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Lab. Anim. Res. 2010: 26(1), 69-74 Proteomic Analysis of Hepatic Ischemia and Reperfusion Injury in Mice Eun-Hae Cho, Jin-Hee Sung and Phil-Ok Koh* Department of Anatomy, College of Veterinary Medicine, Research Insituite of Life Sciences, Gyeongsang National University, Jinju, Korea Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. I/R injury induces serious hepatic dysfunction and failure. In this study, we identified proteins that were differentially expressed between sham and I/R injured livers. Animals were subjected to hepatic ischemia for 1 hr and were sacrificed at 3hr after reperfusion. Serum ALT and AST levels were significantly increased in I/R-operated animals compared to those of sham-operated animals. Ischemic hepatic lobes of I/R-operated animals showed the hepatic lesion with unclear condensation and sinusoidal congestion. Proteins from hepatic tissue were separated using two dimensional gel electrophosresis. Protein spots with a greater than 2.5-fold change in intensity were identified by mass spectrometry. Among these proteins, glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, dynamin-1-like protein, annexin A4, eukaryotic initiation factor 3, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, and proteasome beta 4 levels were significantly decreased in I/R-operated animals compared to those of sham-operated animals. These proteins are related to protein synthesis, cellular growth and stabilization, anti-oxidant action. Moreover, Western blot analysis confirmed that dynamin-1-like protein levels were decreased in I/R-operated animals. Our results suggest that hepatic I/R induces the hepatic cells damage by regulation of several proteins. Key words: Ischemia, liver, reperfusion (Received 17 November 2009; Revised version received 8 March 2010; Accepted 17 March 2010) xx w s» w š., xx w» y y w w e. xx x œ w w, g, ATP š, H +, Na +, Ca w y w s 2+ k (Rhodes and DePalma, 1980). w, xx z w w, w»» ù (Eum et al., 2007; Kuboki et al., 2007).» z 3Ê6 k s x œ reactive oxygen species (ROS)ƒ w s x üv *Corresponding author: Phil-Ok Koh, Department of Anatomy, College of Veterinary Medicine, Gyeongsang National University, 900 Gajwa-dong, Jinju 660-701, Korea Tel: +82-55-751-5809 Fax: +82-55-751-5803 E-mail: pokoh@gnu.ac.kr s š (Jaeschke, 2003). w,» z 18~24 tumor necrosis factor-α, interleukin-1, platelet-activating factor w w y x (neutrophil) x p w, s s x x ùkü (Jaeschke, 2006). xx y j» w s y w y ƒ v w. w x x w s x w w w š ƒ ù w w. z» y wš w. lò x f ICR (35-40 g, 40 ) k g(», ) l w w. x 69

70 Eun-Hae Cho et al. 23±2 o C, 50±5%ƒ y w š, x šx œ w. x w x l x z(institutional Animal Care and Use Committee, IACUC), x w x ³ w. ekl xx Abe et al., (2009) w ww. x pentobarbital sodium (50 mg/kg) w w., w w gš, x (portal vein) (hepatic artery) ƒ wì w (left liver lobe) x œ w xx w. xx w» w. xx w 1 z x t (reperfusion) ww š, 3 z x w. xx w š 1 (laparotomy) w. x» w, w heating pad w 36~37 o C g. en t ALT (aspartate aminotransferase), AST (alanine aminotransferase) e d w. z 3 xw heparin ¼ z 15,000 rpm 2 w x w š, Vet test 8008 (IDEXX-GmbH, Woerrstadt, Germany) w x ü AST ALT e d w. e j xx x 4% neutral buffered paraformaldehyde š w. š w š w z 70, 80, 90, 95, 100% ethanol, xylene w k n y š, paraffin embedding center (Leica, Wetzlar, Germany) s w. q v 4µm š, xylene, 100, 95, 90, 80, 70% ethanol kq v w e z hematoxylin eosin w š, permount w w z, Ÿwx (Leica, Wetzlar, Germany) w. º lysis buffer (8 M urea, 4% CHAPS, 0.2% Bio-Lyte ampholytes, 40 mm Tris-HCl)» ³ y(homogenization)w z» w. z e 10% TCA w q» wš w e Tris HCl (ph 7.6) z 1 g. e sample buffer (8 M urea, 4% CHAPS, 0.2% biolate, 40 mm Tris-HCl, 1% (v/v) pharmalytes, 100 mm DTT) wš, w d. Bradford (Bio-Rad, Hercules, CA, USA) d w. Ò Isoelectric focusing (IEF) w sample buffer (8 M urea, 2% CHAPS, 20 mm DTT, 0.5% IPG buffer, bromophenol blue) w š, 17 cm linear strip (ph 4-7 gradient, Bio-Rad) Protean isoelectric focusing cell (Bio-Rad) w focusing w. w 13 ww š, focusing 250 V 15, 10,000 V 3, 10,000 V 50,000 V¾ ww. Focusing óù strip 1% DTT sww equilibration buffer (6 M urea, 30% glycerol, 2% SDS, 50 mm Tris-HCl, bromophenol blue) 10, 2.5% iodoacetamide sww equilibration buffer 10 k z 7.5-17.5% gradient gel w» w. 4JMWFS º Ò» óù polyacrylamide gel š (12% acetic acid, 50% methanol) 90 š w z 50% ethanol 20 w š, 0.02% sodium thiosulfate 1 k z wš, silver nitrate (0.2% silver nitrate, 0.75 ml/l formaldehyde) 20 k z x (0.2% sodium carbonate, 0.5 ml/l formaldehyde) 3 w z 1% acetic acid g. gel image PDQuest software (Bio rad) w x x w. PDQuest mw k spot reduction (10 mm DTT, 0.1 M NH 4 HCO 3 ) 56 o C 45, alkylation (55 mm iodoacetamide, 0.1 M NH 4 HCO 3 ) 30 g. gel ƒ digestion buffer w gš, digestion w z d w rp z w z Voyager-DETM STR biospectrometry workstation (Applied Biosystem, Forster city, CA, USA)

