This study was approved by the Ethics Committee of Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology in Wuhan, China. Patients provided signed informed consent. Calcific aortic valves samples were obtained from CAVD patients undergoing aortic valve replacement surgery, while control healthy aortic valves were obtained from patients undergoing Bentall surgery. Aortic valves were digested in 2 mg/ml type I collagenase (Sigma-Aldrich, Saint Louis, MO) for 6 h at 37°C, 5% CO₂ after washing with phosphate-buffered saline (PBS). Undigested tissues were removed using the 70 μm nylon cell filters; cells were then seeded in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco Laboratories, Gaithersburg, MD) for primary cultures. We used the osteogenic medium (OM) to induce the osteogenic differentiation of VICs, and AGP was added to inhibit this process. Every experiment was repeated three times for each aortic valve sample (Xu et al., 2019; Liu et al., 2020; Zhou et al., 2020).

Western blotting: cell samples were extracted, quantified, and then boiled at 95 °C for min. The protein sample was separated on an 8% sodium dodecyl sulfate-polyacrylamide electrophoresis gel and then transferred on a polyvinylidene fluoride membrane. Finally, primary antibodies were incubated overnight at 4 °C, followed by the corresponding secondary antibodies for protein expression visualization.

Immunofluorescence: Cell samples were seeded on 24-well confocal culture plates at a density of 10,000 cells/well. Samples were washed twice with PBS, fixed in 4% paraformaldehyde for 15 min, and then washed again with PBS. Cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked for 30 min with goat serum albumin (Boster, Wuhan, China). Then, it was incubated with primary antibodies at 4°C overnight, followed by incubating the fluorescent secondary antibody in the dark for 40 min at room temperature. Finally, the samples were washed twice with PBS and incubated with DAPI (Biofroxx GmbH, Einhausen, Germany) for a few minutes to stain the nuclei; then, they were imaged on the Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany). The following antibodies were used: Runx2 (CST: 8486s, 1:1000), ALP (R&D systems: MAB29092, 1:500), CDK1 (Proteintech: 19532-1-AP, 1:500), KI67 (Proteintech: 27309-1-AP, 1:200), anti-phospho-p38 (Thr180/Tyr182) (CST: 4511, 1:1000), anti-P38 (CST:8690, 1:1000), anti-phospho-p44/42 mitogen-activated protein kinase (Erk) (Thr202/Tyr204) (CST: 4377, 1:1000), anti-Erk (CST: 4695, 1:1000), and E2F1 (Abcam: ab179445, 1:1000).

Total cell RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA). The real-time polymerase chain reaction (PCR) was performed on a StepOne Plus thermal cycler (Applied Biosystems, Foster City, CA) using a 2x SYBR Green qPCR Master Mix (High ROX) (Bimake, Houston, TX) following the manufacturer’s guide. All the primers were referenced from the previous study (Xu et al., 2019). The final data were analyzed by the 2-^ΔΔc _t method. The primers used were as follows: CDK1 (F: 5′-TCC​TCC​AGG​GGA​TTG​TGT​TTT-3′; R: 5′-GCC​AGT​TTG​ATT​GTT​CCT​TTG​TC-3′), E2F1 (F: 5′-ACTTT GGTCTCGAGGAGGGT-3′; R: 5′-TGC​TAT​TCC​AAC​GAG​GCA​GG-3′), and MKI67 (F: 5′-GCC​CCT​GGA​AGA​TTA​TGG​TGG-3′; R: 5′-GGG​TTC​TGA​CTG​GTT​GTG​GTT​GT-3′).

VICs (passage 3) were cultured in 60 mm dishes under different treatments. Then, the samples were trypsinized and re-suspended in PBS at 5*10⁵/ml, followed by fixation in 70% precooled ethanol overnight at 4°C, centrifugation, washing, and staining with PI/RNase staining buffer (BD Biosciences) for 30 min at 4°C. Cell counts at different phases of the cell cycle were analyzed by flow cytometry (FCM) as previously described (Huang et al., 2019).

