Comparison of the chloride channel activator lubiprostone and the oral laxative Polyethylene Glycol

Online Submissions: wigwjgnet.comWorld Castroentero! 2008 October 21; 1439);: 6012-6017wjg@wjgnet.comWorld. Journal of Gastroenterology ISSN 1007-9327doi:10.3748/wig14.60126。2008 The W]G Press. All rights reserved.RAPID COMMUNICATIOINComparison of the chloride channel activator lubiprostoneand the oral laxative Polyethylene Glycol 3350 on mucosalbarrier repair in ischemic-injured porcine intestineAdam J Moeser, Prashant K Nighot, Birgit Roerig, Ryuji Ueno, Anthony T BlikslagerAdam J Moeser, Department of Population Health andlubiprostone stimulates recovery of barrier function inPathobiology, College of Veterinary Medicine, North Carolinaischemic intestinal tissues whereas the PEG laxativeState University, Raleigh NC 27606, United Stateshad deleterious effects on mucosal repair. These resultsPrashant K Nighot, Anthony T Blikslager, Department ofsuggest that, unlike osmotic laxatives, lubiprostoneClinical Sciences, College of Veterinary Medicine, Northstimulates repair of the injured intestinal barrier.Carolina State University, Raleigh NC 27606, United StatesBirgit Roerig, Ryuji Ueno,Sucampo Pharmaceuticals, Inc,⑥2008 The WJG Press. AIl rights reserved.Bethesda MD 20814, United StatesAuthor contributions: Moeser AJ and Blikslager AT wrotethe paper; Moeser AJ and Nighot PK performed the research; ，Key words: Intestinal ischemia; Barrier function;Roerig B and Ueno R contributed new reagents and interpretedPermebility; Laxative; Polyethylene Glycoldata; Moeser AJ and Bikslager AT designed the research.Correspondence to: Dr. Anthony T Blikslager, College ofPeer reviewer: Henrike Hamer, PhD, Department of IntemalVeterinary Medicine, North Carolina State University, 4700Medicine, Division of Gastroenterology (Box 46), MaastrichtHilsborough Street, Raleigh NC 27606,University, PO Box 616, 6200 MD Maastricht, The NetberlandsUnited States. anthony_ _blikslager@ncsu.eduTelephone: +1-919 5137725 Fax: +1-919-5136336Moeser AJ, Nighot PK, Roerig B, Ueno R, Blikslager AT.Received: June 30, 2008Revised: August 18, 2008Accepted: August 25, 2008and the oral laxative Polyethylene Glyeol 3350 on mucosalPublished online: October 21, 2008barrier repair in ischemic-injured porcine intestine. WorldJGastroenterol 2008; 14(39): 6012-6017 Available from: URL:htp://www.wjgnct.com/1007-9327/14/6012.asp DOI: htp//dx.doi.org/10.3748/wjg.14.6012AbstractAIM: To investigate the effects of lubiprostone andPolyethylene Glycol 3350 (PEG) on mucosal barrierINTRODUCTIONrepair in ischemic-injured porcine intestine.METHODS: Ileum from 6 piglets (approximately 15 kgIschemic intestinal disorders including intestinal volvulus,body weight) was subjected to ischemic conditions bythromboembolic disease, and low fow states associatedoccluding the local mesenteric circulation for 45 minwith shock, have a high mortality rate due to the rapidin vivo. Ileal tissues from each pig were then harvestedonset of sepsis and multiple organ failurel"1,. Intestinaland mounted in Ussing chambers and bathed inischemic lesions are characterized by sloughing of theoxygenated Ringer's solution in vitro. Intestinal barrierapical vllus epithelium and rapid breakdown of mucosalfunction was assessed by measuring transepithelialbarrier function，accompanied by increased intestinalelectrical resistance (TER) and mucosal-to-serosalpermeability and subsequent bacterial translocation,fluxes of 3H-mannitol and 'C-inulin. Statistical analysessepsis, and multiple organ dysfunction syndromeof data collected over a 120-min time course induded(MODS)68. Rapid restoration of the compromised2-way ANOVA for the effects of time and treatment onindices of barrier function.intestinal barrier is critical for patient survival. However,RESULTS: Application of 1 umol/L lubiprostone tolimited treatment options are available that targetthe mucosal surface of ischemic-injured ileum in vitromucosal barrier repair'.induced significant elevations in TER compared to non-Lubiprostone. an FDA approved laxative (Amitiza,treated tissue. Lubiprostone also reduced mucosal-to-Suca中国煤化工; previously showaserosal fluxes of 3H-mannitol and "C-inulin. Alternatively,to stCal barrier functionapplication of a polyethylene laxative (PEG, 20 mmol/L)in isYHC N M H G00. Lubiprostone如the mucosal surface of ischemic