As described previously, primary VSMCs were isolated from the thoracic aortas of standard deviation (SD) rats (170–200 g, male) [28]. Briefly, after anesthetization with the intraperitoneal administration of pentobarbital sodium (60 mg/kg), thoracic aortas were removed and washed three times with phosphate buffer saline. The media layer of the aorta was dissected, cut into pieces, and transplanted into a six-well culture plate. Cells were growing at 37°C in a humidified atmosphere containing 5% CO₂ in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin for 2 weeks. The experiments used VSMCs between passages 3 and 5. All animal procedures were conducted in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals and with the approval of the Second Affiliated Hospital of Xi'an Jiaotong University's Ethics Committee.

Next, using the CCK-8 Kit, cell viability was determined (Dojindo, Kumamoto, Japan). Briefly, cells were seeded in 96-well plates and 10 μl of CCK-8 (5 mg/ml) was added to the culture medium in each well. The absorbance was measured at 450 nm using an Exl 800 microplate reader (Bio-tek, Winooski, VT, USA). Cell viability (%) = (experimental group OD value − zero group OD value) / (control group OD value − zero group OD value) × 100%.

VSMC apoptosis was determined using the Tunel assay with a Tunel-specific detection kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100, and then fragmented DNA in VSMCs was end-labeled with FITC. In addition, the Tunel-positive cells were examined using a confocal microscope.

Mitochondrial membrane potential was determined using JC-1 staining (KeyGEN biotechnology, Jiangsu, China) according to the manufacturer's instructions, followed by flow cytometry evaluation (BD Bioscience, San Jose, CA, USA). Specifically, Q2 represents the mitochondrial cells that are healthy, whereas Q3 represents the mitochondrial membrane decline cells.

The ability of VSMCs to migrate was evaluated using the Transwell assay. VSMCs were seeded in the upper chamber of the transwell at a concentration of 1.0 × 10⁵ cells/well in 300 μl. The lower chamber was filled with 600 μl of 10% FBS-containing DMEM. The cells in the upper chamber migrated to the lower chamber after 24 h of incubation. The cells on the surface of the lower chambers were then fixed with 20% methanol for 10 min at room temperature and stained with 1% crystal violet (diluted in methanol) for 15 min at room temperature. Under a light microscope, the migrated cells were then quantified.

Total RNA was isolated using RNAiso Plus (Takara, Shiga, Japan) reagent as directed by the manufacturer. cDNA was synthesized from 500 ng of RNA per sample using the Prime Script Master Mix (Takara). Then, quantitative PCR was conducted using a SYBR Green PCR kit (Takara) in a CFX200 (Bio-Rad, Hongkong, China). Each gene's mRNA level was normalized to those of the housekeeping gene GAPDH. The sequences of the primers are listed in Supplementary Table 1.

In addition, miRNA mimics and inhibitors, in addition to their respective negative controls (NC), Empty vector (NC), and SMAD3 overexpression (SMAD3 OE) plasmids, were acquired from RIBOBIO Co. Ltd. (Guangzhou, China). NC mimics (100 nM; #miR1N0000001-1-10); miR-23b mimics (100 nM; 5-UGGGUUCCUGGCAUGCUGAUUU-3); NC inhibitors (200 nM; #miR2N0000001-1-10); miR-222 inhibitors (200 nM; 5´-AGGAUCUACACUGGCUACUGAG-3´), miR-23b inhibitors (200 nM; 5'-AAAUCAGCAUGCCAGGAACCCA-3´), miR-133a inhibitors (200 nM; 5´-CAGCUGGUUGAAGGGGACCAAA-3´), NC (2 μg), or SMAD3 OE (2 μg) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. After 48 h of transfection, cells were harvested and used in subsequent experiments.

Wt and Mt SMAD3 3' UTR sequences were cloned into the SpeI and HindIII sites of the pMir-Report Luciferase vector following PCR amplification using template and primers (Applied Biosystems, Foster City, CA, USA). Following the manufacturer's instructions, 5 ng of the resulting construct was transfected into 293T cells with 20 nM control mimics or 20 nM miR-23b mimics using Lipofectamine-2000 (Invitrogen). After 24 h of transfection, luciferase activity in the cells was determined using a Luciferase Assay System (Promega, Madison, WI, USA).

