| Object:To explore the expression level of PDE4in lung inflammation and fibrosis induced by Bleomycin (BLM), and to investigate the underlying mechanisms of pulmonary fibrosis.Method:Anesthetized male8-week-old C57BL/6mice were exposed to BLM (2.5mg/kg total,by intratracheal instillation) or saline as control on day0and killed on day3,day7, day14, day21and day28. Bleomycin-induced changes in total and differential cell counts in BALF were observed. Inflammatory cytokins such as IL-1β, IL-6, MIP-2, TGF-β1and TNF-a levels in BALF were evaluated by enzyme-linked immunoassay. Content of collagen and MPO in tissue homogenate at different stage were also detected. The expression of PDE4B protein in lung tissue was evaluated by western blot and the PDE4A, PDE4B, PDE4C and PDE4D mRNA was measured by real-time RCR. Protein of each subtype at different stage in bleomycin-treated groups was located by immunostaining.Results:(1) Histoliogical studies. Infiltration by leukocytes increased in the early stage compared with controls, which indicated bleomycin induced lung inflammation. Bleomycin induced a fibrotic response in lung, with enhanced deposition of collagen and parenchymal remodeling since7days, as visualized by Masson’s trichrome staining.(2) A marked influx of inflammatory cells, particularly of macrophage,into the airways was observed, following intratracheal bleomycin instillation. That reached a peak at3days,75.19±3.89(×105cell/mL).(3)The pulmonary inflammation induced by BLM was significantly serious than that in control group with much higher expression of inflammatory cytokins proteins in BALF. Peak values of expression of IL-1β (218±69pg/ml versus104±26pg/ml in controls, P<0.001) and IL-6(405±119pg/ml versus141±24pg/ml in controls P<0.001) appeared on day3, and those of TGF-β1(390±57pg/ml versus71±25pg/ml in controls, P<0.001) and TNF-a (450±57pg/ml versus131±43pg/ml in controls P<0.001) appeared on day7. In addition, the expression of MIP-2was up-regulated all the time.(4) Changes of collagen content and myeloperoxidase (MPO) activity in tissue homogenate at different stage:Bleomycin induced lung injury was determined by measurement of MPO activity,which increased in the14days (0.838±0.050unit/mg total protein versus0.449±0.070unit/mg total protein in controls, P<0.001) and then descended sharply.A gradual increace in collagen content (36.3±5.95μg/mg total protein versus23.0±3.71μg/mg total protein in controls, P<0.001)μg/mg total protein indicated a fibrotic response in lung.(5) There was a valley form of PDE4A mRNA expression during the bleomycin-induced fibrosis process, the bottom was at7days(0.189±0.053fold of control, P<0.002). PDE4A protein was observed in inflammatory cell by immunohistochemical staining.(6) BLM enhanced the expression of PDE4B mRNA in mice in the fibrosis process. The expression of PDE4B mRNA increased significantly with a1.57fold up-regulation since the14th day (in comparison to control group) and that clamb up to2.57fold up-regulation on28days. Upregulation of PDE4B protein in bleomycin-treated groups was surveyed by the results of wetern blot and tissue immunostaining.(7) There was no significantly change of the mRNA or protein expression of PDE4C(8) There was also an increase in the expression of PDE4D mRNA, with a2.53fold up-regulation on28days. Immunostaining of PDE4D protein has been observed in inflammatory cell and bronchial epithelium and smooth muscle of blood vessel and airway.Conclusion:A valley of PDE4A expression during the bleomycin-induced fibroses process has been observed and that might related with the excretion of TGF-β1and TNF-a. It appears that the high expression of PDE4B and PDE4D was involved in the development of mice BLM-induced lung fibrosis. These results provide further support for a role of PDE4B and PDE4D inhibitor in the prevention and therapy of lung fibrosis. |
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