BACKGROUNDClara cell secretory protein (CC16) is an important lung derived protective factor and may play an important role on the pathogenesis of acute lung injury (ALI) induced by endotoxemia. Growth hormone (GH) is an important anabolism hormone secreted by GH cells of the hypophysis. Previous research showed that GH would significantly exacerbate ALI induced by endotoxemia, but the mechanism is not very clear yet. Whether the effects are related to CC16 or not is undetermined.

METHODSOne hundred and twelve male Sprague-Dawley rats were randomly divided into an ALI group and a GH group. The rats in the two groups were subdivided into seven subgroups, according to injection with lipopolysaccharides (LPS) or not, then according to different intervals of time after LPS injection; 0 hour (pre-injection of LPS, acted as control group), 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours for subgroups. Pulmonary alveolar septa area density (PASAD) and ploymorphonuclear cells (PMN) in the lungs were analyzed morphometrically. The levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6) were determined by radioimmunoassay. To analyze the expression and activation of nuclear factor kappa B (NF-κB), the numbers of NF-κB positive cells in lungs were counted after immunofluorescence staining, and the levels of NF-κB inhibitory protein-α (IκB-α) in lung homogenates of rats were detected by Western blotting. The expression levels of CC16 mRNA in lungs of the rats with ALI were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The levels of CC16 protein in lung homogenates were detected by Western blotting.

RESULTSHalf an hour after LPS injury both the PASAD and PMN numbers in lungs of the rats with ALI began to increase significantly and peaked at 6-hour post-injury. They then began to recover and reached normal levels at 24-hour post-injury. Both the PASAD and PMN numbers in the GH group increased more significantly than those in the ALI group. The levels of TNF in lungs of the rats with ALI homogenates increased significantly 0.5-hour post-injury, peaked at 1-hour and maintained a high level until 6 hours then gradually recovered. The content of TNF in the GH group lung homogenates increased more significantly than in the ALI group post-injury. The contents of IL-6 in rat lung homogenates began to increase significantly at 1-hour post-injury, peaked at 4 hours then gradually returned to normal levels by 6 hours post-injury. The levels of IL-6 in the lung homogenates of the GH group were higher than in the ALI group at different time intervals post-injury. The number of NF-κB positive cells increased dramatically at 0.5-hour post-injury, and the fluorescence intensity was enhanced. Both peaked at 4-hour post-injury. The number of NF-κB positive cells and the enhanced intensity of fluorescence began to decrease from 6-hour post-injury, but the number of NF-κB cells at 24 hours post-injury was still higher than in the control group. The number of NF-κB cells in lungs in the GH group was significantly higher than in the LPS group at the different time intervals post-injury. The IκB-α expression in lungs of the rats with ALI homogenates decreased dramatically 0.5-hour post-injury, reaching a nadir at 4-hour post-injury and then began to recover. The levels of IκB-α in GH group were significantly lower than those in ALI group. Both the levels of CC16 mRNA and protein in lungs of the rats with ALI began to decrease significantly 0.5-hour post-injury, reached a nadir at 6 hours, and then began to recover. Both the expression of CC16 mRNA and CC16 protein in the GH group were significantly lower than those in the ALI group at the different time intervals post-injury. Correlation analysis indicates that CC16 correlates significantly with all the indices mentioned above.

CONCLUSIONSDown-regulation of CC16 expression plays a critical role in the pathogenesis of acute lung injury induced by endotoxemia. The application of GH can exacerbate the lung injury induced by endotoxemia through down-regulating the expression of CC16.