Herbal cardiotonic pills prevent gut ischemia/reperfusion-induced hepatic microvascular dysfunction in rats fed ethanol chronically

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1 PO Box 2345, Beijing , Chin World J Gstroenterol 2005;11(4): Fx: World Journl of Gstroenterology ISSN E-mil: wjg@wjgnet.com ELSEVIER 2005 The WJG Press nd Elsevier Inc. All rights reserved. BASIC RESEARCH Herbl crdiotonic pills prevent gut ischemi/reperfusion-induced heptic microvsculr dysfunction in rts fed ethnol chroniclly Yoshinori Horie, Jing-Yn Hn, Shuk Mori, Mshiro Konishi, Mikio Kjihr, Tkehiko Kneko, Yoshiyuki Ymgishi, Shinzo Kto, Hiroms Ishii, Toshifumi Hibi Yoshinori Horie, Jing-Yn Hn, Shuk Mori, Mshiro Konishi, Mikio Kjihr, Tkehiko Kneko, Yoshiyuki Ymgishi, Shinzo Kto, Hiroms Ishii, Toshifumi Hibi. Deprtment of Internl Medicine, School of Medicine, Keio University, Tokyo Jpn Supported by Grnts From Tsley, Tinjin, Chin Co-correspondents: Jing-Yn Hn Correspondence to: Yoshinori Horie, M.D., or Deprtment of Internl Medicine, School of Medicine, Keio University, 35 Shinnomchi Shinjukuku, Tokyo , Jpn. yhorie@sc.itc.keio.c.jp Telephone: Fx: Received: Accepted: Abstrct AIM: Crdiotonic Pill (CP), n orl herbl medicine tht includes Dnshen (Slvie Miltiorrhize), Pnx notoginseny nd Dyroblnops romtic gertn, hs been cliniclly used for vsculr diseses such s occlusive vsculitis, coronry diseses, therosclerosis, nd cerebrl infrction. The min component, Slvie Miltiorrhize, hs been reported to prevent cerebrl nd intestinl reperfusion injury. However, little is known bout the effect of CP on heptic microcircultion. Thus, this study imed to determine whether CP could ffect heptic microvsculr dysfunction elicited by gut ischemi/ reperfusion (I/R) in rts fed ethnol chroniclly. METHODS: Mle Wistr rts were pir-fed with liquid diet contining ethnol or isocloric control diet for 6 wk. After lprotomy, one lobe of the liver ws exmined through n inverted intrvitl microscope. The rts were exposed to 30 min of gut ischemi followed by 60 min of reperfusion. Rhodmine-6G-lbeled leukocytes in the sinusoids were observed 90 min fter the onset of superior mesenteric rtery occlusion. Plsm tumor necrosis fctor (TNF)-α nd endotoxin levels were mesured 1 h fter the onset of reperfusion. Plsm lnine minotrnsferse (ALT) ctivities were mesured 6 h fter the onset of reperfusion. In nother set of experiments, CP (0.8 g/kg, intrgstriclly) ws dministered 1 nd 24 h before the onset of ischemi. RESULTS: In control rts, gut I/R elicited increses in the number of sttionry leukocytes, nd plsm TNF-α nd endotoxin levels nd plsm ALT ctivities. These chnges were mitigted by pretretment with CP. In ethnol-fed rts, the gut I/R-induced increses in the number of sttionry leukocytes, plsm endotoxin levels nd ALT ctivities were enhnced. Pretretment with CP ttenuted the enhncement of gut I/R-induced responses by chronic ethnol consumption. CONCLUSION: These results suggest tht CP prevents the gut I/R-induced heptic microvsculr dysfunction nd heptocellulr injury. A reduction of inflmmtory responses such s TNF-α production vi reduction of blood endotoxin levels ppers to be involved in the mechnisms. Chronic ethnol consumption enhnces gut I/R-induced heptic microvsculr nd heptocellulr injury. CP lso ttenutes n enhncement of gut I/R-induced responses by chronic ethnol consumption vi the reduction of blood endotoxin levels The WJG Press nd Elsevier Inc. All rights reserved. Key words: Intestinl reperfusion injury; Heptic microvsculr dysfunction; Crdiotonic Pill; Ethnol Horie Y, Hn JY, Mori S, Konishi M, Kjihr M, Kneko T, Ymgishi Y, Kto S, Ishii H, Toshifumi Hibi. Herbl crdiotonic pills prevent gut ischemi/reperfusion-induced heptic microvsculr dysfunction in rts fed ethnol chroniclly. World J Gstroenterol 2005; 11(4): INTRODUCTION A lrge body of evidence