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Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Icariin (ICA), a flavonoid glycoside, has been reported to have several pharmacological effects; however, the anti-inflammatory effects of ICA against AP require further study. Therefore, we aimed to investigate the effect of ICA on cerulein-induced AP. In the present study, AP was induced by intraperitoneally administering a supramaximal concentration of cerulein (50
Acute pancreatitis (AP) is a sudden inflammation of the pancreas and the most common reason for hospitalization among non-malignant gastrointestinal disorders in the United States (
Icariin (ICA) is a natural flavonoid glycoside isolated from plants in the
The purpose of this study was to investigate the effects of ICA against cerulein-induced AP. Pancreas weight/body weight (PW/BW) ratio, serum amylase and lipase levels, histological appearance of the pancreas and lung, myeloperoxidase (MPO) activity and pro-inflammatory cytokine levels were evaluated to determine the attenuating effects of ICA on the severity of cerulein-induced AP. Furthermore, we evaluated the activation of mitogen activated protein kinases (MAPKs, indicated by MAPKs phosphorylation) and nuclear factor κB (NF-κB, indicated by Iκ-Bα degradation) to determine the inhibitory mechanisms of ICA on AP.
ICA, dimethyl sulfoxide (DMSO), cerulein, NaCl, hexadecyl-trimethyl-ammonium bromide (HTAB), and Tris-HCl were purchased from Sigma-Aldrich (Merck KGaA). ICA stock solutions were using DMSO. Easy-Blue™ total RNA extraction kit was purchased from iNtRON Biotechnology. Anti-phosphorylated extracellular signal-regulated kinase (ERK)1/2 (1:1,000; cat. no. 9101L), phosphorylated c-Jun N-terminal kinase (JNK 1/3; 1:1,000; cat. no. 9251L), and p38 (1:1,000; cat. no. 9211L) antibodies and SB203580 (p38 inhibitor) were purchased from Cell signaling Technology, Inc. Total MAPK antibodies against ERK 1/2 (1:1,000; sc-93), JNK 1/3 (1:1,000; sc-474) and p38 (1:1,000; sc-535), inhibitory κ-Bα (Iκ-Bα; 1:1,000; sc-371), and β-actin antibody (1:1,000; sc-1615) were obtained from Santa Cruz Biotechnology, Inc. In addition, horseradish peroxidase (HRP)-conjugated secondary antibodies, including chicken anti-rabbit IgG-HRP (1:5.000; sc-516087) and chicken anti-goat IgG-HRP (1:10.000; sc-516086) were purchased from Santa Cruz Biotechnology, Inc.
All experiments were performed in accordance with the animal care regulations of Wonkwang University set forth and approved by the Wonkwang University Animal Ethics Committee.
All experiments were performed according to the protocols approved by the Animal Care Committee of Wonkwang University. C57BL/6 mice (n=324, 6-8 weeks old, female, weighing 15-20 g), were purchased from Orient Bio. All animals were bred and housed in standard shoebox cages in a climate-controlled room with an ambient temperature of 23±2°C under a 12 h light/dark cycle for 7 days. Animals were fed standard laboratory chow, and water
AP was induced via six intraperitoneal injections (i.p.) of cerulein (50
The appearance of the entire pancreas and the semi-quantitative index based on edema and inflammation were examined from each treatment group. The pancreas and lung tissues were fixed in 4% formalin solution at room temperature overnight, embedded in paraffin, cut into 4
Blood samples for the determination of serum amylase and lipase levels were obtained 6 h after the last injection of cerulein. Mice were sacrificed via CO2 asphyxiation followed by cervical dislocation; blood samples were then withdrawn from the heart. Amylase and lipase activities were determined by an assay kit of Bio Assay Systems.
