Dehydrocorydaline maintains the vascular smooth muscle cell contractile phenotype by upregulating Spta1

Abstract

Vascular smooth muscle cell (VSMC) phenotypic switching plays a crucial role in the initiation and progression of atherosclerosis. Dehydrocorydaline (DHC), a major active component of the traditional Chinese herbal medicine Rhizoma Corydalis, exhibits diverse pharmacological effects. However, its impact on VSMCs remains largely unknown. This study aims to investigate the effects and underlying mechanisms of DHC in phenotypic switching of VSMCs. Our study revealed that DHC increased the mRNA and protein levels of rat VSMC contractile phenotype markers, such as calponin 1 (Cnn1), myosin heavy chain (Myh11, SM-MHC), smooth muscle 22α (Sm22α), and alpha-smooth muscle actin (Acta2, α-SMA) in a time- and dose-dependent manner. Additionally, DHC inhibited platelet-derived growth factor-BB-induced VSMC proliferation and migration. In Apoe^-/- mice, DHC treatment resulted in reduced carotid plaque areas and macrophage infiltration, along with increased contractile phenotype marker expression. RNA sequencing analysis revealed a significant upregulation of spectrin alpha, erythrocytic 1 (Spta1) in DHC-treated rat VSMCs. Strikingly, Spta1 knockdown effectively negated the increase in contractile phenotype marker expression in VSMCs that was initially prompted by DHC. Therefore, DHC preserves the VSMC contractile phenotype through Spta1, thereby attenuating carotid artery atherosclerotic plaques in Apoe^-/- mice. This study provides evidence supporting the potential use of Chinese herbal medicines, particularly those containing DHC such as Rhizoma Corydalis, in the treatment of atherosclerotic cardiovascular disease, thus expanding the clinical application of such herbal remedies.

Keywords: atherosclerosis; dehydrocorydaline; phenotypic switching; vascular smooth muscle cell.

Fig. 1. RNA-sequencing (RNA-seq) analysis shows that dehydrocorydaline (DHC) enhances the expression of contractile phenotype-related genes in rat vascular smooth muscle cells (VSMCs).

Rat VSMCs were exposed to either dimethyl sulfoxide (DMSO) or 100 μM DHC for 24 h, followed by RNA-seq analysis (n = 3). a A volcano plot shows differentially expressed genes, with red and green dots representing significantly upregulated (47) and downregulated genes (51), respectively (P < 0.05, |log2 fold change | > 1). b The top 20 enriched upregulated Gene Ontology pathways. The dot size represents the number of differentially expressed genes in the pathway, and the dot colors correspond to various P values. c The top 20 enriched upregulated Kyoto Encyclopedia of Genes and Genomes pathways. The dot size represents the number of differentially expressed genes in the pathway, and the dot colors correspond to different ranges of P values. d Fragments per kilobase of transcript per million mapped reads (FPKM) values of markers related to VSMC phenotypic switching among differentially expressed genes, including Calponin 1 (Cnn1), smooth muscle 22α (Sm22α), and alpha-smooth muscle actin (Acta2). Data are presented as the mean ± SEM. ***P < 0.001, analyzed using unpaired t-tests.

Fig. 2. DHC maintains the VSMC contractile phenotype.

a, b Rat VSMCs were treated with 100 μM DHC for 0, 12, and 24 h. a mRNA levels of contractile markers (Cnn1, smooth muscle myosin heavy chain (Myh11), Sm22α, and Acta2) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) (n = 4). b Protein levels of contractile markers (Cnn1 and smooth muscle myosin heavy chain, SM-MHC) were detected by Western blotting (n = 3). c, d Rat VSMCs were treated with 0, 50, and 100 μM DHC for 24 h. c mRNA levels of contractile markers (Cnn1, Myh11, Sm22α, and Acta2) were assessed by RT-qPCR (n = 4). d Protein levels of contractile markers (Cnn1 and SM-MHC) were determined by Western blotting (n = 3). e α-SMA (green) expression in rat VSMCs treated with DMSO or 100 μM DHC for 24 h was detected by cyto-immunofluorescence, and 4′,6-diamidino-2-phenylindole (DAPI) (blue) indicates the nucleus (n = 4, scale = 50 μm). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, analyzed using unpaired t-tests.

