Attenuation of graft ischemia-reperfusion injury by urinary trypsin inhibitor in mouse intestinal transplantation

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1 PO Box, Beijing 000, Chin World J Gstroenterol 00;(): World Journl of Gstroenterology ISSN wjg@wjgnet.com EL SEVIER 00 The WJG Press nd Elsevier Inc. All rights reserved. BASIC RESEARCH Attenution of grft ischemi-reperfusion injury by urinry trypsin inhibitor in mouse intestinl trnsplnttion Ji-Ren Yu, Sheng Yn, Xio-Sun Liu, Yi-Jun Wu, Pei-Feng Fu, Li-Hu Wu, Shu-Sen Zheng Ji-Ren Yu, Sheng Yn, Xio-Sun Liu, Yi-Jun Wu, Pei-Feng Fu, Li-Hu Wu, Shu-Sen Zheng, Deprtment of Surgery, the First Affilited Hospitl, College of Medicine, Zhejing University, Hngzhou 000, Zhejing Province, Chin Supported by the Helth Scientific Grnt 00 of Zhejing Province, Chin. No. 00ZX0 Correspondence to: Dr. Ji-Ren Yu, Deprtment of Surgery, the First Affilited Hospitl, College of Medicine, Zhejing University, Qingchun Rod 79, Hngzhou 000, Zhejing Province, Chin. ynsheng_cn@yhoo.com Telephone: Received: Accepted: Abstrct AIM: Ischemi/reperfusion (I/R) injury is one of the mjor obstcles for intestinl trnsplnttion (ITx). Urinry trypsin inhibitor (Ulinsttin, ) suppresses proteses nd stbilizes lysosoml membrnes. We supposed tht Ulinsttin would diminish I/R injury of intestinl grft. METHODS: - treted group nd untreted control group were investigted by histologicl ssessment t.,,, nd 7 h fter ITx. Myeloperoxidse (MPO) ctivity ws used s the ctivity of neutrophils, nd mlondildehyde (MDA) ws used s n index of lipid peroxidtion. TNF nd i-nos mrna expression in grft tissue were mesured by semi-quntittive RT-PCR. CDb + Gr + cells in grft lmin propri were nlyzed by flow cytometry. RESULTS: Histologicl scores of the grft showed tht the tissue injury ws mrkedly ttenuted by tretment t different time points fter ITx, with reduced MPO nd MDA vlue in the grfts. The expression of TNF nd i-nos mrna ws profoundly inhibited, while the infiltrtion of CDb + Gr + cells into the intestinl grft ws decresed in group. CONCLUSION: Urinry trypsin inhibitor ttenutes I/R injury in mouse intestinl trnsplnttion by reducing monocytes infiltrtion nd down-regultion of TNF nd i-nos mrna expression. 00 The WJG Press nd Elsevier Inc. All rights reserved. Key words: Ischemi/reperfusion injury; Ulinsttin; Intestinl trnsplnttion Yu JR, Yn S, Liu XS, Wu YJ, Fu PF, Wu LH, Zheng SS. Attenution of grft ischemi-reperfusion injury by urinry trypsin inhibitor in mouse intestinl trnsplnttion. World J Gstroenterol 00; (): INTRODUCTION Intestinl trnsplnttion (ITx) is the only definitive therpy for ptients with irreversible intestinl filure who hve developed life-thretening complictions of totl prenterl nutrition []. The outcome of clinicl ITx, when compred to other solid orgn trnsplnts, is still fr from cceptble []. Ischemi/reperfusion (I/R) injury hs been one of the mjor obstcles, since the intestinl grft is exceedingly sensitive to ischemi cused by storge nd implnttion []. I/R injury subsequently leds to impirment of mucosl brrier nd bcteril trnsloction, which provoke grft loss, ftl infection, nd even multi-orgn dysfunction in recipients. Therefore, more effective protocols for controlling grft I/R injury re demnded, in order to chieve better outcome for this therpeutic pproch. Urinry trypsin inhibitor (Ulinsttin, ), glycoprotein with moleculr weight of , is protese inhibitor purified from humn urine. It suppresses proteses such s trypsin, chymotrypsin nd elstse, s well s stbilizes lysosoml membrnes nd thereby inhibits the relese of lysosoml enzymes []. Ulinsttin hs been found effective in relieving reperfusion injury in ischemic liver [], intestine [6], nd kidney [7]. In our knowledge, however, its effect on intestinl trnsplnttion hs not been reported. Distinct from other cuses, the ischemic event in trnsplnttion is inevitble, fetured by shorter wrm ischemic nd longer cold ischemic period []. In our previous studies, we hd found quick influx of monocytes, minly neutrophils nd mcrophges, into intestinl grft even in syngeneic combintion. I/R injury is relevnt to the ccumultion nd eruption of these infiltrting monocytes. We supposed tht the suppressive effects on neutrophil protese by Ulinsttin would diminish I/R injury of intestinl grft. In the present study, we exmined the effects of Ulinsttin on I/R injury of intestinl grfts nd the expression of TNF nd i-nos mrna in mouse intestinl trnsplnttion model. MATERIALS AND METHODS Animls Blb/c (H- d ) mice were purchsed from The Centrl Animl Fcility of Medicl School, Zhejing University, nd were used t 8- wk of ge. All nimls were housed under specific

