Korean J Physiol Pharmacol.  2025 Mar;29(2):245-255. 10.4196/kjpp.24.352.

Monotropein resists atherosclerosis by reducing inflammation, oxidative stress, and abnormal proliferation and migration of vascular smooth muscle cells

• Li H
• Ye B
• Tian J
• Wang B
• Zha Y
• Zheng S
• Ma T
• Zhuang W
• Park WS
• Liang J

• KMID: 2565436
• DOI: http://doi.org/10.4196/kjpp.24.352

Abstract

Monotropein is a compound classified into iridoid which is found in herbaceous plants Morindae officinalis. It possesses anti-inflammatory, antioxidant, and anti-osteoarthritic activities. Previous study indicates that monotropein may have the potential to combat cardiovascular disease, although the related mechanism remains unclear. In this study, we constructed the model of atherosclerosis by oxidized low density lipoprotein-induced vascular smooth muscle cells and LDLR –/– mice given high-fat diet to investigate the effects of monotropein on atherosclerosis. Our results showed that monotropein treatment significantly reduced the area of atherosclerotic plaques and necrotic cores in mice, inhibited the proliferation and migration of vascular smooth muscle cells, and reduced inflammatory responses and oxidative stress, which in turn alleviated atherosclerosis. In addition, we found that monotropein reduced the expression levels of P-NF-κB and P-AP-1. In conclusion, our data suggest that monotropein inhibited the proliferation and migration of vascular smooth muscle cells by mediating the activity of NF-κB, AP-1, reducing the level of inflammation and oxidative stress, and thus resisting the development of atherosclerosis. These findings demonstrate the efficacious therapeutic impact of monotropein on atherosclerosis and elucidate its specific target.

Keyword

Atherosclerosis; Migration; Monotropein; Proliferation; Vascular smooth muscle

Figure

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  Fig. 1 Analysis of aortic plaques in mice. (A) Diagram of experimental design. (B, C) Sudan 4 staining of mouse and quantitative analysis. Scale bar, 1 cm. (D–G) H&E and Masson staining of mouse aortic roots and quantitative analysis; scale bar, 50 µm. The black arrow indicates the necrotic cores. 3 male mice were used in this experiment. Bar graphs are mean ± SEM, statistical significance *p < 0.05, **p < 0.01, ***p < 0.001, nsp < 0.05. HFD, high-fat diet; MTP, monotropein.

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  Fig. 2 Monotropein effect on vascular smooth muscle cell proliferation. (A) CCK-8 assay was conducted to assess the effect of different concentrations of monotropein on vascular smooth muscle cell viability. n = 3 independent experiments. (B) EDU assay to determine the proliferation of vascular smooth muscle cells; scale bar, 100 µm. n = 3 independent experiments. (C) Protein levels of PCNA, α-SMA in cells. n = 3 independent experiments. (D) Calculation of proliferation rate of vascular smooth muscle cells. (E) Quantitative analysis of EDU in vascular smooth muscle cells. (F, G) Quantitative analysis of PCNA and α-SMA. (H, I) Quantification of PCNA and Acta2 expressions by qPCR. (J, K) Immunofluorescence staining of mouse aortic root and quantitative analysis of arterial plaques with PCNA (green) and Myh11 (red). Scale bar, 50 µm. n = 3 independent experiments. (L) Expressions of PCNA and Acta2 in the aorta. n = 3 independent experiments. (M, N) EDU proliferation staining and quantification of mouse aorta; scale bar, 50 µm. n = 3 independent experiments. (O, P) Quantitative analysis of PCNA and Acta2 expressions in the aorta. Bar graphs depict mean ± SEM; one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PCNA, proliferative cell nuclear antigen; α-SMA, α-smooth muscle actin; ox-LDL, oxidized low density lipoprotein; HFD, high-fat diet; MTP, monotropein; NCD, normal diet.

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  Fig. 3 Monotropein effect on vascular smooth muscle cell migration. (A) Scratch experiment. Scale bar, 100 µm. n = 3 independent experiments. (B) Immunofluorescence co-staining of mouse aortic roots. The green color in the plaque represents MMP-2, red color represents Myh11. Scale bar, 50 µm. n = 3 independent experiments. (C) MMP-2 protein level in cells. n = 3 independent experiments. (D) Expression of MMP-2 in mouse aorta. n = 3 independent experiments. (E) Blank area statistics of scratch experiments. (F, G) Quantitative analysis of MMP-2 mRNA levels in cells. (H) Quantitative analysis of immunofluorescence staining in mouse aortic roots. (I) Quantitative analysis of MMP-2 protein levels in mouse aorta. Bar graphs depict mean ± SEM; one-way ANOVA *p < 0.05, **p < 0.01. MMP-2, matrix metalloproteinase-2; ox-LDL, oxidized low density lipoprotein; HFD, high-fat diet; MTP, monotropein; NCD, normal diet.

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  Fig. 4 Effect of monotropein on inflammation and oxidative stress in vascular smooth muscle cells. (A–C) Protein levels of TNF-α and 4-HNE in cells and quantitative analysis. n = 3 independent experiments. (D, E) Measurement of ROS activity in cells and quantitative analysis. Scale bar, 100 µm. n = 3 independent experiments. (F, G) mRNA expression levels of TNF-α and 4-HNE in cells and quantitative analysis. (H, I) Immunofluorescence staining of mouse aortic root. Green fluorescence in plaques denotes TNF-α and 4-HNE, and the red fluorescence presents Acta2. Scale bar, 50 µm. n = 3 independent experiments. (J) Protein levels of TNF-α and 4-HNE in mouse aorta. n = 3 independent experiments. (K, L) Quantitative analysis of immunofluorescence staining. (M, N) Quantitative analysis of protein levels of TNF-α and 4-HNE in mouse aorta. Bar graphs depict mean ± SEM; one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001. ROS, reactive oxygen species; ox-LDL, oxidized low density lipoprotein; HFD, high-fat diet; MTP, monotropein; NCD, normal diet.

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  Fig. 5 Effect of monotropein on NF- κB and AP-1 activities. (A–E) NF-κB, P-NF-κB, AP-1, P-AP-1 protein levels in cells and quantification. n = 3 independent experiments. (F–J) NF-κB, P-NF-κB, AP-1, and P-AP-1 protein levels and quantification in mouse aorta. n = 3 independent experiments. Bar graphs depict mean ± SEM; one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001, nsp < 0.05. ox-LDL, oxidized low density lipoprotein; HFD, high-fat diet; MTP, monotropein; NCD, normal diet.

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