Protection exerted by quercetin-loaded nanoparticles on CD36 and β1-integrin, IL-8, and MCP-1 expression induced by the oxysterol mixture.

<p>Gene expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 6 h with 15 µM oxysterol mixture (Mix). Some cells were pretreated for 1 h with 5 µM free quercetin (Q_F) or with 5 µM quercetin loaded into nanoparticles (Q_N) before oxysterol treatment. Untreated cells (Control) were taken as controls, and cells treated with 93.6 mM ethanol (Et-OH) as solvent controls. Cells supplemented with blank nanoparticles (NPs) or with blank nanoparticles plus oxysterol mixture were taken as internal controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of five different experiments. *P<0.05, **P<0.01, and ***P<0.001 vs. control; #P<0.05, ##P<0.01, and ###P<0.001 vs. the specific oxysterol; §§P<0.01 vs. Q_F+ specific oxysterol.</p

Effect of oxysterols on expression of CD36, β1-integrin, IL-8, MCP-1, and MMP-9.

<p>Gene expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 6 h with 5 µM 7β-hydroxycholesterol (7β-OH), 24-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH) or with a 15 µM mixture of these three oxysterols. Untreated cells (Control) were taken as controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of three different experiments. *P<0.05, **P<0.01, and ***P<0.001 vs. control.</p

Cell viability and cell uptake of β-CD-dodecylcarbonate nanoparticles.

<p>A) SH-SY5Y cells were incubated with β-CD-dodecylcarbonate nanoparticles with (Q_N) or without (NPs) being loaded with quercetin (5 µM). Some cells were treated with 5 µM quercetin alone (Q_F). Untreated cells (Control) were taken as controls. Cell viability was measured in terms of release of the enzyme lactate dehydrogenase (LDH), as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096795#s2" target="_blank">Materials and Methods</a> section. Data represent the mean values ± SD of three different experiments. B) SH-SY5Y cells were incubated with fluorescent coumarin 6-β-CD-dodecylcarbonate nanoparticles for the times indicated and then analyzed by confocal laser scanning microscopy (40X/0.75).</p

Protection exerted by quercetin-loaded nanoparticles on CD36 and β1-integrin, IL-8, MCP-1, and MMP-9 expression induced by oxysterols.

<p>Gene expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 6 h with 5 µM 7β-hydroxycholesterol (7β-OH), 24-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH). Some cells were pretreated for 1 h with 5 µM free quercetin (Q_F) or with 5 µM quercetin loaded into nanoparticles (Q_N) before oxysterol treatment. Untreated cells (Control) were taken as controls, and cells treated with 31.2 mM ethanol (Et-OH) as solvent controls. Cells supplemented with blank nanoparticles (NPs) or with blank nanoparticles plus oxysterols, were taken as internal controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of five different experiments. *P<0.05, **P<0.01, and ***P<0.001 vs. control; #P<0.05, ##P<0.01, and ###P<0.001 vs. the specific oxysterol; §P<0.05, §§P<0.01, and §§§P<0.001 vs. Q_F+ specific oxysterol.</p

Average diameter, polydispersity index and zeta potential of the nanoparticle formulations.

<p>Average diameter, polydispersity index and zeta potential of the nanoparticle formulations.</p

Effect of oxysterols on COX-2 synthesis and mPGES-1 expression.

<p>(A) SH-SY5Y cells were treated for 48 h with 5 µM 7β-hydroxycholesterol (7β-OH), 24-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH) or 15 µM oxysterol mixture (Mix). Untreated cells (Control) were taken as controls. COX-2 levels were analyzed by Western blotting. Top: blot representative of three experiments. Bottom: histogram representing mean values ± SD of three experiments. COX-2 densitometric measurements were normalized against the corresponding actin levels and expressed as percentages of control value.**P<0.01 and ***P<0.001 vs. control. (B) mPGES-1 expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 6 h with 5 µM 7β-OH, 24-OH, 27-OH or 15 µM oxysterol mixture. Some cells were pretreated for 1 h with 5 µM free quercetin (Q_F) or with 5 µM quercetin loaded into nanoparticles (Q_N) before oxysterol treatment. Untreated cells (Control) were taken as controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of three different experiments. ***P<0.001 vs. control; ###P<0.001 vs. specific oxysterol.</p

Protective effect of quercetin-loaded nanoparticles on TLR-4 expression induced by oxysterols.

<p>Gene expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 3 h with 5 µM 7β-hydroxycholesterol (7β-OH), 24-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH), or 15 µM oxysterol mixture (Mix). Some cells were pretreated for 1 h with 5 µM free quercetin (Q_F) or with 5 µM quercetin loaded into nanoparticles (Q_N) before oxysterol treatment. Untreated cells (Control) were taken as controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of three different experiments. ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. specific oxysterol; §§§P<0.001 vs. Q_F+ specific oxysterol.</p

Evaluation of serum levels of TGFβ1 and VEGF during colorectal carcinogenesis.

<p>The levels of TGFβ1 (A) and VEGF (B) were measured in controls and patient groups (N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma). TGFβ1 and VEGF were detected by ELISA and values expressed as ng/ml and pg/ml serum, respectively (see materials and methods). A: dots correspond to single TGFβ1 values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 6.3±0.4; TA 7.2±0.45; TVA 6.3±0.6; IAC 6.9±0.3; IIAC 4.9±0.3; IIIAC 4.4±0.3; IVAC 5.7±0.5. *Significantly different versus control group (p<0.05). B: dots correspond to single VEGF values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 227.0±18.7; TA 215.2±31.3; TVA 221.1±36.4; IAC 216.3±34.4; IIAC 205.0±26.6; IIIAC 266.5±38.6; IVAC 295.2±70.2.</p

Evaluation of serum MMP activity and levels during colorectal carcinogenesis.

<p>A: active forms of MMP-2 and MMP-9 are shown in a representative gelatin zymography. B: MMP-9 activation in the serum was evaluated by densitometric analysis as percentage of active MMP-9 compared to controls (taken as 100%). Dots correspond to the activated MMP-9 value for each subject and black lines represent mean values within the experimental groups. Mean values ± SEM: TA 120±3.9; TVA 121±8.5; IAC 151.63±17.2; IIAC 163.81±16.6; IIIAC 164.2±10.6; IVAC 129.0±13.0. *Significantly different versus control group (p<0.01). C: serum levels of MMP-9 protein were detected by ELISA and expressed as ng/ml serum. Dots correspond to the MMP-9 value for each subject and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 1.5±0.2; TA 1.9±0.3; TVA 1.9±0.3; I AC 2.7±0.3; IIAC 2.8±0.3; IIIAC 3.0±0.5; IVAC 2.6±0.4. *Significantly different versus control group (p<0.05). N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma.</p

Evaluation of serum levels of IL-8 and IL-6 during colorectal carcinogenesis.

<p>The levels of IL-8 (A) and IL-6 (B) were measured in controls and patient groups (N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma). IL-8 and IL-6 were detected by ELISA and values expressed as pg/ml serum (see materials and methods). A: dots correspond to IL-8 single values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 17.0±2.5; TA 15.9±3.7; TVA 18.4±2.2; IAC 25.4±4.8; IIAC 38.7±5.1; IIIAC 37.2±5.7; IVAC 30.5±5.7. *Significantly different versus control group (p<0.05). B: dots correspond to single IL-6 values and black lines represent the mean values within experimental groups. Mean values ± SEM: N 247.1±55; TA 375.9±54; TVA 407.9±80; IAC 237.9±68; IIAC 472.3±89; IIIAC 521.3±152; IVAC 436.3±118. *Significantly different versus control group (p<0.05).</p