An observational study on autophagy in mouse lung fibroblasts inhibited by lipopolysaccharide

Objective To evaluate the effects of lipopolysaccharide (LPS) on the expression of autophagy-associated proteins, Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3) in primary mouse lung fibroblasts in vitro. Methods Primary cultured mouse lung fibroblasts were seeded in 6-well plates at a density of 1×104/ml. After adherence, the cells were starved by free-serum culture media overnight and then divided into four groups according to random number table(n=3): a phosphate buffer saline (PBS) control group (group Con), a 250 μg/L LPS group (group LPS250), a 500 μg/L LPS group (group LPS500) and a 1 000 μg/L LPS group (group LPS1 000). The cells were exposed to PBS and the corresponding concentrations of LPS for 30 h before total protein extraction. The expression of Beclin-1 and LC3 were determined by Western blot. Meanwhile, the cells were divided into two groups according to random number table(n=3): group IF-Con and group IF-LPS. The formation of autophagosomes was observed by immunoflourence. Results The amount of LC3 protein was down-regulated as the increase of LPS concentrations. The ratio of LC3-Ⅱ/LC3-Ⅰ was apparently higher in group Con than that in other groups, which was gradually decreased as the concentrations of LPS increased. A lower ratio of LC3-Ⅱ/LC3-Ⅰ was found in group LPS1 000 than that in group Con(P<0.05). There was no obvious difference in Beclin-1 expression among groups Con, LPS250 and LPS500. However, a remarkably decreased amount of Beclin-1 was found in group LPS1 000 than that in group Con(P<0.05). Furthermore, the fibroblasts showed a marked weaker fluorescence expression of autophagosomes in group IF-LPS compared to those in group IF-Con. Conclusions The expression of LC3 and Beclin-1 can be down-regulated by 1 000 μg/L LPS, and the formation of autophagosomes were inhibited by LPS at a concentration of 1 000 μg/L. The findings indicates that LPS inhibits autophagy in mouse lung fibroblasts, which could be part of the internal mechanism of pulmonary fibrosis. Key words: Lipopolysaccharide; Fibroblast; Pulmonary fibrosis; Autophagy