implictes leukocytes s meditors of microvsculr dysfunction nd tissue injury ssocited with reperfusion of ischemic orgns. Severl experimentl strtegies hve been used to demonstrte the contribution of leukocytes to ischemi/reperfusion (I/R) injury, including polyclonl ntibodies tht render niml leukopeni [1-3], dhesion moleculespecific monoclonl ntibodies [1,4-6], nd dhesion molecule deficiency in mice [7,8]. The effectiveness of dhesion moleculespecific monoclonl ntibodies (MAbs) nd dhesion moleculedeficiency in ttenuting I/R-induced tissue injury hve led to the widely held view tht leukocyte-endothelil cell dhesion is rte-determining step in the pthogenesis of this injury process. We developed leukocyte-dependent model of heptocellulr dysfunction elicited by gut I/R [1,7,9]. This murine model llows in vivo ssessment of the effects of I/R on leukocyte sequestrtion in sinusoids of different regions of the liver lobule, leukocyte dherence in postsinusoidl venules, nd the number of perfused sinusoids. Ethnol hs lso been reported to modulte I/R-induced tissue injury [10-12]. In perfused liver model, ethnol enhnced I/R-induced heptotoxicity (n increse in blood levels of liver enzymes) by n enhnced production of rective oxygen species [10]. In n in vivo gut I/R model, ethnol lso enhnced gut I/R-induced neutrophil ccumultion in the intestinl wll [11]. In n in vivo cerebrl I/R model, however, ethnol pretretment ws reported to reduce cerebrl I/R injury [12]. We hve recently reported tht low-dose ethnol ttenutes gut I/R-induced heptic microvsculr dysfunction in the midzonl region nd sequentil heptocellulr injury, wheres high-dose ethnol enhnces heptic microcircultory disturbnces in the pericentrl region nd sequentil heptocellulr injury [13]. Cliniclly, long-term lcohol consumption hs been noted to significntly reduce the incidence of coronry rtery disese [14]. However, chronic lcohol consumption often results in ftty liver nd liver filure, which is of prticulr concern when such ft-lden tissues re used s donor orgns in liver trnsplnttion. This importnt clinicl problem hs drwn ttention to the reltionship between ethnol consumption nd reperfusion

2 512 ISSN CN / R World J Gstroenterol Jnury 28, 2005 Volume 11 Number 4 injury in the liver. Both gut I/R nd chronic consumption of ethnol re known to cuse liver injury vi mechnisms tht involve oxidtive stress nd microcircultory disturbnces tht include leukocyte sequestrtion nd sinusoidl mlperfusion [15]. Gut I/R is known to produce n elevtion in plsm endotoxin levels (9), while chronic ethnol consumption hs been reported to enhnce the heptic microcircultory dysfunction nd heptocellulr injury induced by endotoxins [16-18]. Bsed on these observtions one might expect tht chronic ethnol consumption would led to n exggerted liver injury response to gut I/R. Furthermore, we hve recently reported tht chronic ethnol consumption enhnces heptic leukosequestrtion, impired sinusoidl perfusion, nd heptocellulr injury cused by gut I/R vi expression of intercellulr dhesion molecule (ICAM)-1 [19]. Herbl medicines (trditionl medicines from nturl sources) re recently ttrcting incresed globl ttention. Further, Chinese herbl medicines hve stood the test of time nd mny re widely ccepted s hving relible therpeutic efficcy. Crdiotonic Pill (CP), n orl herbl medicine tht includes Dnshen (Slvie Miltiorrhize), Pnx notoginseny nd Dyroblnops romtic gertn, hs been cliniclly used in Chin, Kore nd Russi for vsculr diseses such s occlusive vsculitis, coronry diseses, therosclerosis, nd cerebrl infrction, which re relted to microvsclr dysfunction. In clinicl cses, the min component, Dnshen, hs been reported to hve curtive efficcy in ischemic cerebrovsculr disese. In niml models, Dnshen hs been reported to prevent cerebrl nd intestinl reperfusion injury [20,21]. In the rt cerebrl