Growth Hormone Releasing Peptide 6 (GHRP6) reduces liver fibrosis in CCl 4. chronically intoxicated rats

Transcription

1 Growth Hormone Relesing Peptide 6 (GHRP6) redues liver firosis in hronilly intoxited rts Jorge Berlng-Aost 1, Dni Vázquez-Blomquist 2, Dny Cirián 1, Yssel Mendoz 1, Mrí E Ohgví 3, Jmilet Mirnd 3, José Suárez 4, Yolnd González-Ferrer 7, José M Vil 7, Angel Areu 7, Dyn Ugrte-Moreno 7, Yolnd Cruz 7, Ivon Howlnd 7, Ros Coro-Antih 8, Olg S León 9, Rirdo Brings 3, Din Grí-del Bro 1, Kreli Cosme-Díz 4, Dniel Plenzuel 2, Julio R Fernández 2, Mrelo Nzl 2, Isel Guillén 2, Alerto Cintdo 2, Lidi Inés 2, Ernesto López-Mol 5, Gerrdo E Guillén-Nieto 6 1 Deprtments of Tissue Repir nd Cytoprotetion, 2 Genomis, 3 Bioinformtis nd 4 Animl Cre; 5 Business Development Group nd 6 Diretion of Biomedil Reserh Center for Geneti Engineering nd Biotehnology, CIGB Ave. 31 / 158 nd 190, Ply, PO Box 6162, CP , Hvn, Cu 7 Center for Medil nd Surgil Reserh 8 Institute of Neurology nd Neurosurgery, INN 9 Center for Biologil Studies, Institute of Phrmy nd Food, University of Hvn, UH E-mil: jorge.erlng@ig.edu.u ABSTRACT Tissue firosis is leding use of moridity nd mortlity. Current tretments for onditions suh s hepti firosis hve een unsuessful. The growth hormone relsing peptide 6 (GHRP6) is endowed with rdioprotetive tions ut its ntifiroti effet hd not een ntiipted. We exmined the GHRP6 ility to prevent nd revert liver irrhosis fter indution in Wistr rts y suutneous dministrtion of. GHRP6 effets were exmined fter onomitnt nd delyed dministrtion to toxi respetively. The perentges of hepti ft, firosis, nodulrity nd septe thikness were histologilly nd morphometrilly determined. Asitis nd portl diltion were judged y ultrsound nd serum iohemil profile nd oxidtive stress prmeters determined. Mehnisti involvement of seletive gene/proteins ws ssessed y RT-PCR nd immunohistohemistry. Mirorrys showed gene expression profiles of GHRP6-treted liver smples on CpitlBio Rt Genome Oligo Arry. GHRP6 onomitnt intervention prevented in more thn 85% prenhyml firoti indurtion (p < ) nd therpeuti dministrtion for only 15 dys llowed for 37% firoti lerne (p = ) with more thn 30% redution of septe thikness (p = ). The 60 dys GHRP6 dministrtion sheme produed 75% redution of the firoti re with more thn 60% redution of nodulrity. GHRP6 redued oxidtive dmge enhning the tivity of ntioxidnt enzymes. Vimentin nd lph smooth musle tin immunodetetion profile indited GHRP6 redued the numer of tivted stellte ells. GHRP6 dministrtion redued firogeni ftors s TGF-β nd CTGF on Kupffer ells. Differentilly expressed genes in the mirorry experiment indited GHRP6 modulte the redox lne nd prenhyml ells response to injury. These evidenes suggest GHRP6 my ontrol the liver s firoplsti response. Keywords: Seretgogue, GHRP6, liver, firosis, irrhosis, ron tetrhloride, ytoprotetion RESEARCH Corresponding uthor Biotenologí Aplid 20;29:60-72 RESUMEN El péptido lierdor de l Hormon de Creimiento-6 (GHRP6) redue l firosis del hígdo en rts intoxids rónimente on. L firosis es us fundmentl de morilidd y mortlidd. Los trtmientos tules hn frsdo. El péptido lierdor de l hormon de reimiento-6 (GHRP6) ejere efetos rdioprotetores, pero no se h desrito su ión ntifiróti. Se exminó l propiedd del GHRP6 pr prevenir y revertir l irrosis hepáti, luego de su induión en rts medinte l dministrión de. Se evluó el porentje de grs hepáti, firosis, nodulridd y grosor septl medinte estudios histomorfométrios y l sitis o diltión portl por ultrsonido. Se determinó el perfil ioquímio y los prámetros de estrés oxidtivo en suero, sí omo l prtiipión de genes y proteíns, medinte reión en den de l polimers on trnsripión invers e inmunohistoquími. Se utilizó un mirorreglo de oligonuleótidos del genom de rt pr estudir el perfil de expresión de los genes induidos por el GHRP6. L intervenión onomitnte on GHRP6 previno l indurión firóti en más del 85% (p < ). L dministrión terpéuti durnte 15 dís permitió l remoión firóti del 37% (p = ), l reduión del grosor septl superó el 30% (p = ). L dministrión durnte 60 dís redujo ls áres firótis en 75%, y l reduión de l nodulridd fue de más del 60%. El GHRP6 redujo el dño oxidtivo porque umentó l tividd de ls enzims ntioxidntes y ls éluls estrellds tivds positivs vimentin y tin lf de músulo liso. Tmién redujo l expresión de ftores firogénios sore ls éluls Kupffer. El perfil de expresión de genes indió que el GHRP6 modul el lne redox y l respuest l dño tisulr en ls éluls prenquimles. Ests evidenis sugieren que el GHRP6 pudier ontrolr l respuest firoplási del hígdo. Plrs lve: Seretgogo, GHRP6, hígdo, firosis, irrosis, tetrloruro de rono, itoproteión

2 Introdution Liver firosis is the finl ommon pthwy of mny humn hepti diseses nd represents mjor soure of moridity nd mortlity worldwide [1]. In ontrst to the trditionl view tht liver firosis is n irreversile disese, reent evidenes otined from niml models nd ptients indite tht dvned irrhosis my e meliorted [2-5]. Hepti firosis is the wound-heling response of the liver to hroni injury [6]. After n ute liver injury, prenhyml ells regenerte nd reple the neroti nd/or poptoti heptoytes. This proess is ssoited with ontrolled inflmmtory response nd limited deposition of extrellulr mtrix (ECM) proteins. If the injury persists, the liver regenertion proess fils nd the prenhyml heptoytes re repled y undnt ECM proteins tht disrupt the hepti rhiteture y forming irrhoti nodules. This indues heptoellulr dysfuntion nd inreses intrhepti resistne to lood flow, whih results in liver insuffiieny nd portl hypertension, respetively [7]. The growth hormone relesing peptide 6 (GHRP6) is six-mino ids syntheti peptide tht elongs to the growth hormone seretgogues (GHS) fmily. Besides its first desried GH-relesing tivity [8], mounting evidenes sustntite tht GHRP6 nd their nlogs exhiit ever expnding phrmologil effets inluding ytoprotetion [9-13]. We hd previously demonstrted tht single GHRP6-prophylti dministrtion prevented heptoytes demise in stringent setting of hepti ishemi [11]. Whether this GHRP6-indued heptoprotetive effet hd impt in liver firosis remined unexplored. Nevertheless, serendipitous oservtions inspired this study, when we oserved tht rts ffeted y doxoruiin-indued dilted myordiopthy nd treted with GHRP6 exhiited fr less