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immunoglobulin antibodies conjugated to Alexa 647 or Ana I. Arroba, Ángela M. Valverde
488 (1:2000; Molecular Probes, Eugene, OR). After
washing, sections were mounted with medium represented the mean ± SEM. Statistical tests were
(Fluoromount G) containing 4-6-diamidino-2-phenylindole performed using SPSS 21.0 for Windows (SPSS Inc. IBM,
(DAPI). Staining was observed with an inverted laser Armonk, NY, USA). Data were analyzed by one-way
confocal microscope LSM710 (Carl Zeiss Microscopy ANOVA followed by Bonferroni t-test or by paired t-test
GmbH, Göttingen, Germany).After washing, sections were when comparisons were among two groups. Differences
mounted with medium (Fluoromount G) containing 4-6- were considered significant at *p= 0.05.
diamidino-2-phenylindole (DAPI). Staining was observed
with an inverted laser confocal microscope LSM710 (Carl 3. RESULTS
Zeiss Microscopy GmbH, Göttingen, Germany).
3.1. LPS-treated Bv-2 cells induced a pro-inflammatory
2.9. Endotoxin detection response that was reduced by co-treatment with M2
cytokines or the sp2-iminosugar derivative R-DS-ONJ
Serum endotoxin concentrations were measured with a
commercial kit (QAYEE-BIO; Deltaclon, Madrid, Spain) We stimulated Bv-2 cells, a mouse microglia cell line,
according to the manufacturer’s instructions. Briefly, with LPS (M1 stimulus) in the absence or presence of M2
blood samples were collected in non-pyrogenic and cytokines (IL4/IL13). LPS was used as a pro-inflammatory
endotoxin-free tubes, centrifuged at 2,500 x g for 10 min stimulus since it induces an environment similar to that
and serum was separated. Samples were diluted (1/5) with found in the obesity-related diabetic context (26). Based
endotoxin-free water and horseradish peroxidase (HRP) on that, Bv-2 cells were cultured in the presence of LPS,
was added. Samples were gently shacked, incubated for 60 IL4/IL13 (M2) and the combination of both (LPS+M2).
min at 37°C and then washed 5 times. Chromogen solution Figure 2A shows that the release of nitrites in the culture
A was added to the samples that were gently shacked and medium in LPS-treated Bv-2 cells was significantly
further incubated for 10 min at 37ºC protected from light. reduced by the co-treatment with IL4/IL13. Likewise,
Finally, stop solution was added and samples were iNOS, which was elevated in LPS-treated Bv-2 cells,
measured at 450 nm. significantly decreased by the co-treatment with M2
cytokines (Figure 2B). Conversely, arginase-1 decreased
2.10. Quantitative real-time PCR (qRT-PCR) by LPS treatment, this effect being ameliorated in the
combination of LPS plus IL4/IL13. The effect of M2
Total RNA was extracted with Trizol® reagent cytokines was also evident by the decrease of LPS-induced
(Invitrogen, Madrid, Spain) and reverse transcribed using a mRNA levels of TNFa, IL1ß, IL6 and iNOS (Figure 2C).
SuperScript™ III First-Strand Synthesis System for RT-
qPCR following the manufacturer’s indications We tested a pharmacological approach using the sp2-
(Invitrogen). RT-qPCR was performed with an ABI 7900 iminosugar dodecylsulfoxide derivative R-DS-ONJ
sequence detector. Primer-probe sets for mouse TNFa, (referred as C5; Figure 3A). As shown in Figure 3B, the
IL6, IL1ß, iNOS, arginase-1 and 18s were purchased as viability of Bv-2 cells exposed to R-DS-ONJ (50 µM) was
predesigned TaqMan gene expression assays (Applied not affected. In Bv-2 cells cultured with LPS plus R-DS-
Biosystems, Spain). ONJ, nitrite production and iNOS expression (mRNA and
protein) were reduced as compared to the LPS condition
2.11. Statistical analysis (Figure 3C, 3D, 3E). R-DS-ONJ also decreased mRNA
levels of the pro-inflammatory cytokines IL1ß and IL6
Densitometry of the Western blots was performed (Figure 3E).
using the Image J program. Values in all graphs
84 @Real Academia Nacional de Farmacia. Spain
