Ghrelin's second life - World Journal of Gastroenterology

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NJ1 10% MeOH 500 mL 1240 mg NJ2 20% MeOH 500 mL 79.1 mg moved from each mouse and fixed in formalin. To measure tissue myeloperoxidase (MPO) activity, an indicator of neutrophil sequestration, and for real-time reverse transcriptase polymerase chain reaction (RT-PCR) studies, the pancreas and lungs were stored at -80 ℃. Histological analysis The pancreases from each treatment group were examined and semi-quantitatively described in terms of necrosis, vacuolization, inflammation, and edema. A tissue 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 disease) on the basis of the number of necrotic acinar cells and the presence of interstitial edema and interstitial inflammation. Measurement of serum amylase and lipase Blood samples to determine serum amylase and lipase were obtained 6 h after inducing 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 After anesthetization, blood was withdrawn from the heart of each mouse into a syringe. Serum amylase and lipase were measured using an assay kit from BioAssay Systems (CA). WJG|www.wjgnet.com Nardostachys jatamansi roots (NJ) Water extract (3.8 g) NJ3 40% MeOH 500 mL 490.7 mg RP C-18 column chromatography (5 cm x 20 cm) NJ4 60% MeOH 500 mL 416.2 mg NJ4-2 3.0 mg NJ5 80% MeOH 500 mL 273.1 mg RP-HPLC (capcell 10 cm x 25 cm) NJ6 100% MeOH 500 mL 67.8 mg Figure 1 Fractionation of the aqueous extract of Nardostachys jatamansi. NJ: Nardostachys jatamansi; HPLC: High-performance liquid chromatography. A B A 254 nm (mAU) 4.00 3.00 2.00 1.00 3 - 13.450 2 - 10.050 5 - 21.058 1 - 8.217 4 - 14.892 6 - 30.133 -0.50 0 13 25 38 50 63 75 88 100 t /min A 254 nm (mAU) 4.00 3.00 2.00 1.00 Bae GS et al . Anti-inflammatory effects of NJ4 -0.50 0 6 12 18 24 30 36 42 48 t /min Figure 2 High-performance liquid chromatography findings of the aqueous extract of Nardostachys jatamansi (A) and the 4th fraction of Nardostachys jatamansi (B). 23.541 28.002 Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) for IL-1β, IL-6, and TNF-α were carried out in duplicate in 96-well plates coated with 100 µL aliquots of anti-mouse IL-1β, IL-6, and TNF-α monoclonal antibodies in phosphate buffered saline (PBS) at pH 7.4 during an overnight incubation at 4 ℃. The plates were washed in PBS containing 0.05% Tween-20 and blocked with PBS containing 10% FBS for 2 h. After additional washes, standards and samples were added and incubated at room temperature for 2 h. Subsequently, the wells were washed, and biotinylated anti-mouse IL-1β, IL-6, and TNF-α were added and incubated at room temperature for 1 h. The wells were washed, avidin-peroxidase was added, and the plates were incubated for 30 min at room temperature before washing again and adding the TMB substrate. Color development was measured at 450 nm by using an automated microplate ELISA reader. Standard samples were run on each assay plate using serial dilutions of recombinant IL-1β, IL-6, and TNF-α. Messenger RNA expression Transcription of target cytokines in mouse pancreatic tissues and acini was analyzed using RT-PCR. Total RNA was isolated from the mouse pancreas using TriZol (In- 3225 July 7, 2012|Volume 18|Issue 25|
Bae GS et al . Anti-inflammatory effects of NJ4 vitrogen, Carlsbad, CA) and subjected to reverse transcription using SuperScript II RT (Invitrogen). TaqMan quantitative RT-PCR using the LightCycler 2.0 detection system was performed according to the manufacturer’s instructions (Roche, Basel, Switzerland). 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 “housekeeping” hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA. Arbitrary expression units were calculated by dividing the expression of the gene of interest by ribosomal protein HPRT mRNA expression. The sequences of forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were as follows: for mouse IL-1β (forward, 5’-TTG ACG GAC CCC AAA AGA T-3’; reverse, 5’-GAA GCT GGA TGC TCT CAT CTG-3’; universal probe, M15131.1, Roche Applied Science), for 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’; universal probe, M20572.1, Roche Applied Science), and for mouse TNF-α (forward, 5’-TCT CTT CAA GGG ACA AGG CTG-3’; reverse, 5’-ATA GCA AAT CGG CTG ACG GT-3’; probe, 5’-CCC GAC TAC GTG CTC CTC ACC CA-3’). For mouse HO-1, we purchased a custom primer from Applied Biosystems (CA). MPO estimation Neutrophil sequestration in the pancreas was quantified by measuring tissue MPO activity. Tissue samples were thawed, homogenized in 20 mmol/L phosphate buffer (pH 7.4), and centrifuged (13 000 rpm, 10 min, 4 ℃). The pellet was resuspended in 50 mmol/L phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich). The suspension was subjected to 4 cycles of freezing and thawing and further disrupted by sonication for 40 s. The sample was then centrifuged (13 000 rpm, 5 min, 4 ℃), and the supernatant was used for the MPO assay. The reaction mixture consisted of the supernatant, 1.6 mmol/L TMB, 80 mmol/L sodium phosphate buffer (pH 5.4), and 0.3 mmol/L hydrogen peroxide. This mixture was incubated at 37 ℃ for 110 s, the reaction was terminated with 2 mol/L H2SO4, and the absorbance was measured at 450 nm. Western blotting Pancreatic tissues and pancreatic acini were homogenized, following which the lysates were boiled in a sample buffer (62.5 mmol/L 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 WJG|www.wjgnet.com enhanced chemiluminesence detection system (Amersham, Piscataway, NJ) according to the manufacturer’s recommended protocol. 