firosis in their mjor prenhyml orgns thn their sline-treted ounterprts. This work demonstrtes for the first time tht GHRP6 intervention sustntilly ttenutes the onset of firoti proess s well s triggers the regression of irrhosis in hronilly intoxited rts. The intervention ppered to mplify hepti ells detoxifition mehnisms whih my ultimtely ttenute hepti stellte ells (HSC) tivtion nd the onset of firogeni progrm. Mterils nd methods Regents The GHRP6 (His-D-Trp-Al-Trp-D-Phe-Lys-NH 2 ) ws purhsed from BCN-Peptides (Brelon, Spin). The produt ws ertified s sterile, pyrogen-free white powder with 95% purity. For niml dministrtion, fresh solutions were lwys prepred y diluting the peptide in sterile norml sline solution. nd minerl oil were purhsed from Merk (Drmstdt, Germny). The ntiodies nti- Trnsforming growth ftor et (TGF-β), nti-p53, nti-cylin D1 nd nti-fsl were purhsed from Snt Cruz Biotehnology In. (USA). The nti- lph smooth musle tin (α-sma) nd nti-vimentin monolonl ntiodies were purhsed from DkoCytomtion (Denmrk). Animls A totl of 75 mle Wistr rts ( g, 9-10 weeks) were purhsed from the Ntionl Center for the Prodution of Lortory Animls (Hvn, Cu). The rts were mintined in ertified room t the Animl Fility of the Center for Geneti Engineering nd Biotehnology (Hvn, Cu), under ontrolled environmentl onditions nd with unrestrited nimls ess to food nd wter. The study protool ws pproved y the institutionl ord on lortory nimls welfre. Firosis indution protool Liver firosis ws indued y the suutneous injetion of twie week (Mondy nd Fridy), for five or seven months. The seleted dose ws 1 ml/kg, diluted s 1:1 proportion with minerl oil efore injetion [14]. Phses nd experimentl groups Seven intt nimls reeived minerl oil for seven months nd were terminted one the study ws ompleted (Intt ontrol group). The 68 remining rts were sujeted to the firosis indution protool s mentioned ove. As this study imed to exmine the potentil effet of the GHRP6 intervention towrd oth firosis prevention nd regression, two experimentl loks were estlished. The first one, developed during the five initil months, inluded the onomitnt dministrtion of GHRP6 with to ssess hepti firosis prevention. The seond ws onduted for the sixth nd seventh months while GHRP6 ws therpeutilly dministered to ssess its potentil in promoting irrhosis regression. Experimentl groups for the prevention tril The groups nmed + GHRP6 nd + Sline reeived s previously desried while reeiving onomitntly two dily intrperitonel (i.p.) doses of GHRP6 (400 μg/kg) or norml sline injetions, respetively, for five months. Both groups were of rts eh nd ll the nimls were utopsied upon onluding the fifth month of the study. Experimentl groups for the regression tril The 44 remining rts lso reeived s previously desried during the five initil months of the study. Afterwrds, ll these nimls were individully sujeted to dignosti lprotomy (see elow). This llowed for lloting the nimls into four lned groups ording to their hepti disese severity. Two groups of 10 rts eh (denominted GHRP6-15d nd Sline-15d) reeived two dily i.p. injetions of GHRP6 (dose 400 μg/kg) or norml sline, respetively, for 15 dys, strting t the eginning of the sixth month. The dministrtion ws interrupted during the pplition of tretments. All these nimls were utopsied when the short time intervention ws ompleted. The lst two groups omprised rts eh, denominted GHRP6-60d nd Sline-60d, respetively. These nimls were treted either with GHRP6 or norml sline s desried for groups GHRP6-15d nd Sline-15d, ut for 60 dys (sixth nd seventh 1. Lefton HB, Ros A, Cohen M. Dignosis nd epidemiology of irrhosis. Med Clin North Am. 2009;93(4): Hmmel P, Couvelrd A, O Toole D, Rtouis A, Suvnet A, Flejou JF, et l. Regression of liver firosis fter iliry dringe in ptients with hroni pnretitis nd stenosis of the ommon ile dut. N Engl J Med. 2001;344(6): Arim M, Tero H, Kshim K, Arit T, Nsu M, Nishizono A. Regression of liver firosis in ses of hroni liver disese type C: quntittive evlution y using omputed imge nlysis. Intern Med. 2004;43(10): Iss R, Zhou X, Constndinou CM, Fllowfield J, Millwrd-Sdler H, G MD, et l. Spontneous reovery from mironodulr irrhosis: evidene for inomplete resolution ssoited with mtrix ross-linking. Gstroenterology. 2004; 6(7): Bourliere M, Khloun A, Gsou- Tessonnier G. Anlogs nd firosis regression in heptitis B. Gstroenterol Clin Biol. 2009;33(10-11): Friedmn SL. Liver firosis -- from enh to edside. J Heptol. 2003;38 Suppl 1:S Gines P, Crdens A, Arroyo V, Rodes J. Mngement of irrhosis nd sites. N Engl J Med. 2004;350(16): Bowers CY, Momny FA, Reynolds GA, Hong A. On the in vitro nd in vivo tivity of new syntheti hexpeptide tht ts on the pituitry to speifilly relese growth hormone. Endorinology. 1984; 114(5): Shen YT, Lynh JJ, Hrgreves RJ, Gould RJ. A growth hormone seretgogue prevents ishemi-indued mortlity independently of the growth hormone pthwy in dogs with hroni dilted rdiomyopthy. J Phrmol Exp Ther. 2003; 306(2): Pned C, Arro AI, Frgo LM, Holm AM, Romer J, Argente J, et l. Growth hormone-relesing peptide-6 inhiits ereellr ell deth in ged rts. Neuroreport. 2003;14(): Cirin D, Ajmieh H, Berlng J, Leon OS, Al JS, Kim MJ, et l. Use of growthhormone-relesing peptide-6 (GHRP-6) for the prevention of multiple orgn filure. Clin Si (Lond). 2006;110(5): Berlng J, Cirin D, Guevr L, Dominguez H, Al JS, Serlen A, et l. Growth-hormone-relesing peptide 6 (GHRP6) prevents oxidnt ytotoxiity nd redues myordil nerosis in model of ute myordil infrtion. Clin Si (Lond). 2007;1(4): Grndo M, Mrtin AI, Lopez-Menduin M, Lopez-Clderon A, Villnu MA. GH-relesing peptide-2 dministrtion prevents liver inflmmtory response in endotoxemi. Am J Physiol Endorinol Met. 2008;294(1):E Ae W, Ikejim K, Lng T, Okumur K, Enomoto N, Kitmur T, et l. Low moleulr weight heprin prevents hepti firogenesis used y ron tetrhloride in the rt. J Heptol. 2007;46(2): Biotenologí Aplid 20; Vol.29, No.2

