Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype - PubMed (original) (raw)

Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype

Xue Zhang et al. J Cell Mol Med. 2018 Jan.

Erratum in

Abstract

Atherosclerosis (AS) is characterized as progressive arterial plaque, which is easy to rupture under low stability. Macrophage polarization and inflammation response plays an important role in regulating plaque stability. Ginsenoside Rb1 (Rb1), one of the main active principles of Panax Ginseng, has been found powerful potential in alleviating inflammatory response. However, whether Rb1 could exert protective effects on AS plaque stability remains unclear. This study investigated the role of Rb1 on macrophage polarization and atherosclerotic plaque stability using primary peritoneal macrophages isolated from C57BL/6 mice and AS model in ApoE-/- mice. In vitro, Rb1 treatment promoted the expression of arginase-I (Arg-I) and macrophage mannose receptor (CD206), two classic M2 macrophages markers, while the expression of iNOS (M1 macrophages) was decreased. Rb1 increased interleukin-4 (IL-4) and interleukin-13 (IL-13) secretion in supernatant and promoted STAT6 phosphorylation. IL-4 and/or IL-13 neutralizing antibodies and leflunomide, a STAT6 inhibitor attenuated the up-regulation of M2 markers induced by Rb1. In vivo, the administration of Rb1 promoted atherosclerotic lesion stability, accompanied by increased M2 macrophage phenotype and reduced MMP-9 staining. These data suggested that Rb1 enhanced atherosclerotic plaque stability through promoting anti-inflammatory M2 macrophage polarization, which is achieved partly by increasing the production of IL-4 and/or IL-13 and STAT6 phosphorylation. Our study provides new evidence for possibility of Rb1 in prevention and treatment of atherosclerosis.

Keywords: Ginsenoside Rb1; M2 macrophages; atherosclerotic plaque stability; inflammation.

© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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Figures

Figure 1

Figure 1

Rb1 induced macrophages M2 polarization. (A) Representative immunoblots of

iNOS

(M1 marker) and Arg‐I (M2 marker) in peritoneal macrophages treated with Rb1 at the different doses for 24 hrs. (B)

iNOS

and Arg‐I expression relative to the β‐actin level. (C) The expression of

CD

206 was examined by flow cytometry. (D) Percentages of

RAW

264.7 cells expressing of

CD

206. (E)

ELISA

for expression of pro‐inflammatory cytokine

MMP

‐9 and anti‐inflammatory cytokine

IL

‐10 in supernatant. Data are presented as means ± S.D.; *P <0.05, compared to

LPS

‐treated group; #P <0.05, compared to control group; n =3.

Figure 2

Figure 2

IL

‐4 and

IL

‐13 involved in Rb1‐mediated macrophage polarization. (A)

ELISA

for

IL

‐4 and

IL

‐13 in supernatant of peritoneal macrophages treated with the indicated concentrations of Rb1 for 1 hr and then treated with

LPS

1 μg/ml for 24 hrs. (B) Representative immunoblots of

iNOS

and Arg‐I protein in peritoneal macrophages treated by 20 μM Rb1 with or without

IL

‐4 and/or

IL

‐13 neutralizing antibody for 24 hrs. Data are presented as means ± S.D.; *P <0.05, compared to

LPS

‐treated group; #P <0.05, compared to control group; n =3.

Figure 3

Figure 3

Rb1 induced macrophage M2 polarization through

STAT

6‐dependent pathway. (A) Representative immunoblots of the p‐

STAT

6 in peritoneal macrophages cells treated by Rb1. (B) Statistics of immunoblots presented as p‐

STAT

6 relative to the

STAT

6 level. (C) Representative immunoblots of the

iNOS

and Arg‐I protein in peritoneal macrophages cells treated by Rb1 for 24 hrs with or without the

STAT

6 inhibitor. (D) Statistics of

iNOS

and Arg‐I expression relative to the β‐actin level. Data are presented as means ± S.D.; *P <0.05, compared to

LPS

‐treated group; #P <0.05, compared to

LPS

+Rb1‐treated group; & P <0.05, compared to control group; n =3.

Figure 4

Figure 4

Rb1 obviously increased

AS

plaque stability of atherosclerotic ApoE−/− mice. (A)(B)(C) Immnohistochemical staining of Oil red‐O staining of lipids,

MOMA

‐2 (macrophage marker), smooth muscle cells. (D) Representative images of Sirius Red‐staining for quantification of the lesion collagen content. (E) Statistics of (A)–(D) measured as area ratio. (F) Vulnerability index of plaque in corresponding groups calculated as (lipid deposit% + macrophages%) / (collagen fibres% +

SMC

s%). Data are means ± S.D.; Scale bar: 100 μm. *P <0.05; n =6. (G) (H) Representative immunohistochemistry and statistics of

MMP

‐9 expression. Quantification of the

MMP

‐9 positive staining area expressed as percentage of total lesion area. Data are presented as means ± S.D.; Scale bar: 20 μm. *P <0.05; n =6.

Figure 5

Figure 5

Effects of Rb1 on macrophage polarization in atherosclerotic lesions of ApoE−/− mice. (A, B) Representative images of

MOMA

‐2+

iNOS

MOMA

‐2+Arg‐I+ macrophages in situ in corresponding groups (

NC

: control group; Rb1: Rb1 treatment group). (C, D) Statistics of the number of

MOMA

‐2+

iNOS

MOMA

‐2+Arg‐I+ macrophages in atherosclerotic lesions in control and Rb1‐treated ApoE−/− mice. Scale bar: 20 μm. *P <0.05; n =6. (E) Representative immunoblots of

iNOS

(M1 marker) and Arg‐I (M2 marker) in vivo. (F) Statistics of

iNOS

and Arg‐I expression relative to the β‐actin level. Data are presented as means ± S.D.; *P <0.05, compared to control group; n =3.

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