| Backgrounds&AimPulmonary fibrosis (PF) is a class of diseases induced by a variety of stimulus,which cause lung inflammation, sustained alveolar injury, repeated destruction, repair, rebuilding and excessive deposition of the extracellular matrix, eventually leading to structural changes and loss of function of the lung. The exact pathogenesis has not been fully elucidated, and it is still lack of specific and effective treatments to prevent or reverse pulmonary fibrosis. Pulmonary fibrosis is still an international problem. Our group found the carboxyamidotriazole (CAI) showed prominent anti-inflammatory effect in a series of acute and chronic inflammation models, and CAI significantly down-regulated the levels of pro inflammatory cytokines. Therefore, we proposed whether CAI, which had anti-cancer and anti-inflammatory effects, be capable of blocking the process of pulmonary fibrosis through inhibiting the initial inflammatory response? What's the mechanism of CAI? We launched a series of studies on these issues.MethodsThe pulmonary fibrosis model of C57BL/6J mice was established by subcutaneous injection of bleomycin (BLM).Three groups were set up, control group (CON), model group(BLM) and drug treatment group (BLM+CAI),to observe the impact of CAI (30mg/kg) on mice with pulmonary fibrosis and explore the possible mechanism of CAI. The experimental period was43days. Body weights, lung coefficients, inflammatory scores, fibrosis scores and the hydroxyproline content were detected. Fluorescent quantitative PCR and ELISA were used to detect the mRNA expression and protein expression levels of proinflammatory cytokines IL-1β, IL-6, TNF-a, Th17type cytokines IL-17A and the fibrogenic cytokine TGF-β1in mouse lung tissue, bronchoalveolar lavage fluid and lavage inflammatory cells. Cell line NR8383and primary rat alveolar macrophages were used to establish models in vitro. Growth curve of NR8383was drawed by cell counting method in a7days period. The viabilities of NR8383cells and primary rat alveolar macrophages after given BLM and CAI were detected by CCK8method. Results1. Subcutaneous injection of bleomycin can successfully establish mouse model of pulmonary fibrosis.2. CAI (30mg/kg) can effectively improve the pulmonary fibrosis induced by bleomycin in C57BL/6J mice. CAI had positive effect on the weights of the mice with pulmonary fibrosis, significantly reduced the mouse lung coefficient, and alleviated the lesions of lungs and the skin pigmentation, indicating that CAI can not only reduce collagen deposition in lung, alleviate the damage of the lung, but also improve the skin pigmentation which is another side effect of bleomycin.3. The inflammatory scores of CON group, BLM group and BLM+CAI group were0.60±0.23,4.07±0.92and2.33±0.74, respectively. The score of BLM group was significantly increased (p<0.01, vs.CON), while the BLM+CAI group was significantly reduced (p<0.01, vs.BLM), indicating that BLM caused serious inflammation and CAI could reduce it.4. The fibrosis scores of CON group, BLM group and BLM+CAI group were0.29±0.07,5.57±1.09and3.24±1.37, respectively. The pulmonary fibrosis level of BLM group was significantly increased (p<0.01, vs. CON group); while that of BLM+CAI group was significantly reduced (p<0.01, vs. BLM group), indicating that BLM caused serious pulmonary fibrosis and CAI could reduce it.5. Ratios (%) of fibrosis area vs. lung slice area of CON group, BLM group and BLM+CAI group were0.02±0.02%,16.04±10.48%and4.74±3.38%, respectively. The ratio of BLM group was significantly increased (p<0.01, vs. CON group); while that of BLM+CAI group was significantly reduced (p<0.05, vs. BLM group), indicating that CAI could reduce the fibrosis in lung caused by BLM.6. The hydroxyproline content of CON group, BLM group and BLM+CAI group were2.72±0.45,3.620.84and2.74±0.31μg/mg dry weight, respectively. The hydroxyproline content of BLM group increased significantly (p<0.05, vs.CON group); while that of BLM+CAI group was significantly decreased (p<0.05vs.BLM group), indicating that CAI can reduce the collagen deposition in lung.7. CAI significantly down-regulated IL-1β protein expression in the lung homogenate (p<0.05), and the transcriptions of IL-6(p-<0.05) and TNF-a (p-<0.05) in BALF cells. 8. CAI significantly down-regulated the transcription of TGF-β1in lung homogenate (p <0.05).9. CAI significantly down-regulated the protein expression of IL-17A in lung homogenate (p-<0.01) and the transcription of IL-17A in BALF cells (p-<0.05).10. Both BLM and CAI could inhibit the proliferation of NR8383cells.11. BLM of higher concentrations (320and640μg/mL) had toxic effects on primary alveolar macrophages of SD rat, while different concentrations of CAI had no effect on viability of primary rat alveolar macrophages.ConclusionsCAI can alleviate the pulmonary fibrosis induced by bleomycin in mice. The results in vivo showed that the alleviation effect of CAI partly due to its anti-inflammatory effect. CAI not only reduced the expression of proinflammatory cytokines IL-1β, IL-6and TNF-a, but also reduced the expression of Th17cytokine IL-17A. CAI blocked the process of fibrosis partly through reducing the inflammation in lung. In addition, CAI also reduced the expression of TGF-β1, suggesting that CAI may have anti-fibrosis effect. CAI not only depressed bleomycin-induced pulmonary fibrosis in mice, but also relieved the other side-effect of bleomycin-pigmentation. It can be speculated that, further studies of CAI on the clinical treatment of pulmonary fibrosis will have potential applications.
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