ischemi model, Dnshen ws reported to improve cerebrl blood flow in the ischemic hemisphere nd to inhibit pltelet ggregtion in rts [20]. However, little is known bout the effect of CP on heptic microcircultion. Hence, the overll objectives of this study ws to ssess the effects of CP on heptic microvsculr dysfunction nd heptocellulr injury induced by gut I/R, nd to determine whether CP could ffect the heptic microvsculr dysfunction elicited by gut I/R in rts fed ethnol chroniclly. MATERIALS AND METHODS Animls Thirty (15 pirs) mle Wistr rts weighing bout 150 g were pir-fed for 6 wk with liquid diet contining ethnol tht provided 36% of the totl dietry clories or n isocloric control diet ccording to the method of Lieber et l. [22]. All rts were fsted for18 h prior to the experiments, which were ll performed ccording to the criteri outlined in the US Ntionl Reserch Council nd the Keio Animl Reserch Guides. Intrvitl microscopy Rts were nesthetized with pentobrbitl sodium (35 mg/kg) intrperitonelly. The left crotid rtery ws cnnulted nd ctheter plced t the ortic rch to monitor blood pressure. The left jugulr vein ws lso cnnulted for drug dministrtion. After lprotomy, lobe of the liver ws observed with n inverted intrvitl microscope (TMD-2S, Diphoto, Nikon, Tokyo, Jpn) ssisted by silicon intensified trget (SIT) cmer (C , Hmmtsu Photonicus, Shizuok, Jpn). The liver ws plced on n djustble Plexigls microscope stge with non-fluorescent coverslip tht llowed for observtion of 2-cm² segment of tissue. The liver ws crefully plced to minimize the influence of respirtory movements, nd the surfce ws moistened nd covered with cotton guze soked with sline. Imges of the microcircultion were observed from the surfce of the liver through 20 fluorescent objective, nd microfluorogrphs were recorded on videotpe using videocssette recorder (S VHS-HQ, Victor, Jpn). Anlysis of leukocyte ccumultion Leukocytes were lbeled in vivo with rhodmine-6g (1 mg ws dissolved in 5 ml of 0.9% sline) using previously described method [12,17,19,23], which ws bsed on method used in rt brin [3]. It ws shown tht rhodmine 6G could selectively stin white blood cells nd pltelets, but not endothelil cells [3]. Thus, the fluorochrome llowed for differentition between dherent leukocytes nd endothelil cells. Rhodmine-6G (0.2 ml/100 g body weight) ws injected prior to ethnol dministrtion with subsequent injections every 30 min. Rhodmine-6G ssocited fluorescence ws visulized by epi-illumintion t nm, using 590 nm emission filter. We selected one of the lobules, which hd well-perfused sinusoids nd the fewest sttionry leukocytes, choosing the furthest lobule from the edge of the liver if ll the conditions were thought to be equivlent. A microfluorogrph of heptic microcircultion, with rhodmine- 6G-lbeled leukocytes in the sinusoids, ws continuously observed for 90 min fter the SMA occlusion nd recorded on digitl video recorder for 1 min t 0, 30, 60, nd 90 min. The number of sttionry leukocytes ws determined off-line during plybck of the videotpe imges. A leukocyte ws considered sttionry within the microcircultion (sinusoids) if it remined sttionry for more thn 10 s. The lobule, which hd the fewest sttionry leukocytes ws selected for observtion t bsl condition. Sttionry leukocytes were quntified in both the midzonl nd pericentrl regions of the liver lobule nd expressed s the number per field of view ( µm 2 ). Experimentl protocols We observed the surfce of the liver for 10 min before ligtion of the superior mesenteric rtery in order to ensure tht ll prmeters mesured on-line were in stedy stte. The superior mesenteric rtery ws then ligted with snre creted from polyethylene tubing for 0 (shm) or 30 min. After the ischemic period, the ligtion ws gently removed. Leukocyte ccumultion ws mesured before ischemi, immeditely following reperfusion