The present study was approved by the Institutional Animal Experimental Ethics Committee of Sichuan University (Chengdu, China). Adult male Wistar rats (n=128; age, 3 months old; body weight, 200–300 g) were purchased from the Experimental Animal Centre of West China, Centre of Medical Sciences of Sichuan University (Chengdu, China) and were used in the present study. Animals were housed in cages under a 12-h light/dark cycle, with free access to food and water. The temperature of the cages were maintained at 21–25°C with 50–60% humidity. To reduce the potential influences of sex differences (12) and transportation (13) on cognitive assessment, only male rats were used in the present study, which were transferred from the rearing room into the behavioral testing room at least 30 min prior to the initiation of each experiment.

Rats were randomly divided into four groups (n=32 per group): Sham group, Sham + berberine group, SAP group and SAP + berberine group. The SAP model was induced via two intraperitoneal injections of 3 g/kg L-arginine (dissolved in normal saline, 1:5, w/v; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at an interval of 1 h (14), whereas rats in the sham groups received equivalent volumes of a saline vehicle. Following the second injection of L-arginine, 100 mg/kg berberine (15) (dissolved in 1 ml normal saline/200 g body weight; Sigma-Aldrich; Merck KGaA) was administered immediately and then once a day for 6 consecutive days. A total of 48 h after the 2nd injection of L-arginine was administered, disease severity of SAP was assessed. A total of 7 days post-SAP induction, the survival rates of rats were calculated and behavioral tests were subsequently performed. Furthermore, surviving rats were sacrificed and hippocampal tissues were isolated for subsequent biochemical analysis.

A total of 48 h post-SAP induction, rats were sacrificed via i.p. injection of 2% sodium pentobarbital (100 mg/kg; Sigma-Aldrich; Merck KGaA) and pancreatic tissues were removed and subsequently fixed in 10% neutral buffer formaldehyde at room temperature for 24 h. Subsequently, tissues were embedded in paraffin and sectioned. The tissue sections (1 mm³) were then stained using hematoxylin and eosin, and were observed under light microscopy. Histological alterations were assessed using a previously established histopathological scoring system (16) with slight modifications: For each pathological section, ten visual fields were randomly selected, visualized under a high-power microscope (magnification, ×100) and then blindly scored by a pathologist. The overall score was presented as the sum of severity of inflammation, extension of inflammation (none, 0; slight, 1; moderate, 2; and severe, 3) and fat/acinar necrosis (none, 0; basal 1/3 damaged, 1; basal 2/3 damaged, 2; only surface epithelium intact, 3; and transmural, 4). In addition, disease severity of SAP was also assessed by measuring the levels of serum amylase. Blood samples were centrifuged at 3000 × g at 4°C for 10 min and serum amylase was subsequently determined by routine colorimetric methods using a commercial kit (cat. no. C016; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and expressed as U/L.

To investigate locomotor activity, which may affect the results of cognitive assessment, an automated rotarod (cat. no. 47750; Ugo Basile SRL, Gemonio, Italy) was used to ensure locomotor activity of rats was completely recovered following the induction of SAP. Locomotor activity assessment was conducted twice, with an accelerating rotational speed of 0.2 rotations/sec (from 4–40 rpm), including two consecutive training trials and one testing trial. One test was performed prior to SAP induction (the baseline latency) and the other was performed on the 7th day prior to cognitive assessment. A blinded observer recorded the average time spent on the bar, which was considered to represent the latency to fall from the rod, with a 10 min interval between each trial in one test.

On the next day following locomotor activity assessment, FCT was performed using a dedicated conditioning chamber (cat. no. 46000; Ugo Basile SRL) as previously described (13) with modifications, including training, context testing and cued testing. During the training period in FCT, rats were placed in the conditioning chamber and were permitted to explore the training environment for 5 min. Following this, an auditory cue (2500 Hz, 75 dB) was presented for 2.5 sec as a conditioned stimulus (CS) that co-terminated with a 0.5 mA electric foot shock, which represented an unconditioned stimulus (US) during the last 2 sec of the auditory cue. Following a 57.5 sec inter-trial interval, the pairing of the auditory cue and the foot shock (CS-US) was again administered and repeated a further 15 times. A total of 24 h post-CS-US testing, all rats were placed back in the same chamber to investigate long-term memory. During the context test, which investigates hippocampus-dependent memory, rats were permitted to freely explore the chamber for 10 min in the absence of CS and US stimulation. Following this, the cued test was performed to determine hippocampus-independent memory functioning, in which rats were placed in a novel context environment with altered olfactory cues and lighting, as well as the attachment of patterned contexts on the walls and floor, received an auditory cue (2500 Hz, 75 dB), 10 times at 57.5 sec time intervals, without the foot shock. Cognitive deficit was investigated via determination of the percentage of time spent completely immobilized except for respiration, which was automatically recorded by a digital video-tracking system.

