Resveratrol may improve bone-like changes, hardening, and cell death in blood vessel muscle cells by affecting JNK/Bax signals

VSMCs were isolated from aortic arteries of male Sprague–Dawley (SD) rats (150 g–180 g) using the explant method. VSMCs were cultured in DMEM [10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL)] at 37 °C in an incubator. VSMCs between passages 5 and 8 were used for the experiments. VSMC calcification was induced by the CM [DMEM, β-glycerophosphate (β-GP, 10 mM), and CaCl₂ (3 mM)]. After the cells were adhered overnight, VSMCs were treated with Res (10 μM; Sigma, St. Louis, MO) in the CM for 10 days (Li et al., 2024; Takemura et al., 2011). The C-jun N terminal kinase (JNK) inhibitor (SP600125, 10 μM; Sigma) or activator (anisomycin, 100 ng/mL; Sigma) was added to the medium 1 h before Res treatment for use in the experiments (Liu et al., 2021; Zhang et al., 2015).

VSMCs in the logarithmic growth stage were inoculated into a 96-well plates (1,000 cells per/well), with five wells in each group. After 24 h of cell inoculation and adherent growth, Res was administered at the final concentrations of 0, 10, 20, 25, 50, 75, and 100 µM (Li et al., 2024). After 24 h, 10 µL of CCK-8 solution was added to each well and incubated in the dark for 30 min. The absorbance (A) value was measured at 450 nm.

After fixed in 4% formaldehyde for 10 min in 6-well plates, VSMCs were treated with 2% alizarin red (pH 4.2) for 5 min. Then, excess dye was removed by deionized water, and an inverted phase contrast microscope was used to visualize staining. Alizarin red dye was eluted with 10% formic acid and quantified by spectrophotometry. As previously described, the calcium content and alkaline phosphatase (ALP) activity was measured (Hou et al., 2016; Liao et al., 2013; Yan et al., 2011). VSMCs were washed with phosphate-buffered saline (PBS) and extracted with 0.6 N HCl for 24 h. The protein concentration was quantified using BCA protein assay (Pierce, United States). The calcium content was normalized to protein concentration and expressed as µg/mg protein. For ALP activity analysis, VSMCs were harvested with 0.1% Triton X-100 in PBS. p-NPP (180 µL) substrate was added to the protein samples, and the reaction was incubated for 15 min at 37 °C. To stop the reaction, NaOH (3 M) was added to the mixture. The absorbance was then measured at 405 nm, and the ALP activity was presented as nmol/mL p-nitrophenol converted per microgram of protein per minute.

Total protein was extracted from VSMCs, and the protein concentration was measured using a BCA protein assay kit (Pierce, United States). Equal amounts of protein samples were loaded and separated by 5% SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad, United States). The membranes were then blocked with 5% non-fat dried milk for 1 h, followed by incubation with primary antibodies including Bax (1:1,000, 2772), JNK (1:1,000, 9252), p-JNK (1:1,000, 9255), and β-actin (1:3,000, ab32572) from Cell Signaling Technology (Beverly, MA, United States), along with Runx2 (1:500, SAB1403638, Millipore, United States) and Osterix2 (1:1,000, ab209484, Abcam, United Kingdom). Then, membranes were incubated with secondary antibodies (1:1,000, 5125, Cell Signaling, United States). SuperSignal West Pico Chemiluminescent Substrate (Pierce, United States) was used to detect the protein signals.

TRIzol reagent (Invitrogen, United States), a reverse transcription kit (Takara Company, China), a StepOne Real-Time PCR system (Applied Biosystems, United States), and SYBR Green mixture were utilized for PCR. PCR primers were as follows: Runx2 (forward): GCC GGG AAT GAT GAG AAC TA, Runx2 (reverse): GGA CCG TCC ACT GTC ACT TT; BMP2 (forward): GTT TGG CCT GAA GCA GAG AC, BMP2 (reverse): CTC GAT GGC TTC TTC GTG AT; β-actin (forward): TGT​CAC​CAA​CTG​GGA​CGA​TA, β-actin (reverse): GGG GTG TTG AAG GTC TCA AA; and Osterix (forward): TCT CCA TCT GCC TGA CTC CT, Osterix (reverse): GGG GCT GAA AGG TCA GTG TA. Housekeeping gene β-actin and the comparative Ct method were used to determine the target gene expression in the experimental group.

The One-Step TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) and Caspase-3/CPP32 Colorimetric Assay Kit (BioVision) were used to detect cell apoptosis according to the manufacturers' instructions. After treatment, VSMCs were fixed in 4% (w/v) paraformaldehyde at 4 °C, followed by incubation with terminal deoxynucleotidyl transferase (TdT) for 1 h. The cell nuclei were stained with DAPI. The TUNEL-positive apoptotic VSMCs were detected using a fluorescence microscope (Olympus). In addition, VSMCs were lysed, and the protein concentration was measured using a BCA protein assay kit (Pierce, United States). Protein (100 µg) was mixed with cell lysis buffer (50 µL) and reaction buffer (50 µL). Then, 5 µL of 4 mM DEVD-qNA substrate was added and incubated at 37 °C for 2 h. The activity of caspase 3 was detected by a microplate reader at 405 nm.

