Expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury

Abstract

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury （ALI） and explore the function of Slit2 and Robo4 in ALI. MethodsForty-eight Sprague-Dawley rats were randomly （random number） divided into control group （n = 24） and ALI group （n = 24）. ALI model was reproduced by cecum ligation and puncture （CLP）. The control group only experienced a simulated operation without CLP. Both groups were further divided into 3 subgroups with 8 rats in each subgroup： 12 h, 24 h, and 48 h subgroups, artery blood gas analysis, lung tissue wet/dry weight （W/D） ratio, lung histopathologic changes, pulmonary microvascular permeability were observed. The serum tumor nocrosis factor-α （TNF-α） was measured with enzyme linked immunosorbent assay （ELISA）. The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry. Date were analyzed by one-way ANOVA with SPSS version 13.0 software. Statistical significance was established at a P value of less than 0. 05. Results Compared with the control group, in ALI rats at different time points, partial pressure of oxygen in arterial blood （PaO2） decreased significantly, lung W/D weight ratio and pulmonary microvascular permeability, the serum TNF-α increased significantly （ all P 〈 0. 05 ）, histopathology of lung revealed signs of injury. The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [ （0.56±0.13）vs. （0.87±0.05）, F=41.39, P〈0.05, （0.42±0.10） vs. （0.85±0.07）, F= 93.54, P〈0.05, （0.26±0.08） vs. （0.89±0.09）, F=227.05, P〈0.05]. but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [ （0.86±0.07） vs. （0.83±0.05）, F=0.695, P〉0.05, （0.82±0.05） vs. （0.89±0.08）, F= 2. 061, P 〉 0.05, （0. 86 ± 0.08） vs. （0. 86 ± 0. 05）, F = 0. 035, P 〉 0. 05 ]. Immunohistoehemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells, while lung epithelial cell nuclei and endoehylema. Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells. Compared with the control group, expression of Slit2 protein in lung tissue in ALl group decreased markedly [ （0.37±0.05） vs. （0.45±0.07）, F=6.82, P〈0.05, （0.32±0.06） vs. （0.47±0.09）, F=23.54, P〈0.05, （0.28±0.07） vs. （0.46±0.06）, F= 28.01, P 〈 0. 05 ]. As good as RT-PCR, there were no significant difference in expression of Robo4 protein in lung tissue between two groups [ （0. 53 ±0.04） vs. （0. 52 ±0.05）, F =0. 155, P 〉0. 05, （0. 53 ± 0.09） vs. （0.50±0.05）, F=0.498, P〉0.05, （0.55±0.06） vs. （0.56±0.07）, F=0.073, P 〉 0. 05 ]. Conclusions Lung tissues of control group rats express Slit2 and Robo4. The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALl caused by CLP may be associated with the occurrence of ALl. Key words: Slit2; Robo4; Acute lung injury; Sepsis; Endothelial permeability; Cecum ligation and puncture; TNF-α; Axon guidance cues