Chemicals and reagents
Avidin-peroxidase, cerulein, hexadecyltrimethylammonium bromide, Triton X-100, and tetramethylbenzidine were purchased from Sigma-Aldrich (St. Louis, MO, United States). Anti-mouse TNF-α and IL-1β antibodies, and recombinant TNF-α and IL-1 were purchased from R-D Systems (Minneapolis, MN, United States).
Preparation of SSM
SSM was purchased from a standard commercial source (Omni Herb, Seoul, South Korea). The identity of the SSM was confirmed by Professor Seung-Heon Hong from Wonkwang University. SSM was prepared by decocting the dried prescription of SSM (100 g) with boiling distilled water (1 L). The decoction time was about 2 h. The water extract was frozen at -80 °C and then freeze-dried to produce a powder form (20.4 g). The yield of extract was 20.4%. The powder was extracted with distilled water and filtered. The filtrates were stored at 4 °C until use.
All experiments were performed according to protocols approved by the Animal Care Committee of Wonkwang University. C57BL/6 mice (age 6-8 wk; weight 15-20 g) were purchased from Orient Bio (Sungnam, KyungKiDo, South Korea). All animals were bred and housed in standard shoebox cages in a climate-controlled environment with an ambient temperature of 23 ± 2 °C and a 12-h light-dark cycle for 7 d. The animals were fed standard laboratory chow, given water, and were randomly assigned to the control or experimental groups. The mice were fasted for 18 h before the induction of AP. Six mice were included in each experimental group.
AP was induced by intraperitoneal injection of supramaximal concentrations of the stable cholecystokinin analog cerulein (50 μg/kg) or saline; injections were performed hourly for 6 h. To verify the prophylactic effects of SSM, SSM (0.1, 0.5, or 1 g/kg) was injected 1 h before the first cerulein injection. Mice were sacrificed 6 h after the last cerulein injection. Blood samples were taken to determine serum amylase, lipase, and cytokine levels. For histological examination and scoring, the entire pancreas and lungs were rapidly removed from each mouse and fixed in formalin. To measure tissue MPO activity, as an indicator of neutrophil sequestration, and to perform real-time reverse transcriptase-polymerase chain reaction (RT-PCR) examinations, 3 portions of both pancreas and lungs were stored at -80 °C.
The entire pancreas of at least 6 mice from each treatment group were examined and semi-quantitatively assessed for levels of necrosis, vacuolization, inflammation, and edema. The entire section, representing a minimum of 100 fields, was examined for each sample and scored on a scale of 0-3 (0 being normal and 3 being severe) on the basis of the number of necrotic acinar cells and the presence of vacuolization, interstitial edema, and inflammatory cells infiltration. These characteristics include the presence of acinar-cell ghosts, vacuolization and swelling of the acinar cells, and/or the destruction of the histoarchitecture of whole or parts of the acini. For scoring the lungs, the sections were examined for the presence of interstitial inflammation and edema.
Measurement of serum amylase and lipase levels
Blood samples, for the determination of serum amylase and lipase levels, were obtained 6 h after induction of pancreatitis. Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (4 mg/kg). After anesthetization, blood was withdrawn from the heart of each mouse into a syringe. The levels of serum amylase and lipase were measured using an assay kit (BioAssay Systems, Hayward, CA, United States).
RT-PCR was performed to measure mRNA transcript levels in the mouse pancreatic tissues and pancreatic acinar cells. Total RNA was isolated from the mouse pancreas using TRIzol (Invitrogen, Carlsbad, CA, United States) and was subjected to reverse transcription using SuperScript II RT (Invitrogen, Garlsbad, CA, United States). TaqMan quantitative RT-PCR using the LightCycler 2.0 detection system was performed according to the instructions of the manufacturer (Roche, Basel, Switzerland). For each sample, triplicate test reactions and a control reaction without reverse transcription were analyzed for expression of the gene of interest, and the results were normalized to those of the “housekeeping” hypoxanthine-guanine phosphoribosyl transferase (HPRT) mRNA. Arbitrary expression units were calculated by dividing the expression level for the gene of interest by the ribosomal protein HPRT mRNA expression level. The sequences of forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were as follows: for mouse TNF-α (forward, 5’-TCTCTTCAAGGGACAAGGCTG-3’; reverse, 5’-ATAGCAAATCGGCTGACGGT-3’; probe, 5’-CCCGACTACGTGCTCCTCACCCA-3’), for mouse IL-1β (forward, 5’-TTGACGGACCCCAAAAGAT-3’; reverse, 5’-GAAGCTGGATGCTCTCATCTG-3’; universal probe, M15131.1-Roche Applied Science).
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assays (ELISAs) for TNF-α and IL-1β were carried out in duplicate in 96-well plates (Nunc, Roskilde, Denmark), which had been incubated with 100 L aliquots of either anti-mouse TNF-α or anti-mouse IL-1β monoclonal antibodies (1.0 μg/mL in phosphate-buffered saline (PBS) at pH 7.4) overnight. The plates were washed in PBS containing 0.05% Tween-20 and blocked with PBS containing 10% fetal bovine serum for 2 h. After additional washes, the standards and the serum, pancreatic homogenates and pancreatic acinar cell supernants were added to the plates and incubated at room temperature for 3 h. To obtain pancreatic homogenates, the pancreas were thawed and then homogenized in PBS. After washing the wells, 0.2 μg/mL of biotinylated anti-mouse TNF-α or IL-1β were added to each well. Incubation was continued at room temperature for 1 h. The wells were washed, avidin-peroxidase was added, and plates were incubated for 30 min at room temperature. Wells were washed again, and 3, 3’, 5, 5’-tetramethylbenzidine substrate was added. Color development was measured at 450 nm using an automated microplate ELISA reader. Standard curves were obtained for each sample by using serial dilutions of recombinant TNF-α and IL-1β.
