Adiponectin protects against lung ischemia-reperfusion injury in rats with type 2 diabetes mellitus

Abstract

Adiponectin (APN) has been associated with the pathogenesis of acute brain, liver and heart injury. However, the role of APN in lung ischemia-reperfusion injury (LIRI) in diabetes mellitus remains unclear. To investigate this, the present study evaluated the effects of APN on lung dysfunction and pathological alterations in rats with type 2 diabetes mellitus via lung ischemia/reperfusion (I/R). The lung‑protective effects of APN globular domain (gAPN) in rats with type 2 diabetes mellitus were also investigated by measuring the oxygenation index, inflammatory cytokines, lung edema, histopathology, oxidative stress, apoptosis and the protein expression levels of phosphorylated 5'adenosine monophosphate‑activated protein kinase (p‑AMPK), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). The results of the present study demonstrated that the diabetes mellitus rats + I/R (DIR) group exhibited greater concentrations of tumor necrosis factor‑α and interleukin‑6, and increases in the wet‑weight to dry‑weight ratio, lung injury score, oxidative stress and cellular apoptosis. These effects were accompanied by lower pulmonary oxygenation compared with the normal rat + I/R (NIR) group (P<0.05). Additionally, all of these alterations were attenuated in the NIR + gAPN and DIR + gAPN groups compared with in the NIR and DIR groups, respectively. In the DIR group, the expression levels of p‑AMPK/AMPK and eNOS were significantly downregulated, and the levels of iNOS were upregulated, compared with those of the NIR group. Treatment with APN activated AMPK, increased eNOS expression and attenuated iNOS expression. The results of the present study demonstrated that APN exerted protective effects against LIRI via its anti‑inflammatory, antioxidative stress and anti‑apoptotic activities. These protective effects of APN were eliminated in rats with type 2 diabetes mellitus, in which LIRI was exacerbated. The present study indicated that APN may be a potential therapeutic agent for LIRI in type 2 diabetes mellitus.

Keywords: adiponectin; diabetes mellitus; lung ischemia-reperfusion injury; 5'adenosine monophosphate-activated protein kinase.

Figure 1.

Diabetes mellitus increases susceptibility to lung I/R injury. Adiponectin improved the IR-induced pathological alterations in lung tissue (hematoxylin and eosin staining, lung injury score, and wet-weight to dry-weight ratio). (A) NS, (B) NIR, (C) NIRA, (D) DS, (E) DIR and (F) DIRA groups. Scale bar=500 µM. (G) Lung injury score and (H) wet-weight to dry-weight ratio were determined. The results are expressed as the mean ± standard deviation. *P<0.05 vs. NS group, ^#P<0.05 vs. NIR group, ^{^}P<0.05 vs. NIRA group, ^$P<0.05 vs. DS group and ^&P<0.05 vs. DIR group. DIR, diabetic I/R group, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; DIRA, diabetic I/R + gAPN group, in which 10 µg gAPN was injected 10 min prior reperfusion; DS, diabetic sham group, in which the left lung hilum was not clamped; NIR, I/R, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; NIRA, I/R + gAPN, in which 10 µg gAPN was injected 10 min prior to reperfusion; NS, Sham group, in which the left lung hilum was not clamped. I/R, ischemia/reperfusion; gAPN, adiponectin globular domain.

Figure 2.

APN reduces the levels of IL-6 and TNF-α, in addition to MPO activity, following ischemia/reperfusion. The results are expressed as the mean ± standard deviation. *P<0.05 vs. NS group, ^#P<0.05 vs. NIR group, ^{^}P<0.05 vs. NIRA group, ^$P<0.05 vs. DS group and ^&P<0.05 vs. DIR group. NIR, I/R, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; NIRA, I/R + gAPN, in which 10 µg gAPN was injected 10 min prior to reperfusion; NS, Sham group, in which the left lung hilum was not clamped; DIR, diabetic I/R group, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; DIRA, diabetic I/R + gAPN group, in which 10 µg gAPN was injected 10 min prior reperfusion; DS, diabetic sham group, in which the left lung hilum was not clamped; MPO, myeloperoxidase; TNF-α, tumor necrosis factor-α; gAPN, adiponectin globular domain; IL-6, interleukin-6; I/R, ischemia/reperfusion.

