The Compatibility of Alisma and Atractylodes Affects the Biological Behaviours of VSMCs by Inhibiting the miR-128-5p/p21 Gene

Objective The compatibility of Alisma and Atractylodes (AA) has been estimated to exhibit antiatherosclerotic effects, but the mechanism remains unclear. This study aimed to identify the role of AA in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cell (VSMC) behaviours and to explore the effects of microRNAs (miRNAs). Methods A scratch wound-healing assay was used to detect the migration of VSMCs, and immunocytochemistry and western blotting for SM22ɑ were used to evaluate phenotypic transformation. Bromodeoxyuridine (BrdU) immunocytochemistry and flow cytometry were applied to detect the proliferation of VSMCs. miRNA microarray profiling was performed using Lianchuan biological small RNA sequencing analysis. VSMCs were transfected with the miR-128-5p mimic and inhibitor, and the migration, phenotypic modulation, and proliferation of VSMCs were investigated. The 3′UTR-binding sequence site of miR-128-5p on the p21 gene was predicted and assessed by luciferase assays. Result AA and the extracellular regulated protein kinase 1/2 (ERK1/2) blocker U0126 markedly inhibited migration, elevated smooth muscle 22α (SM22α) expression, repressed VSMC proliferation, elevated miR-466f-3p and miR-425-3p expression, and suppressed miR-27a-5p and miR-128-5p expression in ox-LDL-induced VSMCs. miR-128-5p targets the tissue inhibitor of metalloproteinases (TIMPs), silent information regulator 2 (SIRT2), peroxisome proliferator-activated receptor (PPAR), and p21 genes, which are linked to the behaviours of VSMCs. The miR-128-5p mimic promoted the migration and proliferation of VSMCs and suppressed p21, p27, and SM22ɑ expression. The inhibitor increased p21, p27, and SM22ɑ expression and repressed the migration, phenotypic transformation, and proliferation of VSMCs. miR-128-5p directly targeted the 3′UTR-binding sequences of the p21 gene, negatively regulated p21 expression, and supported the proliferation of VSMCs. Conclusion Our research showed that the migration, phenotypic transformation, and proliferation of ox-LDL-induced VSMCs were repressed by AA through inhibiting miR-128-5p by targeting the p21 gene, which may provide an effective option for the treatment of atherosclerosis.


Introduction
Atherosclerosis (AS), the main pathological process of arterial lesions, is the cause of the majority of cardiovascular and cerebrovascular diseases. It has been shown that inflammation caused by oxidized low-density lipoprotein (ox-LDL) contributes to the occurrence and development of AS [1], which can promote vascular smooth muscle cell (VSMC) migration, proliferation, and transformation from a contractile to a synthetic phenotype [2][3][4][5][6]. A large number of cytokines, extracellular matrix (ECM), and matrix metalloproteinases (MMPs) are synthesized and secreted by synthetic VSMCs during the progression of AS [7,8]. e migration, phenotypic transformation, and proliferation of VSMCs lead to vascular wall remodeling, which is mediated by activation of extracellular regulated protein kinases 1/2 (ERK1/2) signalling [9].
MicroRNAs (miRNAs) are small noncoding sequences involved in the negative regulation of gene expression. A series of cellular pathophysiological mechanisms involved in AS (e.g., differentiation, proliferation, and signalling pathways) are under the control of miRNAs. miRNAs are recognized as important regulators of lipid metabolism, inflammatory mediators, and VSMC behaviours in the progression of AS [10]. Several studies have shown that miRNAs play multiple roles in the phenotypic transformation, migration, and proliferation of VSMCs by inhibiting ERK1/2 activation [11,12], partly by regulating the tissue inhibitor of metalloproteinases (TIMPs)-MMPs and p21cyclins interactions [13][14][15][16][17][18].
Alisma and Atractylodes (AA) is a classic traditional Chinese medicinal formula that first appeared in the "Synopsis of Prescriptions of the Golden Chamber." AA exhibits multiple pharmacological actions [19][20][21][22]. It has been reported that Alisma orientale has a protective effect on acute lung injury through an anti-inflammatory effect by repressing the NF-kappa B pathway [20]. e extract of Alisma orientale exerted a protective effect on nonalcoholic fatty liver disease and palmitate-induced cellular injury [21,22], suggesting that it could be a potential treatment for abnormal lipid metabolism syndrome. Several studies have shown that AA represses lipid deposition in macrophagederived foam cells [23]. Alisol A 24-acetate, an active extract derived from Alisma orientale, inhibited migration and repressed the transformation from a contractile to a synthetic phenotype in ox-LDL-treated VSMCs by inhibiting ERK1/2 signalling [9]. However, little is known about the mechanisms of AA on VSMC migration, phenotypic transformation, and proliferation. is study aimed to investigate the impact of AA on ox-LDL-induced VSMCs and explore the underlying mechanisms of miRNAs.

