Sodium taurocholate was purchased from Sigma-Aldrich; Merck KGaA. ISO (C₂₉H₃₆O₁₅; FW, 624.59; purity, ≥98%) was obtained from Nanjing TCM Institute of Chinese Matera Medica. ELISA kits for rat TNF-α and IL-6, goat anti-rat TLR4 antibody, anti-rat NF-κB p65 antibody and secondary antibodies were obtained from Abcam. Nuclear-cytosol extraction kits were purchased from Beyotime Institute of Biotechnology. The nitric oxide (NO) assay kit was purchased from AmyJet Scientific, Inc.

A total of 120 male Sprague Dawley (SD) rats (weight, 230–250 g; age, 8 weeks) were obtained from the Experimental Animal Center of Jinling Hospital (Nanjing, China). The rats were housed at a consistent temperature of 23°C with a 12-h light-dark cycle and free access to food and water. The rats were fasted for 12 h prior to induction of SAP; however, the rats had free access to water. All experiments were performed according to protocols approved by the Animal Care Committee of Jinling Hospital (Nanjing, China) and were performed according to established international guiding principles for animal research.

In the first part of the present study, 48 SD rats were randomly divided into six groups (8 rats/group): Sham operation group (sham); SAP group (SAP); SAP+ISO-20 mg/kg treatment group (SAP+ISO20); SAP+ISO-40 mg/kg treatment group (SAP+ISO40); SAP+ISO-60 mg/kg treatment group (SAP+ISO60); and vehicle group (saline). Surgical anesthesia was accomplished by intraperitoneal injection of 10% chloral hydrate (300 mg/kg; Shanghai Shifeng Technology Co., Ltd.), and none of the rats exhibited any signs of peritonitis or discomfort. SAP was induced by retrograde infusion of 5% sodium taurocholate (0.1 ml/100 g body weight) into the pancreatic and biliary duct (19). Sham group animals were administered saline instead of sodium taurocholate into the pancreatic and biliary duct under the same conditions. For the ISO treatment group, ISO was diluted in dimethyl sulfoxide and was administered by intraperitoneal injection at 30 min after the SAP model establishment. Furthermore, the vehicle group was given the same volume of normal saline. The rats were euthanized by exsanguination under anesthesia at 12 h after the SAP model establishment, and anesthesia was accomplished by intraperitoneal injection of 10% chloral hydrate (300 mg/kg), then blood and pancreatic tissue samples were collected immediately. Furthermore, the pancreas samples were rapidly fixed in formalin at 35°C for 48 h for histological examination and scoring. The effect of ISO was determined by evaluating the pathological changes of the pancreas, and the serum levels of amylase (AMY), lipase (LIPA), aspartate-transaminase (AST) and alanine-aminotransferase (ALT).

Based on the first part of the present experiment, the optimal dose of ISO was fixed as 40 mg/kg. Subsequently, 72 SD rats were randomly divided into three groups (24 rats/group): Sham operation group (Sham); SAP group (SAP); and SAP+ISO-40 mg/kg treatment group (SAP+ISO40). Furthermore, 8 rats in each group were sacrificed by exsanguination under anesthesia at 3, 6 and 12 h after SAP model establishment. Subsequently, the blood, pancreas and kidney tissue samples were collected immediately. The collected blood samples were centrifuged at 12,000 × g for 10 min at 4°C, and then the serum was collected. The pancreas and kidney samples were rapidly fixed in formalin at 35°C for 48 h for histological examination and scoring. For western blot analysis, three portions of each kidney sample were stored at −80°C.

The AMY, LIPA, AST, ALT, blood urea nitrogen (BUN) and creatinine (Cr) levels in the serum were measured using an automated biochemical analyzer (Hitachi 7600; Hitachi, Ltd.).

Serum concentrations of TNF-α (ab236712) and IL-6 (ab234570) were measured using commercially available ELISA kits according to the manufacturer's protocols (R&D Systems, Inc.). All samples were assayed three times.

The pancreas and kidney tissue samples were fixed in 4% formalin at 35°C for at least 48 h. The tissues were cut into sections (thickness, 5 µm), and stained with hematoxylin and eosin at 23°C for 2 h. Histopathological evaluation was performed under a light microscope (magnification, ×100) by two experienced laboratory pathologists in a blinded manner. The pathological scores of the pancreas samples were evaluated according to the extent of edema, inflammation, vacuolization and necrosis (20). Histological scores of the kidney were assessed according to the point-counting method described by Paller et al (21). For each kidney, 100 cortical tubules from at least 10 different regions were scored, and repeated scoring of different convolutions of the same tubule was avoided. Higher scores represented more severe damage (maximum score per tubule, 10), with points given for the presence and degree of tubular epithelial cell flattening (1 point), brush border loss (1 point), cell membrane bleb formation (1 or 2 points), interstitial edema (1 point), cytoplasmic vacuolization (1 point), cell necrosis (1 or 2 points) and tubular lumen obstruction (1 or 2 points).

Kidney tissues were fixed in 4% formalin at 35°C for 12 h and embedded in paraffin. Subsequently, kidney tissues were harvested to prepare frozen sections of 6-µm thickness. Following deparaffinization, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min at room temperature. Non-specific adsorption was minimized following incubation of the sections in 5% normal goat serum in PBS (Nanjing Jiancheng Bioengineering Institute) at 37°C for 1 h. Avidin and biotin were used to block endogenous biotin and avidin binding sites, respectively. The sections were incubated overnight with goat anti-rat TLR4 (cat. no. ab22048) and anti-rat NF-κB p65 (cat. no. ab16502) monoclonal antibodies (both 1:100; Abcam) in a humidified incubator at 4°C. Hematoxylin was subsequently used to counterstain the sections for 2 min at room temperature. Stained sections were visualized using a light microscope. PBS was used instead of the primary antibody as a negative control.