3 experimentl months).the dministrtion ontinued in oth groups until the end of the seventh month, when ll these rts were finlly utopsied. Hepti ultrsound All the nimls of the + GHRP6 nd + Sline groups were sujeted to omprtive ultrsoni study fter five months of onomitnt nd tretments dministrtions. Animls from GHRP6-60d nd Sline-60d groups were omprtively evluted y ultrsound t the end of the seventh month. The rts from the Intt ontrol group were onurrently evluted in eh se. Ultrsounds were performed with n Alok pprtus (Jpn) onneted to n 11 MHz trnsduer in previously nesthetized (Ketmine, 50 mg/ kg) nimls. The studied prmeters inluded: portl dimeter (mm), sites, prenhyml nodulrity, ehogeniity inrese nd liver texture evlution. Asites ws sored ording to the following riteri: 0- no sites, 1- smll quntity of sites tht is only detetle y ultrsound, 2- linilly evident sites. For prenhyml nodulrity sle from 0 to 3 ws used to sore the result: 0- no nodules, 1- one or two nodules, 2- fint multinodulrity nd 3- multinodulr imges with lrge size nodules inluded. Ehogeniity inrese ws qulittively grded ording to the following sle: 0- no inrese, 1- moderte inrese nd 2- signifint inrese of prenhyml ehogeniity. Liver texture ws grded s follows: 0- homogeneous texture; 1- heterogeneous, fintly grnulted; nd 2- heterogeneous, grossly grnulted. The ultrsoni firosis index (UFI) ws defined s the totl sum of the vlues otined in these four grdtion sles. To void ised judgment ll the imges from hepti ultrsound were lindly sored. Dignosti study y lprotomy The 44 rts inluded in the firosis regression phse were sujeted to dignosti study y lprotomy one onluding the five initil months of dministrtion. Briefly, smll dominl inision ws performed to previously nesthetized nimls (Ketmine, 50 mg/kg), nd y gentle mnipultion the whole liver mss ws fully exposed for mrosopi inspetion, lssifition nd soring ording to the World Helth Orgniztion sore: mronodulr, mironodulr, mixed nd ft orgn gross pperne [15]. Finlly, the liver ws ppropritely returned to its vity nd the smll wound ws sutured. Blned groups were mde up s judged y the hepti mrosopi spet. Liver histology For histopthology, three liver frgments from ll the nimls were hrvested during the utopsy, eh one from different hepti loe, nd were 10% uffered formlin fixed nd prffin emedded. Slides with semi-thin setions (2-3 μm) were prepred nd stined with hemtoxylin/eosin nd Mllory trihrome. All the histologil evlutions nd morphometri protools were onduted in linded mnner. Mllory stined slides were used to ssess the irrhoti nodulrity for eh niml. For this purpose, the totl numer of irrhoti nodules ws ounted in three eqully rndomly seleted mirosopi fields (5 ) per liver frgment ( totl exmined re of 10 mm 2 y hepti loe). The finl result ws presented s the verge of the nodules/mm 2 mong the three hepti frgments. Histomorphometri nlysis ws onduted using the ImgeJ progrm (NIH, USA). Digitl imges were ptured from the Mllory stined slides in RGB formt, with 24 it true olors nd t pixels resolution, through Crl Zeiss Axiotron mirosope (Germny) oupled to Cnon PC1089 mer (Cnon, Jpn). Ft nd firosis perentges were ssessed for ll the nimls using imges from three eqully rndomly seleted mirosopi fields (10 ) per liver frgment. The finl vlues from the nine ptured imges were verged to otin the representtive perentges of ft nd firosis hepti overed re for eh niml. In order to determinte the verged firoti septum thikness of eh niml, totl of 100 rndomly seleted mirosopi fields (40 ) ws ptured y niml, nd proessed with the lirted ImgeJ softwre. The thikness ws lwys mesured midst the whole septum length nd ws reported in mirons. Serum iohemil determintions Blood smples were olleted from the retro-oritl plexus of previously nesthetized rts from the intt ontrol group, on the first experimentl dy nd from ll the nimls of the + GHRP6 nd + Sline groups fter two months of experiment initition. The finl lood smples were hrvested y myordil puntion during the utopsies. Serum smples were liquoted nd kept t 20 C until proessing. lnine minotrnsferse (ALAT) nd sprtte minotrnsferse (ASAT) serum tivities; s the serum onentrtion of totl proteins (TP), lumin, very low density lipoprotein, holesterol nd triglyerides were ssessed in n utomti nlyzer Hithi 747 (Boehringer Mnnheim, Germny). Commeril kits nd nlytil proedures were onduted ording to the mnufturer s instrutions. Hepti oxidtive stress ssessment Liver entrl loe frgments olleted during the utopsies from ll the rts were used to ssess the hepti oxidtive stress. The protools for tissue homogentes nd superoxide dismutse (SOD) nd tlse enzymes tivities were followed s previously desried [11]. The lipid peroxidtion potentil (LPP) nd mlondildehyde (MDA) were mesured using the Bioxyteh LPO-586 ommeril kit; while the totl hydroperoxide ontent ws ssyed y the Bioxyteh H 2 O ommeril kit, oth ording to mnufturer s instrutions (Bio-Rd Lortories, Germny). The dvned oxidtion protein produts (AOPP) ontent ws ssessed ording to the Witko- Srst desried tehnique [16]. All the hepti iohemil dt were djusted to the totl protein onentrtion determined in the tissue homogentes using ommeril kit (Bio-Rd Lortories, Germny). Immunohistohemistry For immunohistohemistry studies, liver setions (2-3 μm) were mounted on silinized slides (DAKO, Denmrk), het-treted for ntigen exposure, nd proessed ording to the mnufturer s instrutions 15. Anthony PP, Ishk KG, Nyk NC, Poulsen HE, Sheuer PJ, Soin LH. The morphology of irrhosis: definition, nomenlture, nd lssifition. Bull World Helth Orgn. 1977;55(4): Witko-Srst V, Gusson V, Nguyen AT, Toum M, Drueke T, Sntngelo F, et l. AOPP-indued tivtion of humn neutrophil nd monoyte oxidtive metolism: potentil trget for N-etylysteine tretment in dilysis ptients. Kidney Int. 2003;64(1): Biotenologí Aplid 20; Vol.29, No.2

