Targeting of nicotinamide phosphoribosyltransferase enzymatic activity ameliorates lung damage induced by ischemia/reperfusion in rats

Abstract

Background: Emerging evidence reveals that nicotinamide phosphoribosyltransferase (NAMPT) has a significant role in the pathophysiology of the inflammatory process. NAMPT inhibition has a beneficial effect in the treatment of a variety of inflammatory diseases. However, it remains unclear whether NAMPT inhibition has an impact on ischemia-reperfusion (I/R)-induced acute lung injury. In this study, we examined whether NAMPT inhibition provided protection against I/R lung injury in rats.

Methods: Isolated perfused rat lungs were subjected to 40 min of ischemia followed by 60 min of reperfusion. The rats were randomly allotted to the control, control + FK866 (NAMPT inhibitor, 10 mg/kg), I/R, or I/R + FK866 groups (n = 6 per group). The effects of FK866 on human alveolar epithelial cells exposed to hypoxia-reoxygenation (H/R) were also investigated.

Results: Treatment with FK866 significantly attenuated the increases in lung edema, pulmonary arterial pressure, lung injury scores, and TNF-α, CINC-1, and IL-6 concentrations in bronchoalveolar lavage fluid in the I/R group. Malondialdehyde levels, carbonyl contents and MPO-positive cells in lung tissue were also significantly reduced by FK866. Additionally, FK866 mitigated I/R-stimulated degradation of IκB-α, nuclear translocation of NF-κB, Akt phosphorylation, activation of mitogen-activated protein kinase, and downregulated MKP-1 activity in the injured lung tissue. Furthermore, FK866 increased Bcl-2 and decreased caspase-3 activity in the I/R rat lungs. Comparably, the in vitro experiments showed that FK866 also inhibited IL-8 production and NF-κB activation in human alveolar epithelial cells exposed to H/R.

Conclusions: Our findings suggest that NAMPT inhibition may be a novel therapeutic approach for I/R-induced lung injury. The protective effects involve the suppression of multiple signal pathways.

Keywords: Acute lung injury; Ischemia-reperfusion; Nicotinamide phosphoribosyltransferase; Visfatin; pre-B cell colony-enhancing factor.

Fig. 1

Effect of FK866 on pulmonary edema. Lung weight gain (a), K_f (b), lung wet/dry (W/D) weight ratios (c), lung weight/body weight (LW/BW) ratios (d), and protein concentrations in the bronchoalveolar lavage fluid (BALF) (e) significantly increased in the ischemia-reperfusion (IR) group. Treatment with FK866 significantly attenuated the increase in these parameters. Data are expressed as mean ± SD (n = 6 per group). ***p < 0.001, compared with the control group; ⁺⁺⁺ p < 0.001, compared with the IR group

Fig. 2

Effect of FK866 on pulmonary artery pressure (ΔPAP). PAP increased significantly in the ischemia-reperfusion (IR) group. The increase in PAP was attenuated significantly by treatment with FK866. Data are expressed as mean ± SD (n = 6 per group). ***p < 0.001, compared with the control group; ⁺⁺⁺ p < 0.001, compared with the IR group

Fig. 3

Effect of FK866 on NAMPT protein expression in lung tissue. a Immunohistochemistry for NAMPT in the lung (indicated with arrowhead) (200× magnification). b Western blot and densitometry analysis of NAMPT protein in the lung tissue. β-actin served as loading control for cytoplasmic proteins. Representative blots are shown. Ischemia-reperfusion (IR) significantly increased positive staining and protein expression of NAMPT in the lung tissue. FK866 significantly decreased the degree of NAMPT positive staining and protein expression. Data are expressed as mean ± SD (n = 6 per group). **p < 0.01, compared with the control group; ⁺ p < 0.05, compared with the IR group