tissues significantyactivates CIC-2 CI channels resulting in luminal CIincreased flux of 3H-manitol and IKC-inulin.secretion and water movement responsible for itsCONCLUSION: This experiment demonstrates thatlaxative propeties. CIC-2 CI channel activationw际亩数据omMoeser AJ et al. Laxative agents infuence mucosal barrier repair6013by lubiprostone is also the predominant mechanism154; K*, 6.3; CI, 137; HCO;, 24; pH 7.4) containingby which this compound stimulates repair of the5 umol/L indomethacin to prevent endogenoustight junctions and mucosal barrier repair in ischemicprostaglandin production during the stripping procedure.tissueslI0. The mechanism for this action may relate Tissues were then mounted in 1.14 cm2 apertureto co-localization of CIC-2 with tight junctionUssing chambers, as described in previous studies. Forproteins such as occludin0.17. Other commerciallyUssing chamber experiments, ileal tissues from oneavailable laxative agents such as high molecular weight pig were mounted on multiple Ussing chambers andPolyethylene Glycol (PEG 3350) also induce fluidsubiected to different in vitro treatments. Data meansmovement into the lumen via different mechanisms.are representative of 6 Ussing chamber experimentsPolyethylcne laxatives are composed of high molecular(n = 6 animals). Tissues were bathed on the serosalweight PEG which triggers an osmotic gradient in and mucosal sides with 10 mL Ringer's solution. Thethe lumen serving to draw ions and water from theserosal bathing solution contained 10 mmol/L glucose,paracellular space. In addition to its laxative effects, PEGand was osmotically balanced on the mucosal side withagents have been shown to confer mucosal protective10 mmol/L mannitol. Bathing solutions were oxygenatedeffects in vatious gastrointestinal injury animal models(950 mL/L O2/50 mL/L CO) and circulated in water-including 2, 4, 6 titrobenzene sulphonic acid (INBS)-jacketed reservoirs. The spontaneous potential differenceinduced colitis1'l and bacterial translocation and sepsis(PD) was measured using Ringer -agar bridges conectedinduced by surgical stress'.to calomel electrodes, and the PD was short-circuitedGiven the potential alternative beneficial roles ofthrough Ag-AgC1 elecrodes using a voltage clamp thatthese two oral laxatives in intestinal injury and repair,corrected for fluid resistance. Transepithelial electricalthe objective of this study was to compare the effectsresistance (TER) (8.cm) was calculated from theof lubiprostone and PEG 3350 on repair of mucosalspontaneous PD and short-circuit current (I.小. If thebarrier function in ischemic-injured intestine.spontaneous PD was between -1.0 and 1.0 mV, tissueswere current-clamped at士100 μA for5 s and the PDrecorded. I# and PD were recorded at 15 -min intervalsMATERIALS AND METHODSover a 120-min experiment.CompoundsLubiprostone was obtained from R-Tech Ueno (Sanda,Experimental treatmentsJapan). PEG 3350 (Miralax, Schering-Plough Health CareAfter tissues were mounted on Ussing chambers,Products, Inc. Kenilworth NJ) was obtained from thetissues were allowed to acclimate for 30 min to achieveNorth Carolina State University, College of Veterinarystable baseline measurements after which experimentalMedicine pharmacy. H-mannitol and "C inulin weretreatments were added. Lubiprostone (1 μmol/L) orobtained from Sigma Chemical (St. Louis, MO).PEG 3350 (20 mmol/L) were added to the mucosal sideof tissues and TER and Ise were measured at 15-minExperimental animal surgeriesintervals over a 120 min recovery period. The PEG 3350All studies were approved by the North Carolina Statedose was selected as it is the recommended oral dosageUniversity Institutional Animal Care and Use Comitee. for laxative properties and thus would approximate theSix to eight-week- old Yorkshire crossbred pigs ofluminal concentrations attained in wiwo.cither sex were housed individually, and maintainedon a commercial pelleted feed. Pigs were fasted for Mucosal-t-serosal fluxes of radiolabeled paracellular24 h prior to experimental surgery. General anesthesiaprobeswas induced with xylazine (1.5 mg/kg, IM), ketamineTo assess mucosal permeability after experimental(11 mg/kg, IM), and 5% isoflurane vaporized intreatments, 0.2 μCi/ mL 'Hlabeled mannitol (180 kDa)1000 mL/L O2 and was maintained with 2% isoflurane and 0.2 μCi/ mI, "C-labeled inulin (5000 