| Part one BML-111, a lipoxin receptor agonist, inhibits pulmonary fibrosis in mice induced by bleomycinObjective:To investigate whether lipoxin receptor agonist BML-111improves the survival rate of mice instilled with BLM and influence excessive production of the total Collagen, hydroxyproline, transforming growth factor β1(TGFβ1), a-SMA and Fn1of lung tissue.Methods:Male C57BL/6mice were intratracheally administered a single bolus of BLM to make model of pulmonary fiborsis. For survival studies, BLM was dissolved in sterile saline to produce a total dose of2.5mg BLM/kg body weight, mortality was assessed daily until the day21st after BLM instillation. To evaluate the effect of treatment, using the dose of BLM was2mg/kg body weight. Mice were randomly divided into4groups:sham group (Sham), bleomycin (BLM), BLM plus BML-111(BML-111), and BLM plus BML-111and BOC-2(BOC-2). BML-111was intraperitoneally injected every other day starting2h after the BLM injection, and BOC-2group was injected BOC-2(50μg/kg intraperitoneally) before giving BML-11130min. The effects of BML-111on the expression of the total Collagen, hydroxyproline, transforming growth factor β1(TGFβ1), a-SMA and Fnl were analysed by dye-binding method, Chemical colorimetric, ELISA, and Western blotting.Results:BML-111improved mice survival after bleomycin instillation. Compared to the Sham group, the other three groups showed varying degrees of pulmonary fiborsis. Fiborsis in lung in BML-111groups was alleviated compared with BLM group and BOC-2group by HE-staining and massion-staining. The production of the total Collagen, hydroxyproline, transforming growth factor β1(TGFβ1), a-SMA and Fn1in BML-111group was higher than the Sham group but lower than BLM group and BOC-2group.Conclusion:BML-111can ameliorate bleomycin-induced pulmonary fibrosis in mice. Part two Inhibitory effect of BML-111on TGFβ1-induced mouse embryo lung fibroblasts activationObjective:To investigate the effects of BML-111on activation of mouse embryo lung fibroblasts.Methods:To find a suitable concentration of BML-111, different concentrations (1nM,10nM,100nM,200nM or500nM) of BML-111or vehicle (methanol) was co-cultured with the cells for30min before addition of TGFβ1(5ng/ml), leading to a concentration of200nM BML-111used in subsequent experiments. To investigate whether the action of BML-111is related to ALXs,10uM of lipoxin receptor antagonist BOC-2was supplemented to the cells prior to treated with BML-111for30min. The expression of ALX on NIH3T3was determined by PCR, and the effects of TGFβ1and/or BML-111on the expression of a-SMA, Fn1, and the total collagen were analysed by immunofluorescence, Western blotting, dye-binding method. The proliferation of the cells was measured by MTT.Results:Both ALX1/FPR-rsl and ALX2/FPR2were expressed on NIH3T3cells. BML-111and vehicle did not affect NIH3T3cells proliferation. Stimulation of NIH3T3cells with TGFβ1not only induced proliferation of the cells, but also markedly increased a-SMA, Fnl, and the total collagen production. Pretreatment with BML-111significantly inhibited TGFβ1-induced proliferation and production of a-SMA, Fnl, and the total collagen and these effects were inhibited by BOC-2.Conclusion:BML-111can inhibit proliferation and activation of fibroblast cell line NIH3T3via ALX. Part three BML-111inhibit TGFβ1-mediated Smad2/3, Erk, Akt signaling pathways activationObjective: To investigate the effects of BML-111on the activation of TGFβ1mediated signaling pathways, including both Smad-dependent and non Smad-dependent responses.Methods: In all experiments, NIH3T3cells were treated with BML-111or vehicle (0.028%methanol) for30min before addition of5ng/ml TGFβ1under serum-free conditions. The expresson of protein of ERK, Akt, Smad2/3and the phosphorylation of ERK, Akt, Smad2and Smad3was analyzed by Western blotting. Results: The phosphorylation of ERK, Akt, Smad2/3was reduced by BML-111treatment.Conclusion:BML-111downregulates both Smad-dependent and Smad-independent signaling pathways induced by TGFβ1. |
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