Sequestration of neutrophils within the pancreas and lung was evaluated by measuring the MPO activity within tissues. Briefly, tissue samples were weighed, homogenized with 20 mM phosphate buffer (pH 7.4), and centrifuged at 10,000 × g for 10 min, 4°C. The pellets were then re-suspended in 50 mM phosphate buffer (pH 6.0) containing 0.5 % HTAB. The samples were centrifuged at 10,000 × g for 5 min, 4°C, and mixed with 80 mM sodium phosphate buffer (pH 5.4) containing 1.6 mM TMB. The mixture was incubated at 37°C for 110 sec, and the reaction was terminated with 2 M H2SO4. Tissue MPO activity was determined by measuring the absorbance at 450 nm with a Versa Max microplate reader (Molecular Devices, LLC) and was expressed as U/mg protein.
mRNA transcripts of mouse pancreatic tissues were analyzed by RT-qPCR. Total RNA was isolated from the mouse pancreas using Easy-Blue™ and subjected to RT using ABI cDNA synthesis kit according to the manufacturer's protocols (Applied Biosystems; Thermo Fisher Scientific, Inc.). For each sample, triplicate test reactions and a control reaction lacking reverse transcriptase were analyzed for the expression of the gene of interest, and the results were normalized to those of the 'housekeeping' hypoxanthine phosphoribosyltransferase (HPRT) mRNA. qPCR was performed using a standard protocol from the TaqMan™ Universal Master Mix II, no UNG (Applied Biosystems; Thermo Fisher Scientific, Inc.) on StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed at 50°C for 2 min and 95°C for 10 min, followed by 60 cycles of amplification at 95°C for 10 sec and 60°C for 30 sec. Forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.). The forward, reverse and probe oligonucleotide primers for multiplex real-time TaqMan PCR were as follows: For mouse TNF-α forward, 5′-TCT CTT CAA GGG ACA AGG CTG-3′, reverse, 5′-ATA GCA AAT CGG CTG ACG GT-3′; mouse IL-1β forward, 5′-TTG ACG GAC CCC AAA AGA T-3′, reverse, 5′-GAA GCT GGA TGC TCT CAT CTG-3′; mouse IL-6 forward, 5′-TTC ATT CTC TTT GCT CTT GAA TTA GA-3′, reverse, 5′-GTC TGA CCT TTA GCT TCA AAT CCT-3′; mouse HPRT (forward, 5′-GAC CGG TCC CGT CAT GC-3′; reverse, 5′-CAT AAC CTG GTT CAT CAT CGC TAA-3′).
Pancreatic tissues were homogenized and lysed on ice with radioimmunoprecipitation assay lysis buffer (iNtRON Biotechnology). Then, the lysates were boiled in 62.5 mM Tris-HCl buffer, pH 6.8, containing 2% SDS, 20% glycerol, and 10% 2-mercaptoethanol. Proteins were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate-buffered saline with Tween-20 for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. After washing three times, the membrane was incubated with secondary antibodies for 1 h at room temperature. The proteins were visualized using an enhanced chemiluminescence detection system (GE Healthcare) according to the manufacturer's protocols. The bands were detected and quantified by using Quantity One software (version 4.5.2; Bio-Rad Laboratories, Inc.).
Immunofluorescence for the analysis of phosphorylated p38 was performed on pancreas tissue. The pancreas tissues were embedded in frozen section compound, cut into 9
Data were expressed as the mean ± standard error of the mean. Statistical significance of intergroup differences was evaluated using two-way analysis of variance, with time and dose as variables, followed by a Duncan's post-hoc test. All statistical analyses were performed using SPSS version 10.0 statistical analysis software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference. All experiments were conducted in triplicate.
