Protective Effects of Neural Crest-Derived Stem Cell-Conditioned Media against Ischemia-Reperfusion-Induced Lung Injury in Rats

Abstract

Current treatments for ischemia-reperfusion (IR)-induced acute lung injury are limited. Mesenchymal stem cell-conditioned medium (CM) has been reported to attenuate lung injury. Neural crest stem cells (NCSCs), a type of multipotent stem cells, are more easily obtained than mesenchymal stem cells. We hypothesize that NCSC-CM has anti-inflammatory properties that could protect against IR-induced lung injury in rats. In this study, NCSC-CM was derived from rat NCSCs. Typical acute lung injury was induced by 30-min ischemia followed by 90-min reperfusion in adult male Sprague-Dawley rats. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected to analyze the degree of lung injury after the experiment. NCSC-CM was administered before ischemia and after reperfusion. NCSC-CM treatment significantly attenuated IR-induced lung edema, as indicated by decreases in pulmonary vascular permeability, lung weight gain, wet to dry weight ratio, lung weight to body weight ratio, pulmonary arterial pressure, and protein level in BALF. The levels of tumor necrosis factor-α and interleukin-6 in the BALF were also significantly decreased. Additionally, NCSC-CM improved lung pathology and neutrophil infiltration in the lung tissue, and significantly suppressed nuclear factor (NF)-κB activity and IκB-α degradation in the lung. However, heating NCSC-CM eliminated these protective effects. Our experiment demonstrates that NCSC-CM treatment decreases IR-induced acute lung injury and that the protective mechanism may be attributable to the inhibition of NF-κB activation and the inflammatory response. Therefore, NCSC-CM may be a novel approach for treating IR-induced lung injury.

Keywords: acute lung injury; conditioned media; ischemia-reperfusion; neural crest stem cell.

Fig. 1

Hair follicle cells express the NCSC markers, Snail 1, p75, and SOX10. Immunofluorescence image analysis revealed that NCSCs expressed p75, Snail l and SOX10. Nuclei were counterstained with DAPI (blue). A representative image from one of the three experiments is presented. Bar = 50 μm.

Fig. 2

Effect of NCSC-CM on pulmonary artery pressure (ΔPAP). PAP significantly increased in the ischemia-reperfusion (IR) group. The increase in PAP was significantly attenuated by treatment with NCSC-CM but not by treatment with heated NCSC-CM. Data are expressed as mean ± SD (n = 6 per group). *P < 0.05, compared to the control group; #P < 0.05, compared to the IR group; +P < 0.05, compared to the IR+ NCSC-CM group.

Fig. 3

Effect of NCSC-CM on the lung weight gain, ischemia-reperfusion (IR) led to significant lung weight gain (LWG). The increase in the LWG was significantly attenuated by NCSC-CM treatment, but not by heated NCSC-CM treatment. All data are shown as mean ± SD (n = 6 per group). *P < 0.05, compared to the control group; #P < 0.05, compared to the IR group; +P < 0.05, compared to the IR+ NCSC-CM group.

Fig. 4

Effect of NCSC-CM on the indicators of lung edema. Ischemia-reperfusion (IR) significantly increased the K _f (a), the lung weight/body weight (LW/BW) ratio (b), the wet/dry (W/D) weight ratio (c), and the protein concentration in bronchoalveolar lavage (BALF) (d). The increases in these indicators were significantly attenuated by NCSC-CM treatment, but not by heated NCSC-CM treatment. All data are shown as mean ± SD (n = 6 per group). *P < 0.05, compared to the control group; #P < 0.05, compared to the IR group; +P < 0.05, compared to the IR+ NCSC-CM group.

Fig. 5

Effect of NCSC-CM on TNF-α and IL-6 levels in bronchoalveolar lavage fluid (BALF). Ischemia-reperfusion (IR) significantly increased TNF-α and IL-6 levels in BALF. The increase was significantly suppressed by NCSC-CM treatment, but not by heated NCSC-CM treatment. All data are shown as mean ± SD (n = 6 per group). *P < 0.05, compared to the control group; #P < 0.05, compared to the IR group; +P < 0.05, compared to the IR+ NCSC-CM group.

Fig. 6

Effect of NCSC-CM on lung histopathology. Compared to a representative section of lung tissue (× 400 magnification), tissues exposed to ischemia-reperfusion (IR) had a significantly increased number of infiltrating neutrophils and septal thickness (a). NCSC-CM treatment, but not heated NCSC-CM treatment, improved the histological changes. Lung injury scores (b) and the numbers of neutrophils per high power field (× 400 magnification) (c) were significantly greater in the IR group than in the control group. These increases were significantly attenuated by NCSC-CM treatment but not by heated NCSC-CM treatment. All data are shown as mean ± SD (n = 6 per group). *P < 0.05, compared to the control group; #P < 0.05, compared with the IR group; +P < 0.05, compared to the IR+ NCSC-CM group.

Fig. 7

Effect of NCSC-CM on the expression of nuclear NF-κB p65 and cytoplasmic IκB-β in the lung tissues. Treatment with NCSC-CM, but not heated NCSC-CM, increased IκB-β levels (a) and reduced nuclear NF-κB p65 levels (b) in ischemia-reperfusion (IR)-induced lung injury. PCNA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. A representative blot is shown. Data are expressed as mean ± SD (n = 3 per group). *P < 0.05, compared to the control group; #P < 0.05, compared to the IR group; +P < 0.05, compared to the IR+ NCSC-CM group.