Fig. 3. DHC suppresses the platelet-derived growth factor-BB (PDGF-BB)-induced VSMC secretory phenotype transition in vitro.

a–c Rat VSMCs were exposed to 100 μM DHC and/or 20 ng/mL PDGF-BB for 24 h. a mRNA levels of contractile markers (Cnn1, Myh11, Sm22α, and Acta2) were assessed by RT-qPCR (n = 3). b Protein levels of contractile markers (Cnn1 and SM-MHC) were detected by Western blotting (n = 10). c Protein levels of α-SMA (green) were detected by immunofluorescence, and DAPI (blue) indicates the nucleus (n = 4, scale = 50 μm). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, analyzed using one-way ANOVA.

Fig. 4. DHC inhibits PDGF-BB-induced VSMC proliferation and migration.

a–c VSMCs were treated with 100 μM DHC and/or 20 ng/mL PDGF-BB for 36 h. The effects of PDGF-BB and DHC on VSMC proliferation were determined by cell counting a and a Cell Counting Kit-8 (CCK-8) assay (b), n = 3. c The effects of PDGF-BB and DHC on VSMC migration were examined using a wound healing assay (n = 3, scale = 150 μm). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, analyzed using two-way ANOVA (a) and one-way ANOVA (b, c).

Fig. 5. DHC attenuates atherosclerosis in Apoe^−/− mice.

a The strategy for an Apoe^−/− mouse model with carotid artery injury. b Carotid atherosclerotic plaque lesions evaluated by hematoxylin and eosin staining (n = 6/group, scale = 100 μm). c Detection of macrophage infiltration in carotid plaques by F4/80 (red) immunofluorescence staining, with DAPI (blue) indicating the nucleus (n = 8/group, scale = 100 μm/10 μm). Data are presented as the mean ± SEM. *P < 0.05, analyzed using unpaired t-tests.

Fig. 6. DHC maintains the VSMC contractile phenotype in the carotid plaques of Apoe^−/− mice.

a Immunofluorescence detection of α-SMA (red) in carotid plaques, with DAPI (blue) indicating the nucleus (n = 8/group, scale = 50 μm/10 μm). b Immunofluorescence detection of SM-MHC (red) in carotid plaques, with DAPI (blue) indicating the nucleus (n = 8/group, scale = 100 μm or 10 μm). Data are presented as the mean ± SEM. *P < 0.05, analyzed using unpaired t-tests.

Fig. 7. DHC sustains the contractile phenotype of rat VSMCs by upregulating spectrin alpha, erythrocytic 1 (Spta1) expression.

a–c Rat VSMCs were treated with DMSO or 100 μM DHC for 24 h. a FPKM values of Spta1 from RNA-seq analysis (n = 3). b Detection of Spta1 mRNA level using RT-qPCR (n = 3). c Detection of SPTA1 protein level by Western blotting (n = 3). d, e Rat VSMCs were transfected with siNC or siSpta1 for 24 h and then treated with DMSO or 100 μM DHC for 24 h. d mRNA levels of contractile markers (Myh11, Sm22α, and Acta2) were assessed using RT-qPCR (n = 4). e Protein levels of contractile markers (Cnn1 and SM-MHC) were detected by Western blotting (n = 7). f Expression of α-SMA (green) in rat VSMCs was determined by immunofluorescence, with DAPI (blue) indicating the nucleus (n = 4, scale = 50 μm). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, analyzed using unpaired t-tests (a to c) and one-way ANOVA (d to f).

Fig. 8. Schematic illustration of the effects of DHC on VSMCs and atherosclerosis.

DHC from Rhizoma Corydalis promotes the contractile phenotype of VSMCs by upregulating Spta1, thereby inhibiting VSMC proliferation and migration and reducing atherosclerotic plaques in Apoe^−/− mice, suggesting its potential in treating atherosclerotic cardiovascular disease.