MPO activity estimation
Neutrophil sequestration in the pancreas was quantified by measuring the tissue MPO activity. Tissue samples were thawed, homogenized in 20 mmol/L phosphate buffer (pH 7.4), and centrifuged (15 000 revolution/min, 10 min), and the resulting pellet was resuspended in 50 mmol/L phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. The sample was then centrifuged (15 000 revolution/min, 5 min), and the supernatant used for the MPO assay. The reaction mixture consisted of the supernatant, 1.6 mmol/L tetramethylbenzidine, 80 mmol/L sodium phosphate buffer (pH 5.4), and 0.3 mmol/L hydrogen peroxide. The mixture was incubated at 37 °C for 110 s, the reaction was terminated with 2 mol/L of H2SO4, and the absorbance was measured at 450 nm. This absorbance was then corrected for the DNA content of the tissue sample.
Histological and immunohistochemical analysis
Fixed pancreatic tissues were embedded in paraffin, cut into 4-mm sections, and stained with hematoxylin-eosin for standard histological examination. Immunohistochemical (IHC) staining for HMGB-1 was performed using a DAB IHC kit (DAKO, Cytomation, Denmark). The relative intensity was measured using the Leica microscopy software (Wetzlar, Germany).
Acinar cell isolation
Pancreatic acini were isolated from C57BL/6 mice using collagenase digestion. All experiments were performed according to protocols approved by the Animal Care Committee of Wonkwang University. Briefly, pancreatic tissue was minced with scissors and digested for 15 min in solution Q (120 mmol NaCl, 20 mmol HEPES, 5 mmol KCl, 1 mmol MgCl2, 1 mmol CaCl2, 10 mmol sodium pyruvate, 10 mmol ascorbate, 10 mmol glucose, 0.1% bovine serum albumin, 0.01% soybean trypsinogen inhibitor, and 150 units of collagenase/mL). Cells were continuously shaken and gassed with 100% O2 in a 37 °C water bath and subsequently washed in fresh isolation medium. After collagenase digestion, the tissue was gently pipetted. Dispersed acini were filtered through a 150-μm nylon mesh, centrifuged 3 times (each for 90 s at 720 rpm), resuspended in Waymouth medium (Invitrogen, Gibco, CA) and incubated with 95% O2 and 5% CO2 for 4 h.
Cell viability assay
Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert the tetrazolium compound 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) into purple formazan crystals. MTT (5 mg/mL) was dissolved in Kreb’s-Henseleit buffer (115 mmol NaCl, 3.6 mmol KCl, 1.3 mmol KH2PO4, 25 mmol NaHCO3, 1 mol CaCl2, and 1 mol MgCl2), and 50 μL was added to each well. After incubating for 30 min at 37 °C, the suspension was removed, and the formazan crystals formed were dissolved in 200 μL dimethyl sulfoxide. Aliquots from each well were seeded in the wells of a 96-well plate in duplicate and assayed at 540 nm using a microplate ELISA reader. The number of viable cells was expressed as a percentage of the control.
Pancreatic tissues and pancreatic acini were homogenized, following which the lysates were boiled in a sample buffer [62.5 mmol Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 10% 2-mercaptoethanol]. Proteins in the cell lysates were then separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Then, the membrane was blocked with 5% skim milk in PBS-Tween-20 for 2 h at RT and then incubated with primary antibodies overnight. After washing 3 times, each blot was incubated with peroxidase-conjugated secondary antibody for 1 h, and antibody-specific proteins were visualized using an enhanced chemiluminesence detection system (Amersham, Piscataway, NJ) according to the manufacturer’s recommended protocol.
High-performance liquid chromatography sample preparation and conditions
An aliquot of 5.0 mg extract powder was dissolved with 1.0 mL of methanol and then filtered through a 0.45 μm filter membrane before use. A volume of 20 μL was injected into the high-performance liquid chromatography (HPLC) sample injector system. Chromatographic experiments were performed on a SYKAM series HPLC instrument equipped with sample injector and diode-array UV/Vis detector. For all experiments a SHISEIDO CAPCELL PACK C-18 column (4.6 mm × 250 mm; 5 μm) was used as stationary phase and injection volume were set 20 μL, respectively. The mobile phase composed of water (A) and acetonitrile (B), applying gradient program starting from 10 %B to 40 %B in 40 min. The column cleaned with 10 %B for 20 min, and then the system was equilibrated for 20 min with the starting conditions. Flow rate was 0.7 mL/min, and the detection wavelength adjusted to 210 nm. The quantifications of peak are 91% (1st), 4% (2nd), 0.5% (3rd), 4.5% (4th) to total.
The results were expressed as mean ± SE. The significance of change was evaluated using the one-way analysis of variance (ANOVA). Differences between the experimental groups were evaluated by performing ANOVA. P values < 0.05 were considered statistically significant.