Proteomic Analysis of Hepatic Ischemia and Reperfusion Injury in Mice

Eun-Hae Cho; Jin-Hee Sung; Phil-Ok Koh

doi:10.5625/lar.2010.26.1.69

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Cho, Sung, and Koh: Proteomic Analysis of Hepatic Ischemia and Reperfusion Injury in Mice

Original Article

Lab. Anim. Res. 2010;26(1):69-74.

Published online: 17 March 2010

DOI: https://doi.org/10.5625/lar.2010.26.1.69

Proteomic Analysis of Hepatic Ischemia and Reperfusion Injury in Mice

Eun-Hae Cho, Jin-Hee Sung, Phil-Ok Koh^∗

Department of Anatomy, College of Veterinary Medicine, Research Insituite of Life Sciences, Gyeongsang National University, Jinju, Korea

^∗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

Received 17 November 2009 Revised 8 March 2010 Accepted 17 March 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.

Keywords: Ischemia, liver, reperfusion

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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 sham-operated (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 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 sham-and I/R-operated animals.

Figure 4.

Western blot analysis of dynamin-1-like protein (DLP-1) in liver tissue of sham-operated and ischemia/reperfusion-operated 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.

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)

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