Herbal cardiotonic pills prevent gut ischemia/reperfusion-induced hepatic microvascular dysfunction

wjgewignet,comwww.w]gnet.como 2005 The wJG Press and Elsevier Inc. All nights reservedBASIC RESEARCH·Herbal cardiotonic pills prevent gut ischemia/reperfusion-inducedhepatic microvascular dysfunction in rats fed ethanol chronicallyYoshinori Horie, Jing-Yan Han, Shuka Mori, Masahiro Konishi, Mikio Kajihara, Takehiko Kaneko, Yoshiyuki Yamagishi,Shinzo Kato Hiromasa Ishii, Toshifumi HibiYoshinori Horie, Jing-Yan Han, Shuka Mori, Masahiro Konishi, an enhancement of gut I/R-induced responses by chronicMikio Kajihara, Takehiko Kaneko, Yoshiyuki Yamagishi, ethanol consumption via the reduction of blood endotoxin levelsShinzo kato, Hiromasa Ishii, Toshifumi Hibi. Department ofInternal Medicine, School of Medicine, Keio University, Tokyo JapanC 2005 The WJG Press and Elsevier Inc. All rights reserved.Supported by Grants From Tasley, Tianjin, ChinaCo-corresponaey yoshinori Horie, M.D., or Department of Intemal Key words: Intestinal reperfusion injury; Hepatic microvascularg-Yan HanMedicine, School of Medicine Keio University, 35 Shinanomachi Shinjukudysfunction; Cardiotonic Pill; Ethaku, Tokyo 160-8582, Japan. yhorie@ sc itc keio acTelephone:+81-3-3353-121-62298Fax:+81-3-33536247Horie Y, Han JY, Mori S, Konishi M, Kajihara M, KanekoReceived: 2004-02-02 Accepted: 2004-03-16YamagishiY, Kato S, Ishii H, Toshifumi Hibi. Herbal cardiotonicpills prevent gut ischemia/reperfusion-induced hepatiemicrovascular dysfunction in rats fed ethanol chronically. Warld7 Gastroenterol2005;11(4):511-515Abstracthttp://www.wjgnet.com/1007-9327/11/511.aspAIM: Cardiotonic Pill (CP), an oral herbal medidne that indudesDanshen(Salviae Miltiorrhizae), Panax notoginseny andDyroblanops aromatica gaertn, has been clinically used for INTRODUCTIONdiseases, atherosclerosis, and cerebral infarction. The main A large body of evidence implicates leukocytes as mediators ofcomponent, Salviae Mitiomhizae, has been reported to prevent microvascular dysfunction and tissue injury associated withcerebral and intestinal reperfusion injury. However, little reperfusion of ischemic organs. Several experimental strategiesis known about the effect of CP on hepatic microcirculation. have been used to demonstrate the contribution of leukocytesThus, this study aimed to determine whether CP could affect to ischemia/reperfusion(IR)injury, including polyclonalhepatic microvascular dysfunction elicited by gut ischemia/ antibodies that render animal leukopenia[l-3, adhesion moleculereperfusion(I/R)in rats fed ethanol chronically.specific monoclonal antibodies[ 4-6, and adhesion moleculedeficiency in mice. The effectiveness of adhesion moleculeMETHODS: Male Wistar rats were pair-fed with a liquid diet specific monoclonal antibodies(MAbs)and adhesion moleculecontaining ethanol or isocaloric control diet for 6 wk. After deficiency in attenuating IR-induced tissue injury have led tolaparotomy, one lobe of the liver was examined through the widely held view that leukocyte-endothelial cell adhesionan inverted intravital microscope. The rats were exposed is a rate-determining step in the pathogenesis of this injuryto 30 min of gut ischemia followed by 60 min of reperfusion. process. We developed a leukocyte-dependent model ofRhodamine-6G-labeled leukocytes in the sinusoids were hepatocellular dysfunction elicited by gut Ir >. This murineobserved 90 min after the onset of superior mesenteric artery model allows in vivo assessment of the effects of I/r oncausion. Plasma tumor necrosis factor(TNF)r-a and endotoxin leukocyte sequestration in sinusoids of different regions oflevels were measured 1 h after the onset of reperfusion. the liver lobule, leukocyte adherence in postsinusoidal venules,Plasma alanine aminotransferase(alt)activities were and the number of perfused sinusoidmeasured 6 h after the onset of reperfusion. In another setEthanol has also been reported to modulate IR-inducedof experiments, CP(0.8 g/kg, intragastrically )was administered tissue injurylto-12. In a perfused liver model, ethanol enhanced1 and 24 h before the