Effects of N-acetylcysteine on Clara cells in rats with cigarette smoke exposure

Background The number of Clara cells and the Clara cell 16-kDa protein （CC16） levels of the lung decrease in patients with chronic obstructive pulmonary disease （COPD）. N-acetylcysteine （NAC） is a powerful antioxidant and can reduce the frequency of acute exacerbations of COPD. But the exact mechanism is unclear. The present study was designed to investigate the effects of NAC on Clara cells in rats with cigarette smoke exposure. Methods Eighteen adult male Wistar rats were randomly divided into 3 groups, 12 exposed to cigarette smoke （CS） thrice a day, 10 cigarettes for 30 minutes each time for 1 week, without （CS group） or with （CS＋NAC group） oral intake of NAC 80 mg·kg^-1·d^-1, and another 6 rats exposed to fresh air （control group）. Clara cells were observed by an electron microscope. The mRNA expression of CC16 and CC16 protein in lungs were determined by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry respectively. The glutathion （GSH） level in plasma and lung tissue were tested by fluorimetry assay. Results Compared with the controls, the pathologic score of small airways significantly increased in the CS exposed rats （20.3±14.7 vs. 53.7±11.5, P 〈0.05）. The Clara cell particles in cytoplasm decreased in the CS group （P 〈0.05）. The percentage of CC16-positive cells in bronchioles in the CS group （27.8±4.3 and 29.5±2.4 in terminal bronchioles and respiratory bronchioles, respectively） significantly decreased as compared with the control group （37.1±3.8 and 43.8±5.8 in terminal bronchioles and respiratory bronchioles, respectively） （P 〈0.05）. No significant difference was observed in GSH level （（181±26） nmol/L in the control group vs. （170±18） nmol/L in the CS group） between the two groups. After treatment with NAC, the pathologic score of small airways （24.1±17.5） decreased （P 〈0.05）. Clara cell particles in cytoplasm of Clara cells increased and GSH level in plasma （（213±40） nmol/L vs. （170±18） nmol/L in the CS group） increased too （P 〈0.05）, while the increase in the proportions of CC16 positive cells in bronchioles （30.1±6.4 and 34.3±6.3 in terminal bronchioles and respiratory bronchioles, respectively） did not reach the statistical significance （P 〉0.05）. No significant difference was found in the expression of CC16 mRNA among the three groups. Correlation analysis indicated that the percentage of CC16-positive cells in bronchioles negatively correlated with the pathologic score of small airways （r = -0.592, P 〈0.05）, but not with GSH level. Conclusions One-week CS exposure decreased the number of Clara cells and the expression of CC16 in bronchioles in rats. NAC might provide protection of the Clara cells from oxidative damage and possibly through the elevation of the synthesis and secretion of CC16. These data indicate that NAC decreases airway inflammation induced by CS via induction of CC16.

Serum Clara Cell Protein（CC16）：A New Valid Marker of the Distal Airway Damages Caused by Air Pollutants in Rats

Cell count, LDH, total protein, Clara cell protein (CC16) and lysozyme in bronchoalveolar lavage fluid (BAL) and serum were determined in rats. 30 - 50 ppm sulfur dioxide for 4 h, 12 h and 2 week exposure didn't produce any change of above parameters. 1. 0 ppm, 0. 5 ppm, 0. 38 ppm, 0. 25 ppm and 0. 20 ppm ozone for 7 h exposure tests were performed. CC16, total protein and LDH in BAL had significantly changes in BAL in ozone groups. Only serum CC16 showed significant increase among ozone groups. Both BAL and serum CC16 were of the best exposure-response relationship with ozone concentration.The change of BAL fluid reached high peak 18-24 h after ozone exposure.Serum CC16 seemed to be a valid marker of the distal airway damages caused by air pollutants.

Regulation on the expression of Clara cell secretory protein in the lungs of the rats with acute lung injury by growth hormone

Background Clara cell secretory protein （CC16） is an important lung derived protective factor and may play an important role on the pathogenesis of acute lung injury （ALl） 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 ALl induced by endotoxemia, but the mechanism is not very clear yet. Whether the effects are related to CC16 or not is undetermined. Methods One hundred and twelve male Sprague-Dawley rats were randomly divided into an ALl 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-KB）, the numbers of NF-KB positive cells in lungs were counted after immunofluorescence staining, and the levels of NF-KB inhibitory protein-a （IKB-a） in lung homogenates of rats were detected by Western blotting. The expression levels of CC16 mRNA in lungs of the rats with ALl 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. Results Half an hour after LPS injury both the PASAD and PMN numbers in lungs of the rats with ALl 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 ALl group. The levels of TNF in lungs of the rats with ALl 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 ALl 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 ALl group at different time intervals post-injury. The number of NF-KB 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-KB positive cells and the enhanced intensity of fluorescence began to decrease from 6-hour post-injury, but the number of NF-KB cells at 24 hours post-injury was still higher than in the control group. The number of NF-KB cells in lungs in the GH group was significantly higher than in the LPS group at the different time intervals post-injury. The IKB-a expression in lungs of the rats with ALl homogenates decreased dramatically 0.5-hour post-injury, reaching a nadir at 4-hour post-injury and then began to recover. The levels of IKB-Q in GH group were significantly lower than those in ALl group. Both the levels of CC16 mRNA and protein in lungs of the rats with ALl 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 ALl group at the different time intervals post-injury. Correlation analysis indicates that CC16 correlates significantly with all the indices mentioned above. Conclusions Down-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.