Ethical Statement and Animals. All Sprague Dawley (SD) rats, provided by Fujian University of Traditional Chinese
Medicine (Fujian, China), were treated following the Suggested Guidelines for the Care and Use of Laboratory Animals 2006 administered by the Ministry of Science and Technology, China. is study was approved by the Animal Care and Use Committee of the Fujian University of Traditional Chinese Medicine (permission number: 2015-016). All rats were housed in facilities and provided food and water ad libitum.

Preparation of AA-Containing
Serum. AA-containing serum was prepared according to a previous study [23]. In brief, the rats were assigned to 2 groups and then given AA and 0.9% NaCl by oral gavage at 4 mL twice daily for 7 d.
en, blood samples were collected from the rat abdominal aorta following induction of anaesthesia with diazepam/ ketamine (1 : 1) (1 mL/1000 g), and serum was separated by centrifugation at 3000 r·min −1 at 4°C for 10 min. After the bacteria were filtered with a microporous membrane, the serum was inactivated in a 56°C water bath for 30 min and then stored at −20°C for the study.

VSMC Migration
Assay. VSMC migration was assayed by a scratch wound healing assay. Well-functioning VSMCs (1.0 × 105 cells/well) at 60-70% confluence were incubated in a 6-well dish and starved with DMEM/F12 containing 0.5% FBS for 12 h. A linear scratch wound was made at the centre of the cell monolayer with a 200 µL pipette tip and washed twice with PBS. VSMCs in the assigned groups were treated as described above in DMEM/F12 with 20% FBS. Cells were cultured for 24 h at 37°C with 5% CO 2 , and then the images of scratches were observed using a Leica DM IL LED inverted microscope (Wetzlar, Germany) and LAS Interactive Measurement imaging analysis software (Leica Microsystems, Mannheim, Germany).

Detection of Phenotypic Transformation in VSMCs.
Immunofluorescence assays were used to detect the expression of the VSMC phenotypic marker smooth muscle 22α (SM22α). Logarithmic-phase VSMCs (1 × 105 cells/ well) were incubated onto a cover glass in 6-well plates. VSMCs in the assigned groups were treated for 24 h as described above. VSMCs were fixed in 4% paraformaldehyde for 15 min, rinsed with PBS, passed through 0.3% Triton X-100 for 15 min, blocked with goat serum for 30 min, cocultured with anti-rabbit SM22α monoclonal antibody (1 : 200; Proteintech, USA), rinsed with PBS, incubated with secondary antibody (1 : 200; Proteintech, USA) and DAPI dye solution (100 ng·mL-1, BOSTER Biological Technology, China), drained from the dye solution, and observed under a fluorescence microscope. Five fields of vision were randomly photographed from each well plate and comprehensively analysed using imaging processing software (Image-Pro Plus v6.0, Media Cybernetics, Bethesda, MD, USA).

Secretion of MMP-2 and MMP-9 Detected by ELISA.
e secretion of MMP-2 and MMP-9 in VSMCs was detected by ELISA. e cells were treated for 24 h as described above, and the culture supernatants were harvested. According to the manufacturer's instructions, the expression of MMP-9 and MMP-2 was measured in the cell culture supernatant using a Sandwich ELISA kit (BOSTER Biological Technology, China).

