Cold ischemia-induced autophagy in rat lung tissue

Abstract

Autophagy is a highly conserved pathway that permits recycling of nutrients within the cell and is rapidly upregulated during starvation or cell stress. Autophagy has been implicated in the pathophysiological process of warm ischemia‑reperfusion injury in the rat lung. Cold ischemia (CI) preservation for lung transplantation also exhibits cell stress and nutrient deprivation, however, little is known with regard to the involvement of autophagy in this process. In the present study, CI preservation‑induced autophagy and apoptosis was investigated in the lungs of Sprague Dawley rats. Sprague Dawley rat lungs were flushed and preserved at 4˚C (i.e. CI) for various durations (0, 3, 6, 12 and 24 h). The levels of autophagy, autophagic cell death and apoptosis were measured at each time point following CI. The results revealed that autophagy was induced by CI preservation, which was initiated at 3 h, peaked at 6 h after CI and declined thereafter. Additionally, a coexistence of autophagic cell death and apoptosis was observed in rat lung tissues following prolonged CI. These findings demonstrate that autophagy is involved in the pathophysiological process of lung CI. Furthermore, autophagic cell death in addition to necrosis and apoptosis occurs following CI in the lung. CI preservation may therefore be a potential mechanism of lung injury during organ preservation prior to lung transplantation.

Figure 1

Protein expression of LC3B and Beclin-1 in lung tissues following varying time periods of CI preservation. (A) Representative immunoblots of LC3B. (B) Comparisons of LC3B-II/I ratio and Beclin-1. (C) Representative immunoblots of Beclin-1 protein expression in lung homogenates at different time points of CI preservation. (D) Comparisons of ratios of Beclin-1. Protein levels among different groups were determined from six independent experiments. The data represent the mean ± standard deviation of six separate experiments. ^*P<0.05, ^**P<0.01 vs. 0 h group. CI, cold ischemia; LC3B, microtubule-associated protein 1 light chain 3B.

Figure 2

Number of LC3B-positive cells in transverse sections at different time points of CI preservation. (A) Representative immunofluorescence staining of LC3B. Scale bar=50 μm. (B) Number of LC3B-positive cells was compared among different time points of CI preservation. The data represent the means ± standard deviation of 30 transverse sections per time point. ^*P<0.05, ^**P<0.01 vs. 0 h group. CI, cold ischemia; LC3B, microtubule-associated protein 1 light chain 3B.

Figure 3

mRNA expression of Atg5 in lung tissues during CI preservation. The mRNA level of Atg5 was measured by reverse transcription quantitative polymerase chain reaction. The data, expressed as a percentage of the 0 h group, represent the mean ± standard deviation of three separate experiments. ^**P<0.01 vs. 0 h group (one-way analysis of variance followed by Student-Newmans-Keuls test). ATG5, autophagy protein 5; CI, cold-ischemia.

Figure 4

Changes in caspase-3 protein level and Bax/Bcl-2 mRNA ratio in lung tissues during CI preservation. (A) Representative immunoblots of caspase-3 in lung tissues at different time points of CI preservation. (B) Protein levels of cleaved caspase-3 at different time points were analyzed. The data, expressed as a percentage of the 0 h group following the adjustment of β-actin (a loading control), represent the means ± standard deviation of three separate experiments. (C) Ratio of Bax/Bcl-2 mRNA expression following different time periods of CI preservation. ^**P<0.01 vs. 0 h group. CI, cold-ischemia; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 5

Double-immunofluorescence analysis of LC3B and TUNEL in lung tissues following CI preservation. Transverse sections prepared from lung tissues following different time periods of CI preservation were subjected to LC3B immunofluorescent staining and TUNEL assay. 4′,6-Diamidino-2-phenylindole was used to stain the cell nuclei. Compared with the 0 h group, the number of TUNEL-positive cells and LC3B-positive cells evidently increased after 6 h of CI preservation. Scale bar=50 μm. CI, cold ischemia; LC3B, microtubule-associated protein 1 light chain 3B; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 6

Autophagic cell death in lung tissues after 6 h of CI preservation. The LC3B-positive cells were observed in TUNEL-positive cells. The nuclei in TUNEL- and LC3B-positive cells were round, which was consistent with autophagic cell death (arrow). The shrunken or fragmented DNA was observed only in apoptotic nuclei (arrowheads). Scale bar=50 μm in the upper panel images and 10 μm in the lower panel images. CI, cold ischemia; LC3B, microtubule-associated protein 1 light chain 3B; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.