Myricanol was purchased from ChemFaces (Wuhan, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO. Recombinant human PDGF-BB was purchased from Corning Incorporated. EdU kit was purchased from Ribobio. Antibodies of the total levels and phosphorylation of PDGFRα, PDGFRβ, PLCγ1, Src, ERK1/2, JNK, p38, Rb, and p65 were purchased from Cell Signaling Technology. Anti-metalloproteinase 2 (MMP2), anti-matrix metalloproteinase 9 (MMP9), anti-Cyclin D1 (CCND1), and anti-Cyclin E1 (CCNE1) were purchased from Abcam. PCNA, E2F1, P21, P27, P53, Caspase 3, BAX, BCL2, and GAPDH were purchased from proteintech.

Primary VSMCs were isolated from the thoracic aortas of SD rats weighing 150–180 g in an enzymatical way. Rat aortic arteries were removed under sterile conditions. Adventitia from the aorta was removed under a dissecting microscope. The aorta was cut into pieces approximately 1–2 mm sections and digested with Enzyme solution (collagenase type II 3 mg/ml and elastase 1 mg/ml) for 2 h. Then cells were cultured in a flask with DMEM supplemented with 15% FBS, and passage 3–6 were used for experiments (Supplementary Figure 1).

In starvation conditions, VSMCs were treated with indicated concentrations myricanol or not for 30 min, then VSMCs were treated with PDGF-BB (30 ng/ml) for 12 h and cultured into 96-well plates (3 × 10³ cells/well). After incubation with a medium containing EdU for another 2 h, the cells were fixed with 4% paraformaldehyde, and EdU Incorporation Assay was performed according to the manufacturer’s instructions. Images were photographed by using Olympus cellSens Entry.

For the wound healing assay, VSMCs in a 6-well plate with about 90% confluence were treated with indicated concentrations myricanol or not for 30 min in starvation conditions, then VSMCs were treated with PDGF-BB (30 ng/ml). Cell monolayers were scratched by a 200-μl pipette tip and photos were taken under a microscope (Olympus) at 0 and 24 h.

For transwell migration assay, VSMCs were treated with indicated concentrations myricanol or not for 30 min in starvation conditions, then VSMCs were treated with PDGF-BB (30 ng/ml). Then 1 × 10⁵ VSMCs were detached and suspended into a transwell upper surface with 200 μl of DMEM medium with FBS free. 500 μl of FBS-free DMEM medium with PDGF-BB (30 ng/ml) was added in the lower chamber. After 24 h incubation at 37°C, the migrated cells were fixed in 4% paraformaldehyde for 30 min, stained in 0.1% crystal violet for 15 min and photographed by microscope for six randomly assigned fields (Olympus).

The VSMCs were cultured into six-well plates up to 80% confluence. After being pretreated with indicated concentrations of myricanol or vehicle for 30 min, the cells were stimulated by PDGF-BB (30 ng/ml) for 5, 15, 60 min or another 24 h. Western blotting was performed following procedures described previously (Wang et al., 2019).

Primary VSMCs were seeded in a round cell culture dish with a diameter of 60 mm. After the density reached 80% confluence, the cells were preadministrated with vehicle or myricanol at a concentration of 30 μM, and PDGF-BB was administered 30 min later. After 4 h, a nuclear protein and cytoplasmic protein extraction kit (Beyotime, P0027) was used to extract the nuclear and cytosolic proteins.

Primary VSMCs were seeded in a round cell culture dish with a diameter of 15 mm. The cells were preadministrated with vehicle or myricanol at a concentration of 30 μM, and PDGF-BB was administered 15 min later. The cells were fixed in 4% formaldehyde for 30 min and immunostained with p65 (CST, #8242) antibodies overnight at 4°C, then incubated with the indicated secondary antibodies for 1 h at 37°C. Nuclei were stained with DAPI for 20 min at 37°C. Photos were taken under a fluorescence microscope (Olympus).

Animal housing and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology, and IACUC number is 2561. Surgery was performed under sterile conditions. Eight-week-old male C57BL/6 mouses were anesthetized with an intraperitoneal injection of pentobarbital sodium. In ligated animals (n = 6), the left common carotid artery was dissected from the surrounding tissue under a microscope and ligated by using 6–0 silk ligature. The right common carotid artery was dissected without ligation as a sham surgery. Then, mice were randomly divided into two groups on average. Myricanol (5 mg/kg/day) or hydration medium (PEG 400) was intraperitoneally injected into C57BL/6 mice for 14 days. Then mice were euthanized and common carotid arteries were excised. After being fixed with 4% formaldehyde and embedded in paraffin, Cross-sections of the common carotid artery were stained with hematoxylin and eosin (H&E) and elastic Masson trichrome solutions.

SPSS v.20 was used for statistical analyses. All experiments were performed at least three times, and data are given as means ± SEM. The two-tailed unpaired Student’s t-test was used for comparisons of two groups and differences were considered significant at P < 0.05.

When VSMCs are activated by various injury stimuli, they change from a resting state to a proliferative phenotype and migrate under the intima. To investigate the effect of myricanol on PDGF-BB-induced VSMCs proliferation, we performed a EdU assay. The results showed that the ability of cell proliferation was significantly improved under the stimulation of PDGF-BB and was apparently inhibited by myricanol. We found that the effect of myricanol on VSMC proliferation is dose-dependent. For example, 3 μM of myricanol treatment showed no significant influence, whereas both 10 and 30 μM of myricanol can significantly suppressed the proliferation of VSMCs (Figures 1A,B).