VSMC Proliferation Assay.
BrdU immunocytochemistry and flow cytometry were used to measure the proliferation of VSMCs. VSMCs (1 × 105 cells/well) were seeded onto 6-well plates and incubated in DMEM/F12 supplemented with 20% FBS for 24 h. Quiescent VSMCs at 60-70% confluence in DMEM/F12 with 0.5% FBS were grouped as described above and cultivated with BrdU (30 μmol/L) for 24 h following the instructions of the FITC-BrdU cell proliferation assay kit (BOSTER Biological Technology, China) and detected by flow cytometry at a 488 nm excitation wavelength and 520 nm emission wavelength. BrdU expression was assessed by immunocytochemical staining following the manufacturer's instructions. Five fields of vision were randomly photographed from each well plate and comprehensively analysed using image-processing software (Image-Pro Plus v 6.0, Media Cybernetics, Bethesda, MD, USA). e positive cells were detected by BrdU manifesting pale-yellow or deep-tan staining, while negative cells exhibited nonspecific background staining.

miRNA Microarray Assay and miRNA Target Gene
Interactions. Total RNA was extracted from VSMCs. miRNA microarray profiling was performed using Lianchuan Biological Small RNA Sequencing Analysis (Lianchuan Bio, China) according to the manufacturer's recommended protocol. Small RNA sequencing library preparation was performed using the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, USA). After the library preparation work was completed, the constructed library was sequenced using Illumina HiSeq 2000/2500, and the sequencing read length was 1 × 50 bp. e potential target genes of miRNAs were searched with the TargetScan 7.2 database and miRDB. e target gene interactions of miRNAs were determined according to the database.

Statistical Analysis.
e results are presented as the mean ± SD. Statistical analysis was performed using the statistical software SPSS version 21.0 and assessed by oneway analysis of variance (ANOVA) or two-tailed Student's t-test to compare two treatments. A p value < 0.05 was considered statistically significant.

AA and U0126 Suppress the Migration of ox-LDL-Treated
VSMCs. A wound-healing assay was used to evaluate the role of AA on VSMC migration. e control group had a small amount of VSMC migration, and ox-LDL (50 mg·L-1) treatment significantly promoted cell migration ( * P < 0.05, Figure 1). However, both AA and U0126 (10 μmol·L-1) Evidence-Based Complementary and Alternative Medicine treatment remarkably suppressed the migration ability of ox-LDL-treated VSMCs ( # P < 0.05, Figure 1).

AA and U0126 Elevate SM22α Expression in ox-LDL-Treated
VSMCs. SM22α is a marker protein for the contractile phenotype. Immunofluorescence assays revealed that ox-LDL treatment reduced the mean optical density of SM22α expression ( * * P < 0.01, Figure 2). Treatment with AA and U0126 caused elevation of SM22α expression in ox-LDL-induced VSMCs ( # P < 0.05, ## P < 0.01, Figure 2), indicating that AA and U0126 treatment could inhibit the conversion of VSMCs from a contractile to a synthetic phenotype induced by ox-LDL.
e same results were confirmed by WB: the mimic inhibited SM22ɑ expression ( * P < 0.055, Figure 8(a)), and the inhibitor increased SM22ɑ expression ( # P < 0.05, Figure 8(a)), suggesting that miR-128-5p overexpression could induce VSMC transformation from a contractile to a synthetic phenotype. e effect of miR-128-5p on the proliferation of VSMCs was detected by WB and immunocytochemistry staining of incorporated BrdU. e results showed that the miR-128-5p mimic suppressed p21 and p27 expression and that the inhibitor increased p21 and p27 expression compared to that in the NC ( * P < 0.05, # P < 0.05, respectively, Figure 8(a)).
is finding suggests that miR-128-5p can influence the cell cycle and affect VSMC proliferation. Immunocytochemistry staining of BrdU showed that the miR-128-5p mimic increased the number of proliferating VSMCs and the inhibitor repressed the proliferation of VSMCs compared to those in the NC groups ( * P < 0.05, # P < 0.05, respectively, Figure 8(b)). e overexpression of miR-128-5p induced the proliferation of VSMCs, while the inhibitor reversed the results. Furthermore, both AA and U0126 treatment reduced the proliferation of VSMCs induced by mimic transfection compared with that in the mimic group (ΔP < 0.05, Figure 8(b)), suggesting that inhibition of the miRNAs could be a promising therapy for VSMC proliferation and that AA suppressed VSMC proliferation by repressing miR-128-5p expression as well as ERK1/2 inhibitor.