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (Bimake.com, Houston, TX, United States) according to the manufacturer’s instructions. The cells were seeded at a density of 5,000 cells/well in 24-well plates and cultured for 6 days under different treatments. At the end of each time interval, the cell samples were washed with PBS and incubated with a serum-free medium containing 10% CCK-8 reagent for 4 h at 37°C, 5% CO₂, and 10 µl aliquots were pipetted into a 96-well plate and measured at 450 nm using an enzyme-labeling instrument (Thermo Fisher Scientific).

Cell proliferation ability was tested by EdU assay (Ribobio bio, Guangzhou, China) according to the manufacturer’s instructions. The cells were seeded at a density of 10,000 cells/well in 12-well plates. After different treatments, VICs were cultured in a medium containing Edu for 2 h at 37°C, 5% CO₂. The cell samples were fixed with 4% paraformaldehyde for 15 min then washed with PBS several times, stained with apollo staining solution for 30 min, and observed under the fluorescence microscope.

The andrographolide (AGP) compound structure is downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The three-dimensional structure of Erk-2 (PDB ID: 4H3Q) is downloaded from the RCSB Protein Data Bank (www.rcsb.org). Autodock vina 1.1.2 was used for semi-flexible docking. Briefly, the coordinates and box size of the Vina molecule docking were determined, and then the parameter exhaustiveness was set as 20. Except for special instructions, other parameters adopt default values. The best affinity conformation with the lowest docking binding energy is selected as the final docking conformation. Pymol software was used for image construction.

Cells were seeded into 12-well plates cultured in the OM (contained: 10 mM β-glycerophosphate, 100 nM dexamethasone, 50 μg/ml vitamin C, 2% FBS, 100 IU/ml penicillin/streptomycin) with or without AGP for 21 days. The degree of cell calcification was measured by Alizarin Red S stain based on the previous study (Huang et al., 2019,2020 ).

All animals used in this research were purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). The ApoE^−/- mice were fed a high-fat and high-cholesterol diet (42% fat, 0.25% cholesterol) for 24 weeks to induce aortic valves calcification. To treat CAVD, the disease model mice were treated with AGP at a dose of 10 mg/kg by gavage from 16 to 24 weeks. Then, the heart samples of mice were collected and histologically sectioned to isolate the aortic valves. HE staining was used to evaluate the thickness of the valves. Runx2 expression was analyzed by immunofluorescence staining in vivo. The incrassation and Runx2 intensity were measured in the same way as in our previous study (Wang et al., 2021a).

All experimental data were analyzed and expressed as the mean ± standard deviation (SD). Statistical comparisons were made by the analysis of variance to evaluate differences among different groups. The p-value less than 0.05 was considered statistically significant.

We performed western blotting and immunofluorescence staining on aortic valve tissues to detect the proliferation of VICs in aortic valves from the control and CAVD groups. The cell proliferation-related factors, cyclin-dependent kinase 1 (CDK1), and Ki-67 were upregulated in the CAVD group compared to the control group (Figure 1A,C). The expressions of ALP and CDK1 proteins were significantly increased in the CAVD group compared to the control group (*p < 0.05; Figure 1B). The results of immunofluorescence staining were consistent with those of western blotting (*p < 0.05; Figure 1D). Furthermore, cell proliferation-related genes were tested via real-time PCR; CDK1 and MKI67 genes were significantly upregulated in the CAVD group compared to the control group (*p < 0.05; Figure 1E). Briefly, the proliferation of VICs was significantly increased due to the pathophysiological changes in CAVD.