4 from DkoCytomtion LSAB TM + System-HRP ommeril kit. Tissue smples were inuted for 30 min with: nti-αsma (1:100), nti-tgf-β (1:250), ntip53 (1:200), nti-cylin D1 (1:100), nti-fsl (1:200) nd nti-vimentin (1:100). Antiodies were diluted in Dko kground reduing solution. Immunohistohemistry ws omplished on mteril retrieved from three representtive nimls per group purposely seleted ording to the histopthology judgment. Helthy nimls were lso inluded. Slides were ounterstined with hemtoxilin or light green nd were lindly nlyzed y two different investigtors. The numer of Cylin D1 positively leled heptoytes nulei nd FsL positively leled Kupffer ells within the hepti prenhym were quntified in 15 mirosopi fields (20 ), evenly distriuted in the three liver frgments olleted from GHRP6-15d nd Sline-15d groups only. Internl ontrols inluded liver setions from helthy intt rts nd omission or replement of the ommeril primry ntiody y pre-immune isospeies serum. Dt re presented s the verged vlue of the 45 mirosopi fields studied y group (three rts in eh one). Gene expression nlyses y semiquntittive RT-PCR Liver entrl loe frgments olleted from five rndomly seleted rts of the Sline-15d, GHRP6-15d nd Intt ontrol group were proessed to isolte totl RNA using TRI Regent (Sigm, St. Louis, USA). Totl RNA ws digested with RNse-free DNse I (Epientre Tehnologies, USA) ording to the mnufturer s instrutions for DNA ontminnt removl. Afterwrd, one mirogrm of totl RNA ws reverse trnsried using ommeril ville kit (GeneAmp RNA PCR Core Kit. Applied Biosystems, USA) with n oligo-dt primer. PCR were performed using speifi primers nd nneling tempertures referred in tle 1. Finl PCR produts were deteted in 1% (w/v) grose gel nd were quntified using the Kodk ID 3.6 softwre pkge (Kodk In, USA). β-tin ws used s housekeeping gene for normliztion. RNA extrtion for mirorry experiment Rts in Groups GHRP6-60d nd Sline-60d were heked for their perentge of firosis t the end of the seventh month nd firosis redution ws then lulted. Totl RNA isoltion ws rried out s for semiquntittive RT-PCR experiments nd further purified using NuleoSpin RNA len-up kit (Mherey- Ngel, Germny). The qulity of the totl RNA (i.e., the purity nd integrity of the intt RNA) ws ssessed y Nnodrop 1000 (ThermoSientifi, USA) nd Bionlizer Agilent 2100 (Agilent, USA), reporting the onentrtion, sorne 260/280 nm rtio of 1.8 or higher, nd RNA integrity numer equl to or higher thn 7, respetively. Five pired smples from GHRP6-60d nd Sline-60d tht met RNA qulity requirements nd exhiited firosis redution superior to 69% were used for the experiment. Mirorry experiment We hose referene design with five smples per groups GHRP6-60d nd Sline-60d ompred to referene, representing the seven pooled smples from the Intt ontrol group. The mplifition nd leling of mrna were performed using the CpitlBio RNA Amplifition nd Leling kit ording to mnufturing instrutions (CpitlBio, Beijing, Chin). The rt 27 K oligonuleotide mirorry omprises oligo proes of 70-mer (Cpitol-Bio Corportion, Beijing, Chin) from the Operon Compny (Rt Genome Oligo Set, Version 3.0.5). Dul hnnel mirorry hyridiztion ws performed with totl pmol of Cy3- leled ontrol smple nd Cy5-leled test smples (from GPHR6-60d nd Sline-60d) onto mm hips. Hyridiztion nd wshing of slides were rried out ording to the mnufturer instrutions (Cpitol-Bio Corportion, Beijing, Chin).The slides were snned with onfol LuxSn snner (CpitlBio Corp, Chin) nd the rw dt were extrted using LuxSn 3.0 softwre (CpitlBio Corp). For dul-hnnels mirorry dt, the snning setting for Cy3 nd Cy5 hnnels were lned. The signls deteted from housekeeping nd Hex genes, nd those deteted from the exogenous ontrols were used s positive ontrols. Negtive ontrols were t kground levels. Sttistil nlysis All the experimentl dt were initilly evluted for norml distriution using the Kolmogorov-Smirnov test (p < 0.05). When norml distriution ws estlished, n unpired Student s test ws used for omprisons etween the groups of prevention phse; while pired Student s t test ws used for the groups of the regression phse. In se of multiple omprisons, the one wy ANOVA followed y the Student-Newmn- Tle 1. Primers sequenes nd PCR onditions used for gene expression nlyses Gene nme β-tin TGF-β CTGF SODMn MMP13 GenBnk ession # BC NM NM NM M60616 Primer sequenes (5-3 ) sense CCA TGT ACG TAG CCA TCC AGG ntisense GAC AGT GAG GCC AGG ATA GAG C sense TGC CAG AAC CCC CAT TGC TG ntisense TCC ACC TTG GGC TTG CGA CC sense AGA GCT GGG TGT GTG TCC TCC ntisense GCA GCA AAC ACT TCC TCG TGG sense GGA TGG AGT GGT AGA GCC TTT ntisense TCT ACA CCC AAA TGC TGC ACA GG sense AAA GGG GAT AAC AGC CAC TAC AAG G ntisense GAG GGA TTA ACA AAC ATG GTG GAG C Tm (ºC) Produt length (p) No. of Cyles Biotenologí Aplid 20; Vol.29, No.2

5 Keuls test ws used. The perentge vlues were ompred using the Fisher s ext test. A vlue of p < 0.05 ws used to indite signifint differene. The R Limm pkge ( limm) ws used for preproessing nd differentil expression nlysis of mirorry dt [17]. The medin verge intensity of foreground nd kground were extrted from the lsr files. A qulity riterion [18] ws pplied to identify low intensity or high kground spots ssigning weights 0 or 1, whih were lter used s inputs for limm pproh. The normexp+offset method ws seleted for dptively djusting the foreground for the kground intensities. The within-rrys normliztion ws performed with the print-tip loess method using etween hnnels non-differentilly expressed ontrols nd for etween-rry normliztion the sle method using ll ontrol proes. The moderted pired t-test for eh gene ws lulted. Genes with p < [18] nd fold hnge greter thn 1.5 were onsidered for ioinformtis nlysis nd iologil interprettion. Bioinformtis nlysis of differentilly expressed gene Bioinformtis nlysis ws performed using, s input, the list of differentilly expressed genes in rt (GHRP6-60d vs. Sline-60d) nd lso the list of their humn orthologous in order to tke enefit of the funtionl nnottion ville on humn genes iming to predit signifint iologil proess involved in the puttive GHRP6-medited ntifiroti mehnisms. Humn orthologous for rt genes were identified y serhing the Homologene dtse. Whenever orthologous genes were not found in Homologene, sequene similrity serhes of rt s trnsripts ginst the humn RefSeq trnsripts were performed with Blstn. The most similr gene, for every rt gene, ws hosen s the est Blstn hit (gene with lowest E-vlue, E-vlue < ). To identify puttive protein-protein intertions, in whih the protein produts of the input genes re involved, gene networks were onstruted using the Cytospe s [19] plugin BisoGenet [20]. All moleulr intertion dt soures ville t BisoGenet were used to generte the gene networks. These networks were enrihed with trnsription regultion dt extrted from the literture. The Cytospe s plugin BiNGO [21] ws used to identify the Gene Ontology (GO) [22] iologil proesses enrihed in the lists of input genes. The sttistilly signifint GO proesses were determined y using the hypergeometri test nd the Benjmini- Hoherg Flse Disovery Rte (FDR) orretion for multiple testing [23] with threshold of Results