nd every 30 min for one hour therefter. In nother set of experiments, CP (0.8 g/kg, intrgstriclly, Tsley, Tinjin, Chin) ws dministered t 1 nd 24 h before the onset of ischemi to both control nd ethnol-fed rts, nd the sme protocol ws performed. Twenty-five mg (one pill) of CP includes 9 mg of Dnshen (Slvie Miltiorrhize), 1.76 mg of Pnx notoginseny, 0.5 mg of Dyroblnops romtic gertn, nd mg of polyethylene glycol. Liver enzyme, endotoxin nd TNF ssys Sixty min fter the onset of reperfusion, the rts were removed from the microscope stge nd the bdominl wll ws closed. Blood (plsm) smples for endotoxin nd tumor necrosis fctor (TNF)-α level mesurement were collected from the inferior ven cv t point proximl to the heptic vein 1 h fter the onset of reperfusion. For the mesurement of endotoxin levels, blood smples were lso collected from the portl vein. Smples for plsm lnine minotrnsferse (ALT) mesurement were obtined 6 h fter the onset of reperfusion. Plsm ALT ctivity ws determined by conventionl UV method s previously described [24]. Plsm TNF-α concentrtion ws determined in microtiter plte using TNF-α immunossy kit (BioSource Interntionl, Cmrillo, CA) bsed on enzyme-linked immuno sorbent ssy (ELISA). In ccord with our previous report [25,26], plsm endotoxin levels were mesured by endospecy (n endotoxin-specific chromogeic limulus regent; Seikgku Co., Tokyo, Jpn) using n utomted kinetic ssy for endotoxin [27]. Sttisticl nlysis The dt were nlysed using stndrd sttisticl nlyses, i.e.,

3 Horie Y et l. Reperfusion injury nd herbl medicine 513 ANOVA nd Scheffe s (post hoc) test. All vlues were reported s men±sd, with five rts per group. Sttisticl significnce ws set t P<0.05. RESULTS Tble 1 shows the effects of CP tretment on leukostsis in sinusoids of the midzonl nd pericentrl regions nd the terminl heptic venule (THV), plsm ALT ctivities, nd plsm endotoxin nd TNF-α levels. Pretretment with CP per se did not ffect ny vlues of these in control rts. Chronic ethnol feeding per se cused leukostsis in the pericentrl region of the liver, while CP diminished it. Figure 1A illustrtes the effects of CP tretment on gut I/Rinduced leukostsis in sinusoids of the midzonl nd pericentrl regions nd the THV (Pnel A) of the liver lobule, nd the entire liver lobule (sinusoids+thv, pnel B) in the presence or bsence of chronic ethnol consumption In control rts, gut I/R elicited increses in the number of sttionry leukocytes in both heptic sinusoids nd THV. In ethnol-fed rts, the gut I/Rinduced leukostsis ws blunted in the midzonl region (control; 12.6±1.4, ethnol: 7.2±0.7, per field), while exggerted leukostsis ws noted in the pericentrl region (control; 4.3±1.9, ethnol: 6.8±2.2) nd THV (control; 4.0±1.4, ethnol: 13.0±1.6, per field). Although the leukostsis elicited by gut I/R in control rts ws ttenuted by the pretretment with CP (leukostsis in the pericentrl region; 4.0±1.2, THV; 1.8±0.7, per field), the exggerted leukostsis in ethnol-fed rts ws lrgely prevented by pretretment with CP (leukostsis in the pericentrl region; 3.0±0.7, THV; 4.8±1.2, per field). Figure 1B shows the effects of CP tretment on plsm ALT ctivity following gut I/R in the presence or bsence of chronic ethnol consumption. In control rts, gut I/R led to n elevtion of plsm ALT ctivities. Chronic ethnol consumption enhnced the gut I/R-induced increse in plsm ALT ctivities (control; 146±31 IU/L, ethnol: 236±84 IU/L). The increse in plsm ALT ctivities elicited by gut I/R in both control nd ethnol-fed rts ws significntly ttenuted by pretretment with CP (control; 81±19 IU/L, ethnol: 108±58 IU/L). Figure 1C shows the effects of CP tretment on plsm endotoxin levels following gut I/R in the presence or bsence of chronic ethnol consumption. Gut I/R cused slight elevtion Tble 1 Effect of crdiotonic pills on leukostsis in liver, plsm ALT ctivity, nd plsm