Proteomic analysis of liver Injury 71 w. MALDI-TOF MS-FIT (http:// prospector.ucsf.edu/ucsfhtml13.4/msfit.htm) ProFound (http://129.85.19.192/profound_bin/webprofound.exe) mw w š, Sequence database SWISS PROT NCBI w. 8FTUFSOCMPU (30 g) 10% SDS-polyacrylamide gel» ww.» z poly-vinylidene fluoride membrane transferw š, membrane 0.1% Tween-20 sw Tris-buffered saline (TBST) w z immunoblotting ww. w anti-dynamin-1-like protein anti-tubulin (diluted 1:1000, Cell Signaling Technology, Beverly, MA, USA) w g. w HRP-conjugated rabbit IgG (1:5000; Cell Signaling Technology) gš ECL Western blot analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) w x y w. x m Student's t-test w P 0.05 m y w. kl n"45 "-5 xx w x y w» w t x ü AST ALT e d w (Figure 1). AST e 168.5±21.5 U/L ùkûš, x 2850.5±185.5 U/L x AST e AST e x ƒw y w. w, ALT e AST w y, ALT e 102±4.5 U/L ùkû š, x ALT e 3250.5±270.5 U/L, x ALT e ALT e x w ƒw y w. kl ne j xx w sƒ x š, s w jš w, s ƒ yw ùkù s ùküš (Figure 2A). ù x s ù, x q š, x xk ùkû, s w š, s ƒ yw Figure 1. Serum ALT and AST levels in sham-operated (sham) and ischemia/reperfusion-operated (I/R) animals. ALT and AST levels were significantly increased in I/R-operated animals. Data (n=5) are represented as mean±sem. *P<0.01. Figure 2. Histopathological photos of the liver in shamoperated (A) and ischemia/reperfusion-operated (B) animals. There is no lesion in the normal liver of sham-operated animals. Ischemic hepatic lobes of I/R-operated animals showed the hepatic lesion with nuclear condensation (arrows) and sinusoidal congestion (open arrows). Hematoxylin and Eosin stain. Scale bar=100 µm. (Figure 2B). kl n mùº 2-DE» mw x x w. 2-DE 1,200 spot ùkû, x x y 42 spot. x 2.5 intensity 11 spot MALDI-TOF mw. glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, dynamin-1-like protein (DLP-1), annexin A4, eukaryotic initiation factor 3 subunit I, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, proteasome beta 4. x w x (Figure 3, Table 1). Western blot xx x DLP-1 x w x (Figure 4).