2 606 ISSN CN -9/ R World J Gstroenterol Mrch, 00 Volume Number pthogen-free conditions with controlled light/drk cycles nd free ccess to wter nd rodent chow. The investigtions were performed in complince with the policies of the niml cre committee of the locl government. Heterotopic intestinl trnsplnttion in mice Mouse heterotopic intestinl trnsplnttion ws performed s described by Zhong et l [8] with minor modifictions. Blb/c (H- d ) mice were used s donors nd recipients. Briefly, ITx ws crried out under intrperitonel nesthesi using ketmine (0.08 mg/g) nd xylzine (0.0 mg/g). First, the donor jejunum nd proximl prt of the ileum were isolted with ttched superior mesenteric rtery nd portl vein. After luminl irrigtion nd vsculr perfusion, the grft ws hrvested nd stored in Ringer s solution until implnttion. Grft portl vein nd superior mesenteric rtery were nstomosed to recipient s inferior ven cv nd ort in n end-to-side fshion. The distl end of the intestinl grft ws connected to the host jejunum. The proximl grft lumen ws exteriorized s stom. No ntibiotics were dministered periopertively. Experimentl groups nd peri-trnsplnt therpy Three experimentl groups were estblished: Group, Shm opertion (n = 6): Lprotomy nd dissection of SMA nd PV were performed without occlusion of these vessels in this group. Group, -treted group (n = ): (0 000 U/kg, Techpool Bio-Phrm Co. Gungzhou, Chin) ws given to the recipients vi intrvenous injection min before revsculriztion of the intestinl grft. Therefter, it ws given t the sme level of dose every h fter trnsplnttion. Group, untreted controls (n = ): Norml sline insted of ws given to the recipients. At.,,, 7 h fter trnsplnttion, six mice of ech group were killed nd the grfts were hrvested for histologicl grding, FACS nlysis, nd mrna extrction. Histologicl ssessment A -cm segment of the proximl portion of the grft ws divided long its nti-mesenteric side nd wshed in PBS. The smples were fixed by formlin nd embedded in prffin by Swiss rolls with the luminl side fcing outwrds. Then, the rolled smples were cross-sectioned nd stined with hemotoxylin-eosin. All microscopic slides were observed nd ssessed blindly with reference to the histologicl criteri. Briefly, specimens were scored s: (0) less thn 0% of villous tips dmged, () more thn 0% of villous tips dmged; () dmge confined to distl / of villus; () dmge confined to distl / of villus; () dmge including ll of villus, nd () dmge more proximl thn villus. Assy of myeloperoxidse (MPO) ctivity MPO ctivity ws ssyed by spectrophotometriclly mesuring the H O dependent oxidtion of,,, - tetrmethylbenzidine t 60 nm. All the regents in the ssy were fforded by MPO kit, obtined from Jincheng Biotechnology, Nnjing, PR Chin. Assy of mlondildehyde (MDA) The lipid peroxide products, mlondildehyde (MDA) ws used s n index of lipid peroxidtion nd ws expressed s nmol/g protein. It ws determined by TBA ssy, using MDA kit obtined from Jincheng Bio-technology, Nnjing, PR Chin. Mesurement of TNF nd i-nos mrna by semiquntittive RT-PCR Grft RNA ws extrcted by Trizol kit from ech 0-00 mg snp-frozen smples. The concentrtion nd A 60 /A 80 (>.8) ws determined by ultrviolet photospectrometry. Totl RNA ( g) ws reverse-trnscribed to c-dna nd expnded by PCR. The primers of TNF, i-nos nd -ctin re s follows: TNF : Primer : -AGCCCACGTAGCAAACCACCAA- Primer : -ACACCCATTCCCTTCACAGAGC AAT- i-nos: Primer : -ACCCCTGTCTTCCACCAGGAGTTGAA- Primer : -TGCAGCCATGACCTTTCGCATTAGCATGG- -ctin: Primer : -TGGAATCCTGTGGCATCCATGAAAC- Primer : -TAAAACGCAGCTCAGTAACAGTCCG- The expnded products were nlyzed by.