Under the action of extracellular stimulus, the expression of contractile protein in VSMC is significantly reduced, while the expression of cell cycle-related proteins increases rapidly. As shown in Figures 1C,D, the protein level of PCNA, E2F1, phosphorylated Rb, Rb, CCND1, CCNE1, p21, p27, and p53 were detected by western blot analysis. We find that CCND1, CCNE1, PCNA, E2F1, and p-Rb were significantly increased by PDGF-BB stimulation, whereas the myricanol treatment can partially or completely block the effects. Meanwhile, p53 was significantly inhibited by PDGF-BB stimulation, whereas the myricanol treatment can partially recover the effects. Different from the above results, PDGF-BB stimulation had no effect on the expression of p21 and p27, while preadministrated of myricanol increased the expression of p21.

To further confirm the effect of myricanol on cell apoptosis and necrosis, western blot and LDH assays were performed. Western blot results showed that PDGF-BB and myricanol had no effect on the protein level of caspase 3, cleaved caspase3, BAX and BCL2. LDH assay showed that PDGF-BB and myricanol had no effect on LDH release (Supplementary Figure 2). These results indicated that myricanol have no effect on cell apoptosis and necrosis.

To investigate whether myricanol was able to affect the migration in VSMCs, We then assessed the effect by wound healing assay and transwell migration assay (Figures 2A,C). The wound healing assay result showed that 10 and 30 μM myricanol but not 3 μM of myricanol could partially suppress the migration induced by PDGF-BB stimulation, and the result of the transwell migration assay seems to be similar. The migration of VSMCs was shown by the distance of the gap in the wound healing assay (Figure 2B) and the migration cell number in the transwell migration assay (Figure 2D).

In the process of intimal hyperplasia, a large number of proteoglycans and extracellular matrix remodeling related proteins (such as MMP2, MMP9) are synthesized and secreted to promote the migration of VSMCs. Therefore, the protein levels of migration-associated protein MMP2 and MMP9 were detected by western blot analysis, which increased by PDGF-BB stimulation. However, the effect on MMP2 and MMP9 seems to be different. The increasing of MMP9 protein level was significantly suppressed at 3, 10, and 30 μM myricanol, however, increasing of MMP2 protein level can only be significantly suppressed at 30 μM myricanol (Figures 2E,F). Further, consistent with the above results, the activity of MMP2 and MMP9 were increased by PDGF-BB stimulation, while the myricanol treatment can partially or completely block the effects as shown in Zymography assays (Supplementary Figure 3).

At present, it is known that a variety of cell pathways and their effector molecules in VSMC have undergone significant changes, and they jointly participate in the coordinated regulation of VSMC proliferation and migration, of which PDGFRβ pathway is the most important (Levitzki, 2005; Wang Y. et al., 2020). To investigate how myricanol influences the proliferation and migration of VSMC induced by PDGF-BB, we tested if myricanol could suppress the phosphorylation of PDGFRα, PDGFRβ and its downstream PLCγ1, Src and MAPK in VSMC. VSMCs were treated with vehicle or 30 μM myricanol 30 min before exposure to PDGF-BB, and then cells were stimulated by PDGF-BB and the cytolytic products were harvested at different time point after stimulation (5 min, 15 min, 60 min). The expression of p-PDGFRα^Y1018, p-PDGFRβ^Y751, p-PDGFRβ^Y857, p-PDGFRβ^Y1021, p-PLCγ1, p-Src, p-JNK, p-ERK1/2, and p-p38 were increased after PDGF-BB stimulation in 5, 15, and 60 min, and pretreatment with 30 μM myricanol repressed the phosphorylation of these proteins (except p-JNK) at the same time points after PDGF-BB stimulation (Figure 3). The specific inhibitors for PDGFRβ, JNK, ERK1/2 and p38 were provided to verify the effect of myricanol. The results showed that myricanol has weaker inhibitory effect on PDGFRα^Y1018, p-PDGFRβ^Y751, p-PDGFRβ^Y857, p-PDGFRβ^Y1021, p-ERK1/2 and p-p38 than PDGFRβ, JNK, ERK1/2 and p38 inhibitors (Supplementary Figure 4).

In addition, NF-κB is a critical signaling in VSMC proliferation and migration (Lu et al., 2018). Our results exhibited that myricanol can inhibit the phosphorylation level of IκBα and p65 and the nuclear translocation of p65 stimulated by PDGF-BB in VSMC (Figure 4).

To assess the effect of myricanol on intimal hyperplasia after vascular injury, we used the carotid artery wire ligation model. Myricanol (5 mg/kg/day) or hydration medium (PEG 400) was intraperitoneally injected for 14 days after carotid artery ligation. Comparing with the Sham surgery group, the ligation surgery group showed that intimal hyperplasia was well developed. The sections stained with elastic Masson trichrome solutions were used to highlight the media (Figure 5A). Comparing with the ligation surgery group, the myricanol treated ligation surgery group showed significantly reduced intima, both the ratio of intima to media (I/M ratio) and intimal area (Figures 5B,C). Immunofluorescence staining showed that the myricanol-treated ligation surgery group had significantly inhibited Ki67 expression (Supplementary Figures 5A,B). In addition, macrophages are known to play a role in the progression of intimal hyperplasia and other cardiovascular disorders (Zhang et al., 2021). Our results exhibited that the expression of F4/80 significantly increased in the ligation surgery group comparing with the sham surgery group, While the myricanol treatement significantly decreased F4/80 expression (Supplementary Figures 5C,D). These results indicate that myricanol significantly diminishes the neointimal hyperplasia induced by carotid artery ligation.