Journal: Scientific Reports

Article Title: Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis

doi: 10.1038/s41598-017-11450-3

Figure Lengend Snippet: Chol-HCQ inhibits the lung fibroblast proliferation via NF-κB and ERK pathways. The lung fibroblasts were obtained from bleomycin-treated rats as described and the cells were seeded in a 96-well plate. The cells were treated with Chol-HCQ (0-100μΜ) for 24 hours and tested by ( a ) MTT or ( b ) by the percentages of EdU-stained lung fibroblasts. ( c and d ) For the apoptosis analysis, lung fibroblasts were treated with Chol-HCQ for 48 hours followed by Annexin V/PI staining and examined by FACS analysis. Similar results were obtained in three separate experiments. ( e – h ) Chol-HCQ inhibits ERK1/2 and NF-κB phosphorylation in lung fibroblasts. Chol-HCQ decreases ERK1/2 phosphorylation dose-dependently at Thr202/Tyr204 and NF-κB phosphorylation. The data are representative of three separate experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: The rats were administered a single intratracheal instillation of bleomycin sulphate dissolved in saline (5 mg/kg body weight; Melone Pharmaceutical Co., Ltd, Dalian, China) on day 0 of the experiment to induce pulmonary fibrosis as previously described , while an equal volume of saline was injected into the rats from the control group.

Techniques: Staining, Phospho-proteomics

Journal: Scientific Reports

Article Title: Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis

doi: 10.1038/s41598-017-11450-3

Figure Lengend Snippet: Histological examinations of the anti-fibrotic effects and the time kinetic of Chol-HCQ liposomes in bleomycin-induced pulmonary fibrosis. Rats were treated with bleomycin and then injected with Chol-HCQ liposomes (20 mg/kg/day) and HCQ liposomes (8 mg/kg/day) via the tail vein; PBS solution and null liposomes (PC) were used as controls. ( a ) On day 28, the rats were sacrificed and histological examination was performed by H&E staining (up) and Masson’s trichrome staining (below). Original magnification is 200x (n = 6); the data are representative of three separate experiments. ( b ) Bleomycin-induced rats were treated with Chol-HCQ liposomes or PBS as a control. On days 7, 14 and 28, the rats were sacrificed and histological examination was performed by H&E staining (n = 4). ( c ) The hydroxyproline contents in the lung tissues were examined on day 28 (n = 6). ( d ) Lung lavages were collected and examined by differential cell counting in the BALF on day 28 of the experiment; the total cell numbers, neutrophils, macrophages, eosinophils and lymphocytes were counted. Original magnification is 200x; data are representative of three separate experiments. *p < 0.05.

Article Snippet: The rats were administered a single intratracheal instillation of bleomycin sulphate dissolved in saline (5 mg/kg body weight; Melone Pharmaceutical Co., Ltd, Dalian, China) on day 0 of the experiment to induce pulmonary fibrosis as previously described , while an equal volume of saline was injected into the rats from the control group.

Techniques: Liposomes, Injection, Staining, Control, Cell Counting

Journal: Scientific Reports

Article Title: Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis

doi: 10.1038/s41598-017-11450-3

Figure Lengend Snippet: 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.

Article Snippet: The rats were administered a single intratracheal instillation of bleomycin sulphate dissolved in saline (5 mg/kg body weight; Melone Pharmaceutical Co., Ltd, Dalian, China) on day 0 of the experiment to induce pulmonary fibrosis as previously described , while an equal volume of saline was injected into the rats from the control group.

Techniques: Liposomes, Staining, Immunohistochemistry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Expressing, Western Blot, Phospho-proteomics, In Vivo

Journal: Scientific Reports

Article Title: Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis

doi: 10.1038/s41598-017-11450-3

Figure Lengend Snippet: Preliminary safety evaluations of Chol-HCQ liposomes in female rats with bleomycin-induced pulmonary fibrosis. ( a ) Representative H&E images (400x) of vital organs including the heart, liver, spleen and kidney. Chol-HCQ and HCQ liposomes had no obvious toxicity on these tissues. ( b ) The concentrations of Chol-HCQ and HCQ in rat whole blood from 0 to 24 hours after intravenous administration of Chol-HCQ liposomes (total 20 mg/kg, Chol-HCQ 10 mg/kg) and HCQ sulphate (10 mg/kg) were assayed using high performance liquid chromatography (HPLC) (n = 5).

Article Snippet: The rats were administered a single intratracheal instillation of bleomycin sulphate dissolved in saline (5 mg/kg body weight; Melone Pharmaceutical Co., Ltd, Dalian, China) on day 0 of the experiment to induce pulmonary fibrosis as previously described , while an equal volume of saline was injected into the rats from the control group.

Techniques: Liposomes, High Performance Liquid Chromatography