% grose gel electrophoresis nd imged by Kodk digitl imging system. The reltive vlue of mrna ws determined by the rtio between the A of trget gene nd -ctin. Flow cytometry Three-color flow cytometry ws conducted on isolted cells from the lmin propri of the intestinl grfts. Isolted cells from ech preprtion were centrifuged t 000 r/min for min, then wshed twice in FACS buffer (PBS+% FCS+% norml rt serum) nd incubted with primry mabs for 0 min t. The specimens incubted with biotinylted mabs were subsequently developed with streptvidin PercP. After wshing twice, ll smples were nlyzed on FACScn cytometer (Becton Dickinson) to events were collected from ech smple. Dt nlysis ws performed using WinMDI.8 softwre. Sttisticl nlysis Histology score, nd MPO, MDA, i-nos/ -ctin,tnf / -ctin vlue, were clculted for ech group. Student s t- test ws used to determine the significnce of differences in mens. The non-prmetric Mnn-Whitney U-test ws used to compre the medins of groups tht did not follow norml distribution ccording to the Shpiro-Witt test. A P-vlue of less thn 0.0 ws considered significnt. SPSS.0 nd Microsoft Excel softwre were used for the sttisticl nlyses. RESULTS Histologicl finding of the intestinl grfts The histologicl scores of the intestinl grfts in both control nd groups were shown in Figure (A-D). At different time points fter trnsplnttion, the histologicl impirments in group were pprently ttenuted compred to the control group. At the erly stge fter reperfusion (.,, nd h fter trnsplnttion), the mucosl dmge in the group is confined to villous tips, obviously slighter thn the control group, in which the dmge developed in totl villi. The histologicl chnges nerly disppered fter 7 h

3 Yu JR et l. Ulinsttin inhibits ischemi-reperfusion injury of intestinl grfts 607 / h h h NS 7 h Figure At.,,, nd 7 h fter intestinl trnsplnttion, the histologicl scores of tissue injury in the -treted group were pprently less thn the untreted group. MPO (U/g) Figure MPO ctivity in the intestinl grfts in the -treted group remined t lower level when compred with the control group in the erlier time points fter IR (.,, nd h fter trnsplnttion respectively, vs control group). MDA (nmol/g) Figure The MDA level t. nd h fter trnsplnttion ws significntly suppressed in -treted group, vs the untreted control group. in the group, wheres villous impirments remined in the distl prt of the villus in the control group. Moderte congestion nd mrked edem of the intestinl grft t erly time point were found in control groups. In the group, however, no congestion ws found nd the tissue edem ws slighter. Compring with the control group, the infiltrtion of monocytes ws lso reduced in group t ech time point fter trnsplnttion by light microscopic observtion. Myeloperoxidse (MPO) ctivity As shown in Figure, the MPO ctivity of the intestinl grft tissue in the group remined t lower level when compred to the control group in the erlier time points fter I/R (0.6±0. vs 0.6±0.7,.0±0. vs.±0., 0.6±0. vs 0.8±0.8 t.,, nd h fter trnsplnttion respectively, U/g, n = 6 for ech group, ). At h fter trnsplnttion, the MPO ctivity in both nd control groups were reverted to 0.69±0. U/g nd 0.8±0. U/g, no sttisticl difference were found in this time point. Mlondildehye (MDA) level As shown in Figure, the lipid peroxidtion derived MDA level t. nd h fter trnsplnttion ws significntly suppressed by tretment (7.9±0. vs 0.97±0.77, 7.70±0. vs 9.70±0.77 mmol/g, n = 6, ). At the following lter time points ( nd 7 h fter trnsplnttion), however, the grft MDA in both groups reverted to similrly lower level. Grft infiltrtion by CDb + Gr + monocytes As shown in Figure, when the monocyte gte of grft lmin propri ws nlyzed t the third post-trnsplnt dy, the sub-popultion of CDb + Gr + cells were found reduced in the -treted group. The percentge of CDb + Gr + cells, minly neutrophils nd mcrophges, in untreted llogrfts ws 0.±0.7%, nd ws decresed to.0±0.% in the -treted group (P<0.0). It indicted less inflmmtory infiltrtions, prticulrly neutrophils nd mcrophges, into the intestinl grft by therpy.