Hepti ultrsound explortion At the fifth month, the omprtive ultrsoni study etween + GHRP6 nd + Sline groups demonstrted signifint differene (p = ) etween the lulted UFI vlues, thus suggesting tht GHRP6 prevented firosis (Tle 2). The nimls of the GHRP6-60d nd Sline-60d groups were lso sujeted to omprtive study y ultrsound t the seventh month. At this time point, the Sline-60d Tle 2. Results from hepti ultrsound studies Experimentl groups Intt ontrol + GHRP6 + Sline GHRP6-60d Sline-60d N 7 UFI ± ± 0.79*** 4.80 ± ± 0.29*** Portl dimeter (mm) hieved the lrgest UFI vlue registered (Tle 2), whih ws signifintly different (p = ) to tht of GHRP6-60d group; thus inditing tht proess of firosis regression hd een set forth. The portl vein ppered dilted over 60% in the nimls from the + Sline group t the fifth month, s ompred to the Intt ontrol group (p = ; tle 2). In ontrst, the verged portl dimeter for the + GHRP6 group did not differ from the norml vlues (p = ). At the seventh month, one the regression phse onluded, the GHRP6-60d group showed signifint redution of portl diltion s ompred to the Sline-60d group (p = ). As shown in tle 2, signifint redution of the nimls ering sites ws deteted in the onomitnt (p = ) nd the therpeuti GHRP6 for 60 dys (p = ). No sites ws deteted for the GHRP6-15d nd Sline-15d groups during the short time of therpeuti intervention. Liver histopthology After five months of ontinuous dministrtion, the nimls from the +Sline group showed dense ollgen undles surrounding irrhoti nodules (Figure 1A) whih represented out 17% of hepti re overed y firosis (Tle 3). However, the onomitnt intervention with GHRP6 prevented in more thn 85% the firoti indurtion (p < ; figure 1B). In line with this, the + GHRP6 group showed fr less irrhoti nodules nd verged lesser septum thikness s ompred to the +Sline group (oth p < ). These findings sustin the ultrsound evidenes of GHRP6-medited nti-firoti response. Moreover, the perent of firosis, the numer of irrhoti nodules, nd the verged septum thikness of the sline-treted nimls within the reversion tril for 15 dys were very lose to those of the + Sline group (ll p > 0.05); whih indited tht no relevnt spontneous firosis resolution took ple during the 15 dys in whih the ws not injeted (Figures 1C nd D; tle 3). The therpeuti dministrtion of GHRP6 for 15 dys in the first reversion protool, llowed for 37% of firosis lerne (p = ). It ws minly due to redution of more thn 30% of septe thikness (p = ). No differenes were found in the numer of irrhoti nodules (p = ) etween the groups (Tle 3). After seven months of ontinuous dministrtion, the + Sline-60d group rehed the lrgest Clinil sites (%) Ultrsound sites (%) 0.71 ± ± (8) 2(17) 1. ± 0.14* 5(42) 8(67) 0.96 ± (25) 1.34 ± 0.13* 5(42) 8(67) Ultrsounds to + GHRP6 nd + Sline groups were onduted t the fifth experimentl month while to GHRP6-60d nd Sline-60d groups were t the seventh month. Portl dimeter vlue reported for Intt ontrol group ws otined t seventh month. UFI: Ultrsoni firosis index. Dt from UFI nd portl dimeter re presented s verge ± SEM y group. Asites results re indited s the totl numer of nimls nd s perentge, y group. Ultrsound sites dt lso inlude the linil sites. (*/***) indite signifint differenes etween the GHRP6-treted nimls nd their ounterprt Sline groups for t lest p < Smyth GK. Liner models nd empiril yes methods for ssessing differentil expression in mirorry experiments. Stt Appl Genet Mol Biol. 2004;3:Artile Simon R, Korn E, MShne L, Rdmher M, Wright G, Zho Y. Design nd Anlysis of DNA Mirorry Investigtions. New York: Springer-Verlg; Shnnon P, Mrkiel A, Ozier O, Blig NS, Wng JT, Rmge D, et l. Cytospe: softwre environment for integrted models of iomoleulr intertion networks. Genome Res. 2003;13(11): Mrtin A, Ohgvi ME, Rs LC, Mirnd J, Fernndez-de-Cossio J, Brings R. BisoGenet: new tool for gene network uilding, visuliztion nd nlysis. BMC Bioinformtis. 2010;11: Mere S, Heymns K, Kuiper M. BiN- GO: Cytospe plugin to ssess overrepresenttion of gene ontology tegories in iologil networks. Bioinformtis. 2005;21(16): Ashurner M, Bll CA, Blke JA, Botstein D, Butler H, Cherry JM, et l. Gene ontology: tool for the unifition of iology. The Gene Ontology Consortium. Nt Genet. 2000;25(1): Benjmini Y, Hoherg Y. Controlling the flse disovery rte: prtil nd powerful pproh to multiple testing. J R Stt So Ser B. 1995;57(1): Biotenologí Aplid 20; Vol.29, No.2

6 perent of firosis whih ws signifintly different from the + Sline group (p = ) t the fifth experimentl month, inditing progression in the disese severity due to sustntil inrese in septl thikness. As shown in tle 3, the therpeuti dministrtion of GHRP6 y 60 dys hieved 75% redution of the firoti re when ompred to the Sline-60d group (p < ), even though the injetions were not interrupted (Figure 1F). Tle 4 shows the redution in firosis from eh of the twelve pirs of liver from GHRP6-60d nd Sline- 60d groups. A redution higher thn 69% is oserved in pirs 3, 4, 7, 8, 10, 11, nd higher thn 80% in pirs 3, 7 nd 8. Aordingly, the GHRP6-60d group showed signifint redutions in the irrhoti nodules/ mm 2 nd the septe thikness s ompred to the Sline-60d group (oth p < ). The + GHRP6 group exhiited two-fold inrese of hepti ft perentge with respet to the + Sline group (p = ; tle 3). At the seventh month, the GHRP6-60d group showed 38% inrese of ft deposition ompred to the Sline-60d group (p = ). In ontrst, in the rts from the GHRP6-15d group, where the injetions were interrupted, notorious redution of ft ws deteted s ompred to the Sline-15d group (p < ). A C E B D F Serum iohemistry In the firosis prevention tril the serum iohemil nlysis ws done fter the seond nd the fifth month of ontinuous nd GHRP6/Sline dministrtions. At the seond month, the + Sline group showed the highest ASAT nd ALAT levels deteted s ompred to the Intt ontrol group (oth p < 0.001; figures 2A nd B). These vlues dropped y the fifth experimentl month lthough remined signifintly superior to the helthy nimls (oth p < 0.001). A similr iphsi ehvior for oth trnsminses ws oserved for the + GHRP6 group; whih resulted signifintly lower thn those deteted for the + Sline group t the seond month (oth p < 0.001). At the fifth month, the ASAT level of the + GHRP6 group ws lso signifintly lower thn the + Sline group (p < 0.001) while similr vlues for ALAT were oserved for oth groups. The -indued liver dmge ws ssoited to redution of the orgn s iosyntheti funtion in oth groups (Figures 2C to F). However, t the two evlution time points, the + GHRP6 group exhiited signifintly etter liver funtion s ompred to the + Sline group for ll the evluted prmeters (t lest p < 0.05). In the firosis regression tril, serum hemil prmeters in the GHRP6-15d nd Sline-15d groups were evluted one the short therpeuti sheme ws ompleted. As showed in tle 5, signifint differenes for ALAT (p < 0.01) nd ASAT (p < 0.05) vlues were deteted etween these groups refleting the GHRP6-indued heptoprotetive effet. Despite this, no signifint differenes were deteted etween the GHRP6-15d nd the Sline-15d groups in ny of the evluted hepti synthesis inditors, whih remined lower thn those of the Intt ontrol group (t lest p < 0.05). The serum iohemil prmeters from the GHRP6-60d nd the Sline-60d groups were Figure 1. Histopthologil imges from liver rts iopsy. Imges orrespond to Mllory stining slides (10 ) nd re representtive from the experimentl groups: A) + Sline; B) + GHRP6; C) Sline-15d; D) GHRP6-15d; E) Sline-60d; F) GHRP6-60d. A more intense degree of firosis (lue stined) is oserved in ll the Sline-groups in omprison with their ounterprt GHRP6-treted rts. Tle 3. Histomorphometri results Experimentl groups + GHRP6 + Sline GHRP6-15d Sline-15d GHRP6-60d Sline-60d N Septe thikness ± 1.66*** ± ± 4.62** ± ± 4.36*** ± 8.14 Nodules/mm 2 Firosis (%) Ft (%) 0.82 ± 0.23*** 1.99 ± 0.31*** ± 2.23*** 5.58 ± ± ± ± ± 1.15*** 7.01 ± 0.60*** 5.00 ± ± ± ± 0.42*** 5.90 ± ± 0.65*** ± ± 1.94** ± 1.23 The histomorphometri nlyses were onduted using Mllory stining slides. Dt re presented s verge ± SEM y group. The septum thikness ws determined in mirons. (**/***) indite signifint differenes etween GHRP6-treted nimls nd their respetively Sline groups for t lest p < Tle 4. Perentge of firosis redution in twelve pirs of livers* Pir Firosis redution (%) * It is shown the perentge of firosis redution in pired nimls (1 to ) fter the tretment with GHRP6. Pirs 3, 4, 7, 8 nd 10 to showed the highest redutions in firosis (> 69%). 65 Biotenologí Aplid 20; Vol.29, No.2

7 A Intt ontrol + Sline + GHRP6 B ASAT (U/L) ASAT (U/L) Dy 1 2 months 5 months 30 Dy 1 2 months 5 months C Cholesterol (mmol/l) D Triglyerides (mmol/l) Dy 1 2 months 5 months 0.30 Dy 1 2 months 5 months E VLDL (mmol/l) (U/L) F Totl proteins (g/l) Dy 1 2 months 5 months 30 Dy 1 2 months 5 months Figure 2. Serum iohemil ssessment of firosis prevention phse. Dt re presented s the verge ± SEM y group: Intt ontrol group, + Sline nd + GHRP6. Sttistil nlyses were onduted t the seond nd fifth months, respetively. Different letters indite signifint differenes for t lest p < A) Asprtte minotrnsferse (ASAT); B) Alnine minotrnsferse (ALAT); C) Cholesterol; D) Triglyerides; E) Very low density lipoprotein (VLDL); F) Totl proteins. lso ssessed following utopsy (Tle 6). No signifint differenes were deteted etween these groups for the serum trnsminses s for ny of the orgn s funtionl prmeters (t lest p < 0.05). Hepti oxidtive stress An etiopthogeni ingredient of the heptotoxi mehnism is the oxidtive dmge to liver ells [24]. It ws onfirmed y the deteted inrese of the evluted oxidtive stress mrkers (totl hydroperoxide ontent, MDA, LPP nd AOPP) in ll the experimentl groups, s ompred to the Intt ontrol group (t lest p < 0.001; tle 7). The GHRP6 intervention signifintly ttenuted ll these oxidtive mrkers s ompred with eh respetive sline group (t lest p < 0.01), in the three interventionl pprohes ssessed. Conurrently, the GHRP6-treted rts exhiited remrkle inrese of Ctlse nd SOD tivities s ompred to their ounterprt sline groups (t lest p < 0.05). Immunohistohemil results The α-sma protein is moleulr mrker rodly used to detet tivted HSC [25, 26]. Although sed on qulittive judgment, nimls intervened with GHRP6 either under onomitnt (dt not shown) or therpeuti pprohes exhiited fr less α-sma leling thn their sline ounterprts (Figures 3A Tle 5. Serum iohemil ssessments of the 15 dys firosis regression sheme* Prmeters GHRP6-15d group (n = 10) Sline-15d group (n = 10) Intt ontrol group (n = 7) ALAT (U/L) ASAT (U/L) Cholesterol (mmol/l) Triglyerides (mmol/l) Alumin (g/l) Totl proteins (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.01 *Serum iohemil prmeters were ssessed fter 15 dys of GHRP6 or Sline firosis regression tretments. Dt re presented s the verge ± SEM y group. Different letters indite signifint differenes for t lest p < ASAT: sprtte minotrnsferse; ALAT: lnine minotrnsferse. 66 Biotenologí Aplid 20; Vol.29, No.2

8 Tle 6. Serum iohemil ssessments of the 60 dys firosis regression sheme* Prmeters ALAT (U/L) ASAT (U/L) Cholesterol (mmol/l) Triglyerides (mmol/l) VLDL (mmol/l) Alumin (g/l) Totl proteins (g/l) GHRP6-15d group (n = 10) ± ± ± ± ± ± ± 1.33 Sline-15d group (n = 10) Intt ontrol group (n = 7) ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.59 * Serum iohemil prmeters were ssessed fter 60 dys of GHRP6/Sline firosis regression tretments. Dt re presented s the verge ± SEM y group. Different letters indite signifint differenes for t lest p < ASAT: sprtte minotrnsferse; ALAT: lnine minotrnsferse; VLDL: very low density lipoprotein. to D). The immuno-detetion of the α-sma ntiody ppers prtiulrly restrited to the externl sides of the firoti septe where it hs een reported tht tivted HSC re onfined [27]. It is worthy to highlight tht n ppreile redution in the numer of leled ells ws hllmrk in the GHRP6-derived smples, inluding to those fields in whih the firoti septe ppered similr in terms of thikening nd ellulr density (Figures 3C nd D). Similrly, TGF-β ppered fr less expressed in those smples derived from GHRP6-treted rts from oth preventive nd regression phses (Figures 3E to H). As disussed elow, our immunostining for TGF-β lso ppered restrited to the firoti septe where it is nhored to the ECM proteins [28]. Vimentin is lso used s moleulr mrker for tivted HSC nd Kupffer ells [29, 30]. GHRP6- treted nimls within the prevention tril showed fr less Vimentin positive ells (in the firoti septe s within the hepti prenhym) thn the sline group (Figures 4A nd B). This result ws lso similr for the smples of the firosis regression