endotoxin nd TNF-α level in control nd ethnol-fed rts Leukostsis Pericentrl THV ALT ctivity Endotoxin TNF-α Group midzonl (per field) (per field) (per field) (IU/L) (pg/ml) (pg/ml) 1.8± ± ± ± ± ±8.9 Ethnol 1.4± ± ± ± ± ± ± ± ± ± ± ±10.1 Ethnol 1.6± ± ± ± ± ±6.2 CP: crdiotonic pills, P<0.05 vs. A Plsm ALT ctivity 300 (IU/I) c c e e Sttionry leukocytes (per field) e Midzonl Pericentrl THV A Plsm endotoxin level (pg/ml) Totl e e ec Sttionry leukocytes (per field) B C D e e e e c c e c Plsm TNF-lph level (pg/ml) Figure 1 Effects of chronic ethnol consumption nd/or Crdiotonic Pill (CP) on the number of sttionry leukocytes, plsm ALT ctivities, plsm endotoxin concentrtion, nd plsm TNF-α concentrtion fter gut I/R. Ech group consisted of five nimls. P<0.05 vs control, c P<0.05 vs +I/R, e P<0.05 vs ethnol+i/r. A: Effects of chronic ethnol consumption nd/or CP on the number of sttionry leukocytes in ech region (the midzonl nd pericentrl regions) (pnel A) nd the entire (combined) liver lobule (pnel B) fter 30 min of gut ischemi nd 60 min of reperfusion; B: Effects of chronic ethnol consumption nd/or CP on plsm ALT ctivities t 6 h fter gut I/R; C: The effects of chronic ethnol consumption nd/or CP on plsm endotoxin concentrtion fter 30 min of gut ischemi nd 60 min of reperfusion; D: The effects of chronic ethnol consumption nd/or CP on plsm TNF-α concentrtion fter 30 min of gut ischemi nd 60 min of reperfusion.

4 514 ISSN CN / R World J Gstroenterol Jnury 28, 2005 Volume 11 Number 4 of plsm systemic nd portl endotoxin levels in control rts, while chronic ethnol consumption enhnced the gut I/R-induced increse in plsm endotoxin levels (control; 26.2± 27.1 pg/ml, ethnol: 93.2±51.4 pg/ml). The exggerted elevtion of plsm endotoxin levels in ethnol-fed rts ws lrgely prevented by the pretretment with CP (endotoxin levels; 21.3± 1.4 pg/ml). Figure 1D summrizes the effects of CP tretment on gut I/Rinduced increse in plsm TNF-α levels in the presence or bsence of chronic ethnol consumption. In control rts, gut I/R elicited significnt increse in plsm TNF-α levels. Although chronic ethnol consumption did not ffect gut I/R-induced increses in plsm TNF-α levels, CP tretment reduced plsm TNF-α in both control nd ethnol-fed rts. DISCUSSION Our previous study [19] hs demonstrted tht leukocyte-endothelil cell dhesion is n importnt determinnt of the exggerted microvsculr dysfunction nd tissue injury fter gut I/R in the liver of rts fed ethnol chroniclly. The min component of CP is Dnshen. Seven wter-soluble components hve been isolted from the Dnshen root, Rdix Slvie Miltiorrhize. Using HPLC, t lest 10 peks were resolved bsed on its ffinity to α 1-cid glycoprotein [28]. One of the components, slvinolic cid, ws reported to protect cerebrl I/R injury in rts [20]. Althogh CP hd protective effect on crbon tetrchlorideinduced heptocellulr injury [29], the effect of CP on either heptic I/R injury or heptic microcircultion hs not been reported. In this study, we demonstrted the protective effects of CP on gut I/R-induced liver injury in rts fed ethnol chroniclly. Reperfusion of the ischemic intestine in control rts resulted in n ccumultion of dherent leukocytes in sinusoids nd THV, reduction in the number of perfused sinusoids, nd the relese of liver enzyme (ALT) into the blood strem. In control rts, gut I/R-induced leukostsis in the pericentrl region nd THV were not noted fter pretretment with CP. This pretretment lso ttenuted the gut I/R-induced increse in plsm ALT nd TNF-α levels. An interesting finding in the present study is tht the gut I/R-induced increse in plsm endotoxin level ws not seen in control rts fter the pretretment with CP. Gut I/R ws reported to elevte plsm endotoxin levels, which ppered to be derived from the gut [19]. CP hs been reported to blunt mesenteric I/R injury [21]. Endotoxin hs been