脂多糖诱导内源性高迁移率族蛋白B1分泌活化核因子-κB通路促进肺成纤维细胞异常增殖

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

目的观察脂多糖(lipopolysaccharide, LPS)刺激小鼠原代培养肺成纤维细胞后内源性高迁移率族蛋白B1(high mobility group box-1 protein, HMGB1)细胞内移位及细胞外分泌的情况,并明确其在LPS诱导肺成纤维细胞异常增殖过程中的重要作用。 方法①将原代培养的小鼠肺成纤维细胞采用随机数字表法分为2组(每组3个孔),即PBS对照组(Con组)和LPS组(100 μg/L),Western blot检测LPS刺激12 h后HMGB1乙酰化修饰的情况,同时采用免疫荧光检测HMGB1细胞内的定位情况。②将原代培养的小鼠肺成纤维细胞采用随机数字表法分为4组(每组3个孔),即PBS对照组(Con组)、100 μg/L LPS组(LPS100组)、250 μg/L LPS 组(LPS250组)和500 μg/L LPS(LPS500组),于LPS刺激24 h后采用ELISA法检测上清液中HMGB1的含量。③采用随机数字表法将小鼠原代肺成纤维细胞分为4组(每组6个孔),NF-κB通路抑制剂吡咯烷二硫代甲酸铵(ammonium pyrrolidinedithiocarbamate, PDTC)预处理细胞30 min后再分别使用LPS、HMGB1刺激细胞0、12、24 h和48 h,即PBS对照组(Con组)、 LPS组(或HMGB1组)、PDTC组、LPS+PDTC组(或HMGB1+PDTC组),Cell Counting Kit-8(CCK-8)实验检测细胞增殖情况。 结果①与Con组比较,LPS刺激细胞12 h后,LPS组乙酰化HMGB1/总HMGB1明显升高(P<0.05),同时细胞质中HMGB1表达明显增加。② LPS刺激细胞24 h后,LPS各组上清液中HMGB1含量较Con组明显升高(P<0.05)。③ LPS刺激细胞24 h和48 h 后,PDTC+LPS组细胞的光密度(D)值较LPS组明显降低(P<0.05);HMGB1刺激细胞12、24 h和48 h后,PDTC+HMGB1组D值较HMGB1组均明显降低(P<0.05)。 结论LPS可促进小鼠肺成纤维细胞核内HMGB1的主动分泌,进而通过活化NF-κB信号通路加速细胞增殖,可能是LPS诱导肺成纤维细胞异常增殖的重要机制。

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.