onset of ischemiaIR-induced hepatotoxicity(an increase in blood levels of liverenzymes)by an enhanced production of reactive oxygenRESULTS: In control rats gut I/R elicited increases in the species to. In an in vivo gut I/R model, ethanol also enhancednumber of stationary leukocytes, and plasma TNF-a andIR-induced neutrophil accumulation in the intestinal wall]n.endotoxin levels and plasma ALT activities. These changes In an in vivo cerebral I/R model, however, ethanol pretreatmentwere mitigated by pretreatment with CP In ethanol-fed ratwas reported to reduce cerebral I/R injury al. We have recentlythe gut I/R-induced increases in the number of stationary reported that low-dose ethanol attenuates gut IR-inducedleukocytes, plasma endotoxin levels and ALT activities were hepatic microvascular dysfunction in the midzonal region andenhanced Pretreatment with CP attenuated the enhancementequential hepatocellular injury, whereas high-dose ethanolof gut I/R-induced responses by chronic ethanol consumption. enhances hepatic microcirculatory disturbances in the pericentralential hepatocellular injury s).CONCLUSION: These results suggest that CP prevents中国煤化工 mption has been notedthe gut IR-induced hepatic microvascular dysfunction and to sioronary artery diseasehepatocellular injury. A reduction of infammatory responses HoweCNMH Gon often results in fattysuch as TNF-a production via reduction of blood endotoxin liver and liver failure, which is of particular concem when suchlevels appears to be involved in the mechanisms. Chronic fat-laden tissues are used as donor organs in liver transplantation.ethanol consumption enhances gut I/R-induced hepatic This important clinical problem has drawn attention to themicrovascular and hepatocellular injury. CP also attenuates relationship between ethanol consumption and reperfusion512ISSN 1007-9327 CN 14-1219/R World J Gastroenterol January 28, 2005 Volume 11 Number 4injury in the liver. Both gut I/R and chronic consumption of Analysis of leukocyte accumulationethanol are known to cause liver injury via mechanisms that Leukocytes were labeled in vivo with rhodamine-6G (1 mg wasinvolve oxidative stress and microcirculatory disturbances that dissolved in 5 mL of 0.9% saline)using a previously describedinclude leukocyte sequestration and sinusoidal malperfusion! 51. method 2. 17, 19, 23), which was based on a method used in ratGut VR is known to produce an elevation in plasma endotoxin brain 3.It was shown that rhodamine 6G could selectively stainlevels(9), while chronic ethanol consumption has been reported white blood cells and platelets, but not endothelial cells. Thusto enhance the hepatic microcirculatory dysfunction and the fluorochrome allowed for differentiation between adherenthepatocellular injury induced by endotoxins 6-8. Based on these leukocytes and endothelial cells. Rhodamine-6G(0.2 mL/100 gobservations one might expect that chronic ethanol consumptionody weight) was injected prior to ethanol administration withwould lead to an exaggerated liver injury response to gut IR. subsequent injections every 30 min Rhodamine-6G associatedFurthermore, we have recently reported that chronic ethanol fluorescence was visualized by epi-illumination at 510-560 nmconsumption enhances hepatic leukosequestration, impaired using a 590 nm emission filter. We selected one of the lobulessinusoidal perfusion, and hepatocellular injury caused by gut which had well-perfused sinusoids and the fewest stationaryIR via expression of intercellular adhesion molecule (CAM-1 9. leukocytes, choosing the furthest lobule from the edge of theHerbal medicines(traditional medicines from natural sources) liver if all the conditions were thought to be equivalent. Aare recently attracting increased global attention. Further, microfluorograph of hepatic microcirculation, with rhodamineare widely accepted as having reliable therapeutic efficacy. observed for 90 min after the SMA occlusion and recorded onCardiotonic Pill( CP), an oral herbal medicine that includes a digital video recorder for I min at 0, 30, 60, and 90 min. TheDanshen(Salviae Miltiorrhizae), Panax