30 research outputs found

    Regulatory effect of miR-148a on total bile acid (TBA) secretion induced by estradiol.

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    <p>(A)The expression of miR-148a was upregulated by estradiol in a dose-dependent for 24 h. (B)The expression of miR-148a was upregulated by estradiol in a time-dependent manner. (C)The transfection of miR-148a-siRNA inhibited the effect of estradiol on miR-148a expression. (D) The transfection of miR-148a-siRNA decreased TBA level in the medium. Values represent the mean ± standard error of the mean (n = 3). **<i>P</i> < 0.01 vs. N-siRNA plus estradiol (500 nmol/L, 12 or 48 h); <sup>++</sup><i>P</i> < 0.01 vs. N-siRNA.</p

    Involvement of MRP3 in total bile acid (TBA) secretion induced by estradiol.

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    <p>(A) LV-miR-148a-siRNA regulated MRP3 mRNA expression induced by estradiol (500 nmol/L, 12 h). (B) LV-miR-148a-siRNA regulated MRP3 protein expression induced by estradiol (500 nmol/L, 12 h). (C) Rifampin (10 μmol/L) reversed the effect of estradiol on MRP3 mRNA expression. (D)Rifampin (10 μmol/L) reversed the effect of estradiol on MRP3 protein expression. Values represent the mean ± standard error of the mean (n = 3). <sup>+</sup><i>P</i> < 0.05 vs. control group, *<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. estradiol (500 nmol/L, 12 h).</p

    Effect of estradiol on the secretion of total bile acid (TBA) in LO2 cells.

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    <p>(A) TBA secretion was induced by estradiol in a dose-dependent for 24 h. (B) TBA secretion was induced by estradiol in a time-dependent manner. *<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control group (n = 3).</p

    Data_Sheet_1_An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid.XLSX

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    <p>Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKβ/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products.</p

    Expression of recombinant <i>Mj</i>Gal (r<i>Mj</i>Gal), r<i>Mj</i>Gal<sup>Δ102–106</sup> and their bacteria binding assay by Western blot.

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    <p>(A) Lane 1, purified r<i>Mj</i>Gal; lane 2, purified r<i>Mj</i>Gal<sup>Δ102–106</sup>; lane 3, purified Trx-His which was used as the control;lane 4, protein markers. (B) Schematic diagram of the five deleted residues (Cys102-Cys106) in mutant <i>Mj</i>Gal<sup>Δ102–106</sup>. (C) Purified r<i>Mj</i>Gal (500 µg/ml) was pre-incubated with either lactose (200 mM), glucose (200 mM), or TBS alone for 1 h, and suspensions of four Gram-positive (G<sup>+</sup>) bacteria (<i>S. aureus</i>, <i>B. subtilis</i>, <i>B. megaterium</i> and <i>B. thuringiensis</i>) and four Gram-negative (G<sup>−</sup>) bacteria (<i>V. anguillarum</i>, <i>K. pneumoniae</i>, <i>E. coli</i> and <i>P. aeruginosa</i>) were added and incubated at room temperature for 1 h. The bacteria were centrifuged, washed four times with TBS, and treated with 7% SDS for 1 min. The eluate was collected by centrifugation, subjected to SDS-PAGE, and the eluted r<i>Mj</i>Gal detected by Western blot using anti-His antibody. (D) The purified r<i>Mj</i>Gal was pre-incubated with excess LPS or LTA before the bacteria were added, and the binding activity for bacteria was tested by western blot. (E) Purified r<i>Mj</i>Gal<sup>Δ102–106</sup> or Trx-His were also used as controls for the bacteria binding activity.</p

    Temporal and spatial expression of <i>MjGal</i>.

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    <p>Total RNAs from different tissues of unchallenged and <i>V. anguillarum</i> or PBS challenged shrimp were reverse transcribed to cDNAs that served as templates for PCR amplification. (A) Expression of <i>MjGal</i> in different tissues of naïve animals as assessed by RT-PCR. The time-course expression of <i>MjGal</i> was upon <i>V. anguillarum</i> challenge was analyzed by qRT-PCR in hemocytes (B) and hepatopancreas (C), with β-actin serving as the reference gene. The histograms show the statistical analysis of the quantitative real-time PCR results. The asterisks indicate significant differences (**P<0.01, ***P<0.001) between the <i>V. anguillarum</i>-challenged and PBS-injected group. Error bars represent ± SD of three independent PCR amplifications and quantifications.</p

    <i>Mj</i>Gal promoted bacterial clearance from hemolymph.

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    <p>Bacteria phagocytosis assay: fluorescently labeled <i>V. anguillarum</i> (5×10<sup>7</sup> cells) were coated with either Trx-His (A, the control) or r<i>Mj</i>Gal (B) and injected into shrimp. The hemocytes were collected after 30 min and placed onto the glass slides. Subsequently, trypan blue solution (2 mg/ml PBS) (Amresco) was added to quench the fluorescence of non-phagocytosed bacteria. The phagocytosis by hemocytes was observed at 400× magnification, and phagocytosed bacteria counted at 200×magnification under the fluorescence microscope. (A) Hemocytes from shrimp injected with <i>V. anguillarum</i> coated with Trx-His. (B) Hemocytes from shrimp injected with <i>V. anguillarum</i> coated with r<i>Mj</i>Gal. (C) The phagocytic percentages of two groups (*<i>P</i><0.05). Bar = 50 µm. (D) RNA interference assay: shrimp were injected with dsRNAs twice to silence <i>Mj</i>Gal expression, and or with GFP dsRNAs as control. <i>Vibrio anguillarum</i> (2×10<sup>7</sup> cells) were injected into shrimp after the second injection. Hemolymph was collected from the two groups at 5, 15, 60 min post-infection, and serially diluted with PBS. Fifty µl of each sample was plated onto 2216E agar plates and incubated at 37°C overnight. RNA was extracted from hemocytes and gills of two groups, and tested by RT-PCR to confirm silencing of <i>Mj</i>Gal expression. (E) The bacterial counts in hemolymph samples collected at 5, 15 and 60 min from shrimp from the ds<i>Mj</i>Gal and dsGFP groups.</p
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