4 608 ISSN CN -9/ R World J Gstroenterol Mrch, 00 Volume Number 0 M S C T C T C T C T 0 Gr CDb -treted bp 0 0 TNF-lph bet-ctin Gr CDb Cell % in lmin propri Figure The sub-popultion of CDb + Gr + cells in the monocyte gte of grft lmin propri ws found reduced in the -treted group on the third post-trnsplnt dy. TNF-lph/bet-ctin bp 0/89 7 inos/bet-ctin M S C T C T C T C T 0. 7 bet-ctin inos Figure In group, the expression of i-nos mrna ws pprently inhibited t.,,, nd 7 h fter intestinl trnsplnttion. The upper bnds show the representtive imges of the PT-PCR results for i-nos. M: bnd for moleculr mrker; S: Shm opertion group; C-C: bnds for untreted group t.,, nd 7 h fter trnsplnttion respectively; T-T: bnds for -treted group t.,, nd 7 h fter trnsplnttion, respectively. Expression of i-nos, TNF mrna in intestinl grfts In the shm-operted mice, the expression of i-nos nd TNF mrna in non-ischemic intestine is minor. However, the expression of the intestinl grft elevted drmticlly due to I/R events fter trnsplnttion. The expression of i-nos mrna reched.8±0.8 to.7±0.8 (TNF / -ctin, n = 6), nd the TNF mrna were.7±0. to.67±0.7 (i-nos/ -ctin, n = 6) within the first 7 h fter trnsplnttion. However, in group, the expression of i-nos mrna ws pprently inhibited in ech time point, nd the level of TNF mrna ws lso lower thn the control group t. nd h fter trnsplnttion. It indicted profound suppression of i-nos nd TNF expression by tretment, when chllenged by trnsplnt-ssocited I/R. Figures, 6 displyed i-nos nd TNF mrna expression t different time points fter trnsplnttion nd representtive imges. Figure 6 The expression of TNF mrna ws inhibited t. nd h fter intestinl trnsplnttion in -treted group, while no difference ws found t nd 7 h fter trnsplnttion. The upper bnds show the representtive imges of the PT-PCR results for TNF. M: bnd for moleculr mrker; S: Shm opertion group; C- C: bnds for untreted group t.,, nd 7 h fter trnsplnttion respectively; T-T: bnds for -treted group t.,, nd 7 h fter trnsplnttion, respectively. DISCUSSION Intestine is one of the most vulnerble orgns to I/R injury in solid orgn trnsplnttion []. The high morbidity nd mortlity fter intestinl trnsplnttion re unexceptionlly ssocited to grft impirments due to I/R injury. Therefore, effective mesures with miniml risk of evoking llogeneic rejection should be developed for mitigting the intestinl impirments cused by I/R response [9]. The most common model for investigtions of intestinl I/R is performed by temporry occlusion of superior mesenteric rtery (SMA) [0]. Esy s it is, however, it is unble to feture the I/R events occurring in intestinl trnsplnttion. First, I/R injury, in trnsplnttion, is minly due to longer cold ischemic preservtion, while the SMA occlusion model cn merely mimic shorter wrm ischemi. Second, collterl vsculr supply cnnot be excluded by SMA occlusion, which usully ppers s sub-mucosl hemorrhge tht is bsent in trnsplnttion. Other fctors ffecting the investigting outcomes, such s irrigtion of the grft lumen during orgn procurement, cnnot be imitted by SMA occlusion either []. Thus, we took the more complicted mouse trnsplnttion model to estimte the effect of, in order to void possible errors. Constitutive NO produced by c-nos is well-known meditor in severl physiologicl processes. When insulted by inflmmtory stimuli such s ischemi, i-nos expression is promoted nd lrger mount of NO is produced by i-nos ctlysis. Insted of being signl trnsductor in lower levels, the highly concentrted NO cts s free rdicls nd eventully leds to locl tissue dmge []. Therefore, over expression of i-nos is the key point for the NO medited inflmmtory rection. TNF is nother crucil meditor in the onset nd sustining of the I/R response in the gut [].