notoginseny and number of stationary leukocytes was determined off-line duringDyroblanops aromatica gaertn, has been clinically used in China, playback of the videotape images. A leukocyte was consideredKorea and Russia for vascular diseases such as occlusive stationary within the microcirculation(sinusoids) if it remainedvasculitis, coronary diseases, atherosclerosis, and cerebral stationary for more than 10 s. The lobule, which had the fewestinfarction, which are related to microvasclar dysfunction. In stationary leukocytes was selected for observation at a basalclinical cases, the main component, Danshen, has been reported condition. Stationary leukocytes were quantified in both theto have curative efficacy in ischemic cerebrovascular disease. midzonal and pericentral regions of the liver lobule andIn animal models, Danshen has been reported to prevent expressed as the number per field of view(2.1x10 um?)cerebral and intestinal reperfusion injury. 2. In the rat cerebralhemia model, Danshen was reported to improve cerebral ExpeExperimental protocolblood flow in the ischemic hemisphere and to inhibit platelet We observed the surface of the liver for 10 min before ligationaggregation in rats D. However, little is known about the effect of of the superior mesenteric artery in order to ensure that allCP on hepatic microcirculation. Hence, the overall objectives of parameters measured on-line were in a steady state. The superiorthis study was to assess the effects of CP on hepatic microvascular mesenteric artery was then ligated with a snare created fromdysfunction and hepatocellular injury induced by gut IR, and polyethylene tubing for 0 (sham)or 30 min. After the ischemicto determine whether CP could affect the hepatic microvascular period, the ligation was gently removed. Leukocyte accumulationdysfunction elicited by gut IR in rats fed ethanol chronically. was measured before ischemia, immediately following reperfusionand every 30 min for one hour thereafterIn another set of experiments, CP (0.8 g/kg, intragastrically,MATERIALS AND METHODSTasley, Tianjin, China)was administered at 1 and 24 h beforeAnimalsthe onset of ischemia to both control and ethanol-fed rats, andhirty (15 pairs)male Wistar rats weighing about 150 g were the same protocol was performed. Twenty-five mg(one pill) ofpair-fed for 6 wk with a liquid diet containing ethanol that CP includes 9 mg of Danshen(Salviae Miltiorrhizae), 1.76 ofdiet according to the method of Lieber et al. 221, All rats were and 13.74 mg of polyethylene g/yao anops aromatica gaertn,rovided% of the total dietary calories or an isocaloric control Panax notoginseny, 0.5 mg of Dyrobfasted for18 h prior to the experiments, which were all performedccording to the criteria outlined in the US National Research Liver enzyme, endotoxin and TNFassaysCouncil and the Keio Animal Research GuidesSixty min after the onset of reperfusion, the rats were removedfrom the microscope stage and the abdominal wall was closedintravital microscopyBlood(plasma)samples for endotoxin and tumor necrosis factorRats were anesthetized with pentobarbital sodium(35 mg/kg) ( TNF)-a level measurement were collected from the interiorintraperitoneally. The left carotid artery was cannulated and a vena cava at a point proximal to the hepatic vein 1 h after thecatheter placed at the aortic arch to monitor blood pressureonset of reperfusion. For the measurement of endotoxin levelsThe left jugular vein was also cannulated for drug administration. blood samples were also collected from the portal vein SamplesAfter laparotomy, a lobe of the liver was observed with an for plasma alanine aminotransferase(ALt)measurement wereinverted intravital microscope(TMD-2S, Diaphoto, Nikon, obtained 6 h after the onset of reperfusion. Plasma ALT activitycamera(C-2400-08, Hamamatsu Photonicus, Shizuoka, Japan). described 24. Plasma TNF-a concentration was determined in aThe liver was placed on an adjustable Plexiglas microscope mIcItage with a non-fluorescent coverslip that allowed for Inte中国煤化工: enzyme-linked immunobservation of a 2-cm? segment of tissue. The liver