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Objective: To explore the effect and mechanism of exosome derived from lipopolysaccharide (LPS)-induced mouse macrophage (RAW264.7) on acute lung injury. Methods: RAW264.7 were cultured in vitro and divided into 2 groups: control group and LPS-induced group. The exosomes were extracted from the two groups of cell supernatant by ultracentrifugation and classified into 2 groups: C-EXO group and LPS-EXO group. In vivo, random allocation was used to averagely divide the eighteen male C57BL/6 mice into 3 groups: control group, EXO-control group and EXO-LPS group. All mice were sacrificed after 12 h. The lung tissue was used for HE staining to assess the degree of acute lung injury as well as immunohistochemical staining for interleukin (IL) -1β and tumor necrosis factor (TNF)-α. The tissue protein expression levels of IL-1β, TNF-α, β-catenin, E-cadhein, ZO-1 and Occludin were measured by Western blot. In vitro, alveolar type Ⅱ epithelial cells (MLE-12) were cultured and divided into 3 groups: C-control group, EXO-control-induced group and EXO-LPS-induced group. The tissue protein expression levels of IL-1β, TNF-α, and Occludin were measured by Western blot after 12 h. Results: The two samples of C-EXO group and LPS-EXO group was proved to be exosomes. Under a light microscope, the lung tissue of EXO-LPS group showed inflammatory cell infiltration, hemorrhage, interstitial and alveolar edema, and the thickness of alveolar septum. The tissue protein levels of IL-1β and TNF-α in EXO-LPS group were obviously higher than the control group, EXO-control group (1.331±0.203 and 0.274±0.018, 0.892±0.074; 0.800±0.096 and 0.596±0.025, 0.441±0.061; all P <0.05). While the tissue protein levels of Occludin showed the opposite phenomenon (0.251±0.021 and 0.862±0.029, 0.453±0.013; all P <0.05). In vitro, Compared with the C-control group and the EXO-control-induced group, the expression levels of IL-1β and TNF-α increased significantly in the EXO-LPS-induced group (0.900±0.033 and 0.320±0.030, 0.661±0.028; 0.739±0.045 and 0.151±0.024, 0.360±0.037; all P <0.05). whereas the protein levels of Occludin expression were reversed in MLE-12 (0.585±0.082 and 0.941±0.090, 0.732±0.083; all P <0.05). Conclusion: Exosomes derived from LPS-induced RAW264.7 can induced the acute lung injury by affecting barrier function, which probably is related to the low degree of Occludin in alveolar type Ⅱ epithelial cells.

Objective: To explore the mechanism of Yes-associated protein 1 (Yap1) in angiotensinⅡ(AngⅡ)-induced pulmonary fibrosis. Methods: In vivo, 18 male Wistar rats were randomly divided into three equal groups with 6 rats in each group, including control group, bleomycin-treated group (BLM), and BLM+ AngⅡ group. 28 days later, the lung tissues in all groups were harvested for the HE and Masson staining as well as the immunohistochemical (IHC) staining for Yap1. In vitro, the isolated fibroblasts were treated with 10(-7) mmol/L AngⅡor the AngⅡ-targeted inhibitor irbesartan for the scheduled time for mRNA and protein expressions of Yap1, PDZ-binding motif (TAZ), and collagen Ⅰusing PCR and Western blot, as well as the translocation test from the nucleus to the cytoplasm of Yap1 and TAZ. Subsequently, the fibroblasts were assigned into 4 groups: the empty plasmid (vector) group, the vector+ AngⅡ group, the Yap1 shRNA group, and the Yap1 shRNA+ AngⅡ group. Western blot was used to detect the relative expressions of Yap1, TAZ, Smad3 and collagen Ⅰ. The CCK-8 and EdU assays were performed to determine the proliferative capacity. Results: In vivo, severe lung fibrosis and increased Yap1 expression of IHC staining were found in BLM group. Additionally, more severe lung fibrosis and higher Yap1 expression were detected in the BLM+ AngⅡ group than the BLM group (both P <0.05). In vitro, both the mRNA and protein relative expressions of Yap1, TAZ and collagenⅠ were markedly higher in AngⅡ-treated groups than the control group (all P <0.05). Meanwhile, the relative expression of phosphorylated Yap1 reached its peak at 2 h after AngⅡ stimulation. In the protein translocation tests, after treated with AngⅡ for 24 h, the relative protein levels of Yap1 and TAZ in the nucleus of the AngⅡ group were significantly higher than those in the control group (0.382±0.007 vs 0.031±0.001, 1.097±0.030 vs 0.357±0.015). However, the relative protein expressions in the cytoplasm of the AngⅡ group were obviously less than that in the control group (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015). Compared with the AngⅡ group, the expressions of Yap1 and TAZ in the AngⅡ+ irbesartan group were higher in cytoplasm (0.598±0.060 vs 0.323±0.058, 1.495±0.052 vs 0.858±0.059), while lower in the nucleus (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015) (all P <0.05). Furthermore, the relative protein expressions of Yap1, TAZ, Smad3 and collagenⅠin Yap1 shRNA+ AngⅡ group were distinctly lower than the vector+ AngⅡ group (all P <0.05). In the cell proliferation tests, the absorbance and the percentage of EdU positive cells of vector+ AngⅡ group exceeded that of vector group (both P <0.05). However, the absorbance and the percentage of EdU positive cells in the Yap1 shRNA+ AngⅡgroup were less than the vector+ AngⅡ group (both P <0.05). Conclusion: AngiotensinⅡ promoted the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the Yap1 activity, leading to the progress of fibrosis.