known to cuse heptic microvsculr dysfunction nd heptocellulr injury [17,18,24]. Tken together, these results nd evidence from the literture suggest tht CP cn reduce blood endotoxin levels by protecting the intestinl mucosl brrier from I/R injury, thereby preventing the subsequent heptic microvsculr dysfunction nd heptocellulr injury. Chronic ethnol consumption exggerted gut I/R-induced leukostsis in the liver, gut I/R-induced increse in plsm endotoxin levels, nd the subsequent heptocellulr injury (ALT elevtion). The findings in the present study lend support to the possibility tht elevted plsm levels of endotoxin contribute to the exggerted inflmmtory nd tissue injury responses seen in the liver fter gut I/R in rts chroniclly fed ethnol. The portl endotoxin level in rts fed ethnol chroniclly ws higher thn tht in controls [19]. This result suggested tht intestinl mucosl permebility incresed in ethnol-fed rts fter gut I/R. However, systemic endotoxin levels were much lower thn portl endotoxin levels in control rts, wheres there ws no significnt difference between systemic nd portl endotoxin levels in ethnol-fed rts fter gut I/R [19], suggesting tht clernce of endotoxin ws impired in ethnol-fed rts. Thus, both n increse in intestinl mucosl permebility nd impired clernce of endotoxin in ethnol-fed rts could be involved in the enhncement of plsm endotoxin levels. In the present study, pretretment with CP substntilly reduced the exggerted increse in plsm endotoxin levels in ethnol-fed rts to lmost the sme level s tht in untreted rts (without I/R) This result suggests tht CP might blunt the enhnced increse in intestinl mucosl permebility nd /or improvesed the impired clernce of endotoxin in ethnol-fed rts. Although chronic ethnol consumption enhnced the gut I/R-induced increse in plsm endotoxin levels, it did not ffect the gut I/R-induced increse in plsm TNF-α levels. Chronic ethnol consumption enhnced the gut I/R-induced increse in plsm ALT ctivities with prllel increse in leukostsis in the liver. These results suggest tht leukostsis per se or leukocyte-derived oxidnts my ply more importnt role in gut I/R-induced liver (heptocellulr) injury thn cytokines. Another likely interprettion is tht cytokines other thn TNF-α re involved in the enhnced responses fter gut I/R in rts fed ethnol chroniclly. In the present study, however, CP decresed the gut I/R-induced increse in plsm ALT ctivities with prllel decrese in plsm TNF-α levels. Moreover, CP lso ttenuted the enhncement of gut I/R-induced increse in plsm ALT ctivities in ethnol-fed rts with prllel ttenution of plsm TNF-α levels. These results indicte tht CP cn reduce the production of TNF-α independent of endotoxin levels. CP ws lso reported to blunt TNF-α-induced endothelil cell injury vi NF-κB ctivtion [30]. Therefore, CP my prevent gut I/R-induced heptic microvsculr dysfunction vi protection of endothelil cells. Thus, CP cn protect gut I/R-induced heptic microvsculr dysfunction nd heptocellulr injury. Although further studies nd clinicl trils re required, CP ppers to hve therpeutic usefulness ginst reperfusion injury in the liver. REFERENCES 1 Horie Y, Wolf R, Miysk M, Anderson DC, Grnger DN. Leukocyte dhesion nd the heptic microvsculr responses to intestinl ischemi-reperfusion. Gstroenterology 1996; 111: Jeschke H, Frhood A, Smith CW. Neutrophils contribute to ischemi/reperfusion injury in rt liver in vivo. FASEB J 1990; 4: Romson JL, Hook BG, Kunkel SL, Abrms GD, Schork MA, Lucchesi BR. Reduction of the extent of ischemic myocrdil injury by neutrophil depletion in the dog. Circultion 1983; 67: Frhood A, McGuire GM, Mnning AM, Miysk M, Smith CW, Jeschke H. Intercellulr dhesion molecule 1 (ICAM-1) expression nd its role in neutrophil induced ischemireperfusion injury in rt liver. 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