was carefully sorbetplaced to minimize the influence of respiratory movements, andN M H Gured by endospecy (athe surface was moistened and covered with cotton gauze endotoxin-specific chromogeic limulus regent; Seikagaku Cosoaked with saline. Images of the microcirculation were observed Tokyo, Japan)using an automated kinetic assay for endotoxinfrom the surface of the liver through a x20 fluorescent objective,and microfluorographs were recorded on videotape using a Statistical analysiswy号数r( -HQ. actor, JapanThe data were analysed using standard statistical analyses, i.eHorie Y et a/ Reperfusion injury and herbal medicine513ANOVA and Scheffe's(post hoc)test. All values were reported 12.6*1. 4, ethanol: 7.2 *0.7, per field), while exaggeratedas meanSD, with five rats per group. Statistical significance leukostasis was noted in the pericentral region(control; 43+1. 9,was set at P<0. 05ethanol:68±22) and THV(contro;4.0±14, ethanol:l3.0±1.6,per field). Although the leukostasis elicited by gut IR in controlats was attenuated by the pretreatment with CP (leukostasis inRESULTSthe pericentral region; 4.0*1. 2, THV; 1.8=+0.7, per field), theTable 1 shows the effects of CP treatment on leukostasis in exaggerated leukostasis in ethanol-fed rats was largely preventedsinusoids of the mid zonal and pericentral regions and the by pretreatment with CP (leukostasis in the pericentral region;terminalhepatic venule(THv), plasma ALT activities, and plasma 3.0H0.7, THV; 4.8+1. 2, per field)endotoxin and TNF-a levels. Pretreatment with CP per se didFigure 1B shows the effects of CP treatment on plasma ALtnot affect any values of these in control rats Chronic ethanol activity following gut I/R in the presence or absence of chronicfeeding per se caused leukostasis in the pericentral region of ethanol consumption. In control rats, gut IR led to an elevationthe liver. while cP diminished it.of plasma ALT activities. Chronic ethanol consumptionFigure lA illustrates the effects of CP treatment on gut IR- enhanced the gut IR-induced increase in plasma ALT activitiesinduced leukostasis in sinusoids of the midzonal and pericentral (control; 146+31 IU/L, ethanol: 236+84 IU/L). The increase inregions and the THV(Panel A)of the liver lobule, and the entire plasma ALT activities elicited by gut VR in both control andliver lobule(sinusoids+THV, panel B)in the presence or absence ethanol-fed rats was significantly attenuated by pretreatmentof chronic ethanol consumption In control rats, gut IR elicited with CP(control; 81+19 IU/L, ethanol: 108*58 IU/L).increases in the number of stationary leukocytes in bothFigure IC shows the effects of CP treatment onhepatic sinusoids and THV. In ethanol-fed rats, the gut I/R- endotoxin levels following gut I/R in the presence orinduced leukostasis was blunted in the midzonal region(control; of chronic ethanol consumption. Gut IR caused a slight elevationTable 1 Effect of cardiotonic pills on leukostasis in liver, plasma ALT activity, and piasma endotoxin and TNF-a level in controland ethanol-fed ratsTHVALT actiGroup midzonal (per field(per field(per field)/mL)Control1.8±0.71.0±0.70.5±0531.6±12.095±53Ethanoi14±0.53.2±0.51.2±0.740.6±l05192±10.823.1±15.1Control+CP14±1.228,4±11.893±53133±10.1Ethan+CP1.6±0.50.8±050.6±1.036.6±72136±55106±6.2CP: cardiotonic pills, p<0.05 us ControlControl口 Midzonal A2 ControlTotalEtOHEtOH■THeTohaecStationary leukocytesStationary leukocytes(per field(per field)COtrolcEtoHENOH+CPEtoH+CPBioH+CPPlasma ALT activityPlasma endotoxin levelPlasma TNF-alpha level(U/r)(pg/mL)(pg/mL)Figure 1 Effects of chronic ethanol consumption and/or Cardiotonic中国煤化工 hary leukocytes. plasmaALT activities, plasma endotoxin concentration, and plasma TNF-a ceanimals.p<0.05 as control, p<0.05 us Control+I/R,P<0.05 vs ethanol+I/RCNMH Ggroup consisted of fivethe number of stationary leukocytes in each region(the midzonal and pericentral regions)(panel A)and the entire(combined)liver lobule(panel B)after 30 min of gut ischemia and 60 min of reperfusion; B: Effects of chronic ethanol consumption and/ or CPon plasma aLT activities at 6 h after gut I/R; C: The