5 Yu JR et l. Ulinsttin inhibits ischemi-reperfusion injury of intestinl grfts 609 The present study revels profound inhibitive effect of UIT towrd the expression of i-nos mrna in intestinl grft, which ws chllenged by long-term cold ischemi nd followed reperfusion. The decresed levels of i-nos mrna nd TNF mrna in the grfts re well consistent with the ttenuted villous injury, s well s the reduced neutrophil index (MPO) nd improved membrne stbility (MDA). It suggested tht the inhibition of TNF nd i-nos, thereby, breking down the cytokine cscde nd the NO-induced tissue injury could be the underlying mechnism of the protective effects of in intestinl trnsplnttion. Mcrophges nd other monocytes re the mjor source of TNF nd i-nos mrna, [] therefore we isolted these infiltrted cells from the intestinl grfts nd mesured them by flow cytometry. Gr is usully expressed on grnulocytes nd mcrophges, nd CDb hs the similr tendency while it lso ppers on ctivted nd mture lymphocytes []. The subpopultion, CDb + Gr + cells, including most neutrophils nd mcrophges, were found mrkedly reduced in -treted grfts when compred to untreted grfts t the third post-trnsplnt dy. Hence, the lower level of TNF nd i-nos mrna in UIT-treted group might owe to reduced infiltrtion of mcrophges nd neutrophils. On the other hnd, the suppressive effect of on TNF nd i-nos expression, if there is ny, my lessen the severity of grft infiltrtion by bting the cytokine or chemotctic response. Shortly, prevention of mcrophges nd other monocytes into the grft by is possible mechnism for its lleviting effects on I/R injury. As sfe nd well-tolerted gent, hs not found ny rejection-relted dverse effects in clinicl trnsplnttion []. The reveled mechnism of s well s our findings, such s inhibition of TNF nd i-nos production, theoreticlly excludes the potentil risk of provoking lloimmune response [6]. Consequently, could be n idel gent in protection of intestinl grft from IR injury. Further investigtions for in other solid orgn trnsplnts nd clinicl trils re required. REFERENCES Pltell CF, Coster J, McCuley RD, Hll JC. The mngement of ptients with the short bowel syndrome. World J Gstroenterol 00; 8: -0 Fishbein TM, Gondolesi GE, Kufmn SS. Intestinl trnsplnttion for gut filure. Gstroenterology 00; : 6-68 Newell KA, Fishbein TM. Experimentl models of smll bowel trnsplnttion. Curr Opin Orgn Trnsplnt 00; 8: 09-6 Kobyshi H, Gotoh J, Fujie M, Tero T. Chrcteriztion of the cellulr binding site for the urinry trypsin inhibitor. J Biol Chem 99; 69: Ymguchi Y, Ohshiro H, Ngo Y, Odwr K, Okbe K, Hidk H, Ishihr K, Uchino S, Furuhshi T, Ymd S, Mori K, Ogw M. Urinry trypsin inhibitor reduces C-X-C chemokine production in rt liver ischemi/reperfusion. J Surg Res 000; 9: 07-6 Li XK, Suzuki H, Kimur T, Kwbe A, Uno T, Hrd Y. Ulinsttin, protese inhibitor, ttenutes intestinl ischemi/reperfusion injury. Trnsplnt Proc 99; 6: - 7 Nkhm H, Obt K, Sugit M. Ulinsttin meliortes cute ischemic renl injury in rts. Ren Fil 996; 8: Zhong R, Zhng Z, Qun D, Grci B, Duff J, Stiller C, Grnt D. Intestinl trnsplnttion in the mouse. Trnsplnttion 99; 6: Newell KA. Trnsplnttion of the intestine: is it truly different? Am J Trnsplnt 00; : - 0 Pilli SB, Luquette MH, Nowicki PT, Besner GE. Segmentl intestinl ischemi: n improved method of producing smll bowel injury. J Invest Surg 998; : -8 Hssoun HT, Weisbrodt NW, Mercer DW, Kozr RA, Moody FG, Moore FA. Inducible nitric oxide synthse medites gut ischemi/reperfusion-induced ileus only fter severe insults. J Surg Res 00; 97: 0- Ymmoto S, Tnbe M, Wkbyshi G, Shimzu M, Mtsumoto K, Kitjim M. The role of tumor necrosis fctorlph nd interleukin-bet in ischemi-reperfusion injury of the rt smll intestine. J Surg Res 00; 99: - Kim JY, Jeong HG. Down-regultion of inducible nitric oxide synthse nd tumor necrosis fctor-lph expression by bisphenol A vi nucler fctor-kppb inctivtion in mcrophges. Cncer Lett 00; 96: Willim E, Pul M. Fundmentl Immunology th edition: Lippincott Willims & Wilkins Publishers, 00 Wtnbe T, Sto Y, Ichid T, Ymmoto S, Oy H, Nktsuk H, Kobyshi T, Htkeym K. Comprison of urinry ulinsttin levels between donors nd recipients immeditely following dult living relted donor liver trnsplnttion. Trnsplnt Proc 00; : Itbshi K, Ito Y, Tkhshi T, Ishii K, Sto K, Kkit A. Protective effects of urinry trypsin inhibitor () on heptic microvsculture in hypotensive brin-ded rts. Eur Surg Res 00; : 0-8 Science Editor Li WZ Lnguge Editor Elsevier HK