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Insituite of Life Sciences, Gyeongsang National University, Jinju, Korea Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery.

1 Lab. Anim. Res. 2010: 26(1), 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 , Korea Tel: Fax: 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

2 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 % 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.

3 Proteomic analysis of liver Injury 71 w. MALDI-TOF MS-FIT ( prospector.ucsf.edu/ucsfhtml13.4/msfit.htm) ProFound ( 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 ±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 ±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.

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 % 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 P / ±0.02* 2 Dynamin-1-like protein Q8K1M / ±0.01* 3 Glutaredoxin-3 Q9CQM / ±0.01* 4 Eukaryotic initiation factor 3 Q9QZD / ±0.02* 5 Spermidine synthase Q / ±0.03* 6 Annexin A4 P / ±0.02* 7 Proteasome subunit alpha 1 Q9R1P / ±0.01* 8 26S proteasome Q9CX / ±0.03* 9 Proteasome subunit beta 4 P / ±0.02* 10 Peroxiredoxin-3 P / ±0.02* 11 Glyoxalase I Q9CPU / ±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.

5 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< 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

6 74 Eun-Hae Cho et al. initiation factor 3, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, proteasome beta 4 x w y w. s, y,, w, w y w, xx w s»., xx w w yƒ š,» w. q 2007 ( ) w w w (KRF E00538). k Abe, Y., Hines, I.N., Zibari, G., Pavlick, K., Gray, L., Kitagawa, Y. and Grisham, M.B. (2009) Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo. Free Radic. Biol. Med. 46, 1-7. Chattopadhyay, M.K., Tabor, C.W. and Tabor, H. (2003) Polyamines protect Escherichia coli cells from the toxic effect of oxygen. Proc. Natl. Acad. Sci. USA. 100, Eum, H.A., Cha, Y.N. and Lee, S.M. (2007) Necrosis and apoptosis: Sequence of liver damage following reperfusion after 60min ischemia in rats. Biochem. Biophys. Res. Commun. 358, Hom, J.R., Gewandter, J.S., Michael, L., Sheu, S.S. and Yoon, Y. (2007) Thapsigargin induces biphasic fragmentation of mitochondria through calcium-mediated mitochondrial fission and apoptosis. J. Cell Physiol. 212, Jaeschke, H., Bautista, A.P., Spolarics, Z. and Spitzer, J.J. (1992) Superoxide generation by neutrophils and Kupffer cells during in vivo reperfusion after hepatic ischemia in rats. J. Leukoc. Biol. 52, Jaeschke, H. (2003) Molecular mechanisms of hepatic ischemiareperfusion injury and preconditioning. Am. J. Physiol. Gastrointest. Liver Physiol. 284, Jaeschke, H. (2006) Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cellinjury during hepatic ischemiareperfusion and other acute inflammatory conditions. Am. J. Physiol. Gastrointest. Liver Physiol. 290, Koo, A., Komatsu, H., Tao, G., Inoue, M., Guth, P.H. and Kaplowitz, N. (1992) Contribution of no-reflow phenomenon to hepatic injury after ischemia-reperfusion: evidence for a role for superoxide anion. Hepatology, 15, Kuboki, S., Schuster, R., Blanchard, J., Pritts, T.A., Wong, H.R. and Lentsch, A.B. (2007) Role of heat shock protein 70 in hepatic ischemia-reperfusion injury in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 292, Kumagai, T., Nangaku, M. and Inagi, R. (2008) Pathophysiological role of the glyoxalase system in renal hypoxic injury. Ann. NY. Acad. Sci. 1126, Kumagai, T., Nangaku, M., Kojima, I., Nagai, R., Ingelfinger, J.R., Miyata, T., Fujita, T. and Inagi, R. (2009) Glyoxalase I overexpression ameliorates renal ischemia-reperfusion injury in rats. Am. J. Physiol. Renal. Physiol. 296, Minton, K.W., Tabor, H. and Tabor, C.W. (1990) Paraquat toxicity is increased in Escherichia coli defective in the synthesis of polyamines. Proc. Natl. Acad. Sci. USA. 87, Pitts, K.R., Yoon, Y., Krueger, E.W. and McNiven, M.A. (1999) The dynamin-like protein DLP-1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells. Mol. Biol. Cell. 10, Pronobesh, C., Dagagi, A.V., Pallab, C. and Kumar, W.A. (2008) Protective role of the calcium channel blocker amlodipine against mitochondrial injury in ischemia and reperfusion injury of rat liver. Acta. Pharm. 58, Rhodes, R.S. and DePalma, R.G. (1980) Mitochondrial dysfunction of the liver and hypoglycemia in hemorrhagic shock. Surg. Gynecol. Obstet. 150, Suen, D.F., Norris, K.L. and Youle, R.J. (2008) Mitochondrial dynamics and apoptosis. Genes Dev. 22, Thornalley, P.J. (2003) Glyoxalase I-structure, function and a critical role in the enzymatic defence against glycation. Biochem. Soc. Trans. 31, Thornalley, P.J. (2008) Protein and nucleotide damage by glyoxal and methylglyoxal in physiological systems-role in ageing and disease. Drug Metabol. Drug Interact. 23, Wang, X., Su, B., Fujioka, H. and Zhu, X. (2008) Dynamin-like protein 1 reduction underlies mitochondrial morphology and distribution abnormalities in fibroblasts from sporadic Alzheimer's disease patients. Am. J. Pathol. 173, Wang, X., Su, B., Lee, H.G., Li, X., Perry, G., Smith, M.A. and Zhu, X. (2009a) Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J. Neurosci. 29, Wang, H., Lim, P.J., Karbowski, M. and Monteiro, M.J. (2009b) Effects of overexpression of huntingtin proteins on mitochondrial integrity. Hum. Mol. Genet. 18, Wu, H., Min, J., Ikeguchi, Y., Zeng, H., Dong, A., Loppnau, P., Pegg, A.E. and Plotnikov, A.N. (2007) Structure and mechanism of spermidine synthases. Biochemistry 46, Zhao, Y.J., Xu, C.Q., Zhang, W.H., Zhang, L., Bian, S.L., Huang, Q., Sun, H.L., Li, Q.F., Zhang, Y.Q., Tian, Y., Wang, R., Yang, B.F. and Li, W.M. (2007) Role of polyamines in myocardial ischemia/reperfusion injury and their interactions with nitric oxide. Eur. J. Pharmacol. 562, Zwacka, R.M., Zhang, Y., Halldorson, J., Schlossberg, H., Dudus, L. and Engelhardt, J.F. (1997) CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver. J. Clin. Invest. 100,

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16(1)-3(국문)(p.40-45).fm w wz 16«1y Kor. J. Clin. Pharm., Vol. 16, No. 1. 2006 x w$btf3fqpsu'psn û w m w Department of Statistics, Chonnam National University Eunsik Park College of Natural Sciences, Chonnam National University