Li Wen, Xu Qiaoyi, Deng Yuxiao, Yang Zhongwei, Xing Shunpeng, Zhao Xianyuan, Zhu Ping, Wang Xiangrui, He Zhengyu, Gao Yuan
Department of Critical Care Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Department of Anesthesiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Lab Invest. 2015 Jun;95(6):635-47. doi: 10.1038/labinvest.2015.44. Epub 2015 Apr 13.
The mechanism underlying lipopolysaccharide (LPS)-induced aberrant proliferation of lung fibroblasts in Gram-negative bacilli-associated pulmonary fibrosis is unknown. High-mobility group box 1 (HMGB1) is a ubiquitous nuclear protein that is released from the nuclei of lung fibroblasts after LPS stimulation. It can exasperate LPS-induced inflammation and hasten cell proliferation. Thus, this study investigated the effects of LPS- and/or HMGB1-stimulating murine lung fibroblasts on gene expression using various assays in vitro. Thiazolyl-diphenyl-tetrazolium bromide (MTT) assay data showed that either LPS or HMGB1 could induce lung fibroblast proliferation. Endogenous HMGB1 secreted from lung fibroblasts was detected by enzyme-linked immunosorbent assay (ELISA) 48 h after LPS stimulation. Pretreatment with an anti-HMGB1 antibody inhibited the proliferative effects of LPS on lung fibroblasts. DNA microarray data showed that the NF-κB signaling genes were upregulated in cells after stimulated with LPS, HMGB1, or both. Secretion of matrix metalloproteinase (MMP)-2 and MMP-9, and tissue inhibitor of metalloproteinase 2 (TIMP-2) was significantly upregulated after treatment with LPS, HMGB1, or their combination. However, an NF-κB inhibitor was able to downregulate levels of these proteins. In addition, levels of Toll-like receptor 4 (TLR4), Toll-like receptor 2 (TLR2), and receptors for advanced glycation end products (RAGE) mRNA and proteins were also upregulated in these cells after LPS treatment and further upregulated by LPS plus HMGB1. In conclusion, the data from the current study demonstrate that LPS-induced lung fibroblast secretion of endogenous HMGB1 can augment the proproliferative effects of LPS and, therefore, may play a key role in exacerbation of pulmonary fibrosis. The underlying molecular mechanisms are related to the activation of the TLR4/NF-κB signaling pathway and its downstream targets.
革兰氏阴性杆菌相关性肺纤维化中，脂多糖（LPS）诱导肺成纤维细胞异常增殖的潜在机制尚不清楚。高迁移率族蛋白B1（HMGB1）是一种普遍存在的核蛋白，在LPS刺激后从肺成纤维细胞核中释放出来。它可加剧LPS诱导的炎症并加速细胞增殖。因此，本研究使用多种体外试验研究了LPS和/或HMGB1刺激小鼠肺成纤维细胞对基因表达的影响。噻唑基二苯基溴化四氮唑（MTT）试验数据表明，LPS或HMGB1均可诱导肺成纤维细胞增殖。LPS刺激48小时后，通过酶联免疫吸附测定（ELISA）检测到肺成纤维细胞分泌的内源性HMGB1。用抗HMGB1抗体预处理可抑制LPS对肺成纤维细胞的增殖作用。DNA微阵列数据显示，在用LPS、HMGB1或两者刺激后，细胞中的NF-κB信号基因上调。用LPS、HMGB1或其组合处理后，基质金属蛋白酶（MMP）-2和MMP-9以及金属蛋白酶组织抑制剂2（TIMP-2）的分泌显著上调。然而，NF-κB抑制剂能够下调这些蛋白的水平。此外，在LPS处理后，这些细胞中Toll样受体4（TLR4）、Toll样受体2（TLR2）以及晚期糖基化终产物受体（RAGE）的mRNA和蛋白水平也上调，并且在LPS加HMGB1处理后进一步上调。总之，本研究数据表明，LPS诱导肺成纤维细胞分泌内源性HMGB1可增强LPS的促增殖作用，因此可能在肺纤维化加重中起关键作用。潜在的分子机制与TLR4/NF-κB信号通路及其下游靶点的激活有关。