Discussion
As an ancient classical traditional Chinese medicinal formula, the compatibility of Alisma orientalis and Atractylodes macrocephala was first described in the Eastern Han Dynasty and exhibited a wide range of bioactivities in diverse cells [9,[19][20][21][22][23]. Until now, its effect on the migration, phenotypic transformation, and proliferation of VSMCs induced by ox-LDL remained unclear.
Several studies have reported that phenotypic transformation of VSMCs is a crucial process and promotes VSMC proliferation and migration in the process of AS5. Our study verified that the migration, phenotypic transformation, and proliferation abilities of ox-LDL-treated VSMCs were higher than those of control VSMCs. Previous studies demonstrated that AA could inhibit the phosphorylation of ERK1/29, and AA suppressed the migration, transformation, and proliferation abilities of ox-LDL-treated VSMCs as well as ERK1/2 blockers in the present study, suggesting that AA could affect the biological behaviours of VSMCs, which is associated with the inhibition of the ERK1/ 2 signalling pathway. miRNAs are conserved, small and single-stranded noncoding RNAs that negatively regulate gene expression at the posttranscriptional level and therefore, repress protein expression. Accumulating evidence reveals that miRNAs    # P < 0.05, ## P < 0.01 versus control; * P < 0.05, * * P < 0.01 versus ox-LDL, @ P < 0.05, @@ P < 0.01 versus ox-LDL.   Figure 6: Transfection of VSMCs with miR-128-5p mimic and inhibitor. (a) e expression of miR-128-5p was detected by qRT-PCR. ese data are representative of 6 experiments. e results are described as the mean ± SD (n � 6). * P < 0.05 versus mimic NC and # P < 0.05 versus inhibitor NC. (b) e migration of VSMCs was detected after transfection. ese data are representative of 6 experiments. e results are described as the mean ± SD (n � 6). * P < 0.05 versus mimic NC and # P < 0.05 versus inhibitor NC. 8 Evidence-Based Complementary and Alternative Medicine induced VSMC migration, transformation, and proliferation by regulating the expression of these miRNAs, which could be associated with the ERK1/2 pathway. Cyclins are recognized as important mediators in the cell cycle [24]. As negative regulators of cyclins, p27 and p21 exert a central role in cell cycle arrest [25,26]. e activity of cyclin-dependent kinases (CDKs) is strongly linked to the expression of cyclins, which can initiate DNA synthesis, promote the cell cycle, and play an important role in cell proliferation [27][28][29]. P21 prevents DNA replication from inhibiting cell proliferation through the control of CDK2, CDK4, and CDK6. Cyclins and CDKs are the target genes of miR-466f-3p and miR-425-3p, and p21 is the target gene of miR-128-5p according to the TargetScan7.1 database. Our study showed that miR-128-5p directly targeted the 3′UTRs of the p21 gene, suppressed p21 expression and induced the proliferation of VSMCs. Inhibition of miR-128-5p increased the levels of p21 and p27 expression and then inhibited proliferation. AA and U0126 treatment suppressed the increasing number of VSMCs induced by miR-128-5p overexpression. Treatment with AA and ERK1/2 inhibitor effectively suppressed the proliferation of VSMCs by inhibiting miR-128-5p in ox-LDL-induced VSMCs, indicating that AA could inhibit VSMC proliferation by suppressing miR-128-5p expression, which is closely linked to the ERK1/2 signalling pathway.

