Keratinocyte Growth Factor-2 Is Protective in Oleic Acid-Induced Acute Lung Injury in Rats

Abstract

Objective: The aim of this study was to examine the role of keratinocyte growth factor-2 (KGF-2) in oleic acid-induced acute lung injury (ALI) in rats.

Methods: Forty-five healthy adult male Sprague Dawley rats were divided into 3 groups. Rat ALI model was established by injection of 0.01 mL/kg oleic acid into the tail vein. Rats in the control group were injected with the same amount of normal saline (NS). In the ALI + KGF-2 group, 5 mg/kg of KGF-2 was instilled into the airway of rats 72 hours before the model preparation, and the control group and the ALI model group were instilled with the same amount of NS. The lung permeability index (LPI) and lung wet/dry weight (W / D) ratios were measured 8 hours after the model preparation. The permeability of pulmonary microvascular endothelium was evaluated by Evans blue leakage test. Histopathological changes were observed under light microscope and the ALI pathology score (LIS) was calculated. Ultrastructural changes of lung tissue were observed under electron microscope. The apoptosis was detected by TUNEL assay. The expression of Claudin-5, ZO-1, and VE Cadherin in lung tissue was qualitatively and quantitatively analyzed by immunohistochemistry, Western Blot, and qRT-PCR, respectively.

Results: The ALI model group had severe lung injury and obvious pathological changes, including alveolar septal thickening and inflammatory cell infiltration. TUNEL assay showed that the apoptosis of ALI group was significantly increased. The LIS score, lung W/D ratio, LPI, and Evans blue leakage were significantly higher than those in the control group; electron microscopy showed that the alveolar-capillary barrier was severely damaged in the ALI group. Compared with the control group, the expression of Claudin-5, ZO-1, and VE cadherin in the lung tissue of the ALI model group was significantly attenuated. After pretreatment with KGF-2, the degree of lung tissue damage was significantly reduced and the pathological changes were significantly improved. TUNEL assay showed that the apoptosis of ALI group was decreased. Lung W/D ratio, LPI, and Evans blue leakage decreased; electron microscopy showed that the alveolar-capillary barrier of ALI group recovered significantly. Compared with the ALI model group, the expression of Claudin-5, ZO-1, and VE cadherin in the lung tissue of the KGF-2 pretreatment group increased.

Conclusion: The results indicate that KGF-2 may attenuate oleic acid-induced ALI in rats by maintaining the pulmonary microvascular endothelial barrier, which is an effective ALI preventive measure.

Figure 1

(a) Pathological changes of lung tissue in each group were observed under light microscope. (A) The alveolar structure of the control group was intact (middle magnification); (D) there was no obvious inflammatory cell infiltration in the control group and no significant change in the pulmonary interstitial (high magnification); (B) alveolar septal thickening in the ALI group, alveolar fusion (middle magnification); (E) inflammatory cells in the ALI group were extensively infiltrated and a large amount of protein exuded (high magnification); (C) mild fusion of alveolus in the ALI+KGF-2 group (middle magnification); (F) a small amount of inflammatory cell infiltration in ALI+KGF-2 group and a small amount of protein exudation (high magnification) HE staining. (b) Quantitative analysis of lung injury scores in each group (∗< 0.01 versus control group, #< 0.01 versus ALI group).

Figure 2

Ultrastructural changes in lung tissue of rats in each group under transmission electron microscopy (10,000 times). (a) Control alveolar-capillary barrier integrity (red arrow); (b) alveolar-capillary barrier was severely impaired in ALI group and alveolar type II epithelial cells were degenerated, endothelial cell apoptosis (black arrow) and basement membrane exposure; (c) ALI+KGF-2 group reduced damage; alveolar-capillary barrier was basically intact (red arrow).

Figure 3

(a) Changes in apoptosis of lung tissue in each group. (A) DAPI in the control group; (B) DAPI in the ALI group; (C) DAPI in the ALI+KGF-2 group; (D) number of apoptotic cells in the control group; apoptotic cells were sparse and scattered; (E) apoptotic cells in ALI group, diffuse distribution of apoptotic cells; (F) number of apoptotic cells in ALI+KGF-2 group, scattered number of apoptotic cells; (G) control group mixed images; (H) mixed images of ALI group; (I) mixed images of ALI+KGF-2 group medium magnification. (b) Statistical analysis of apoptosis of lung tissue of each group (∗< 0.01 versus control group, #<0.01 versus ALI group).

Figure 4

Shows a significant increase in EB content, W/D value, and LPI value in the ALI group compared with the control group and improved after KGF-2 intervention. (a) Evans blue content in the lung. (b) Lung wet-to-dry weight ratio. (c) Lung permeability index (∗< 0.01 versus control group; #<0.01 versus ALI group; Δ< 0.05 versus ALI group ).

Figure 5

Immunohistochemical staining to determine Claudin-5, ZO-1, and VE cadherin expression in rat lung tissue. (a) Claudin-5 expression in the lung tissue of the control group was strongly positive (dark brown); ALI group Claudin-5 expression was negative in lung tissue (light yellow); Claudin-5 expression was weakly positive (brown) in lung tissue of ALI+KGF-2 group; ZO-1 expression in lung tissue of control group strong positive (dark brown); ZO-1 expression was negative in the lung tissue of ALI group (light yellow) and showed that ZO-1 expression was weakly positive in lung tissue of ALI+KGF-2 group (brown yellow); VE cadherin expression was strongly positive in the lung tissue of the control group (dark brown); VE cadherin expression was negative in the lung tissue of the ALI group (light yellow); ALI+KGF-2 group lung tissue VE cadherin expression is weakly positive (brownish yellow). Medium magnification. Positive expression of brown stained strip is indicated by the arrow; (b) statistical analysis of positive expression of each group (∗< 0.01 versus control group, #<0.01 versus ALI group, and Δ< 0.05 versus ALI group ).

Figure 6

Western blot analysis of Claudin-5, ZO-1 and VE Cadherin expression in rat lung tissue. (a) Claudin-5 protein polyacrylamide gel electrophoresis and protein expression level (∗< 0.01 versus control group; #< 0.01 versus ALI Group); (b) ZO-1 protein polyacrylamide gel electrophoresis and protein expression level (∗< 0.01 versus control group; #< 0.01 versus ALI Group); (c) VE cadherin protein polyacrylamide gel electrophoresis and protein expression level (∗< 0.01 versus control group; #< 0.01 versus ALI Group).

Figure 7

qRT-PCR analysis of Claudin-5, ZO-1 and VE cadherin expression in rat lung tissue. (a) Detection of Claudin-5 mRNA expression levels in rat lung tissue by qRT-PCR assay (∗< 0.01 versus control group; #<0.01 versus ALI group); (b) detection of ZO-1 mRNA expression levels in rat lung tissue by qRT-PCR assay (∗< 0.01 versus control group; #<0.01 versus ALI group); (c) detection of VE Cadherin mRNA expression levels in rat lung tissue by qRT-PCR assay (∗< 0.01 versus control group; #<0.01 versus ALI group).