tril (dt not shown). As judged y ell morphology nd topogrphi lotion (ore of the firoti septe nd hepti prenhym), lineges immunoleled with the nti-p53 orrespond to reruited round mononuler ells, Kupffer nd HSC. Remrkly, the most intense signl nd mount of positive nti-p53 ells were deteted in the GHRP6 treted nimls; in oth prevention (dt not shown) nd regression trils (Figures 4C nd D). Although doule immunohistohemistry ws not onduted, FsL expression speifilly mthed to Kupffer ells, s suggested y its morphology nd lotion. A lrger inrese of FsL positively leled Kupffer ells ws deteted in the Sline-15d group (14.22 ± 6.17 vs ± 2.; p < ). Conversely, the nimls of the GHRP6-15d group showed remrkle inrese of Cylin D1 positively leled heptoytes nulei, s ompred to Sline-15d group (8.65 ± 2.36 vs ± 1.59; p < ), whih is likely n inditive of GHRP6-indued prenhyml regenertion [31]. A similr result of FsL nd Cylin D1 expression ptterns ws otined in the remining experimentl groups (dt not shown). RT-PCR nlysis The trnsriptionl profile of firosis-ommitted trget genes ws studied in the GHRP6-15d nd Sline- 15d groups (Figure 5). In omprison to intt rts, the sline treted nimls showed signifint enhnement on the trnsriptionl expression of TGF-β nd onnetive tissue growth ftor (CTGF; oth p < 0.001), two growth ftors with well-hrterized role in the firogeni proess [28]. A signifintly shrp expression redution for oth firogeni ytokines ws redily oserved in the GHRP6-15d group s ompred to the sline ounterprts (t lest p < 0.05). The lso indued signifint derese of the superoxide dismutse mngnese enzyme (SODMn) trnsriptionl expression in the Sline-15d group s ompred to the intt ontrol group (p < 0.05; figu-re 5). However, the SODMn trnsriptionl expression of the GHRP6-treted rts did not differ from the onstitutive levels found in the Intt ontrol group nd ws signifintly superior to the Sline-15d group (p < 0.01). Mtrix metlloprotese-13 (MMP13) is the mjor interstitil ollgense tht redues liver firosis y degrding the ECM proteins [32]. A drmti expression enhnement of MMP13 trnsriptionl 24. Bisi F, Alno E, Chirpotto E, Corongiu FP, Pronzto MA, Mrinri UM, et l. In vivo nd in vitro evidene onerning the role of lipid peroxidtion in the mehnism of heptoyte deth due to ron tetrhloride. Cell Biohem Funt. 1991;9(2): Crpino G, Morini S, Ginnni Corrdini S, Frnhitto A, Merli M, Siilino M, et l. Alph-SMA expression in hepti stellte ells nd quntittive nlysis of hepti firosis in irrhosis nd in reurrent hroni heptitis fter liver trnsplnttion. Dig Liver Dis. 2005;37(5): Tomnovi N, Borii I, Brsn D. Immunohistohemil nlysis of lph-sma nd GFAP expression in liver stellte ells. Vojnosnit Pregl. 2006;63(6): Rmm GA, Nir VG, Bridle KR, Shepherd RW, Crwford DH. Contriution of hepti prenhyml nd nonprenhyml ells to hepti firogenesis in iliry tresi. Am J Pthol. 1998;153(2): Gressner OA, Weiskirhen R, Gressner AM. Evolving onepts of liver firogenesis provide new dignosti nd therpeuti options. Comp Heptol. 2007;6: Wu HH, To LC, Crmer HM. Vimentin-positive spider-shped Kupffer ells. A new lue to ytologi dignosis of primry nd metstti heptoellulr rinom y fine-needle spirtion iopsy. Am J Clin Pthol. 1996;106(4): Tle 7. Hepti oxidtive stress results Groups THP AOPP MDA LPP Ctlse SOD Intt ontrol +Sline +GHRP6 Sline-15d GHRP6-15d Sline-60d GHRP6-60d.1 ± ± ± 2.7*** 86.6 ± ± 2.6*** ± ± 3.5*** 3.9 ± ± ± 0.7*** 26.8 ± ± 0.4*** 31.8 ± ± 1.4*** 0.22 ± ± ± ± ± ± ± ± ± 0.2*** 2.2 ± 0.2*** ± 38.8*** ± 3080*** 3.0 ± ± ± ± ± 0.1*** 2.2 ± 0.1*** ± 38.8*** ± 909*** 6.4 ± ± 0.3*** 2.8 ± ± 0.2** ± ± ± 35.8* ± 1648*** Dt re presented s the verge ± SEM of ll the nimls y group. THP: totl hydroperoxide; AOPP: dvned oxidtion protein produts; MDA: mlondildehyde; LPP: lipid peroxidtion potentil; SOD: superoxide dismutse. The vlues of THP, AOPP, MDA nd LPP re reported s nmoles/mg of totl proteins. The enzymti tivities of tlse nd SOD re reported s U/min per grm of tissue. */**/*** indite signifint differenes etween the GHRP6-treted nimls nd their ounterprt Sline groups for p < 0.05, p < 0.01 nd p < 0.001, respetively. 67 Biotenologí Aplid 20; Vol.29, No.2

9 levels ppered with the GHRP6 intervention s ompred to the sline group (p < 0.01). A B Mirorry nd ioinformti nlyses Mirorry experiment ws rried out ompring pired smples 4, 7, 8, 11, in Groups GHRP6-60d nd Sline-60d with the referene smple from the Intt ontrol group. The 1.5 -fold differentilly-expressed rt genes nd their homologous humn genes (ORT-Humn), reported in Homologene or identified y Blstn re shown in tle 8. The moleulr intertion network tht ws generted with BisoGenet, using differentilly-expressed rt genes s input dt, ontined only 41 genes nd 25 moleulr intertions. However, similr network generted from ORT- Humn genes inluded 386 genes nd 1883 intertions. After gene enrihment nlysis performed with BiNGO, the most iologilly signifint proesses were undersored s onsequene of the GHRP6 intervention. These were: oxidtion-redution nd response to wounding (Tle 9). For the former, memers of the ytohrome P450 fmily s CYP2A13, CYP2C18, CYP2C19 nd CYP2C9; ldoketoredutse (AKR1C1) nd UDP gluuronosyltrnsferse, lso ommitted in drugs nd xenoioti metolism pthwys, were inluded. Cysteine dioxygense type I (CDO1), prtiipting in redox proess, nd the NADP + -dependent isoitrte dehydrogense 1 (IDH1)/Pipeoli id oxidse (PIPOX), whih prtiipte in peroxisome pthwy, were lso inorported within the oxidtion-redution proess. Besides, Cdo1, lph-1-inhiitor 3 (A1i3), ogultion ftor X (F10), histidine-rih glyoprotein (Hrg), serine (or ysteine) peptidse inhiitor, lde A, memers 3N (Serpin3n) nd 3M (Serpin3m), trnsferrin (TF) nd hepidin ntimiroil peptide (HAMP) genes were relted to response to wounding. Disussion hroni dministrtion indued n overt irrhoti disese to otherwise norml rts, whih engendered systemi disturnes for the niml homeostsis. Herein, we provide the first evidenes suggesting tht lssi memer of the Bower s syntheti seretgogue peptides, GHRP6, is not solely endowed with rdioprotetive tions ut lso with nti-firoti effet. The evidenes derived from these experiments provide the fundmentls to onsider tht the GHRP6 intervention prevented the progression of liver firogeni proess nd lso triggered its regression. This result sustntites previous