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. 2018 Feb 9;9:72.
doi: 10.3389/fphar.2018.00072. eCollection 2018.

The Flavonoid Quercetin Ameliorates Liver Inflammation and Fibrosis by Regulating Hepatic Macrophages Activation and Polarization in Mice

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Free PMC article

The Flavonoid Quercetin Ameliorates Liver Inflammation and Fibrosis by Regulating Hepatic Macrophages Activation and Polarization in Mice

Xi Li et al. Front Pharmacol. .
Free PMC article

Abstract

At present, there are no effective antifibrotic drugs for patients with chronic liver disease; hence, the development of antifibrotic therapies is urgently needed. Here, we performed an experimental and translational study to investigate the potential and underlying mechanism of quercetin treatment in liver fibrosis, mainly focusing on the impact of quercetin on macrophages activation and polarization. BALB/c mice were induced liver fibrosis by carbon tetrachloride (CCl4) for 8 weeks and concomitantly treated with quercetin (50 mg/kg) or vehicle by daily gavage. Liver inflammation, fibrosis, and hepatic stellate cells (HSCs) activation were examined. Moreover, massive macrophages accumulation, M1 macrophages and their related markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1) in livers were analyzed. In vitro, we used Raw 264.7 cells to examine the effect of quercetin on M1-polarized macrophages activation. Our results showed that quercetin dramatically ameliorated liver inflammation, fibrosis, and inhibited HSCs activation. These results were attributed to the reductive recruitment of macrophages (F4/80+ and CD68+) into the liver in quercetin-treated fibrotic mice confirmed by immunostaining and expression levels of marker molecules. Importantly, quercetin strongly inhibited M1 polarization and M1-related inflammatory cytokines in fibrotic livers when compared with vehicle-treated mice. In vitro, studies further revealed that quercetin efficiently inhibited macrophages activation and M1 polarization, as well as decreased the mRNA expression of M1 macrophage markers such as TNF-α, IL-1β, IL-6, and nitric oxide synthase 2. Mechanistically, the inhibition of M1 macrophages by quercetin was associated with the decreased levels of Notch1 expression on macrophages both in vivo and in vitro. Taken together, our data indicated that quercetin attenuated CCl4-induced liver inflammation and fibrosis in mice through inhibiting macrophages infiltration and modulating M1 macrophages polarization via targeting Notch1 pathway. Hence, quercetin holds promise as potential therapeutic agent for human fibrotic liver disease.

Keywords: Notch1; hepatic fibrosis; hepatic stellate cells (HSCs); macrophages; polarization; quercetin.

