Figure 4

Chol-HCQ liposomes suppress bleomycin-induced pulmonary fibrosis through anti-inflammatory effects and by inhibiting the CTGF/ERK signalling pathways. (a, up) Specific esterase staining of neutrophils in rat lung sections on day 7 of the experiment. The rats were treated with bleomycin before administration (400×). (b) Neutrophils were counted in 5 random 200 s fields. (a, below, and c)Immunohistochemistry analysis of CTGF levels in the lung tissues from BLM-induced pulmonary fibrosis rats. Original magnification is 200 s. Positive cells were countered in 5 random fields. (d and e) TNF-α and TGF-β1 contents in plasma from rats on day 7 were determined by ELISA kits. (f) Isolated alveolar macrophage cells were stimulated with bleomycin (25 μg/ml) and treated with Chol-HCQ (10 μM) or HCQ (10 μM) and etanercept (5 μg/ml) in the presence of brefeldin A in 24-well plate at 37 °C for 24 h. Flow cytometry was conducted to detect macrophage intracellular TNF-α expression. (g–j) On day 14, rats were sacrificed and lung tissues lysate were made for Western blot analysis. Chol-HCQ inhibits ERK1/2 (Thy202/Thr204) and NF-κB phosphorylation in vivo. Data are representative of three separate experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.