11 research outputs found

    Effects of PAPep on cytokine production in the AqH during EIU.

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    <p>Vehicle (PBS), PAPS (10 µg/eye), PAPep (1, 5, 10 µg/eye) or dexamethasone (10 µg/eye) was applied intravetreally to both rat eyes 1 h before LPS (200 µg) administration. TNF-α (A) and IL-6 (B) levels were assessed in the AqH 24 h after EIU. Data are expressed as mean±SD (n = 8 per group). ##, P<0.01 compared with control group; **, P<0.01 compared with vehicle-treated group. Dex, dexamethasone; N.D., Not detected.</p

    The effects of PAPep on protein leakage and cellular infiltration into the aqueous humor during EIU.

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    <p>The rats were treated with vehicle (PBS), PAPS (10 µg/eye), PAPep (1, 5, 10 µg/eye) or dexamethasone (10 µg/eye) 1 h before LPS (200 µg) injection. Protein levels (A) and cell count (B) were assessed in the AqH 24 h after EIU. Data are expressed as mean±SD (n = 8 per group). ##, P<0.01 compared with control group; **, P<0.01 compared with vehicle-treated group. Dex, dexamethasone.</p

    The properties of the three newly identified peptides.

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    <p>The properties of the three newly identified peptides.</p

    Anti-inflammatory effects of PAP peptides (P1–P3) in EIU rats.

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    <p>The rats were treated with PAP peptides (P1–P3) in different concentrations 1 h before LPS (200 µg) injection, and were evaluated 24 h after LPS challenge. Clinical scores (A) were graded in a blinded fashion. Protein levels (B) and cell count (C) were assessed in the AqH 24 h after EIU. Data are expressed as mean±SD (n = 8 per group). ##, P<0.01 compared with control group; *, P<0.05 or **, P<0.01 compared with vehicle-treated group; §, P<0.01 compared with P2-treated group.</p

    Western blot analysis of protein levels of NF-κB p65.

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    <p>(A) The total and phosphorylation levels of NF-κB p65 was analyzed by Western blot in ICB and retina complex of EIU rats for indicated periods. Lane 1: control; lane 2: LPS and vehicle; lane 3: LPS and PAPS (10 µg/eye); lane 4: LPS and PAPep (10 µg/eye). (B) RAW264.7 cells were pretreated with PAPep (1, 10, 50 µM) or PAPS (50 µM) for 1 h, then stimulated with LPS (100 ng/ml) for 30 min. Total and phosphorylation levels of NF-κB p65 was analyzed by Western blot. Lane 1: control; lane 2: LPS; lane 3: LPS and 50 µM PAPS; lane 4: LPS and 1 µM PAPep; lane 5: LPS and 10 µM PAPep; lane 6: LPS and 50 µM PAPep. (C) HUVEC were incubated with PAPep (1, 10, 50 µM) or PAPS (50 µM) for 1 h, then stimulated with TNF-α (10 ng/ml) for 30 min. Total and phosphorylation levels of NF-κB p65 was analyzed by Western blot. Lane 1: control; lane 2: TNF-α; lane 3: TNF-α and 50 µM PAPS; lane 4: TNF-α and 1 µM PAPep; lane 5: TNF-α and 10 µM PAPep; lane 6: TNF-α and 50 µM PAPep. ##, P<0.01 compared with control group, **, P<0.01 compared with LPS or TNF-α group. Data are expressed as mean±SD of three independent experiments, each performed in duplicates.</p

    Amino acid sequence of human PAP.

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    <p>The secondary structure is depicted above the primary sequence. Red arrows above the sequences correspond to helical regions. Blue arrows above the sequences correspond to beta-strand regions. Signal sequence is indicated underlined. The blue boxed region corresponds to the N-terminal PAP domain. The C-type lectin domain is indicated in italic. The yellow boxed region is the C-type lectin consensus sequence. Cysteines involved in the formation of the three disulfide bonds are highlighted in red. Three distinct regions from which peptides are processed out are boxed in red as follows: P1, PAP[52–67]; P2, PAP[79–94]; P3 (PAPep), PAP[147–162].</p

    PAPep inhibited LPS-induced TNF-α and IL-6 mRNA expression in RAW264.7 cells.

