Lipopolysaccharide-induced endogenous high mobility group box-1 protein secretion promotes aberrant proliferation of lung fibroblasts through nuclear factor-κB pathway

Objective To observe the nuclear-cytoplasmic translocation and extracellular secretion of endogenous high mobility group box-1 protein (HMGB1) in primary mouse lung fibroblasts stimulated by lipopolysaccharide (LPS), and identify the role of HMGB1 in the proliferation of LPS-induced lung fibroblasts. Methods ① Primary mouse lung fibroblasts were randomly divided into two groups (n=3): a PBS control group (Con group) and a LPS group (100 μg/L). Endogenous HMGB1 acetylation followed by LPS stimulation for 12 h was detected by Western blot, while HMGB1 intracelullar translocation was detected by immunofluorescence assay. ② Primary mouse lung fibroblasts were randomly divided into four groups (n=3): a PBS control group (Con group), a 100 μg/L LPS group (LPS100 group), a 250 μg/L LPS group (LPS250 group) and a 500 μg/L LPS group (LPS500 group). The content of HMGB1 in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA) after stimulation of LPS for 24 h. ③ Primary mouse lung fibroblasts were randomly divided into four groups (n=6): a PBS control group (Con group), a LPS group (or HMGB1 group), a PDTC group, a LPS+PDTC group (or HMGB1+PDTC group). After pretreated with nuclear factor-κB (NF-κB) pathway inhibitor ammonium pyrrolidinedithiocarbamate(PDTC) for 30 min, Cell Counting Kit-8 (CCK-8) assay was adopted to detect the effects of PDTC in the proliferation of HMGB1-induced lung fibroblasts at 0, 12, 24 h and 48 h, respectively. Results ① Compared with the Con group, the ratio of ace-HMGB1/total HMGB1 and the expression of HMGB1 in cytoplasm were significantly increased in the LPS group after 12 h (P<0.05).② The content of HMGB1 in the supernatant of the LPS group was significantly higher than that in the Con group at 24 h (P<0.05). ③ Besides, the D value of LPS+PDTC group was significantly decreased at both 24 h and 48 h compared with the LPS group (P<0.05), and the D value of the HMGB1+PDTC group was significantly decreased at 12, 24 h and 48 h compared with HMGB1 group(P<0.05). Conclusions LPS could induce the endogenous HMGB1 protein secretion in mouse lung fibroblasts and promote cell proliferation through NF-κB signaling pathway, which may be one of the internal mechanisms for LPS-induced abnormal lung fibroblasts proliferation. Key words: Lipopolysaccharide; Lung fibroblast; High mobility group box-1 protein; Nuclear factor-κB; Cell proliferation