Objective: To explore the mechanism of angiotensin-converting enzyme 2 (ACE2) overexpression improving collagen synthesis in lung. Methods: Lung fibroblasts of mice over-expressing ACE2 and the wild type (WT) were cultured in vitro and divided into 5 groups: WT-control, WT-AngiotensinⅡ (AngⅡ), ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ and ACE2(+ /+) -AngⅡ+ A779. The protein relative expression levels of ACE2, collagen Ⅰ, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), autophagy-related protein (Beclin1), ubiquitin-binding protein p62 (P62), microtubule-associated proteins light chain 3-Ⅱ (LC3-Ⅱ) were measured by Western blot and triphosadenine (ATP) level was measured by ATP Assay Kit. Fibroblasts over-expressing ACE2 were pretreated with or without the autophagy inhibitor and were separated into 4 groups: ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ, ACE2(+ /+) -AngⅡ+ 3-MA and ACE2(+ /+) -3-MA. In vivo, random allocation was used to averagely divide mice into four groups: WT-control, WT-Bleomycin (BLM), ACE2(+ /+) - control, ACE2(+ /+) -BLM. Wild type and ACE2 over-expressing mice were instilled with bleomycin endotracheally (3.5 mg/kg) or the same volume saline. All mice were sacrificed after 28 days and the lung tissue were used for HE and Masson staining as well as immunohistochemical staining for NOX4, P62 and LC3. Results: The vimentin in lung fibroblasts isolated from mice was proved to be positive by both immunohistochemical and immunofluorescence. The ACE2 protein level of lung fibroblasts over-expressing ACE2 was higher than the wild type (0.202±0.062 and 0.067±0.040, P <0.05). The protein levels of collagenⅠ, NOX4 and NLRP3 in WT-AngⅡ group were obviously higher than the WT-control group (0.861±0.129 and 0.417±0.076, 0.432±0.036 and 0.318±0.058, 0.367±0.125 and 0.045±0.012, all P <0.05). The difference of collagenⅠand NLRP3 between ACE2(+ /+) -AngⅡ group and ACE2(+ /+) -control group had no statistical significance (all P >0.05). CollagenⅠand NOX4 protein level in ACE2(+ /+) -AngⅡ+ A779 group were observably higher than ACE2(+ /+) - AngⅡ group (0.707±0.155 and 0.458±0.108, 0.299±0.038 and 0.149±0.090, all P <0.05). The autophagy related protein levels of Beclin1, P62 and LC3-Ⅱ in ACE2(+ /+) -control group were distinctly higher than WT-control group (0.834±0.051 and 0.274±0.018, 0.467±0.078 and 0.093±0.025, 0.494±0.065 and 0.150±0.054, all P <0.05). However, these protein levels in ACE2(+ /+) -AngⅡ+ A779 group were lower than ACE2(+ /+) -AngⅡ group (1.331±0.203 and 1.565±0.069, 0.298±0.096 and 0.438±0.077, 0.464±0.093 and 0.768±0.071, all P <0.05). ACE2(+ /+) -AngⅡ+ 3-MA group had higher collagenⅠ (0.383±0.125 and 0.032±0.013, P <0.05) and lower LC3-Ⅱ protein level (1.177±0.140 and 1.387±0.183, P <0.05) than AngⅡ group. In bleomycin induced lung fibrosis in mice, ACE2(+ /+) -BLM mice exhibited milder lung fibrosis and lower NOX4 protein level but higher LC3-Ⅱprotein level compared with WT-BLM mice. Conclusion: ACE2 over-expression ameliorated collagen synthesis through enhancing autophagy in lung.

Objective: To evaluate the incidence of pulmonary embolism in patients with acute exacerbations of chronic obstructive pulmonary disease (COPD)., Methods: Comprehensive searches as of June 2012 were performed in PubMed (1966-), Embase (1974-), Chinese Biomedical Literature Database (1978-), Chinese Journal Full-text Database (1979-) and VIP Database (1989-) for literatures on the incidence of pulmonary embolism in patients with acute exacerbations of COPD. Meta-analysis was conducted with Stata version 11.0., Results: Among 2273 articles identified, 5 studies met the inclusion criteria (4 in English, 1 in Chinese). The total sample size was 762 patients, among whom 145 were diagnosed with pulmonary embolism. The incidence of pulmonary embolism ranged from 3.3% to 33.0%. Meta-analysis showed that the combined incidence was 15.8% (95%CI: 5.1%-26.4%). Among patients with acute exacerbations of COPD of unknown etiology, the incidence was 29.0% (95%CI: 20.8%-37.1%)., Conclusions: There is a high incidence of pulmonary embolism in patients with acute exacerbations of COPD, especially among those of an unknown etiology. More attention should be paid to this population.