Convallatoxin Promotes M2 Macrophage Polarization to Attenuate Atherosclerosis Through PPARγ-Integrin αvβ5 Signaling Pathway

Abstract

Introduction: As the primary immune cells, macrophages play a key role in atherosclerotic progression. M2 macrophage polarization has been reported to promote tissue repair and attenuate plaque formation upon the expression of anti-inflammatory factors. Convallatoxin (CNT) is a natural cardiac glycoside with anti-inflammatory pharmacological properties. However, whether CNT protects against atherosclerosis (AS) and underlying mechanisms is unknown. This work was designed to explore the potential effects of CNT on atherosclerosis.

Methods: In this study, Apolipoprotein E deficiency (ApoE^-/-) mice fed with high-fat diet were established, and CNT (50 or 100 μg/kg) were intragastrically administrated for 12 weeks every day. In vitro, RAW264.7 macrophages stimulated with ox-LDL were treated with CNT (50 or 100 nM) for 24 h. The specific PPARγ antagonist, GW9662, was used to block the PPARγ signaling pathway in vitro. Then, the atherosclerotic lesions, macrophage polarization markers, inflammatory cytokines and PPARγ signaling pathway were examined in further examinations.

Results: Our results showed that the atherosclerotic lesions were reduced by CNT, as demonstrated by the downregulation of serum lipid level and aortic plaque area in AS mice. Furthermore, we found that CNT treatment promoted the expression of M2 macrophage markers (Arg1, Mrc1, Retnla and Chi3l3), and decreased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), accompanied by the increase of anti-inflammatory factor (IL-10) in aortic vessels of AS mice. In ox-LDL-induced RAW264.7 cells, CNT administration also facilitated macrophages polarizing towards M2 subtype and inhibited inflammatory responses. Furthermore, both the in vivo and in vitro experiments showed CNT could increase the expression of PPARγ, Integrin α_v and Integrin β₅, and GW9662 could block CNT-induced M2 macrophage polarization.

Conclusion: Taken together, these data suggest that CNT may promote M2 macrophage polarization to exert an anti-atherosclerotic effect, partially through activating PPARγ-Integrin α_vβ₅ signaling pathway.

Keywords: PPARγ-Integrin αvβ5 signaling pathway; atherosclerosis; convallatoxin; macrophage polarization; ox-LDL.

Figure 1

The chemical structure of Convallatoxin and flow diagram for experimental groups in vivo. (A) The chemical structure of Convallatoxin (CNT). (B) The flow diagram of animal experimental treatment.

Figure 2

CNT attenuated atherosclerotic lesions in vivo. (A) Body weight. (B–D) Serum levels of TC, TG and LDL-C. (E) Oil red O staining and quantification of plaque areas in the aortic roots. The corresponding right panels showed the partially enlarged plaque areas labeled with black boxes. Bar=500 μm. Data were expressed as mean ± SD.

Figure 3

CNT promoted M2 macrophage polarization in vivo. (A) Immunofluorescence staining of the co-localization of CD68 (green) and CD163 (red) in the aortic roots. The right panels were the corresponding partially enlarged images in white boxes. Bar=50 μm. (B) qPCR analysis of the relative mRNA levels of M2 markers in the aortic tissues, including Arg1, Mrc1, Retnla, and Chi3l3. (C) Contents of IL-6, TNF-α and IL-10 in the aortic tissues were determined by ELISA. Data were expressed as mean ± SD.

Figure 4

CNT reduced ox-LDL-induced foam cell formation. (A) Representative images of Oil red O staining in ox-LDL-treated RAW264.7 cells. Bar=50 μm. (B) TC contents in ox-LDL-treated cells. Data were expressed as mean ± SD.

Figure 5

CNT facilitated macrophage polarizing towards M2 phenotype in vitro. (A) Flow cytometry analysis and quantification of the percentage of CD163 positive cells. (B) qPCR results of the relative expressions of M2 markers, including Arg1, Mrc1, Retnla, and Chi3l3. (C) ELISA assays of the levels of IL-6, TNF-α and IL-10 in cell supernatants. Data were expressed as mean ± SD.

Figure 6

PPARγ antagonist, GW9662, blocked CNT-enhanced M2 macrophage polarization. (A and B) Western blot analysis and quantification of PPARγ, Integrin α_v and Integrin β₅ in the aortic tissues (A) and RAW264.7 cells (B). (C) Representative images of Oil red O staining in macrophages. Bar=50 μm. (D) Levels of TC in cells were measured by available kits. (E) Arg1, Mrc1, Retnla and Chi3l3 mRNA levels were assessed by qPCR. (F) IL-6, TNF-α and IL-10 contents in cell supernatants were tested using ELISA. Data were expressed as mean ± SD.

Figure 7

The schematic diagram of the potential molecular mechanism of the anti-atherosclerotic role of Convallatoxin.