72 Eun-Hae Cho et al. Figure 3. Two-dimensional SDS-PAGE analysis of proteins in liver tissue of sham-operated (A) and ischemia/reperfusion-operated (B) animals. Isoelectric focusing was performed at ph 4-7 using IPG strips, followed by second-dimensional separation on 7.5-17.5% gradient SDS gels stained with silver. Squares indicate the protein spots that were differentially expressed between shamand I/R-operated animals. Table 1. List of identified proteins that were significantly differentially expressed in sham- and I/R-operated groups Spot No. Protein name Accession No. Mw (kda) pi Mass matched Coverage (%) Ratio of I-R / Sham 1 Eukaryotic initiation factor 4A P60843 46125 5.32 8/74 25 0.32±0.02* 2 Dynamin-1-like protein Q8K1M6 82606 6.61 13/141 24 0.33±0.01* 3 Glutaredoxin-3 Q9CQM9 37754 5.42 6/114 31 0.32±0.01* 4 Eukaryotic initiation factor 3 Q9QZD9 36438 5.38 9/89 28 0.33±0.02* 5 Spermidine synthase Q64674 33973 5.31 7/85 25 0.32±0.03* 6 Annexin A4 P97429 35967 5.43 7/132 25 0.31±0.02* 7 Proteasome subunit alpha 1 Q9R1P4 29528 6.00 9/145 37 0.30±0.01* 8 26S proteasome Q9CX56 30007 6.03 12/155 45 0.31±0.03* 9 Proteasome subunit beta 4 P99026 29097 5.47 9/116 39 0.32±0.02* 10 Peroxiredoxin-3 P20108 28109 7.15 9/123 26 0.29±0.02* 11 Glyoxalase I Q9CPU0 20796 5.24 11/102 26 0.18±0.02* Ratio is described as spots intensity of I/R-operated to spots intensity of sham-operated. *P<0.05 (vs. sham) x œ œ v w, z» jš y j w e. z 3» y proteomic» k w. ALT AST s mg s ùkü t, ALT AST eƒ x w x ƒw, xx w y w. w, w s œsx, x x, ƒ ùkû, y z» ùkù ew (Jaeschke et al., 1992; Koo et al., 1992; Zwacha et al., 1997). z» glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, DLP- 1, annexin A4, eukaryotic initiation factor 3, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, proteasome beta 4 x x w. DLP-1 GTPase dynamin family w wù mg w l w. m g xk w ³x w mg» w w w

Figure 4. Western blot analysis of dynamin-1-like protein (DLP- 1) in liver tissue of sham-operated and ischemia/reperfusionoperated animals. Each lane represents an individual experimental animal. Densitometric analysis is represented as an arbitrary unit (A.U.), normalized by tubulin. Data (n=5) are represented as mean±sem. *P<0.05.. DLP-1 s w s» xk» w w w (Pitts et al., 1999). DLP-1 s xk ƒw y w s w (Pitts et al., 1999). Alzheimer s disease huntington disease nw y mg w l ³x š š(wang et al., 2008; 2009a; 2009b), Alzheimer s disease y s oxidative stress w ƒ reactive oxygen species (ROS) DLP-1 x g xk w (Hom et al., 2007; Wang et al., 2008). w Alzheimer s disease β-amyloid w mg l cytochrome c s ü w apoptosis w, mg density g m g xk ùkü š (Suen et al., 2008). DLP-1 x(over-expression) β-amyloid w mg s j š ý» y g (Wang et al., 2009a). DLP-1 y w. DLP-1 mg w s w š. xx w w x w s y w š, xx n x mg Proteomic analysis of liver Injury 73 ƒ t š š, (cristae) ƒ x w mg ùkü (Pronobesh et al., 2008). v l» m w DLP-1 xš w Western blot mw DLP-1 x w y w. DLP-1ƒ w š, DLP-1 xk g s w. Glyoxalase I glyoxalase system z methylglyoxal lactate yw, methylglyoxal w y (AGEs, advanced glycation endproducts) w w w. glyoxalase I methylglyoxal jš y(glycation) g y ƒ k (Thornalley et al., 2003). w, y (aging process) oxidative stress glyoxalse I y jš y ƒ g s k š (Thornalley et al., 2008). gq xx glyoxalase I y methylglyoxal ƒƒ š š, glyoxalase I overexpression methylglyoxal g s l z ùký x (Kumagai et al., 2008; 2009). xx w s y w š, glyoxalase I x w x y w. glyoxalase I methylglyoxal ƒ w s w. Spermidine spermine sww polyamines s w, spermidine synthase w š (Wu et al., 2007). w, spermidine synthase» w polyamine v kƒ s w š, oxidative stress ù š š (Minton et al., 1990; Chattopadhyay et al., 2003). xx polyamine ww s w e, polyamine n» wš s (cell death) g s yz ù kþ (Zhao et al., 2007). xx s y w š, spermidine synthase x w x y w. spermidine synthase polyamine w s w. xx s glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, DLP-1, annexin A4, eukaryotic

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