Fig. 4

Effect of FK866 on CINC-1, TNF-α, and IL-6 levels, and total cell counts in bronchoalveolar lavage fluid (BALF). CINC-1 (a), TNF-α (b) and IL-6 (c) levels, and total cell counts (d) in the BALF significantly increased in the ischemia-reperfusion (IR) group. Treatment with FK866 significantly attenuated these increases in the BALF. Data are expressed as mean ± SD (n = 6 per group). ***p < 0.001, compared with the control group; ⁺⁺⁺ p < 0.001, compared with the IR group

Fig. 5

Effect of FK866 on protein carbonyl contents, MDA levels, and MPO-positive cells in lung tissue. MPO-positive cells (a), carbonyl contents (b), and MDA levels (c) in lung tissue significantly increased in the ischemia-reperfusion (IR) group. FK866 treatment significantly attenuated these increases. a Immunohistochemistry for MPO in the lung (indicated with arrowhead) (200× magnification). Data are expressed as mean ± SD (n = 6 per group). ***p < 0.001, compared with the control group; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, compared with the IR group

Fig. 6

Effect of FK866 on lung pathology. As shown by a representative micrograph of lung tissue (400× magnification) (a), neutrophil infiltration and septal edema were increased in the ischemia-reperfusion (IR) group. FK866 treatment significantly attenuated these histopathological changes, the numbers of neutrophils per high power field (400× magnification) (b), and the lung injury scores (c). Data are expressed as mean ± SD (n = 6 per group). ***p < 0.001, compared with the control group; ⁺⁺⁺ p < 0.001, compared with the IR group

Fig. 7

Effect of FK866 on the expression of caspase-3 and Bcl-2 in lung tissue. a Immunohistochemistry for active caspase-3 in the lung (indicated with an arrowhead) (200× magnification). b Western blot analysis of Bcl-2 protein in the lung tissue. β-actin served as a loading control for cytoplasmic proteins. Representative blots are shown. Ischemia-reperfusion (IR) significantly decreased Bcl-2 protein expression and induced caspase-3 activation in the lung tissue. FK866 treatment significantly increased Bcl-2 protein expression and attenuated the signals for active caspase-3 in the IR group. Representative blots are shown. Data are expressed as mean ± SD (n = 6 per group). ***p < 0.01, compared with the control group; ⁺⁺⁺ p < 0.01, compared with the IR group

Fig. 8

Effect of FK866 on MAPK and MKP-1 expressions in lung tissue. The phosphorylation of ERK (a), JNK (b), and p38 (c) was enhanced in the ischemia-reperfusion (IR) group. FK866 treatment attenuated these effects. In contrast, the expression of MKP-1 protein (d) was decreased in the IR group but reversed by FK866 treatment. β-actin served as the loading control. A representative blot is shown. All data are shown as mean ± SD (n = 6 per group). *p < 0.05, ***p < 0.001, compared with the control group; ⁺ p < 0.05, ⁺⁺ p < 0.01, compared with the IR group

Fig. 9

Effect of FK866 on NF-κB activation and Akt phosphorylation in lung tissues. FK866 reduced Akt phosphorylation (a) and nuclear NF-κB p65 levels (b), and increased IκB-α levels (c) in ischemia-reperfusion (IR)-induced lung injury. PCNA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. Representative blots are shown. Data are expressed as mean ± SD (n = 6 per group). *p < 0.05, ***p < 0.001, compared with the control group; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, compared with the IR group

Fig. 10

Effect of FK866 on A549 cells subjected to hypoxia-reoxygenation (H/R). a A representative Western blot of NF-κB nuclear translocation in the lung tissue. β-actin served as the loading control. FK866 significantly reduced the increase of degradation of IκB-α (b), phosphorylated NF-κB p65 (c) at 2 h and 4 h, and IL-8 production (d) at 4 h in A549 cells exposed to H/R. Data are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group. ⁺ p < 0.05, ⁺⁺⁺ p < 0.001, compared with the H/R group