A total of 100 male Sprague-Dawley (SD) rats (Laboratory Animal Center, Xiangya Hospital, Central South University, China), weighing 250±50 g, were used in the present study. All animals were housed in a controlled temperature environment (25°C; 50% humidity) with a 12-hour day/night rhythm with free access to standard laboratory chow and water. All animals received humane care in compliance with the National Institutes of Health standards (Guide for the Care and Use of Laboratory Animals, revised 1996) (24). The present study was approved by the Ethics Committee of the Xiangya School of Medicine, Central South University (Changsha, China).

Rutaecarpine (purity, >98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Capsazepine (a competitive VR antagonist) (purity, >98%) and 45% sodium taurocholate were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dimethyl sulfoxide and ethanol were mixed at a ratio of 1:4 and used to dissolve rutaecarpine. A vehicle containing 8% ethanol, 2% dimethyl sulfoxide and 90% saline was used to dissolve capsazepine.

SD rats were randomly divided into 10 different groups (all n=10). The groups included were as follows: i) Sham-operated group (Sham), AP was not induced during surgery; ii) AP group (AP), 5% sodium taurocholate solution (1.0 ml/kg) was injected into rats to induce AP during surgery; iii) Sham-operated + rutaecarpine (100 µg/kg) group (Sham+Rut), rats were injected with 100 µg/kg rutaecarpine into the sublingual vein 20 min prior to surgery, during which AP was not induced; iv) AP + rutaecarpine (30 µg/kg) group (AP+Rut L), rats received an injection of 30 µg/kg rutaecarpine into the sublingual vein 20 min prior to surgery, in which AP was induced; v) AP + rutaecarpine (100 µg/kg) group (AP+Rut M), rats were injected with 100 µg/kg rutaecarpine into the sublingual vein 20 min prior to surgery, in which AP was induced; vi) AP + rutaecarpine (300 µg/kg) group (AP+Rut H), rats were injected with 300 µg/kg rutaecarpine into the sublingual vein 20 min prior to surgery, in which AP was induced; vii) sham-operated + capsazepine group (Sham+Cap), animals were injected with 3 mg/kg capsazepine into the sublingual vein 30 min prior to surgery, in which AP was not induced; viii) AP + capsazepine group (AP+Cap), animals were injected with 3 mg/kg capsazepine into the sublingual vein 30 min prior to surgery; ix) AP + capsazepine + rutaecarpine (100 µg/kg) group (AP+Cap+Rut), mice were injected with 3 mg/kg capsazepine into the sublingual vein 30 min prior to surgery and were subsequently injected with 100 µg/kg rutaecarpine into the sublingual vein 20 min prior to surgery, in which AP was induced; and x) vehicle control group (AP+Sol), mice were injected with the vehicle [a mixture of dimethyl sulfoxide and ethanol (1:4) in a volume of 0.125 ml/kg] into the sublingual vein 20 min prior to surgery, in which AP was induced.

SD rats were fasted for 12 h prior to surgery. Subsequently, rats were administered 3% pentobarbital sodium (40 mg/kg; Sigma-Aldrich; Merck KGaA) intraperitoneally to induce anesthesia. Following a conventional disinfection (first with 2.5% iodine inunction, followed by drying with 70% alcohol twice) and towel spreading, an incision (~1.5 cm) along the white line of abdomen was created. Freshly prepared 5% sodium taurocholate solution was used to induce AP by retrograde infusion through the cholangiopancreatic duct following laparotomy (25). Instead of 5% sodium taurocholate solution, an equivalent volume of normal saline solution was administered to the Sham group. The incision was closed with a continuous silk suture. All rats were sacrificed 24 h after surgery and arterial blood and pancreatic tissue were collected. The serum was collected following centrifugation (4°C, 1,500 × g for 5 min) and stored at −20°C. Pancreatic tissue was fixed in 4% phosphate-buffered formaldehyde at 4°C prior to histopathological examination.

The abdomen was opened following euthanasia of the rats (all rats were sacrificed 24 h after surgery), ascites were removed from the abdominal cavity using a syringe and a measuring cylinder was used to measure the volume of the ascites.

Formaldehyde-fixed pancreatic tissues obtained from the rats were embedded in paraffin, sectioned at 4 µm thick, and were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol and then washed in PBS. Sections were stained with hematoxylin (4 min), washed with H₂O, and then 0.5% eosin (1 min) at room temperature. Two independent pathologists, who were blinded to this experiment, evaluated the sections under a light microscope. As outlined by the histopathological features scoring criteria (26), the severity of pancreatitis was scored according to edema, vacuolization, necrosis and inflammation.

Serum amylase activity was measured using a Hitachi 7170A full-automatic biochemical analyzer (Hitachi, Ltd., Tokyo, Japan) according to the manufacturer's protocol (α-Amy-DR; Autec Diagnostics, Bötzingen, Germany).