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/L NaCl, 20 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 10 mmol/L sodium pyruvate, 10 mmol/L ascorbate, 10 mmol/L glucose, 0.1% BSA, 0.01% soybean trypsinogen inhibitor, and 150 units of collagenase/mL). Cells were continuously shaken and gassed with 100% O2 in a 37 ℃ 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/L NaCl, 3.6 mmol/L KCl, 1.3 mmol/L KH2PO4, 25 mmol/L NaHCO3, 1 mol/L CaCl2, and 1 mol/L MgCl2), and 50 µL was added to each well. After incubating for 30 min at 37 ℃, 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. Statistical analysis Results were expressed as means ± SE. The significance of differences was evaluated using a two-way analysis of variance (ANOVA) with time and dose parameters. Differences between the experimental groups were evaluated using ANOVA. P values < 0.05 were considered statistically significant. RESULTS Effect of NJ4 on cerulein-induced AP To examine the effect of NJ4 on the development and severity of AP, mice pretreated with saline or NJ4 were injected intraperitoneally with a supramaximal dose (50 µg/kg) of cerulein. Intraperitoneal injection of cerulein resulted in significant pancreatic parenchyma destruction, inflammation, edema, and necrosis. However, NJ4-treated 3226 July 7, 2012|Volume 18|Issue 25|
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World Journal of Gastroenterology W
Page 3 and 4:
Phillip S Oates, Perth Ross C Smith
Page 5 and 6: Deepak N Amarapurkar, Mumbai Abhiji
Page 7 and 8: Trine Olsen, Tromsø Eyvind J Pauls
Page 9 and 10: Conor P Delaney, Cleveland Sharon D
Page 11 and 12: S Contents EDITORIAL TOPIC HIGHLIGH
Page 13 and 14: Contents ACKNOWLEDGMENTS APPENDIX A
Page 15 and 16: Verhulst PJ et al . Ghrelin and glu
Page 27: Online Submissions: http://www.wjgn
Page 31: Ishikawa T. Zinc during interferon
Page 35 and 36: Sitarz R et al . Gastroenterostoma
Page 37 and 38: Sitarz R et al . Gastroenterostoma
Page 39 and 40: Morais TC et al . Gastrointestinal
Page 47 and 48: Song MJ et al . Usefulness of PET/C
Page 49 and 50: A Sensitivity B Sensitivity C Sensi
Page 55: Bae GS et al . Anti-inflammatory ef
Page 59 and 60: B Myeloperoxidase activity (U/mg pr
Page 61 and 62: A Time (h) - 1 3 6 NJ4 B Relative H
Page 63 and 64: A C Time (h) - 1 3 6 NJ4-2 B Relati
Page 65 and 66: Bae GS et al . Anti-inflammatory ef
Page 70 and 71: A Collagen staining (fold change) C
Page 72 and 73: CCl4/YGJ CCl4-13 wk CCl4-7 wk Contr
Page 74 and 75: CCl4/YGJ CCl4-13 wk CCl4-7 wk Contr
Page 76 and 77: liver and have potent proliferative
Page 78 and 79: A 8 wk 9 wk 10 wk 13 wk CCl4/YGJ CC
Page 80 and 81: Cheng JC. A Chinese Herbal Decoctio
Page 82 and 83: Singh R, Neo EN, Nordeen N, Shanmug
Page 84 and 85: etter patient tolerance with regard
Page 86: Key words: Intestinal alkaline phos
Page 89 and 90: Molnár K et al . iAP in pediatric
Page 97 and 98: Chung WC et al . Ulcer after gastre
Page 99: Yang HY et al . Early resection of
Page 102 and 103: indicate a later timing for radical
Page 104 and 105: Zhao WC, Zhang HB, Yang N, Fu Y, Qi
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level, or rapid enlargement of lesi
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Table 3 Area under the receiver ope
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Zhao WC et al . Prognostic factors
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Online Submissions: http://www.wjgn
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mV 0 2 4 6 8 cpm FD patients are sh
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COMMENTS Background Gastric motilit
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thirds partial hepatectomy (PH) in
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A Serum ALT (U/L) C Serum alkaline
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REFERENCES 1 Schibler U. Circadian
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positive patients were found to hav
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21 Tosolini M, Kirilovsky A, Mlecni
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Diao TJ et al . NO and hepatopulmon
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Gallegos M et al . Lymphogranuloma
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A Hadžić N et al . Diagnosis in B
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Hadžić N et al . Diagnosis in BAA
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A B Chung HJ et al . Choledocholith
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Chung HJ et al . Choledocholithiasi
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Instructions to authors ISSN and EI
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Instructions to authors AIM (no mor
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Instructions to authors Guidelines
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