kDa) weredelivered wia an endotracheal tube. Pigs were placed onadded to the mucosal side of tissucs mounted in Ussinga heating pad and ventilated with 1000 mL/L O2 usingchambers. After a 15 min equilibration period, standardsa volume limited, time-cycled ventilator (Hallowell,were taken from the mucosal side of each chamber andPittsfield, MA). Lactated Ringers solution wasa 60-min fux period was established by taking 0.5 mLadministered iv at a maintenance rate of 15 mL/kg persamples from the serosal compattment. The presence ofhour. The ileum was approached via a ventral midline'H and "C was established by measuring β-emission inincision. Ileal segments were delineated by ligating the a liquid -cinilltiono counter (LKB Wallac, Model 1219intestine at 10-cm intervals, and subjected to ischemia byRack Beta, Perkin Elmer Life and Analytical Sciences,occluding the local mesenteric blood supply for 45 min. .Inc, Boston, MA). Unidirectional mannitol fuxes frommuco中国煤化工d using standardUsing chamber studiesequatiFollowing the 45-min ischemic period, tissues wereMHCNMHG.harvested from the pig and the mucosa was stripped Histological examIhauon?from the seromuscular layer in oxygenated (950 mL/LTissues were taken at 0 and 120 min for routineO2/50 mL/L CO2) Ringer's solution (mmol/L; Na",histological evaluation. Tissues were sectioned (5 μm)6014 ISSN 1007-9327 CN 14-1219/R World J GastroenterolOctober 21, 2008 Volume 14 Number 39” LschemiaA 0.8I Cortrol (non-lshemic)■Ischemia ，- Ischemla + PEG 3350 (20 mmolL)0.6G Ischen;+ PEG 3350 ((20 mmo/L)76050卜I..I子40Add T0.30， 2(Treatment100 1530456075 90 105 120B 0.006 r■Control (non-lschemic)t/min0.005号Ischemnia + Lubostone (1 mo/)口Ishemla + PEG 3350 (20 mmo/)B 800.004e↓↓0.0020.0010.000-20-40Figure 2 A: Mucosal t-serosal fux of Hmanitol in porcine ileum. P < 0.050153045607590105120ischemia VS control, ischemialubiprostone; 'P < 0.05 ischemia + PEG3350Vs control, ischemia, ischemia + lubiprostone; B: Mucosal-to-serosal flux of"C inulin in porcine ileum. P < 0.05 ischemia Vs control, ischemialubiprostone;Flgure 1 TER (A) and short cruit curent (Uw) (B) in ischemic porcine ileum"P< 0.05 ischemia + PEG3350 Vvs control, ischemia, ischemialubiprostone.reated with lubipostone and PEG 3350.lubiprostone to the mucosal side of ischemic-injuredand stained with hematoxylin and eosin. For each tssue,mucosa induced rapid clevations in TER that attained3 sections were evaluated. Four well-oriented villi andnon-ischemic control tissues within 15 min of its additioncrypts were identified in each section, Villus length wasand TER continued to increase 45 min post- treatment.obtained using a micrometer in the eye piece of a lightLubiprostone stimulated rapid elevations in Iu (an indexmicroscope.of electrogenic ion transport) that peaked at 15 min post-treatment (peak△I,= 66 μA/ cm) and remained elevatedStatistical analysisthroughout the remainder of the experiment. MucosalAll data were reported as mean土SE. TER and Ieaddition of 20 mmol/L PEG 3350 stimulated a transientdata were analyzed by using an ANOVA for repeatedincrease in TER measured 15 min after treatment;measures. Radiolabeled fAux data was analyzed by using ahowever, TER returned to ischemic control levels withinstandard one-way ANOVA (Sigmastat, Jandel Scientific,30 min post-tteatment. PEG 3350 stimulated significantSan Rafael, CA). A Tukey's test was used to determineclevations in Ie (peak OI, = 35 μA/cm) compared withdifferences between treatments following ANOVA.ischemic control tissues. In non-ischemic ileal tissues,lubiprostone and PEG 3350 induced similar elevations inTER (ATER = 30%土7% and 36%土8% in lubiprostoneRESULTSand PEG 3350-treated tssues) (data not shown). .TER and Ie in ischemia-injured porcine ileal mucosatreated with lubiprostone or PEG 3350Mucosal-to-serosal fux of paracellular probesPorcine ileum was subjected to 45 min of acuteMucosal-to-serosal flux of both medium molecularmesenteric ischemia and mounted on Ussing chambersweight (H- mannitol, 180 kDa) and large molecular weightfor measurement of TER and Le over a 120-min recovery("C-In中国煤化工bes in ilel tsesperiod. Ischemic-injured mucosa had significantlymoun: conducted as anlower starting TER values (by approximately 40%)alternaHC N M H Germability In linecompared with non. ischemic control tissue (Figure 1),with TER responses, ischemuc tissues had greater (P