Figures

FIGURE 1
FIGURE 1
Quercetin inhibited liver inflammation and fibrosis in CCl4-treated mice. (A) Quercetin powder and chemical structure of quercetin (3,3′,4′,5,7-pentahydroxyflavone). (B) Histological examination of liver sections from each group (H&E staining, original magnification: ×100). Scale bar = 100 μm. (C) Sirius red staining of liver sections (original magnification: ×100). Scale bar = 100 μm. (D) The hepatocellular damage observed in H&E-stained liver sections were analyzed and scored as described under the section “Materials and Methods” (n = 10/group). (E) Hepatic fibrotic area based on Sirius red staining. (F) Assessment of liver fibrosis based on Scheuer’s scoring system. ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 2
FIGURE 2
Quercetin inhibited liver fibrotic markers expression in CCl4-induced mouse fibrotic liver model. (A) Representative microscopy images of Collagen III and Collagen IV immunohistochemistry in the liver (original magnification, ×100). Scale bar = 100 μm. (B) Quantitative analysis of Collagen III- and Collagen IV-positive area by ImageJ software (NIH). n = 5/group. (C) Expression of fibrotic markers (Col3 α1, Col4α1, Ctgf, and Timp-1) was examined by quantitative RT-PCR in whole liver samples from each group (n = 6/group). Results were normalized to GAPDH mRNA and expressed as fold change compared to DMSO/oil mice. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 3
FIGURE 3
Quercetin inhibited hepatic stellate cells (HSCs) activation in CCl4-treated mice. (A) Representative microscopy images of desmin staining (magnification: ×200) in the liver. Scale bar = 100 μm. (B) Quantification of desmin-positive area by ImageJ software (NIH). Results mean of six fields and n = 5/group. (C) Western blotting analysis of desmin expression in lysed liver tissues, with results normalized relative to the expression of GAPDH (n = 3). (D) Expression of desmin and vimentin mRNA was determined in the liver by quantitative RT-PCR (n = 6). Results were normalized relative to GAPDH expression and expressed as mean ±SD fold change over normal control mice. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 4
FIGURE 4
Quercetin inhibited massive macrophage recruitment into the fibrotic livers of CCl4-induced mice. (A) Immunohistochemical detection of F4/80- and CD68-positive cells in liver sections from each group (original magnification: ×200). Insert (magnification: ×400) shows typical morphology of positive macrophages. Scale bar = 100 μm. (B) Quantification of F4/80- and CD68-positive cells in liver sections. Results mean of six fields and n = 5/group. (C) Gene expression of macrophage marker F4/80 and CD68 was determined in livers by quantitative RT-PCR, and the results are shown as fold change compared with sham-treated control and GAPDH served as loading control (n = 6). P < 0.05; ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 5
FIGURE 5
Effect of quercetin on M1 macrophage polarization and expression of inflammatory properties in fibrotic livers. (A) Representative immunostaining of IL-12, CD11c, and IRF5 in liver sections (original magnification: ×200). Scale bar = 100 μm. (B) Quantification of IL-12, CD11c, and IRF5 immunostaining in the liver from each group. (C) Hepatic M1 polarization genes of TNF-α, IL-1β, IL-6, and MCP-1 were determined by quantitative RT-PCR, and results are shown as fold change compared with sham-treated control and GAPDH served as loading control (n = 5). P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 6
FIGURE 6
Effect of quercetin on M2 macrophages polarization and expression of immunosuppressive genes in fibrotic livers. (A) Representative immunostaining of Ym-1 and CD163 in liver sections (original magnification: ×200). Scale bar = 100 μm. (B) Quantification of Ym-1 and CD163 immunostaining in the liver from each group. (C) M2-polarized gene expression of Arginase I (Arg I) and Ym-1 was determined by quantitative RT-PCR, and the results are shown as fold change compared with sham-treated control and GAPDH served as loading control (n = 5). P < 0.05; ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 7
FIGURE 7
Quercetin treatment suppressed M1 polarization of macrophages in vitro. (A) Representative fluorescence microscopic images of RAW macrophages with anti-IL12 and anti-IRF5 whole-mount staining. Undifferentiated RAW macrophages conditioned medium, and using LPS (100 ng/mL) to induce M1 differentiation. Quercetin- (50 μM) or DMSO-treated M1-differentiated macrophages conditioned medium. Bars represent mean ±SD of at least three independent experiments. Scale bar = 200 μm. (B) Effect of quercetin (50 μM) on the cell viability of macrophages. Cell viability was then determined by the CCK-8 assay as described in the “Materials and Methods” section. (C) Western blotting analysis of M1-markers IL12 and IRF5 protein expression in macrophages RAW 264.7 cells, with results normalized relative to the expression of β-actin or GAPDH (n = 3). (D) Quantification gene expression analysis of M1-specific markers TNF-α, IL-1β, IL-6, and NOS2. The mRNA levels were normalized to GAPDH mRNA levels and presented as fold stimulation (mean ± SD) versus vehicle-treated control. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; “NS” indicates not significant.
FIGURE 8
FIGURE 8
Quercetin inhibited hepatic Notch1 expression in CCl4-treated mice. (A) Immunofluorescent double staining of Notch1 in liver sections from each group. Livers were double stained for Notch1 (green) and F4/80 (Red) macrophages; DAPI as blue nuclear counterstain. Scale bar = 100 μm. (B) Western blotting analysis of Notch1 expression in lysed liver tissues, with results normalized relative to the expression of GAPDH (n = 3). (C) Hepatic Notch1 mRNA expression was measured by quantitative RT-PCR. Results are shown as fold change compared with oil-treated control and GAPDH served as loading control (n = 5). ∗∗P < 0.01; ∗∗∗P < 0.001.
FIGURE 9
FIGURE 9
Quercetin inhibited M1-macrophages polarization through regulating the expression Notch1 on macrophages in vitro. (A) Undifferentiated RAW 264.7 macrophages conditioned medium or LPS-stimulated M1-differentiated macrophages, quercetin (50 μM)-treated RAW macrophages, DMSO as vehicle-treated control. Scale bar = 200 μm. (B) Western blotting analysis for Notch1 in RAW 264.7 cells. (C) The levels of Notch1 mRNA expression in RAW 264.7 cells were measured by quantitative RT-PCR. The mRNA levels were normalized to GAPDH mRNA levels and presented as fold stimulation (mean ±SD) versus DMSO. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; “NS” indicates not significant.

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