VSMCs were incubated with the antibodies Runx2 (1:100, SAB1403638, Millipore), p-JNK (1:200, 9255, Cell Signaling, United States), and Bax (1:200, 2772, Cell Signaling, United States), followed by staining with FITC-conjugated secondary antibody. VSMCs were then double-stained with DAPI for visualizing the nuclei and viewed with a Nikon Eclipse 80iEpi-fluorescence microscope equipped with a digital camera (DS-Ri1, Nikon).

Docking compound Res (compound CID: 445154) was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/↗), and JNK (ID: P49185) was obtained from the UniProt database (https://www.uniprot.org/↗). The processing and optimization of the molecular docking process were performed by the Glide module in the Schrodinger Maestro software. For screening in the Glide module, the prepared receptors were imported, the protein protoligand was selected as the binding site of the protein, and the box size was set to 10Å × 10Å × 10Å. The complexes of the JNK proteins and Res were visualized by PyMOL 2.1.

All animal experimental procedures in this study were performed in accordance with the requirements of the Ethics Committee for Heze Medical College. The vascular calcification model was established in 8–10-week-old male C57BL/6 mice (weighing 20 g–25 g). The mice were randomly divided into a vehicle group (n = 6), a vitamin D₃ group (Vit D group, n = 6), and a vitamin D₃ + resveratrol group (Vit D + Res group, n = 6). Briefly, the mice received subcutaneous injection of vitamin D₃ (cholecalciferol, 5 × 10⁵ IU/kg/day) once a day for 3 consecutive days, as previously described (Bhat et al., 2020; Zeng et al., 2021; Zhang et al., 2021). In the vehicle control group, the mice were treated with an injection of matched vehicle (5% v/v ethanol). In the vitamin D₃ + resveratrol group (Vit D+ Res group), the mice received the same vitamin D₃ regimen. Starting 1 day after vitamin D₃ injection, these mice were co-treated with Res (50 mg/kg/day) by intraperitoneal injection for 7 consecutive days. All mice were sacrificed at the end of the experimental period, and the aortic vessels were harvested for subsequent experiments.

Frozen sections of the aorta were allowed to rewarm at room temperature for 2 h and then were hydrated in 1× histochemical PBS. A sufficient amount of 5% AgNO₃ solution was applied onto the vascular tissue to completely cover the tissue and then irradiated under UV light for 30 min–60 min. After discharging the AgNO₃ solution, the sections were washed with distilled water and 5% Na₂S₂O₃ solution for 2 min, respectively. After staining with the hematoxylin solution, the sections were successively dehydrated with 70%, 80%, 90%, 95%, 100%, and 100% ethanol and then made transparent with xylene. The sections were sealed with neutral gum and baked to dryness in an incubator at 37 °C.

All data were expressed as the mean ± SD and analyzed using the software package SPSS 17.0. Statistical differences between the two groups were analyzed by Student's t-test, and the differences between more than two groups were compared by one-way ANOVA. A value of p < 0.05 was considered statistically significant.

To investigate vascular calcification, we established a cell calcification model using CM to treat VSMCs. Alizarin red staining confirmed the successful induction of the cell calcification model, with visible calcium deposition (Figures 1A, B). Calcium concentration in CM-treated VSMCs significantly increased over time (Figure 1C). VSMCs exhibited increased ALP activity after CM treatment (Supplementary Figure S1A). qRT-PCR and Western blotting results showed that the mRNA and protein levels of the calcification markers Runx2, BMP2, and Osterix increased significantly in CM-stimulated VSMCs (Supplementary Figure S1B, D). Given that VSMC apoptosis via calcifying apoptotic bodies is a key driver of vascular calcification (Cui et al., 2020; Proudfoot et al., 2000), we next examined whether CM induces VSMC apoptosis. TUNEL staining results showed a significant increase in the proportion of TUNEL-positive cells after CM treatment (Figures 1D, E). The activity of caspase-3 was markedly elevated in CM-treated VSMCs (Figure 1F). These results confirmed that apoptosis is a key event in CM-treated VSMCs and directly contributes to calcification initiation.

