The Effects of N-acetylcystein and Epigallocatechin-3-Gallate in Ischemia-Reperfusion Injury of Rat Lungs

Seokjin Haam; Gu Lee Jin; Sungsoo Lee; Chae Paik Hyo; Jin Lim Beom

doi:10.4285/jkstn.2015.29.3.130

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Haam, Jin, Lee, Hyo, and Beom: The Effects of N-acetylcystein and Epigallocatechin-3-Gallate in Ischemia-Reperfusion Injury of Rat Lungs

Original Article

J Korean Soc Transplant 2015;29(3):130-138.

Published online: 10 September 2015

DOI: https://doi.org/10.4285/jkstn.2015.29.3.130

The Effects of N-acetylcystein and Epigallocatechin-3-Gallate in Ischemia-Reperfusion Injury of Rat Lungs

Seokjin Haam, M.D.¹, Gu Lee Jin, M.D.², Sungsoo Lee, M.D.¹, Chae Paik Hyo, M.D.², Jin Lim Beom, M.D.³

¹Department of Thoracic and Cardiovascular Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea

²Department of Thoracic and Cardiovascular Surgery, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea

³Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea

Corresponding author: Hyo Chae Paik, Department of Thoracic and Cardiovascular Surgery, Severance Hospital, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea Tel: 82-2-2228-2140, Fax: +82-2-393-6012 E-mail: hcpaik@yuhs.ac

Received 14 July 2015 Revised 16 July 2015 Accepted 16 July 2015

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

Background

Ischemia-reperfusion injury (IRI) is a major cause of early graft dysfunction after lung transplantation. The aim of this study was to assess the effects of N-acetylcystein (NAC) and epigallocatechin-3-gallate (EGCG) on IRI of rat lungs.

Methods

Sprague-Dawley rats were divided into four groups. Sham group (n=6) did not receive IRI. Rats in the control group (n=6), NAC group (n=6), and EGCG group (n=6) were treated with an intraperitoneal injection of normal saline, NAC, and EGCG, respectively, prior to IRI. In the latter three groups, IRI was induced by clamping the left pulmonary artery, vein, and main stem bronchus for a period of 60 minutes. After ischemia, reperfusion and ventilation of the lung was allowed for a period of 180 minutes. The expression levels of inducible nitric oxide synthase (iNOS), hemeoxygenase-1 (HO-1), AMP-activated protein kinase-α(AMPK), and caveolin-1 in lung tissues were evaluated by Western blot. The pathological findings and the extent of pulmonary edema after IRI were compared among the groups.

Results

The expression levels of iNOS decreased in the Sham and EGCG groups. The expression level of HO-1 was significantly higher in the EGCG group (P=0.0001). Although the expression levels of AMPK and caveolin-1 showed no differences, the extent of phosphorylation of AMPK and caveolin-1 was higher in the EGCG and NAC groups, respectively. In hematoxylin-eosin staining, the lungs in the NAC and EGCG groups showed fewer alveolar injuries and less hemorrhagic congestion compared with the control group.

Conclusions

NAC and EGCG enhanced the phosphorylation of caveolin-1 and AMPK, respectively, and attenuated lung injury induced by ischemia-reperfusion.

Keywords: Lung transplantation, Reperfusion injury, Epigallocatechin gallate, Acetylcysteine

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Fig. 1.

(A) The expression levels of inducible nitric oxide synthase (iNOS) in lung tissues. Sham and epigallocatechin-3-gallate (EGCG) group showed lower the expression levels but, these were not significant (P=0.477). (B) The expression levels of hemeoxygenase-1 (HO-1) in lung tissues. The expression levels of HO-1 had the significant differences (P=0.001). EGCG group showed the higher level than N-acetylcystein (NAC) group (P=0.005; n=6 in each group; mean±SD). Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 2.

(A) The expression levels of AMP-activated protein kinase (AMPK) in lung tissues. There were no differences in the expression levels of AMPK. (B) The extents of phosphorylations of AMPK. The extents of phosphorylation in sham and epigallocatechin-3-gallate (EGCG) group were higher (P=0.002), but there was no difference between control and N-acetylcystein (NAC) group (P=1.000; n=6 in each group; mean±SD). Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 3.

(A) The expression levels of caveolin-1 (CAV-1) in lung tissues. There were no differences in the expression levels of CAV-1 among groups (P=0.784). (B) The extents of phosphorylations of CAV-1. In N-acetylcystein (NAC) group, the extent of phosphorylation of CAV-1 was higher (P=0.010). Although epigallocatechin-3-gallate (EGCG) group showed the higher extent of phosphorylation than control group, it was not significant (P=0.753; n=6 in each group; mean±SD). Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 4.

Pathologic findings of lung after ischemia-reperfusion injury (HE stain, ×40). (A) Sham group: the alveolar structure was maintained well and there was no congestion. (B) Control group: the most of alveolar structure was destroyed and severe neutrophil infiltration and congestion were found. (C) Epigallocatechin-3-gallate (EGCG) group: although mild neutrophil infiltration and congestion were found, alveolar structure was maintained relatively well. (D) N-acet-ylcystein group: the finding was similar to that of EGCG group.

Fig. 5.

Lung injury severity (LIS) scoring. Lung injuries such as congestion, hemorrhage and neutrophil infiltration were less in epigallocatechin-3-gallate (EGCG) and N-acetylcystein (NAC) group (P=0.0001; n=6 in each group; mean±SD).

Fig. 6.

Wet/dry ratio. Wet/dry ratio in control group was higher than other groups (P=0.045). This result means that lung edema in control group was more severe (n=6 in each group; mean±SD). Abbreviations: EGCG, epigallocatechin-3-gallate; NAC, N-acety-lcystein.

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