Quercetin Inhibits LPS-Induced Inflammation and ox-LDL-Induced Lipid Deposition

Abstract

Aberrant activation of inflammation and excess accumulation of lipids play crucial role in the occurrence and progression of atherosclerosis (AS). Quercetin (QCT) has been tested effectively to cure AS. It is widely distributed in plant foods and has been proved to have potential antioxidative and anticancer activities. However, the underlying molecular mechanisms of OCT in AS are not completely understood. In the present study, we stimulated murine RAW264.7 cells with lipopolysaccharide (LPS) or oxidized low-density lipoproteins (ox-LDL) to mimic the development of AS. The data show that QCT treatment leads to an obvious decrease of multiple inflammatory cytokines in transcript level, including interleukin (IL)-1α, IL-1β, IL-2, IL-10, macrophage chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX-2) induced by LPS. Moreover, expressions of other factors that contribute to the AS development, such as matrix metalloproteinase-1 (MMP-1) and suppressor of cytokine signaling 3 (SOCS3) induced by LPS are also downregulated by QCT. Furthermore, we found that QCT suppressed LPS-induced the phosphorylation of STAT3. Meanwhile, QCT could ameliorate lipid deposition and overproduction of reactive oxygen species induced by ox-LDL, and block the expression of lectin-like oxidized LDL receptor-1 (LOX-1) in cultured macrophages. Taken together, our data reveal that QCT has obvious anti-inflammatory and antioxidant virtues and could be a therapeutic agent for the prevention and treatment of AS.

Keywords: Mongolian medicine; atherosclerosis (AS); inflammation; lipid deposition; quercetin (QCT).

FIGURE 1

Quercetin (QCT) inhibited inflammation in LPS-treated RAW264.7 cells. RAW 264.7 cells were grown in six-well plates for 24 h and treated with 20 μM QCT for 24 h, and then stimulated with 500 ng/ml of LPS for another 6 h. The cells were harvested and transcript levels of the following cytokines were examined by qRT-PCR. Relative mRNA levels were shown in (A) IL-1α, (B) IL-1β, (C) IL-2, (D) IL-10, (E) MCP-1, and (F) COX-2.^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the LPS-treated group. One-way ANOVA analysis was used to calculate P-values. The bars represent mean ± SD. All data represent at least three independent experiments.

FIGURE 2

Quercetin treatment inhibits LPS-induced activation of STAT3 in RAW264.7 cells. (A) QCT suppresses SOCS3 and MMP-1 expression induced by LPS. RAW264.7 cells were grown in six-well plates for 24 h and treated with 20 μM QCT for 24 h, and then stimulated with 500 ng/ml of LPS for another 6 h. The cells were harvested and transcript levels of the following cytokines were examined by qRT-PCR. ^∗P < 0.05 vs. the control group; ^#P < 0.05 vs. the LPS-treated group. The bars represent mean ± SD. (B) Representative immunoblot showing phosphorylated STAT3 (p-STAT3) and total STAT3 (t-STAT3) protein levels in RAW264.7 cells treated with DMSO, QCT (20 μM), LPS (500 ng/ml), LPS (500 ng/ml) + QCT (20 μM), and LPS (500 ng/ml) + S3I-201 (100 μM). β-actin as a loading control. (C) Densitometry was used to quantify relative p-STAT3 protein levels normalized to t-STAT3 protein levels ^∗P < 0.05 vs. control group; ^#P < 0.05 vs. LPS-induced group. One-way ANOVA analysis was used to calculate P-values. The bars represent mean ± SD. All data represent at least three independent experiments.

FIGURE 3

Quercetin ameliorated oxidized low-density lipoproteins (ox-LDL)-induced inflammation in RAW264.7 cells. RAW264.7 cells growing in six-well plates were pre-incubated with 20 μM of QCT for 24 h before treatment with 50 μg/ml of ox-LDL. Cells were harvested and transcript levels of IL-1β (A) and LOX-1 (B) were measured by qRT-PCR. ^∗P < 0.05 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. ox-LDL-induced group. One-way ANOVA analysis was used to calculate P-values. The bars represent mean ± SD. All data represent at least three independent experiments.

FIGURE 4

Quercetin decreased ox-LDL-induced lipid deposition in RAW264.7 cells. (A) Murine RAW264.7 cells treated with ox-LDL (20 μg/ml) and QCT (20 μM) were fixed with formaldehyde for 15 min followed by stained with Oil Red O solution for an additional 15 min and then photographed randomly. Representative photographs of Oil Red O stained cells in different groups were shown. (B) The percentage of Oil Red-positive cells to total cells was calculated using Image-Pro Plus Image Analysis Software (Media Cybernetics, USA). (C) and (D) RAW264.7 cells were treated with ox-LDL and QCT as the method described above. Total cholesterol concentrations (C) and free cholesterol concentrations (D) of cells in different groups were quantitatively analyzed by using cholesterol quantitation kit. ^∗∗∗P < 0.001 vs. the control group; ^##P < 0.01, ^###P < 0.001 vs. ox-LDL-induced group. One-way ANOVA analysis was used to calculate P-values. The bars represent mean ± SD. All data represent at least three independent experiments.

FIGURE 5

Quercetin abrogated ox-LDL-induced overproduction of reactive oxygen species (ROS) in RAW264.7 cells. (A) Murine RAW264.7 cells treated with ox-LDL (50 μg/ml) and QCT (20 μM) were processed for DCFH-DA treatment for 60 min. ROS production in response to oxidation and the subsequent treatment of QCT in RAW264.7 cells was detected by spectrofluorimetry and quantified. (B) The expression of SOD-1 in RAW264.7 cells from different groups was detected by qRT-PCR. ^∗P < 0.05, ^∗∗∗P < 0.001 vs. control group; ^#P < 0.05, ^###P < 0.001 vs. ox-LDL-treated group. One-way ANOVA analysis was used to calculate P-values. The bars represent mean ± SD. All data represent at least three independent experiments.