Subsequently, using the RIPA buffer, proteins were extracted from cells for immunoblotting. 15–50 μg of total protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocked with 5% skimmed milk, membranes were then probed with anti-Bax (1:2,000 dilution; #ab3203; Abcam, Cambridge, UK), anti-Bcl-2 (1:1,000; #ab32124; Abcam), anti-cleaved-caspase3 (1:500; #ab32042; Abcam), anti-cleaved-caspase9 (1:1,000; #ab2324; Abcam), anti-α-SMA (1:1,000; #ab5694; Abcam), anti-SM22 (1:1,000; #ab14106; Abcam), anti-SM-MHC (1:2,000; #ab133567; Abcam), anti-vimentin (1:2,000; #ab92547; Abcam), anti-collagen I (1:1,000; #ab270993; Abcam), anti-SMAD3 (1:2,000; #ab40854; Abcam), and anti-β-actin (1:5,000; #ab8226; Abcam) at room temperature for 1.5 h. Then, membranes were incubated with the appropriate secondary antibody conjugated to HRP. Then, the BM chemiluminescence blotting system (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize protein bands, and ImageJ Software (NIH, Bethesda, MD, USA) was used to quantify protein bands.

All values are presented using the mean ± SD format. The data were compared using the unpaired t-test, or one-way ANOVA followed by Tukey's test, as appropriate. The normal distribution of data was analyzed using the Kolmogorov–Smirnov normality test. Using Bonferroni's correction for multiple comparisons. When p < 0.05, differences were considered significant.

VSMCs were treated with lactate (0, 2, 4, or 8 mM) for 3 days. Lactate treatment caused the VSMCs to become less spindle-shaped and to develop the irregular morphology characteristic of synthetic VSMCs (Fig. 1A). The analysis of cell viability revealed that lactate treatment decreased the viability of VSMCs after 24 h of treatment and then significantly increased cell viability in a dose-dependent manner, despite the presence of 6 mM lactate (Fig. 1B). We hypothesized that the cytotoxicity induced by a high lactate concentration (6 mM) may have contributed to this persistent reduction in cell viability. Lactate (4 mM) treatment significantly decreased the apoptotic rate in VSMCs (Fig. 1C), whereas it significantly increased JC-1 signaling in VSMCs (Fig. 1D, E). Furthermore, Western blotting revealed that lactate treatment (4 mM) significantly decreased the levels of Bax, cleaved-caspase-3, and cleaved-caspase-9 in VSMCs, while increasing the level of Bcl-2 (Fig. 1F). These results suggest that lactate treatment may promote the proliferation of VSMCs. In addition, lactate treatment (4 mM) increased VSMC migration as measured by the transwell assay (Fig. 1G). Moreover, lactate treatment (4 mM) for 3 days decreased mRNA levels for markers of the contractile phenotype, including α-SMA, SM22, and SM-MHC, while increasing mRNA levels for markers of the synthetic phenotype, such as vimentin and collagen I (Fig. 1H). These findings suggest that lactate promotes the synthetic phenotype switch of VSMC.

In order to determine whether miRNA is involved in lactate-induced regulation of the VSMC phenotype switch, the reported miRNAs involved in the VSMC phenotype switch were screened. As shown in Fig. 2A, 15 miRNAs were detected, and miR-222, miR-23b, and miR-133a were the top three miRNAs that decreased in lactate-treated VSMCs relative to untreated VSMCs. VSMCs were then transfected with their respective miRNA inhibitors to inhibit miR-222, mi-23b, and miR-133a expression (Fig. 2B). Then, phenotypic markers, including α-SMA, SM22, SM-MHC, vimentin, and collagen I, were then detected in VSMCs transfected with miRNA inhibitors. The qRT-PCR revealed that miR-222, miR-23b, and miR-133a inhibitors inhibited the levels of contractile markers (α-SMA, SM22, and SM-MHC) but increased the levels of synthetic markers (vimentin and collagen I), with the miR-23b inhibitor having the most significant effect (Fig. 2C–E). Consequently, miR-23b was chosen for the subsequent experiments. Overexpression of miR-23b inhibitors significantly increased the viability and migration of VSMCs compared to the NC group, as determined by additional analyses (Fig. 2F, G). These findings suggested that lactate may promote the VSMCs’ switch to a synthetic phenotype via miR-23b downregulation.