The 2D and 3D structures of AGP are shown in Figure 2A. The toxicity of AGP on VICs was assessed by determining the IC50 values, which showed that AGP has significant cytotoxicity at concentrations >10 mΜ (Figure 2B). The results indicated that 10 μΜ is the suitable AGP concentration for our experiments. In addition, the CCK-8 assay was used to analyze cell proliferation after treatment with AGP for 5 days. The results indicated that the cell viability of the AGP group significantly decreased starting from day 3 when compared with the control group (*p < 0.05; Figure 2C). Ki-67 staining indicated that cell proliferation was significantly inhibited after treatments with 5 and 10 μmol/L AGP for 48 and 72 h, respectively (*p < 0.05; Figure 2D,E). Correspondingly, cell cycle analysis showed that the numbers of VICs involved in the S-phase were decreased when cultured in 5 and 10 μM AGP medium for 72 h (*p < 0.05; Figure 2F,G).

VICs were treated with AGP for 2 h at doses of 5 and 10 μmol/L. Compared to the control group, expression levels of cell proliferative genes were inhibited by AGP in VICs (Figure 3A). AGP significantly downregulated the mRNA levels of MKI67 (*p < 0.05), CDK1 (*p < 0.05), and E2F1 (*p < 0.05). Similar to the gene expression after treatment, AGP inhibited the CDK1 protein synthesis and downregulated E2F1 expression (*p < 0.05; Figure 3B,C). Based on the immunofluorescence staining of the EdU assay (Figure 3E), 5 and 10 μmol/L AGP inhibited cell proliferation activity relative to the control group, consistent with the levels of gene and protein expression (*p < 0.05; Figure 3D). SwissTarget prediction provided more than 10 potential molecular targets for AGP (Figure 3F), and Erk protein was selected for further molecular docking analysis, based on the previous literature on AGP (Figure 3G). The results indicated that AGP successfully bound to ERK (extracellular regulated protein kinases) using the residues HOH581, HOH542, HOH524, and HOH545 (red dot), via hydrogen bond interactions and residues ASP106, ARG79, GLU81, and ILE83.

Based on the target analysis above, we focused on the MAPK-ERK signaling pathway. The osteogenesis-specific gene Runx2 and cell proliferative genes CDK1 and MKI67 were tested in VICs under an OM culture with or without AGP for 48 h. The results demonstrated that the OM culture significantly increased the expression of CDK1, MKI67, and Runx2. However, after the addition of AGP to the OM culture, CDK1, MKI67, and Runx2 were significantly downregulated (* vs control group p < 0.05; # vs OM + DMSO group p < 0.05; Figure 4A). The results of the Ki-67 staining were consistent with the PCR results (Figure 4C). AGP significantly inhibited the OM-induced cell proliferative activity (* vs control group p < 0.05; # vs OM + DMSO group p < 0.05; Figure 4B). We detected the protein expression levels of phosphorylated ERK and P38 after OM treatment with or without AGP and found that AGP effectively reversed the OM-induced upregulation of phosphorylated ERK and P38 protein expression (Figure 4D,G). After being cultured in OM for 21 days, VICs were tested for calcification using Alizarin Red S staining. OM + DMSO and OM + AGP groups stained positively for Alizarin Red S, showing significant differences compared to the control group (Figure 4E). Semi-quantitation of stain intensity indicated an approximately 1-fold decrease in the OM + AGP group compared to the OM + DMSO group (* vs control group p < 0.05; # vs OM + DMSO group p < 0.05; Figure 4F).

We used the high-fat diet-fed animal model to verify the effect of AGP on CAVD in vivo. Hematoxylin and eosin (HE) staining and Runx2 immunofluorescence staining were performed on the aortic valve samples of mice (Figure 5A,B). The results demonstrated that the aortic valves of high-fat diet-fed mice were significantly thickened, while AGP feeding ameliorated the aortic valve incrassation (* vs control group p < 0.05; # vs HF (high-fat diet) group p < 0.05; Figure 5C). Immunofluorescence staining showed that AGP significantly reduced the Runx2 expressions induced by high-fat diet-feeding in vivo (* vs control group p < 0.05; # vs HF group p < 0.05; Figure 5D).