findings of our group [11] nd others [13] in terms of GHRP6-medited hepti tissue protetion. In this study three experimentl settings were estlished. The prophylti pproh ttempted to rerete linil ondition in whih ptient is thretened to evolve to firosis following triggering event. Further, the therpeuti intervention tril ws split in two linil shemes, one in whih the hepti hllenge ws interrupted nd n lterntive one relted to hroni liver insult. This work seems to e the first prelinil study in whih firosis regression effet is exmined in homogeneously lloted groups ording to sle of liver gross pthology [15]. C E G Figure 3. Immunohistologil detetion of lph smooth musle tin (α-sma) nd trnsforming growth ftor et (TGF-β) expression on firoti septe. Representtive imges of the lph-sma expression on the experimentl groups: A) GHRP6-60d; B) Sline-60d; C) GHRP6-15d; nd D) Sline- 15d. Representtive imges of the TGF-β expression on the experimentl groups: E) GHRP6-15d; F) Sline-15 d; G) +GHRP6; nd H) +Sline. Imges A, B, G nd H were ptured with 5 mgnifition, nd imges C, D, E, nd F with 40 mgnifition. A C Figure 4. Immunohistologil detetion of Vimentin nd p53 leled ells. Representtive imges of the Vimentin expression on the experimentl groups: A) + GHRP6 nd B) + Sline; 20 mgnifition. Representtive imges of the p53 expression on the experimentl groups: C) GHRP6-15d nd D) Sline-15d; 40 mgnifition. D F H B D 68 Biotenologí Aplid 20; Vol.29, No.2

10 A Intt ontrol Sline-15d GHRP6-15d TGF-β CTGF SODMn MMP13 β-tin B Expression rtio Intt Control Sline-15d CTGF TGF-β GHRP6-15d C Expression rtio Intt Control Sline-15d SODMn MMP13 GHRP6-15d Groups Groups Figure 5. RT-PCR nlyses of the TGF-β, onnetive tissue growth ftor (CTGF), superoxide dismutse mngnese enzyme (SODMn) nd mtrix metlloprotese-13 (MMP13) trnsripts. RNA smples from the livers of five rts from the group Sline- 15d, GHRP6-15d nd Intt ontrol groups were reverse trnsried. A) PCR mplifition produts. The expression results were normlized ginst β-tin nd represented in the grphis (B nd C) s the verge ± SEM y group, n = 5. Different letters indite signifint differenes for t lest p < The histomorphometri ssessment s the ultrsound study onurrently indited less firosis in ll the GHRP6 intervened nimls. The most remrkle result in terms of firosis mteril redution ws ssoited to the GHRP6 preventive sheme, where the liver prenhym ppered spred of nodulr orgniztion. Aordingly, the lowest UFI vlue ws lulted for this group. This ft highlights the heptoprotetive effet indued y GHRP6 in order to prevent prenhyml ells downfll nd susequent firogeni response. The short term therpeuti intervention (15 dys) ounted for signifint regression of liver firosis, minly expressed in firoti septe involution. Importntly, this effet ws ttined in senrio where no spontneous firoti resolution took ple in the sline group. Prenhyml nodules lerne long with septe thikness redution ws lso onfirmed in the 60 dys GHRP6-therpeuti sheme. It is noteworthy tht this effet ppered in senrio of ontinuous liver ggression s the dministrtion ws intentionlly mintined. One of the methodologil limittions of this study is the lk of portl venous pressure/flow mesurements. Alterntively, the most relile pproh undertken ws to ssess portl diltion y ultrsound s desried elsewhere [33, 34]. Generlly speking, the inidene of sites nd portl diltion ws srely registered within the GHRP6 groups, thus suggesting lesser mount of firoti umultion nd more physiologi hemodynmi performne. We do not rule out however, the hypothetil involvement of the GHRP6 indution of endothelil nitri oxide relese s key ftor ontrolling portl hemodynmi lne [35]. Ft quntifition results suggest tht mgnifition of the ftty liver phenotype is ssoited to the o-dministrtion of nd GHRP6 nd not to the GHRP6 intervention lone. Long term linil interventions with the ognte GHRP2 hve proved to e sfe in dwrf hildren [36]. In line with this, our long-term systemi dministrtion toxiity studies in helthy rts proved tht GHRP6 does not hrm the hepti tissue (Cosme-Díz K; unpulished dt). The Cylin D1 expression profile inites to speulte tht GHRP6 stimultes heptoytes mitosis. Whether this mitogeni response is diretly triggered y GHRP6 itself nd/or through the GH/Insulin-like growth ftor 1 xis remins to e exmined [37]; moreover, regenertion of the hepti mss ould hypothetilly explin the inrese of ft storge in onomitntly + GHRP6-treted rts. It ould lso explin the notiele ft storge redution in those nimls solely exposed to GHRP6 without orrelte inrese in ft serum mrkers. In generl terms, ALAT nd ASAT serum levels exhiited iphsi ehvior. The ute dmge phse, histologilly expressed s hepti stetosis nd nonprenhyml ells retivity (dt not shown), orrelted with the highest trnsminses levels. From this point onwrd, however, firosis severity inrese ppered ssoited to drsti trnsminses drop-down, irrespetive to the medition, whih hs een previously reported [38, 39]. The ft tht ALAT levels from the GHRP6-preventively treted 30. Geerts A, Elisson C, Niki T, Wielnt A, Veyens F, Pekny M. Formtion of norml desmin intermedite filments in mouse hepti stellte ells requires vimentin. Heptology. 2001;33(1): Stey DW. Cylin D1 serves s ell yle regultory swith in tively proliferting ells. Curr Opin Cell Biol. 2003;15(2): Wtne T, Niiok M, Hozw S, Kmeym K, Hyshi T, Ari M, et l. Gene expression of interstitil ollgense in oth progressive nd reovery phse of rt liver firosis indued y ron tetrhloride. J Heptol. 2000;33(2): Liu Y, Li L, Yu Z, Liu Q, Li Z, Wng Y, et l. Correltive study etween portl vein pressure nd portl hemodynmis in ptients with portl hypertension. Zhonghu Gn Zng Bing Z Zhi. 2002;10(2): Quintnilh LF, Mnnheimer EG, Crvlho AB, Predes BD, Dis JV, Almeid AS, et l. Bone mrrow ell trnsplnt does not prevent or reverse murine liver irrhosis. Cell Trnsplnt. 2008;17(8): Fiorui S, Antonelli E, Morelli A. Nitri oxide nd portl hypertension: nitri oxide-relesing derivtive of ursodeoxyholi id tht seletively releses nitri oxide in the liver. Dig Liver Dis. 2003;35 Suppl 2:S Meriq V, Cssorl F, Slzr T, Avil A, Iniguez G, Bowers CY, et l. Effets of eight months tretment with grded doses of growth hormone (GH)-relesing peptide in GH-defiient hildren. J Clin Endorinol Met. 1998;83(7): Biotenologí Aplid 20; Vol.29, No.2