Effect of bleomycin and lipopolysaccharide on proliferation and fibroblast-to-myofibroblast differentiation of rat lung fibroblasts

Abstract

Objective To investigate the effect of bleomycin (BLM) and lipopolysaccharide (LPS) on the proliferation and fibroblast-to-myofibroblast differentiation of rat lung fibroblasts and the possible mechanism. Methods Primary lung fibroblasts were isolated and purified from new born Sprague Dawley rats.Fibroblasts were divided into control group, BLM-treated group and LPS-treated group.In the experimental groups, the fibroblasts were exposed to the concentration of 0.000 1, 0.001, 0.01, 0.1, 1 mg/L BLM and 0.01, 0.1, 1, 10, 100 mg/L of LPS respectively.The proliferation of fibroblasts and the secretion of transforming growth factor-β1 (TGF-β1) were evaluated by methyl thiazolyl tetrazolium (MTT) assay and enzyme linked immunosorbent assay, respectively.The concentration of 0.001/0.1 mg/L and 1/10 mg/L were further defined as low-dose and high-dose in BLM/LPS group according to the MTT assay.The expression of α-smooth muscle actin (α-SMA), ultrastructural morphology and DNA damage of fibroblasts were assessed by Western blot, transmission electron microscopy and single cell gel electrophoresis in the above groups. Results HE staining showed primary cultured cells had typical morphology of fibroblast with a spindle shape.Immunohistochemical staining showed the expression of vimentin was positive.Compared with control group, the proliferation of fibroblasts was elevated at certain concentrations in BLM and LPS groups (P<0.05), this effect was significantly greater in BLM-treated group.The level of TGF-β1 was increased in low-dose of BLM group (P<0.05), which was decreased as the concentration became higher (P<0.05). However, the level of TGF-β1 was unchanged in different concentrations of LPS-treated groups.The expression of α-SMA was significantly increased in the low-dose of BLM/LPS group (P<0.05), but markedly reduced in the high-dose of BLM/LPS group (P<0.05), which was more obvious in BLM-treated group.Transmission electron microscopy showed that the nucleus was shapeless in the low-dose of BLM group, the organelles were evidently dilated in the high-dose of BLM group.However, the dilated organelles and shapeless nucleus were noted in low/high-dose of LPS group.Single cell gel electrophoresis showed that the length of the tail was significantly increased in the low-dose of BLM/LPS group and there was no significant difference between them.However, high-dose of BLM/LPS group displayed a more accentuated comet tail, especially in BLM-treated group. Conclusions BLM and LPS can promote the proliferation of fibroblasts and fibroblast-to-myofibroblast differentiation at a certain concentration, and the former has more obvious effect than the latter.BLM and LPS can both induce DNA damage of fibroblasts to a different extent, which may be related to the proliferation and differentiation of fibroblasts. Key words: Bleomycin; Endotoxins; Lung fibroblast proliferation and fibroblast-to-myofibroblast differentiation; DNA damage