Evidence-Based Complementary and Alternative Medicine
TIMPs play a central role in suppressing the activation of MMPs. TIMPs and MMPs have been identified as key molecules in vascular remodeling and are linked to VSMC proliferation and migration [30][31][32][33]. SM22α is a contractile marker of VSMCs that affects the proliferation of VSMCs and the development of AS5. We detected that treatment with AA and U0126 effectively increased the expression of SM22α and suppressed the levels of MMP-2 and MMP-9 in VSMCs exposed to ox-LDL. It has been reported that several miRNAs contribute to an SMC-specific transcriptional program in regulating VSMC phenotypic transformation and proliferation [34]. In the present study, AA and U0126 obviously suppressed the expression of miR-27a-5p and miR-128-5p, and elevated miR-466f-3p and miR-425-3p expression in ox-LDL-treated VSMCs. MMP-11, MMP-13, and MMP-19 are negatively regulated by miR-466f-3p and miR-425-3p. TIMP-3, TIMP-4, PPARɑ, PPARδ, and SIRT2 are the target genes of miR-27a-5p and miR-128-5p according to the TargetScan 7.1 database. Inhibition of SIRT2 represses the proliferation and synthetic phenotypic transformation of VSMCs [35]. Activation of PPARɑ and PPARc can attenuate VSMC proliferation and migration [36][37][38] and exert a protective effect on AS [39]. Furthermore, miR-27a and miR-128 suppress the LDL receptor and dysregulate cholesterol homeostasis, which is involved in cholesterol efflux and represses the progression of AS [40,41]. erefore, we assumed that AA could suppress VSMC migration, phenotypic transformation, and proliferation by altering the expression of these miRNAs and exert multiple roles in the process of AS.
It has been reported that overexpression of miR-128 can significantly decrease VSMC migration, phenotypic transformation, and proliferation by targeting Kruppel-like factor 4 (KLF4) [42]. KLF4 acts as a key repressor of VSMC  inhibition of miR-128-5p elevated SM22α expression and suppressed the migration and proliferation of VSMCs, suggesting that inhibition of miR-128-5p may be a promising treatment for the progression of AS. AA and U0126 treatment reduced the increase in VSMC proliferation induced by miR-128-5p mimic transfection compared with that in the mimic group, indicating that AA and U0126 could inhibit VSMC proliferation by repressing miR-128-5p expression. e p21 protein is involved in cell proliferation, and the 3′UTR of the p21 gene contains the binding sequence sites of miR-128-5p. Luciferase assays showed that p21 was the direct target gene of miR-128-5p. e miR-128-5p mimic induced overexpression of miR-128-5p, blocked p21 expression, and increased VSMC proliferation. AA and ERK1/2 blockers suppressed VSMC proliferation by repressing miR-128-5p expression. is finding indicates that miR-128-5p can effectively inhibit the proliferation of VSMCs by targeting p21 expression and that AA alters the behaviours of VSMCs by arresting miR-128-5p expression by targeting the p21 gene.

Conclusion
In conclusion, the results of our study verify that treatment with AA and ERK1/2 blockers inhibits ox-LDL-stimulated VSMC migration, phenotypic transformation, and proliferation by regulating the expression of miR-466f-3p, miR-425-3p, miR-27a-5p, and miR-128-5p, especially by suppressing miR-128-5p and by targeting the p21 gene. is study provides new insights into how AA regulates the biological behaviours of VSMCs and exerts multiple roles in the process of AS.
Data Availability e data that support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure
is manuscript has been submitted as a preprint [44] according to the following link: https://www.researchsquare. com/article/rs-68415/v1 and DOI: 10.21203/rs.3.rs-68415/ v1. However, it has not been previously published and is not under consideration in the same or substantially similar form in any other peer-reviewed media.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  Figure 9: e p21 gene is the direct target of miR-128-5p. (a) e predicted miR-128-5p binding sequences in the p21 gene and the designed mut sequence (p21-mut). (b) e miR-128-5p mimic was cotransfected with the wild-type or mutated p21 3′UTR sequence vectors in HEK 293T cells. e relative firefly luciferase activity was calculated. ree independent experiments were repeated. * P < 0.05 versus the respective NC.
Evidence-Based Complementary and Alternative Medicine 11