Kidney tissues were thawed and homogenized in phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide. NO production was determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.

Kidney tissue samples were homogenized, and the lysate was boiled in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 20% glycerol and 10% 2-mercaptoethanol). The protein concentration was determined using a Bradford protein assay kit (Thermo Fisher Scientific, Inc.) with bovine serum albumin (Nanjing Jiancheng Bioengineering Institute) as the standard. Proteins (50 mg) were separated via 10% SDS-PAGE and transferred to PVDF membranes. Following the transfer of proteins, the membrane was blocked with 5% skimmed milk in PBS with 0.1% Tween-20 (PBST) for 2 h at room temperature, and then incubated overnight with antibodies (all 1:1,000; Abcam) against TLR4 (ab22048), NF-κB p65 (ab16502) or β-actin (cat. no. ab8227) at 4°C. Subsequently, the membranes were washed with PBST containing 0.1% Tween. Following three washes in PBST, each membrane was incubated with peroxidase-conjugated secondary antibody (anti-mouse IgG; 1:5,000; cat. no. ab131368; Abcam) for 1 h at 37°C. Labeled proteins were visualized using an Odyssey infra-red scanner (LI-COR Biosciences). Signals were assessed by densitometry and normalized to the β-actin signals. An enhanced chemiluminescence detection system (Amersham; Cytiva) was used to visualize the antibody-specific proteins according to the manufacturer's recommended protocol.

Data are presented as the mean ± standard deviation. One-way analysis of variance with Bonferroni's test was used to analyze the differences among multiple groups. Statistical analysis was performed using SPSS v19.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

The operation was completed successfully on all rats, and no rats died during the experimental period between the first injection and the time of euthanasia in the present study.

The present study evaluated the serum levels of AMY, LIPA, ALT and AST in SAP rats that were treated with multiple doses of ISO (20, 40 and 60 mg/kg) in order to determine the optimum dose of ISO. The three doses of ISO significantly decreased the levels of AMY and LIPA compared with those in the SAP group (P<0.05; Fig. 1A). However, the SAP group treated with ISO was the only ISO-treated group in which both AST and ALT levels were decreased significantly compared with those in the SAP group (P<0.05; Fig. 1B). Therefore, 40 mg/kg ISO was determined to be the optimum dose for treatment of SAP. Additionally, 3 h following induction of the SAP model was identified to be the best time of medication to reduce inflammation.

Compared with those in the sham group, the serum levels of AMY, LIPA, BUN and Cr in the SAP group were significantly increased at either designated time point (P<0.05; Fig. 2). However, the serum levels of AMY, LIPA, BUN and Cr in the ISO-treated group were markedly decreased compared with those in the SAP group (P<0.05; Fig. 2). Furthermore, the serum levels of AMY peaked at the 6 h time point in the SAP group (Fig. 2A), and the serum levels of LIPA, BUN and Cr peaked at the 12 h time point (Fig. 2B-D).

As shown in Fig. 3, a significant increase in serum levels of TNF-α and IL-6 was observed in the SAP group compared with the sham group (P<0.05), and the levels of these cytokines were progressively upregulated between 3 and 12 h. Furthermore, the serum levels of TNF-α and IL-6 were decreased following treatment with ISO (P<0.05). However, the levels of these cytokines in the ISO group were still higher than those in the sham group at each time point (P<0.05).

Representative histological sections of the pancreas and kidney are shown in Figs. 4 and 5. The pancreas tissues of the sham group exhibited a normal histological structure (Fig. 4A). Samples in the SAP group were characterized by typical histological signs of pancreatic edema, inflammatory leukocyte infiltration, hemorrhage and necrosis (Fig. 4B). However, there was a significant decrease in the severity of the pancreatic histological injuries in the ISO-treated group (P<0.05; Fig. 4C and D).

The pathological changes in kidney samples were analyzed to examine the effect of ISO on AKI induced by SAP (Fig. 5). The kidney samples of the sham group exhibited a normal histological structure of the renal glomerulus, tubules and interstitium (Fig. 5A). By contrast, samples in the SAP group were characterized by typical histological signs of glomerular and tubular damage, and the most severe kidney injury was detected at the 12 h time point (Fig. 5B and D). ISO treatment attenuated the severity of kidney injury, as demonstrated by decreased glomerular and tubular damage and decreased inflammatory cell infiltration (Fig. 5C). Additionally, the kidney pathohistological scores were significantly decreased in the ISO-treated group compared with the SAP group at each time point (P<0.05; Fig. 5D).

Localization of TLR4 and NF-κB p65 in the kidney is shown in Fig. 6. There was almost no TLR4 and NF-κB p65 expression in the sham group. In the SAP group, marked positive TLR4 and NF-κB p65 expression was observed compared with the sham group. However, following treatment with ISO, the expression levels of TLR4 and NF-κB p65 were markedly decreased compared with those in the SAP group at the corresponding time points (P<0.05).

NO activity in the kidney was detected to examine the infiltration of neutrophils (Fig. 7). NO activity was markedly elevated in the SAP group compared with the sham group (P<0.05). However, treatment with ISO significantly decreased NO activity in the kidney compared with the SAP group at each time point (P<0.05).

Western blotting was used to determine the effects of ISO on the expression levels of TLR4 and NF-κB p65 in the kidney (Fig. 8). Compared with those in the sham group, the expression levels of TLR4 and NF-κB p65 were upregulated in the SAP group (P<0.05). Following treatment with ISO, the expression levels of TLR4 and NF-κB p65 were downregulated compared with those in the SAP group at each time point (P<0.05).