SAP group, berberine administration was observed to have a protective effect against ileal mucosal injury, and the ilea of rats in the SAP+ber group exhibited relatively normal villi and mucosal integrity. The pathological scoring also demonstrated that berberine treatment attenuated the pancreatitis-induced mucosal injury compared with that observed in the SAP group (1.417 vs. 1.833 for the SAP+ber and SAP groups, respectively; P<0.05).

TJs have a critical role in maintaining the function of the intestinal barrier (17,18). The impairment of intestinal barrier function is directly associated with the aberrant expression of TJ proteins (19). qPCR and western blot analyses were performed to detect the expression of the TJ proteins ZO-1 and occludin. Berberine was not observed to influence ZO-1 and occludin mRNA expression in the SAP-induced rats (Fig. 4A). Furthermore, occludin protein expression did not differ significantly among the three groups; however, ZO-1 protein expression was observed to be significantly higher in the SAP+ber group than that in the SAP group (Fig. 4B and C). Overall, these results suggest that berberine exerts few effects on TJ proteins in the ilea of SAP rats.

It is well established that MLCK-mediated phosphorylation of MLC has a significant role in the physiological and pathophysiological regulation of intestinal epithelial TJs and paracellular leak pathways (20,21). In the present study, although berberine was observed to have little effect on ZO-1 and occludin mRNA and protein expression, berberine was hypothesized to ameliorate SAP-induced barrier dysfunction through MLC phosphorylation, which, to the best of our knowledge, has not been previously investigated. As shown in Fig. 5, western blot analysis of MLC and pMLC revealed that berberine treatment had no significant effect on total MLC expression in SAP-induced rats. However, significantly higher pMLC levels were observed in rats in the SAP group compared with those in the Sham group (6.175-fold increase, P<0.05). This increase was significantly attenuated with berberine treatment, with pMLC levels in the SAP+ber group reduced to 0.349-fold those in the SAP group (P<0.05). These data suggest that the inhibition of SAP-induced MLC phosphorylation may be one of the mechanisms responsible for the beneficial effect of berberine on intestinal barrier function during SAP-induced damage.