To assess whether Res can regulate VSMC calcification and osteogenic differentiation, we initially performed cytotoxicity assays for Res in VSMCs. After Res treatment at different concentrations, the CCK-8 assay revealed no significant alteration in VSMC viability across varying Res concentrations, indicating that Res had almost no cytotoxicity (Figure 2A). Based on these findings, we selected Res at a concentration of 10 μM for further experimental studies, which is consistent with earlier studies. Res treatment markedly reduced the deposition of calcium phosphate minerals in CM-induced VSMCs (Figures 2B, C). Calcium quantification analysis confirmed that Res treatment significantly reduced the CM-induced increase in calcium content (Figure 2D). Furthermore, we investigated the effect of Res on early osteogenic differentiation markers. The activity of ALP was significantly enhanced after CM treatment, but it decreased upon co-treatment with CM and Res (Figure 2E). Co-treatment with Res markedly suppressed the mRNA and protein levels of Runx2, BMP2, and Osterix compared to that with CM alone (Figures 2F, G). Immunofluorescence further demonstrated a significant enhancement in the fluorescence intensity of Runx2 following treatment with CM. Res effectively counteracted the above effect on Runx2 expression (Figure 2H). Importantly, Res reversed CM-induced apoptosis, as indicated by reduced TUNEL-positive cells and the decreased activity of caspase-3 (Figures 2I, J).

The JNK/Bax signaling pathway is involved in the regulation of VSMC calcification and osteogenic differentiation. To explore whether Res inhibits VSMC calcification and osteogenic differentiation by regulating JNK/Bax signaling, we performed molecular docking of Res and JNK. The lowest-energy docking conformations are shown in Figure 3A, with a binding energy of −7.783 kcal/mol between Res and JNK. The docking calculations showed that Res could form four conventional hydrogen bonds with MET111, GLU109, ASN156, and GLY38 at the active center of JNK, forming a close interaction (Figure 3A). This finding indicated that Res effectively binds to and modulates JNK activity in VSMCs. Subsequently, we validated these findings using Western blot and immunofluorescence assays in CM-treated VSMCs. Western blot results confirmed that CM treatment elevated the protein levels of JNK and Bax, which was inhibited by Res co-treatment (Figure 3B). Furthermore, the results of the immunofluorescence assay demonstrated a significant enhancement in the fluorescence intensity of JNK and Bax following treatment with CM. Conversely, Res co-treatment resulted in a decrease in JNK and Bax fluorescence intensity (Figures 3C,D).

To explore the role of the JNK/Bax signaling pathway in Res-mediated inhibition of vascular calcification, we treated VSMCs with CM or GM for 10 days in the presence of JNK inhibitor SP600125 (10 μM). Western blot assay revealed that CM-induced increase of JNK and Bax was markedly reduced by SP600125, and no significant difference of JNK and Bax was observed following GM and SP600125 co-treatment (Figure 4A). Alizarin red staining confirmed that SP600125 markedly prevented CM-induced calcium deposition in VSMCs (Figures 4B, C). At the same time, quantitative analysis of the cellular calcium content corroborated these findings (Figure 4D). We found that ALP activity substantially increased in the CM group but was significantly reduced following SP600125 treatment (Figure 4E). Western blotting revealed that SP600125 treatment suppressed CM-induced increases in Runx2, BMP2, and Osterix protein levels (Figure 4F). TUNEL staining and caspase-3 activity assay also demonstrated that CM-induced cell apoptosis was suppressed by SP600125 treatment (Figures 4G, H).

Given the preliminary evidence of Res's inhibitory effects on VSMC calcification and the JNK/Bax pathway, we hypothesize that Res regulates VSMC calcification by blocking the activation of the JNK/Bax pathway. As shown in Figure 5A, treatment with the JNK activator (anisomycin, 100 ng/mL) significantly reversed Res's inhibitory effects on the expression of p-JNK and Bax proteins (Figure 5A). In CM-induced VSMCs, the downregulation of calcium deposition, calcium content, and ATP by Res was abolished upon co-treatment with anisomycin (Figures 5B–E). Similarly, the inhibitory effect of Res on the Runx2, BMP2, and Osterix levels was antagonized when Res was co-administered with anisomycin (Figure 5F). Additionally, Res treatment reduced TUNEL-positive cells and caspase-3 activity, and the combined treatment with anisomycin counteracted its effects on these parameters (Figures 5G, H).

We further sought to determine whether Res could inhibit vascular calcification in an animal model of vascular calcification. Vascular calcification was induced in mice via subcutaneous injection of vitamin D₃. Under the stimulation of vitamin D₃, alizarin red staining revealed orange–red deposits, while von Kossa staining showed black–brown precipitates, indicating calcium deposition in the mouse aorta. Res intervention significantly attenuated vitamin D₃-induced aortic calcification, as evidenced by the diminished orange–red alizarin red staining and reduced dark–brown von Kossa staining (Figures 6A, B). We lysed the mouse aortae for calcium quantification and confirmed that Res significantly inhibited the vitamin D₃-induced increase in calcium content (Figure 6C). At the same time, we examined the activity of ALP and the expression of Runx2 as markers of osteogenic differentiation. The results revealed that Res treatment resulted in a decrease in the activity of ALP and Runx2 protein levels in mouse aorta (Figures 6D, E). Importantly, Western blot analysis demonstrated that Res treatment effectively suppressed the expressions of p-JNK and Bax in the aorta of vitamin D3-induced mice (Figure 6F).