Figure 3.

APN attenuates oxidative stress induced by I/R. The results are expressed as mean ± standard deviation. *P<0.05 vs. NS group, ^#P<0.05 vs. NIR group, ^{^}P<0.05 vs. NIRA group, ^$P<0.05 vs. DS group and ^&P<0.05 vs. DIR group. DIR, diabetic I/R group, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; DIRA, diabetic I/R + gAPN group, in which 10 µg gAPN was injected 10 min prior reperfusion; DS, diabetic sham group, in which the left lung hilum was not clamped; NIR, I/R, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; NIRA, I/R + gAPN, in which 10 µg gAPN was injected 10 min prior to reperfusion; NS, Sham group, in which the left lung hilum was not clamped. I/R, ischemia/reperfusion; gAPN, adiponectin globular domain; SOD, superoxide dismutase; MDA, malondialdehyde; NO, nitric oxide.

Figure 4.

APN inhibits I/R-induced apoptosis, as demonstrated via TUNEL staining. TUNEL-positive cells observed under a light microscope were identified by brown-stained nuclei. (A) NS, (B) NIR, (C) NIRA, (D) DS, (E) DIR and (F) DIRA groups. Scale bar=200 µM. (G) Percentages of TUNEL-positive cells. The results were expressed as the mean ± standard deviation. *P<0.05 vs. NS group, ^#P<0.05 vs. NIR group, ^{^}P<0.05 vs. NIRA group, ^$P<0.05 vs. DS group and ^&P<0.05 vs. DIR group. DIR, diabetic I/R group, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; DIRA, diabetic I/R + gAPN group, in which 10 µg gAPN was injected 10 min prior reperfusion; DS, diabetic sham group, in which the left lung hilum was not clamped; NIR, I/R, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; NIRA, I/R + gAPN, in which 10 µg gAPN was injected 10 min prior to reperfusion; NS, Sham group, in which the left lung hilum was not clamped; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labeling; I/R, ischemia/reperfusion; gAPN, adiponectin globular domain.

Figure 5.

APN inhibits I/R-induced caspase-3 expression. Caspase-3-positive cells observed under a light microscope were identified by brown-stained nuclei. (A) NS, (B) NIR, (C) NIRA, (D) DS, (E) DIR and (F) DIRA groups. Scale bar=200 µM. (G) Percentages of caspase-3-positive cells. The results are expressed as the mean ± standard deviation. *P<0.05 vs. NS group, ^#P<0.05 vs. NIR group, ^{^}P<0.05 vs. NIRA group, ^$P<0.05 vs. DS group and ^&P<0.05 vs. DIR group. DIR, diabetic I/R group, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; DIRA, diabetic I/R + gAPN group, in which 10 µg gAPN was injected 10 min prior reperfusion; DS, diabetic sham group, in which the left lung hilum was not clamped; NIR, I/R, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; NIRA, I/R + gAPN, in which 10 µg gAPN was injected 10 min prior to reperfusion; NS, Sham group, in which the left lung hilum was not clamped. I/R, ischemia/reperfusion; gAPN, adiponectin globular domain.

Figure 6.

APN activates p-AMPK and eNOS expression and attenuates iNOS expression. The results are expressed as mean ± standard deviation. *P<0.05 vs. NS group, ^#P<0.05 vs. NIR group, ^{^}P<0.05 vs. NIRA group, ^$P<0.05 vs. DS group and ^&P<0.05 vs. DIR group. DIR, diabetic I/R group, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; DIRA, diabetic I/R + gAPN group, in which 10 µg gAPN was injected 10 min prior reperfusion; DS, diabetic sham group, in which the left lung hilum was not clamped; NIR, I/R, in which the lung was subjected to 90 min of ischemia and 4 h of reperfusion; NIRA, I/R + gAPN, in which 10 µg gAPN was injected 10 min prior to reperfusion; NS, Sham group, in which the left lung hilum was not clamped. p-AMPK, phosphorylated 5′ adenosine monophosphate-activated protein kinase; I/R, ischemia/reperfusion; gAPN, adiponectin globular domain; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase.