Abstract
Background. Atherosclerosis (AS) is a common vascular disease, and its main influencing factor is endothelial damage caused by oxidized low-density lipoprotein (ox-LDL). As one of the main active ingredients of ginseng, ginsenoside Rb3 has anti-inflammatory and anti-oxidative effects. However, the role of ginsenoside Rb3 in endothelial injury induced by ox-LDL is not clear.
Objectives. This study aimed to evaluate the effect and potential mechanism of ginsenoside Rb3 action on ox-LDL-treated human aortic endothelial cells (HAECs).
Materials and methods. The HAECs treated with ox-LDL were used to establish an in vitro AS model. The viability of the HAECs was analyzed with Cell Counting Kit-8 (CCK-8). Flow cytometry was performed to assess the apoptosis. Oxidative stress, inflammation and endothelial dysfunction were evaluated using enzyme-linked immunosorbent assay (ELISA) and western blotting. The levels of miR-513a-5p were assessed using quantitative real-time polymerase chain reaction (qPCR). A dual-luciferase assay was performed to analyze the relationship between miR-513a-5p and a zinc finger and BTB domain-containing protein (ZBTB20).
Results. Exposure of HAECs to ox-LDL (50 µg/mL) reduced cell viability, superoxide dismutase (SOD) activity and endothelial nitric oxide synthase (eNOS) expression, while increasing the levels of malondialdehyde (MDA), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and soluble intercellular adhesion molecule-1 (sICAM-1). The pretreatment with Rb3 markedly enhanced cell viability and decreased ox-LDL-induced oxidative stress, inflammation and endothelial dysfunction in HAECs. The ox-LDL decreased the level of miR-513a-5p, which was reversed by Rb3 pretreatment. The ZBTB20 was a target of miR-513a-5p in HAECs, and ox-LDL upregulated ZBTB20 expression, which was reversed by Rb3 pretreatment. The protective effect of Rb3 on ox-LDL-induced HAECs was diminished by miR-513a-5p inhibition, which was reversed by ZBTB20 knockdown.
Conclusions. Ginsenoside Rb3 reduces the effects of ox-LDL on HAECs by regulating the miR-513a-5p/ZBTB20 axis, which provides a theoretical basis for the treatment of AS.
Key words: atherosclerosis, oxidized low-density lipoprotein, miR-513a-5p, ZBTB20
Background
Atherosclerosis (AS) is a condition characterized by a lipid metabolic imbalance and persistent inflammation.1 It is a major cause of cardiovascular disease (CVD), which is the greatest cause of death worldwide.2 Atherosclerosis lays down the groundwork for future strokes and heart attacks.3 Aberrant human aortic endothelial cell (HAEC) injuries are regarded as one of the pathological characteristics in the progression of AS.4 According to research, oxidized low-density lipoprotein (ox-LDL)-induced proliferation, migration and apoptosis of HAECs are linked to the progression of AS.5, 6
Ginsenosides Rb1, Rd, F1, Rg1–Rg3, compound K, and total ginsenosides (TG) have all been shown to be anti-AS.7 For example, Rb2 via the GPR120/AMPK/HO-1 pathway suppressed human umbilical vein endothelial cell (HUVEC) apoptosis induced by lipopolysaccharide (LPS) and inhibited THP-1 cell adhesion to HUVEC, resulting in a reduction in inflammation and endoplasmic reticulum (ER) stress.8 Ginsenoside Rb3 (PubChem CID: 12912363) is one of ginseng’s most common active components, and has shown anti-inflammatory and anti-oxidative properties.9 The Rb3 strongly suppresses angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) proliferation by arresting the cell cycle,10 implying that it may play a positive function in CVD. However, the role of Rb3 in preventing AS is still unclear.
Non-coding RNA (ncRNA) has increasingly been recognized for its role in a range of illnesses, including CVD.11 It has been established that ncRNA plays important regulatory roles in vascular inflammation and smooth muscle cells, thus constituting a novel treatment for AS.12 MicroRNA (miRNA) is reported to inhibit gene expression resulting in mRNA degradation or translation inhibition.13 Existing research indicates that miRNA is directly associated with inflammation, oxidative stress and apoptosis, which suggests its involvement in the underlying mechanism of AS development.14 Interestingly, Rb1 and Rb2 have shown anti-aging and anti-inflammatory properties through the modulation of miRNA expression.15 Based on these findings, ox-LDL-induced HAECs were used to construct an in vitro AS model in order to investigate the regulation of miRNA by Rb3 in AS.
Objectives
This study aimed to evaluate the effect and potential mechanism of ginsenoside Rb3 action on ox-LDL-treated HAECs.
Materials and methods
Cell treatment
The HAECs (Cat. No. 6100 from 3 individuals; ScienCell Research Laboratories, Carlsbad, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, Waltham, USA) at 37°C in a 5% CO2 humidified incubator with penicillin and streptomycin (Sigma-Aldrich, St. Louis, USA), at doses of 100 U/mL and 100 mg/mL, respectively. The HAECs were then cultured in a medium containing various concentrations (0–100 μg/mL) of ox-LDL (Biosynthesis Biotechnology Company, Beijing, China) for 24 h, or 50 μg/mL of ox-LDL for 0 h, 12 h, 24 h, or 48 h to establish the in vitro AS model. For Rb3 treatment, HAECs were treated with various concentrations (0–80 µM) of Rb3 (Cat. No. A0236; ≥98%; Must Biotechnology, Chengdu, China) for 24 h. For the combined treatment, HAECs were treated with Rb3 (20 µM or 40 µM) for 24 h, and then Rb3-treated HAECs were treated with ox-LDL (50 μg/mL) for 24 h.
Cell viability
Following the appropriate treatment, Cell Counting Kit-8 (CCK-8) reagent (10 μL) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to the HAECs (5×104 cells/well) in a 96-well plate for 1 h, followed by the measurement of cell viability with the use of a LB960 microplate reader (450 nm; Titertek-Berthold, Pforzheim, Germany).
Cell apoptosis
Flow cytometry was used to determine the apoptosis rate after HAECs (4×105 cells/well in 6-well plates) were stained with fluorescein-5-isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI; Keygen, Nanjing, China) for 10 min in the dark at room temperature.
Enzyme-linked immunosorbent assay
To measure the content of malondialdehyde (MDA), superoxide dismutase (SOD), soluble intercellular adhesion molecule-1 (sICAM-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the culture media, enzyme-linked immunosorbent assay (ELISA) kits (Beyotime, Beijing, China) were used according to the manufacturer’s protocol, followed by measuring the absorbance at 450 nm with the use of a LB960 microplate reader (Titertek-Berthold).
Western blot analysis
After lysing HAECs, the protein content was determined using a bicinchoninic acid (BCA) assay, and then the cells were transferred to a polyvinylidene difluoride (PVDF) membrane and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prior to detection using Femto enhanced chemiluminescence substrates (Thermo Fisher Scientific, Waltham, USA) and a ChemiDoc MP Imaging System (Bio-Rad, Hercules, USA), the membranes were blocked with 5% non-fat milk at room temperature for 1 h, followed by the addition of anti-endothelial nitric oxide synthase (anti-eNOS) and anti-zinc finger and BTB domain-containing protein (anti-ZBTB20) antibodies, as well as an alkaline phosphatase-conjugated secondary antibody.
Microarray data and differential expression analysis
The microarray expression profiling analysis GSE137580 (platform GPL24741; https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE137580) was downloaded from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The GEO annotation file for GPL21265 was obtained. Using the online analysis program GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r), the expression profiles of ox-LDL-treated HAECs (n = 3) and untreated HAECs (n = 3) were compared to identify differentially expressed miRNAs using the following criteria: |log2FC (fold change)| > 1 and p < 0.05.
Quantitative real-time polymerase chain reaction
Using the TRIzol reagent purchased from Invitrogen (Waltham, USA), RNA was extracted from the HAECs, and a RT First Strand Kit was subsequently used to synthesize cDNA, which was utilized as a template for quantitative real-time polymerase chain reaction (qPCR) amplification to examine miRNA expression using miRNA-specific primers (Ribobio, Guangzhou, China). Moreover, after using PrimeScriptTM RT Master Mix for mRNA expression of ZBTB20, the qPCR was carried out with a SYBR Green qPCR mix (Yeasen Biotechnology Co., Ltd., Shanghai, China) on an ABI 7500 system (Applied Biosystems, Foster City, USA). The miRNA expression was standardized to U6 while standardizing mRNAs to GAPDH. The 2−ΔΔCt technique was used to examine the data.
Transfections
RiboBio developed and manufactured the miR-513a-5p mimics/inhibitors/negative control (NC), and Genepharma (Shanghai, China) provided small interfering RNAs (siRNAs) specific for ZBTB20 (si-ZBTB20, 5′- GAC TAG TTA AAT GGC GGA AGA TAA A-3′) and non-specific siRNA (si-NC) for the NC. Lipofectamine 2000 (Invitrogen) was used to transfect the HAECs.
Dual-luciferase assay
After using a QuikChange Kit (Qiagen, Hilden, Germany) to perform site-directed mutagenesis of the ZBTB20 3’-UTR within the probable miR-513-5p binding region, the ZBTB20-wt or ZBTB20-mut was amplified and cloned into a pMIR-REPORT luciferase vector (Ambion, Austin, USA). The HAECs were seeded in 24-well plates (2×105 cells per well) and co-transfected with ZBTB20-wt/ZBTB20-mut and miR-513-5p mimic/NC. The relative firefly luciferase activity was measured 48 h after transfection.