ELISA was performed to measure the serum concentrations of interleukin (IL)-6 (Rat IL-6 ELISA kit; cat. no. JER-04), IL-10 (Rat IL-10 ELISA kit; cat. no. JER-05) and tumor necrosis factor (TNF)-α (Rat TNF-α ELISA kit; cat. no. JER-06), according to the manufacturer's protocol (all products were purchased from Joyee Biotechnics Co., Ltd., Shanghai, China). Absorbance values were used to determine cytokine concentrations using a standard curve. All samples were tested three times.

Plasma was collected when rats were sacrificed 24 h after surgery by centrifugation at 1,000 × g for 30 min at 4°C. Subsequently, CGRP concentration in plasma was measured using a radioimmunoassay kit, according to the manufacturer's protocols.

All results are presented as the mean ± standard deviation and were analyzed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). Results were compared between all groups using one-way analysis of variance and least significant difference t tests were used to compare two different groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the protective role of rutaecarpine in a rat model of AP, histopathological changes of the pancreas in rats from each group were assessed by evaluating H&E-stained tissue. Representative histological sections are presented in Fig. 2. There were no evident histopathological changes identified in the pancreas of mice from the Sham, Sham+Rut and Sham+Cap groups (Fig. 2A, C and G); however, the interlobular septum was slightly broadened in Sham+Rut group. There were varied degrees of pathological changes in the pancreatic acinar, including acute inflammation, necrosis, dilated intercellular spaces and interlobular septum in the other groups (Fig. 2B, D-F and H-J). Conspicuous hemorrhagic necrosis, pancreatic edema, interstitial leukocyte and erythrocyte infiltration, and acinar cell vacuolization were observed in the AP and AP+Sol groups (Fig. 2B and J). Compared with the AP group, the extent and severity of pancreatic injuries were markedly alleviated in the AP+Rut L, AP+Rut M and AP+Rut H groups (Fig. 2D-F and K). Rutaecarpine (30, 100 and 300 µg/kg) significantly decreased the severity of pathological changes in a dose-dependent manner (Fig. 2D-F and K). As presented in Fig. 2K, pancreatic histological scores were highest in the AP+Cap and AP+Cap+Rut groups; they were even significantly higher than in the AP group (P<0.05). Coagulative necrosis was observed in these two groups, which also exhibited widened interlobular septums and the disappearance of acinic structures (Fig. 2H and I).

In the Sham, Sham+Rut and Sham+Cap groups, ascite volume in the abdominal cavity of rats was low and barely detectable (Fig. 3) and serum amylase activity remained low in these 3 groups (Fig. 4). Ascite volume significantly increased in the AP and AP+Sol groups (P<0.05; Fig. 3). Furthermore, compared with the Sham groups, the AP groups exhibited markedly increased levels of serum amylase (P<0.05; Fig. 4). However, pre-treatment with 30, 100 and 300 µg/kg rutaecarpine significantly reduced the volume of ascites and serum amylase activity in a dose-dependent manner (P<0.05). Capsazepine, a competitive VR1 antagonist reversed these effects (Figs. 3 and 4).

Cytokines serve an important function in the systemic response in AP; therefore, changes in the levels of IL-10, IL-6 and TNF-α were assessed in the serum of the rats to identify the mechanism by which rutaecarpine protects against rutaecarpine in AP. Groups in which AP was induced by injection of 5% sodium taurocholate exhibited a marked increase of IL-6 and TNF-α serum concentrations (Fig. 5). IL-6 and TNF-α are two pro-inflammatory cytokines expressed in response to local damage to the pancreas. Pre-treatment with 30, 100 and 300 µg/kg rutaecarpine significantly reduced IL-6 and TNF-α levels compared with the AP group in a dose-dependent manner (P<0.05; Fig. 5). Furthermore, pre-treatment with rutaecarpine significantly increased serum concentrations of IL-10 (P<0.05; Fig. 6), an anti-inflammatory cytokine that may attenuate pancreatic damage (27). However, rats injected with capsazepine prior to surgery exhibited significantly higher IL-6 and TNF-α concentrations (P<0.05) and significantly lower serum IL-10 concentrations (P<0.05) compared with those treated with rutaecarpine. This indicates that rutaecarpine suppresses the inflammatory response in AP, an effect that is reversed by capsazepine.

Pre-treatment with 30, 100 or 300 µg/kg rutaecarpine significantly upregulated CGRP concentrations in a dose-dependent manner (P<0.05; Fig. 7). Contrastingly, treatment with capsazepine led to a significant decrease in plasma CGRP levels even when rutaecarpine was administered (P<0.05; Fig. 7). Taken together, these results indicate that capsazepine may attenuate the effect of rutaecarpine on CGRP concentration.