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    <p>RAW264.7 cells were pretreated with PAPep (1, 10, 50 µM), PAPS (50 µM), or dexamethasone (10 µM) for 1 h, and then stimulated with LPS (100 ng/ml) for 6 h. TNF-α (A) and IL-6 (B) mRNA induction levels were quantified by real-time PCR and normalized to GAPDH mRNA expression. All samples were analyzed in duplicate and repeated at least three times. Data are expressed as mean±SD. ##, P<0.01 compared with control group; *, P<0.05 or **, P<0.01 compared with LPS group. Dex, dexamethasone.</p

    Effect of PAPep on nuclear translocation of NF-κB in RAW264.7 cells.

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    <p>(A) The intracellular location of NF-κB p65 was determined in RAW264.7 cells by immunofluorescence using an anti-NF-κB p65 antibody with Alexa Fluor 555 labelling (red fluorescence), and the nuclei were counterstained with DAPI (blue fluorescence). (a) Untreated cells exhibit the localization of NF-κB in the cytoplasm. Cells stimulated with 100 ng/ml LPS (b) and 50 µM PAPS(c) display a significant increase in the translocation of NF-κB into the nucleus. (d) Stimulated cells in the presence of 50 µM PAPep maintained predominantly cytoplasmic NF-κB immunostaining, indicating inhibition of NF-κB translocation. Results are displayed individually or as merged images (confocal fluorescence microscopy). Data represent one of three experiments with similar results. Scale bars, 10 µm. (B) Nuclear NF-κB was quantitated by visual fluorescent microscopy. The number of cells with p65 nuclear translocation in six random fields were counted in a masked fashion and expressed as a percentage of the number of translocated cells in comparison to that of total cells. ##, P<0.01 compared with control group, **, P<0.01 compared with LPS group.</p

    The effects of PAPep on clinical development of EIU.

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    <p>The rats were treated with vehicle (PBS), PAPS (10 µg/eye), PAPep (1, 5, 10 µg/eye) or dexamethasone (10 µg/eye) 1 h before LPS (200 µg) injection, and were evaluated 24 h after LPS challenge with biomicroscope examination. Clinical manifestation of EIU in Wistar rats are shown in Figure A–E. Rats of control group (A) showed no inflammation in the anterior chamber. Severe inflammation was observed in vehicle-treated rats (B) and rats treated with PAPS (C). Note the fibrinous pupillary membrane (arrow). In the group of EIU rats treated with PAPep (10 µg/eye; D), attenuation of inflammation was observed compared with vehicle-treated rats. A similar inhibition was also found for intravitreal pretreatment with dexamethasone (E). (F) Effect of various dosages of PAPep on clinical scores in EIU rats, which were graded in a blinded fashion 24 h after EIU. Data are expressed as mean±SD (n = 8 per group). ##, P<0.01 compared with control group; *, P<0.05 or **, P<0.01 compared with vehicle-treated group. Dex, dexamethasone.</p

    Western blot analysis demonstrating the expression of ICAM-1 protein in HUVECs.

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    <p>HUVEC were incubated with PAPep (1, 10, 50 µM) or PAPS (50 µM) for 1 h, then stimulated with TNF-α (10 ng/ml) for another 6 h. Protein levels of ICAM-1 was analyzed by Western blot. GAPDH was used as loading control. Lane 1: control; lane 2: TNF-α; lane 3: TNF-α and 50 µM PAPS; lane 4: TNF-α and 1 µM PAPep; lane 5: TNF-α and 10 µM PAPep, and lane 6: TNF-α and 50 µM PAPep. ##, P<0.01 compared with unstimulated cells, **, P<0.01 compared with TNF-α-stimulated cells. Data are expressed as mean±SD of results from three independent experiments, each performed in duplicate.</p
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