Department of Anesthesiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
PLoS One. 2012;7(4):e35926. doi: 10.1371/journal.pone.0035926. Epub 2012 Apr 26.
Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.
肺纤维化的特征是肺成纤维细胞增殖和胶原分泌。在脂多糖（LPS）诱导的急性肺损伤（ALI）中，肺成纤维细胞的异常增殖发生在疾病早期阶段，但潜在机制尚不清楚。在这项研究中，我们使用 TLR4-siRNA-慢病毒在培养的小鼠肺成纤维细胞中敲低 Toll 样受体 4（TLR4）表达，以研究 LPS 挑战对肺成纤维细胞增殖、磷酸肌醇 3-激酶（PI3K）-Akt 途径激活和磷酸酶和张力蛋白同源物（PTEN）表达的影响。BrdU 测定法检测到的肺成纤维细胞增殖在 1 μg/mL LPS 挑战下 24 小时内不受影响，但在 72 小时时，细胞增殖显著增加。这种增殖被 siRNA 介导的 TLR4 敲低或 PI3K 抑制剂 Ly294002 处理所抑制。此外，siRNA 介导的 TLR4 敲低抑制了 LPS 诱导的 TLR4 上调、PTEN 下调和 PI3K-Akt 途径（磷酸化 Akt 的过表达）激活，这是通过实时 PCR 和 Western blot 分析检测到的。用 PTEN 抑制剂 bpV(phen)处理导致 PI3K-Akt 途径的激活。PI3K 激活状态既不影响肺成纤维细胞中 PTEN 的基础表达，也不影响 LPS 诱导的下调。PTEN 抑制足以发挥 LPS 对肺成纤维细胞增殖的作用，而 PI3K-Akt 途径抑制可以逆转这一过程。总的来说，这些结果表明，LPS 可以通过涉及 PTEN 表达下调和 PI3K-Akt 途径激活的 TLR4 信号机制促进肺成纤维细胞增殖。此外，PI3K-Akt 途径的激活是 PTEN 抑制的下游效应，在肺成纤维细胞增殖中起着关键作用。这种机制可能有助于并可能加速 ALI/ARDS 早期的肺纤维化。
