Background: Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro. However, its effects on the process of lung fibroblast activation and fibrosis initiation during LPS induced acute lung injury (ALI) remain unknown. The goal of the present study was to determine the effect of inhibiting TLR4 on LPS-induced ALI and fibrosis in vivo. Methods: The ALI model was established by intraperitoneal injection of LPS in mice. TLR4-small hairpin RNA (shRNA) lentivirus was injected intravenously into the mice to inhibit TLR4 expression. mRNA and protein levels were detected by real-time PCR and Western-blot analysis, respectively. The contents of the C-terminal propeptide of type I procollagen (PICP) in bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the degree of fibrosis was detected by van Gieson collagen staining, the hydroxyproline assay, and alpha smooth muscle actin (α-SMA) immunohistochemical staining. Results: Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected. Moreover, the degree of fibrosis was shown to increase by ELISA analysis of PICP in BALF, van Gieson collagen staining, the hydroxyproline assay, and α-SMA immunohistochemical staining. All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus. Conclusions: Inhibiting TLR4 signaling could ameliorate fibrosis at the early stage of ALI induced by LPS.

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.

Acute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process. Recombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo. Features of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro. HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.