effects of chronic ethanol consumption and /or CP on plasma endotoxinconcentration after 30 min of gut ischemia and 60 min of reperfusion; D: The effects of chronic ethanol consumption and /or CPon plasma TNF-a concentration after 30 min of gut ischemia and 60 min of reperfusion514ISSN 1007-9327 CN 14-1219/R World J Gastroenterol January 28, 2005 Volume 11 Number 4of plasma systemic and portal endotoxin levels in control rats,present study, pretreatment with CP substantially reduced thewhile chromicethanol consumption enhanced the gut IR-induced exaggerated increase in plasma endotoxin levels in ethanol-fedincrease in plasma endotoxin levels(control: 26.2- 27. 1 pg/mLrats to almost the same level as that in untreated rats(withoutethanol:932+51.4 pg/mL). The exaggerated elevation of plasma IR) This result suggests that CP might blunt the enhancedendotoxin levels in ethanol-fed rats was largely prevented by increase in intestinal mucosal permeability and /or improvesedthe pretreatment with CP(endotoxin levels; 21.3+ 1. 4 pg/mL). the impaired clearance of endotoxin in ethanol-fed ratsFigure ID summarizes the effects of CP treatment on gut IR-Although chronic ethanol consumption enhanced the gutinduced increase in plasma TNF-a levels in the presence or IR-induced increase in plasma endotoxin levels, it did not affectabsence of chronic ethanol consumption. In control rats, gut IR the gut I/R-induced increase in plasma TNF-a levels. Chronicelicited a significant increase in plasma TNF-CL levels. Althoughethanol consumption enhanced the gut I/R-induced increasechronic ethanol consumption did not affect gut I/R-induced in plasma ALT activities with a parallel increase in leukostasisincreases in plasma TNF-a levels, CP treatment reduced plasmain the liver. These results suggest that leukostasis per se orTNF-a in both control and cthanol-fed ratsleukocyte-derived oxidants may play a more important role ingut IR-induced liver(hepatocellular)injury than cytokinesAnother likely interpretation is that cytokines other than TNF-aare involved in the enhanced responses after gut IR in rats fedOur previous study has demonstrated that leukocyte-endothelial ethanol chronically In the present study, however, CP decreasedcell adhesion is an important determinant of the exaggeratedthe gut I/R-induced increase in plasma ALt activities with amicrovascular dysfunction and tissue injury after gut IR in the parallel decrease in plasma TNF-a levels. Moreover, CP alsoliver of rats fed ethanol chronically. The main component of CP attenuated the enhancement of gut I/R-induced increase inis Danshen. Seven water-soluble components have been plasma ALT activities in ethanol-fed rats with a parallelisolated from the Danshen root, Radix Salviae Miltiorrhiae. attenuation of plasma TNF-a levels. These results indicate thatUsing HPLC, at least 10 peaks were resolved based on its CP can reduce the production of TNF-a independent ofaffinity to o 1-acid glycoprotein 2. One of the components, endotoxin levels. CP was also reported to blunt TNF-o-jnducedsalvianolic acid, was reported to protect cerebral IR injury in endothelial cell injury via NF-KB activation 5o. Therefore, CPrats. Althogh CP had a protective effect on carbon tetrachloride- may prevent gut I/R-induced hepatic microvascular dysfunctioninduced hepatocellular injury 2, the effect of CP on either hepatic via protection of endothelial cellsIR injury or hepatic microcirculation has not been reported InThus, CP can protect gut IR-induced hepatic micRovascularthis study, we demonstrated the protective effects of CP ondysfunction and hepatocellular injury. Although further studiesgut IR-induced liver injury in rats fed ethanol chronicallyand clinical trials are required, CP appears to have therapeuticReperfusion of the ischemic intestine in control rats resulted usefulness against reperfusion injury in the liverin an accumulation of adherent leukocytes in sinusoids andTHV, a reduction in the number of perfused sinusoids, and the REFERENCESrelease of liver enzyme(ALT) into the blood stream. 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