To evaluate the prophylactic effects of ICA against cerulein-induced AP, histological examination of the pancreas and lung was carried out. In the DMSO-treated mice, histological features of the pancreas and lung showed typical normal architecture. In the DMSO-treated mice with AP, histological features of damaged pancreas tissue were observed, as characterized by interstitial edema and inflammatory cell infiltration. However, treatment with ICA ameliorated the pancreatic histological damage in a dose-dependent manner (
Neutrophil migration into target tissue is increased when inflammation occurs (
To investigate the inhibitory mechanisms of ICA on AP, the activation of MAPKs and NF-κB was examined in the pancreas. Mice were administered ICA (20 mg/kg) or DMSO for 1 h and then stimulated with cerulein for 0, 15, 30 and 60 min. Cerulein treatment triggered the phosphorylation of MAPKs and the degradation of Iκ-Bα. However, administration of ICA inhibited the phosphorylation of p38, but not of ERK 1/2, JNK, and degradation of Iκ-Bα (
To determine whether inhibition of p38 could affect the reduction of inflammatory responses, a p38 inhibitor (SB203580, 1 mg/kg) was employed, after which the severity of AP was evaluated. As presented in
To examine the therapeutic effects of ICA in cerulein-induced AP, we applied ICA after the onset of AP. Post-treatment of ICA at 1 h but not 3 and 5 h after first cerulein injection significantly reduced the PW/BW ratio, serum amylase and lipase activities, and histological injury of the pancreas and lung compared with the AP mice, which suggests that ICA could exhibit therapeutic properties in the early phase of AP (
AP is an acute inflammatory disease that occurs in the pancreas, and its incidence is ~300 in 1 million (
The diagnosis of AP is based on the increasing levels of serum amylase and lipase. This is due to the fact that the levels of these digestive enzymes, which increase during AP, contribute to acinar cell injury, further promoting the inflammatory process, including the release of cytokines and chemokines (
Neutrophils have a central role in the development of AP by mediating local tissue damage and remote organ injury (
Severe AP often leads to MODS, and the most common and earliest target organ is the lung (
In regards to the anti-inflammatory activities of ICA, which were determined to be mediated by MAPKs and the NF-κB pathway in a mouse model (
In the present study, we have demonstrated that ICA exhibited protective and therapeutic effects against ceru-lein-induced AP and AP-associated lung injury. Furthermore, ICA inhibited the activation of p38 MAPK in cerulein-induced AP. Collectively, our findings suggest that ICA is a potential therapeutic agent for the treatment of AP.
Not applicable.
This study was supported by the National Research Foundation of Korea grant funded by the Korea government (MEST; grant nos. NRF-2017R1C1B2010031, NRF-2017R1D1A1B03032371 and NRF-2017R1A5A2015805).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
DUK, GSB, HJS and SJP made substantial contributions to the design of the study. DUK, GSB, MJK, JWC and DGK performed the experiments. MJK and DGK contributed to the statistical analysis of data. DUK and GSB wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.
All experiments were carried out in accordance with the animal care regulations set forth and approved by the Wonkwang University Animal Ethics Committee.
Not applicable.
The authors declare that they have no competing interests.
Effects of ICA on the severity of cerulein-induced AP. (A) Scheme for pretreatment experiment. Mice were pretreated with ICA (5, 10 or 20 mg/kg) or DMSO 1 h prior to the induction of AP with cerulein (50
Effects of ICA on MPO activity and the pancreatic cytokines production in cerulein-induced AP. Mice were pretreated with ICA (5, 10 or 20 mg/kg) or DMSO 1 h prior to the induction of AP with cerulein (50
Effects of ICA on the activation of MAPK. (A) Scheme for applied in the study. Mice were pretreated with ICA (20 mg/kg) or DMSO (control) 1 h prior to the induction of AP with cerulein (50
Effects of p38 inhibition by SB203580 on the severity of cerulein-induced AP. (A) Scheme for p38 inhibitor experiment. Mice were pretreated with ICA (20 mg/kg), SB203580 (1 mg/kg) or DMSO 1 h prior to the induction of AP with cerulein (50
Effects of p38 inhibition by SB203580 on MPO activity and pancreatic cytokine production in cerulein-induced AP. Mice were pretreated with ICA (20 mg/kg), SB203580 (1 mg/kg) or DMSO 1 h prior to the induction of AP with cerulein (50
Therapeutic effects of ICA against cerulein-induced AP. (A) Scheme for posttreatment experiment. Mice were treated with ICA (20 mg/kg) or DMSO 1, 3 or 5 h after the first cerulein injection. Mice were sacrificed 6 h after the last cerulein injection. Representative H&E-stained sections of the (B) pancreas and (D) lung (original magnification, ×200). Histological scores for (C) pancreatic edema and inflammation, (E) pulmonary wall thickening and inflammation. (F) PW/BW ratio, and serum levels of (G) amylase and (H) lipase. Data are presented as the mean ± standard deviation, n=6. Results are representative of three experiments. *P<0.05 vs. DMSO treatment alone; †P<0.05 vs. cerulein treatment alone. Scale bar, 20