A miR-23b mimic was used to determine whether miR-23b contributes to lactate's effects on VSMC phenotype switch. Figure 3A demonstrates that the miR-23b mimic increased miR-23b levels in VSMCs. Thus, miR-23b mimic diminished the effects of lactate (4 mM) on cell viability (Fig. 3B). In addition, miR-23b mimics inhibited lactate-induced apoptosis and JC-1 signaling (Fig. 3C, D). In addition, overexpression of the miR-23b mimic attenuated the lactate-induced migration enhancement (Fig. 3E). In addition, Western blot analysis demonstrated that the overexpression of the miR-23b mimics significantly reversed the effects of lactate on reducing the expression of α-SMA, SM22, and SM-MHC while increasing the expression of vimentin and collagen I (Fig. 3F). These findings suggest that inhibition of miR-23b contributes to the effects of lactate on VSMC synthetic phenotype switch promotion.

miR-23b target genes were predicted by miRDB, TargetScan, ENCORI, and GO 0007050 (cell cycle arrest). Four candidates were screened using a Venn analysis (Fig. 4A). Compared to the NC group, the miR-23b mimic reduced the mRNA levels of WEE1, HMGA2, and SMAD3, with VSMCs showing the greatest reduction (Fig. 4B). Fig. 4C indicates that SMAD3 is a potential target of miR-23b based on the correlation between the protein levels detected by the Western blot and the mRNA levels. Dual-luciferase reporter assay was used to confirm this result. Fig. 4D depicts the complementary sequences between the 3'UTR of SMAD3 and miR-23b. Either wild-type or mutant 3'UTRs containing putative miR-23b binding sites were cloned into a reporter plasmid, and their responsiveness to miR-23b in cells was evaluated. The results demonstrated that miR-23b inhibited luciferase activity in SMAD3 wild-type 3'UTR constructs but had no effect when the miR-23b binding sites were mutated (Fig. 4E, F). Western blot analysis revealed that lactate treatment significantly increased SMAD3 protein expression, whereas miR-23b mimic clearly reversed this upregulation (Fig. 4G). These results indicated that SMAD3 is a miR-23b target.

In order to determine whether it contributes to the effects of lactate on the VSMC phenotype switch, VSMCs were transfected with SMAD3, and miR-23b mimics individually. miR-23b was significantly upregulated in miR-23b mimic overexpressed VSMCs, whereas lactate treatment significantly reversed this upregulation (Fig. 5A). Moreover, SMAD3 expression was significantly increased after transfection with SMAD3 OE plasmid, miR-29b mimic significantly reversed this accumulation, and lactate significantly attenuated miR-29b's effects, thereby increasing SMAD3 expression (Fig. 5A). Lactate treatment reversed the effect of miR-23b to increase the cell viability of VSMCs. In contrast, SMAD overexpression significantly increased the cell viability of VSMCs (Fig. 5B). Meanwhile, SMAD3 decreases the apoptotic rate and promotes JC-1 signaling in VSMCs; however, miR-29b mimic significantly reverses these changes; and lactate treatment clearly reverses the effects of miR-23b to enhance SMAD3 effects on apoptosis and JC-1 signaling in VSMCs (Fig. 5C, D). In addition, miR-23b mimic inhibited the migration enhancement of VSMCs induced by SMAD3 expression, whereas lactate treatment abolished miR-23b's effects on SMAD3-mediated migration enhancement (Fig. 5E). Additionally, the expression of α-SMA, SM22, SM-MHC, vimentin, and collagen I detect the effects of lactate on phenotype switch. The findings revealed that miR-23b significantly inhibited the SMAD3-mediated decrease of α-SMA, SM22, and SM-MHC, as well as the increase of vimentin and collagen I in VSMCs. Conversely, lactate could reverse the effect of miR-23b and enhance the effect of SMAD3 in VSMCs (Fig. 5F). These findings supported the hypothesis that lactate promotes the switch of VSMC to a synthetic phenotype via regulation of the miR-23b/SMAD3 axis.