A total of 48 h post-SAP induction, 3 ml/kg 2% Evans blue (EB) dye in PBS was administrated into the tail vein to evaluate the integrity of the BBB. A total of 1 h post-administration, rats were sacrificed via i.p. injection of 2% sodium pentobarbital (100 mg/kg) and perfused through the left heart ventricle with PBS until the outflow from the right auricle was colorless. Following this, brain tissues were harvested and homogenized in formamide solution buffer and then centrifuged at 10,000 × g for 20 min at 4°C. The extracted supernatant was investigated at a wavelength of 655 nm using a spectrophotometer based on external standards in the same solvent as the control, and data are expressed as ng/mg of total brain tissue.

Following cognitive assessment, rats were sacrificed via i.p. injection of 2% sodium pentobarbital (100 mg/kg). The brains of each group were harvested and hippocampal tissues were isolated, which were subsequently snap-frozen in liquid nitrogen and stored at −80°C prior to further experimentation. Levels of TNF-α (cat. no. CSB-E11987r; Cusabio Biotech Co. Ltd., Wuhan, China) and IL-1β (cat. no. CSB-E08055r, Cusabio Biotech Co. Ltd., Wuhan, China) were determined by ELISA in accordance with the manufacturer's protocols. Briefly, the stored brain tissues were homogenized in normal saline using a sonicator (120 times; 1 time for 0.8 sec, then 1 sec with 2 sec intervals) at room temperature, and the supernatants were subsequently harvested and centrifuged at 3000 × g for 15 min at 4°C. The supernatant was subsequently collected and incubated with biotinylated antibodies (included in the ELISA kits) for 30 min at 37°C. Subsequently, the supernatant was incubated with streptavidin-horseradish peroxidase solution for 1 h at 37°C and the protein concentrations were subsequently quantified using the Bradford protein assay (cat. no. P0010; Beyotime Institute of Biotechnology, Haimen, China). Following incubation with supplied chromogen solution and stop solution, protein concentrations were determined at an absorbance of 450 nm. Levels of TNF-α and IL-1β were calculated using a standard curve and expressed as pg/ml.

Following cognitive assessment, rats were sacrificed via i.p. injection of 2% sodium pentobarbital (100 mg/kg). Hippocampal tissues were harvested following cognitive assessments to investigate the protein expression levels of anti-cleaved caspase-3 (1:500; cat. no. 9661; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-receptor-interacting protein kinase (RIP)1 (1:750; cat. no. ab106393; Abcam, Cambridge, UK) and anti-RIP3 (1:750; cat. no. ab62344; Abcam) via western blotting. Briefly, the frozen hippocampal tissue samples were homogenized in cell lysis buffer (Cell Signaling Technology, Inc.) The samples were then centrifuged at 3000 × g for 15 min at 4°C, the supernatant was collected and total protein concentrations of the supernatants were determined using a Bradford protein assay (P0010, Beyotime Institute of Biotechnology). Equal amounts of protein (0.1 mg/µl) extracts were heated, denatured and separated by electrophoresis on a NuPAGE 8–15% Bis-Tris gel (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and subsequently transferred to polyvinylidene difluoride membranes. Following blocking using 5% non-fat dry milk for 90 min at room temperature, these membranes were probed with primary antibodies overnight at 4°C and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. 7076; Cell Signaling Technology, Inc.) for 1 h at room temperature. β-actin (1:1,000; cat. no. 8457; Cell Signaling Technology, Inc.) was used as an internal housekeeping protein. Proteins were visualized using an enhanced chemiluminescence detection kit (cat. no. sc-2048; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and densitometric analysis was performed using ImageJ software (version 1.52; National Institutes of Health, Bethesda, MD, USA).

