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.

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We report a novel strategy of simultaneous in situ extraction and fabrication of surface-enhanced Raman scattering substrate (IE-SERS) to perform selective and reliable on-site determination of thiram residue in soil, fruits, and vegetables. In this protocol, the thiram residue on complex surfaces can facilely diffuse into the solvent (dichloromethane (DCM)) and specifically bind to gold nanoparticles (AuNPs), affording the SERS substrate through the embedding of the thiram-trapped AuNPs into the cellulose p-toluenesulfonates (CTSAs) film through the evaporation of DCM. SERS signals of the specifically prepared CTSAs could be used as an internal standard to calibrate the absolute signal of thiram, which can avoid the fluctuation of SERS intensities caused by uneven and irregular morphology of SERS substrate. Thus, reliable quantitation of thiram through SERS detection and superior reproducibility in the SERS measurement (RSD = 4.21%) were achieved. As for directly sensing the thiram residue in soil, the established method shows strong anti-interference ability and a good linear response from 0.1 to 12 μg/g with a low limit of detection (LOD) of 50 ng/g, which is lower than that of all the previously reported methods. The recoveries range from 91.76 to 112.3% for thiram in paddy soils, indicating that the established IE-SERS method is reliable and applicable to the detection of thiram residue in real soil samples. In addition, the measurement of the residual thiram on strawberry and cucumber surface was also successfully accomplished by this strategy, indicating that the established method also has great potential in the in situ ultrasensitive detection of thiram on irregular fruits and vegetables.

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