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. 2024 Mar 12;10(6):e28021.
doi: 10.1016/j.heliyon.2024.e28021. eCollection 2024 Mar 30.

Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo

Yin-Fang Yan  1   2 , Yue Feng  1   2 , Si-Min Wang  1   2 , Fei Fang  1   2 , Hong-Yan Chen  1   2 , Ming-Xia Zhen  1   2 , Yu-Qiang Ji  1   2 , Song-Di Wu  1   2
Affiliations

Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo

Yin-Fang Yan et al. Heliyon. .

Abstract

Vascular calcification (VC) is an accurate risk factor and predictor of adverse cardiovascular events; however, there is currently no effective therapy to specifically prevent VC progression. Capsaicin (Cap) is a bioactive alkaloid isolated from Capsicum annuum L., a traditional medicinal and edible plant that is beneficial for preventing cardiovascular diseases. However, the effect of Cap on VC remains unclear. This study aimed to explore the effects and related mechanisms of Cap on aortic calcification in a mouse and on Pi-induced calcification in vascular smooth muscle cells (VSMCs). First, we established a calcification mouse model with vitamin D3 and evaluated the effects of Cap on calcification mice using von Kossa staining, calcium content, and alkaline phosphatase activity tests. The results showed that Cap significantly improved calcification in mice. VSMCs were then cultured in 2.6 mM Na2HPO4 and 50 μg/mL ascorbic acid for 7 days to obtain a calcification model, and we investigated the effects and mechanisms of Cap on VSMCs calcification by assessing the changes of calcium deposition, calcium content, and subsequent VC biomarkers. These results showed that Cap alleviated VSMCs calcification by upregulating the expressions of TRPV1. Moreover, Cap reduced the expression of Wnt3a and β-catenin, whereas DKK1 antagonised the inhibitory effect of Cap on VSMC calcification. This study is the first to offer direct evidence that Cap inhibits the Wnt/β-catenin signaling pathway by upregulating the expression of the TRPV1 receptor, resulting in the decreased expression of Runx2 and BMP-2, thereby reducing VSMC calcification. Our study may provide novel strategies for preventing the progression of VC. This could serve as a theoretical basis for clinically treating VC with spicy foods.

Keywords: Capsaicin; TRPV1; Vascular calcification; Wnt/β-catenin signaling pathway.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Cap treatment attenuates VC in mice; A) Chemical structure of Cap. B) Von Kossa staining assay showing the effect of Cap on calcium deposition in calcification mice. C) Effect of the Cap on calcium content in calcification mice assessed with a calcium colorimetric assay kit. D) Effect of the Cap on ALP activity in calcification mice assessed using an alkaline phosphatase assay kit; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Model; ns: p > 0.05 vs Control; p > 0.05 vs Model.
Fig. 2
Fig. 2
Cap treatment suppresses VSMC calcification induced by high Pi concentrations. A) Alizarin Red staining assay showing the effect of Cap on calcium deposition in VSMCs. B) Effect of the Cap on calcium content in VSMCs assessed using a calcium colorimetric assay kit. C) RT-qPCR revealing the effect of Cap on the expression of osteogenesis-specific markers in VSMC calcification. D) RT-qPCR illustrating the effect of Cap on the expression of phenotypic markers in VSMC calcification. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively, and VSMCs treated with different concentrations of Cap in the pro-calcifying medium were used as the treatment group (Pi+1 μM, 5 μM, 10 μM, 20 μM Cap); Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ns: p > 0.05 vs Control; p > 0.05 vs Pi. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Cap inhibits Pi-induced calcification of VSMCs by activating the TRPV1 receptor. A) RT-qPCR showing the mRNA levels of TRPV1; B) Western blot depicting the protein expression levels of TRPV1; C) Alizarin Red staining assay; D) Calcium content in VSMCs assessed using a calcium colorimetric assay kit. E) RT-qPCR illustrating the expression of osteogenesis-specific and phenotypic markers in VSMCs calcification. F) Western blot revealing the protein expression levels of osteogenesis-specific and phenotypic markers in VSMCs calcification. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively; Pi + Cap: VSMCs cultured in the pro-calcifying medium with 20 μM Cap; Pi + Cap + CPZ: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 10 μM CPZ; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ※: p < 0.05, ※※: p < 0.01, ※※※: p < 0.001 vs Pi + Cap; ns: p > 0.05 vs Control; p > 0.05 vs Pi; p > 0.05 vs Pi + Cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Cap inhibits Pi-induced calcification of VSMCs by inhibiting the Wnt/β-catenin signaling pathway by upregulation of TRPV1 receptor expression. A) RT-qPCR showing the mRNA levels of Wnt3a and β-catenin; B) Western blot illustrating the protein expression levels of Wnt3a and β-catenin; C) Alizarin Red staining assay; D) Calcium content in VSMCs assessed using a calcium colorimetric assay kit. E) RT-qPCR showing the expression of osteogenesis-specific and phenotypic markers in VSMC calcification. F) Western blot depicting the protein expression levels of osteogenesis-specific and phenotypic markers in VSMC calcification; G) Western blot showing the protein expression of β-catenin after treatment with DKK1. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively; Pi + Cap: VSMCs cultured in the pro-calcifying medium with 20 μM Cap; Pi + Cap + CPZ: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 10 μM CPZ; Pi + Cap + DKK1: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 20 ng/mL DKK1; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ※: p < 0.05, ※※: p < 0.01, ※※※: p < 0.001 vs Pi + Cap; ns: p > 0.05 vs Control; p > 0.05 vs Pi; p > 0.05 vs Pi + Cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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