Data are presented as the means ± standard error of the mean of three repeated experiments, and were analyzed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). One-way analysis of variance followed by the Bonferroni test was used to analyze the differences among the groups. Survival rates were analyzed using the Kaplan-Meier method and subsequently compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

It has been reported that repeated injections with high doses of L-arginine may induce AP in rodents with well-defined, gradually progressive pancreatic necrosis (14). In the present study, L-arginine was used to induce SAP, and disease severity was assessed by investigating alterations in pancreatic histopathology and serum amylase levels. Following treatment with L-arginine for a total of 48 h, all typical features of SAP, including significant isolation of pancreatic lobes, marked inflammatory cell infiltration, hemorrhaging and acinar cell necrosis, were observed in the SAP group (Fig. 1A). Following treatment with berberine, pancreatic morphology was markedly attenuated in the SAP + berberine group compared with the SAP group (Fig. 1A). These pancreatic morphological alterations at 48 h were reflected by the pancreatic histopathological scores (Fig. 1B-D). Pancreatic tissues obtained from the SAP + berberine group exhibited a significantly decreased overall histopathological score compared with the SAP group (mean score, 2.66 vs. 6.49; P<0.05; Fig. 1B), and significantly decreased neutrophil infiltration and acinar necrosis scores compared with the SAP group (Fig. 1C and D). As presented in Fig. 1E, serum amylase levels were significantly increased in the SAP group compared with the Sham group, and significantly decreased in the SAP + berberine group compared with the SAP group. In the present study, the survival rate of rats in the SAP group over the 7 day time period was investigated, and the results revealed that the survival rate of rats with SAP was ~50%, which was consistent with the findings of a previous study (17). Following repeated treatment with berberine, there was a significant increase in the survival rate of rats in the SAP + berberine group compared with the SAP group (75 vs. 50%; Fig. 2; P<0.05).

To ensure that the locomotor activity of rats was completely recovered following SAP, an automated rotarod was used to investigate the locomotor activity of rats, which may have affected the results of cognitive assessment. Compared with the baseline locomotor activity, there was no significant difference among all groups prior to cognitive assessment (P>0.05; Fig. 3A). The clinical presentation of cognitive impairments induced by SAP is not specific, and is characterized by functional disabilities and long-term cognitive impairment (2). In the present study, FCTs were performed to investigate SAP-induced cognitive impairments. The FCT is one of the most reliable and widely used behavioral paradigms used to assess associative fear learning and memory in rodents (13,18), which predominantly includes context and cued FCTs. Context fear conditioning requires the presentation of complex associations of stimuli and an aversive stimulus, which is processed by the hippocampus. Therefore, context FCT is used to investigate hippocampus-dependent memory, whereas cued FCT is performed to investigate hippocampus-independent memory. The results revealed that the freezing time exhibited by rats in the SAP group during the context test was significantly decreased compared with the Sham group, thus suggesting that SAP may induce hippocampus-dependent long-term memory deficits, whereas this effect was significantly attenuated following treatment with berberine (Fig. 3B). In addition, there were no significant differences in the freezing times exhibited by rats in all four groups during the cued test (Fig. 3C), which suggested that the hippocampus-independent memory of rats was not affected by SAP.

BBB breakdown has been revealed to represent an important factor associated with the development of sepsis-associated cognitive impairment (19). The permeability of the BBB was investigated via determination of EB content in rat brain tissues. A total of 48 h post-induction of SAP, the rats in the SAP group exhibited significantly increased levels of extravasation of EB into brain tissues compared with the Sham group; however the extravasation of EB was significantly decreased in the SAP + berberine group compared with the SAP group (Fig. 4A).

Levels of TNF-α and IL-1β proinflammatory cytokines in rat hippocampal tissues during the development of SAP were investigated via ELISA analyses. The results revealed that levels of TNF-α and IL-1β were significantly increased in the brain tissues of rats in the SAP group compared with rats in the Sham group. Therefore, the results suggested that treatment with berberine attenuates elevated levels of IL-1β and TNF-α in rat hippocampal tissues (Fig. 4B and C).

It has been well established that neuronal apoptosis and necroptosis represent crucial factors associated with the development of neurodegeneration during brain dysfunction (20,21). To further investigate the hippocampus-dependent memory impairment exhibited by rats in the SAP group, as demonstrated by the aforementioned FCT, western blotting was performed to determine the expression levels of proteins associated with apoptosis and necroptosis in hippocampal tissues (Fig. 5A). The results demonstrated that the protein expression levels of caspase-3, an inducing apoptosis effector, were significantly increased in the hippocampal tissues of SAP rats compared with rats belonging to the Sham group; however, this effect was significantly attenuated following treatment with berberine (Fig. 5B). RIP1 and RIP3 are the predominant inducers of neuronal necroptosis (22). In comparison with the Sham group, SAP-induced rats exhibited significantly increased expression levels of RIP1 and RIP3 in hippocampal tissues. Notably, rats in the SAP + berberine group exhibited significantly decreased expression levels of RIP1 and RIP3 in hippocampal tissues compared with the SAP group (Fig. 5C and D).