Statistical analyses
IBM SPSS v. 22.0 software (IBM Corp., Armonk, USA) was used to examine the data, which were expressed as mean and 95% confidence intervals (95% CIs). Each experiment was performed on 3 samples for each group, and each experiment was repeated in triplicate for every sample. To determine statistical significance, Student’s t-test was used to compare the 2 groups. For the comparison of 3 or more groups, a bootstrap analysis of variance (ANOVA) with a bootstrap post hoc test was utilized. The distribution of data was analyzed using the Shapiro–Wilk test. Additionally, ‘0’ in Figure 1 represents the drug concentration of 0, i.e., untreated conventional HAECs. The heatmap was generated using the R package ComplexHeatmap (R Foundation for Statistical Computing, Vienna, Austria). The Venn diagram was generated using the R package Venn. A threshold p-value lower than 0.05 indicated a significant difference.
Results
Effects of ox-LDL and Rb3 treatment on HAECs
The ox-LDL is a major risk factor for AS, causing endothelial cell death and promoting the onset and progression of the disease.16 The viability of HAECs was lowered in a dose- and time-dependent manner after exposure to ox-LDL (Figure 1A,B), and the subsequent experiment used 50 μg/mL of ox-LDL to stimulate HAECs for 24 h in order to establish the in vitro AS model. Moreover, we found little effect of Rb3 on HAEC viability at different concentrations (0–80 µM) for 24 h (Figure 1C,D). However, the effects of ox-LDL on HAEC activity and apoptosis were restored by Rb3 (20 µM or 40 µM, 2 h; Figure 1E,F).
Rb3 attenuated the ox-LDL-induced injury of HAECs
Subsequently, ox-LDL-induced oxidative stress and inflammation in HAECs were found, with the increased production of MDA, the enhanced level of pro-inflammatory cytokines and the decreased activity of SOD, while the activity of SOD was attenuated by Rb3 pretreatment (Figure 2A–D). Additionally, ox-LDL increased the secretion of sICAM-1 and downregulated the expression of eNOS (Figure 2E,F). The Rb3 pretreatment alleviated to a certain extent the endothelial dysfunction of HAECs caused by ox-LDL (Figure 2E,F).
Rb3 upregulates miR-513a-5p
in ox-LDL-treated HAECs
We used the online analysis program GEO2R to evaluate the differentially expressed miRNAs between ox-LDL-treated HAECs and untreated HAECs from the GEO database. In total, 7 downregulated miRNAs were discovered, including miR-7110-5p, miR-1202, miR-6749-5p, miR-154-3p, miR-513a-5p, miR-6068, and miR-4697-5p (Figure 3A). Moreover, ox-LDL-treated HAECs showed a significantly reduced expression of miR-154-3p, miR-513a-5p and miR-7110-5p in our experiments (Figure 3B). However, miR-513a-5p was markedly upregulated in ox-LDL-treated HAECs after Rb3 pretreatment (Figure 3C). Moreover, miR-513a-5p inhibitor (inhib-miRNA) transfection prevented Rb3-induced elevation of miR-513a-5p in ox-LDL-treated HAECs (Figure 3D–E).
ZBTB20 as a target gene was regulated by miR-513a-5p in HAECs
Bioinformatics analysis conducted using miRDB (https://mirdb.org/), TargetScan (https://www.targetscan.org/vert_72/) and miRTarBase (https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php) (Figure 4A), as well as the dual-luciferase reporter assay (Figure 4B) confirmed that miR-513a-5p binds to the 3’UTR of ZBTB20 mRNA, and its mimics successfully upregulate the levels of miR-513a-5p in HAECs (Figure 4C) while decreasing the expression of ZBTB20 mRNA and proteins. The transfection with an inhibitor had the opposite effects (Figure 4D,E). Moreover, Rb3 treatment reduced ZBTB20 expression in ox-LDL-treated HAECs (Figure 4F,G). Additionally, the upregulation of ZBTB20 expression in ox-LDL-treated HAECs enhanced by the miR-513a-5p inhibitor was abolished after Rb3 treatment (Figure 4F,G). Furthermore, ox-LDL upregulated the expression of ZBTB20 in HAECs, which was reversed by Rb3 treatment (Figure 4H,I).
The effect of Rb3 on endothelial dysfunction of HAECs induced by ox-LDL is mediated by miR-513a-5p/ZBTB20 axis
As shown in Figure 5A, ZBTB20 siRNA transfection markedly downregulated the expression of ZBTB20 in HAECs. The inhibition of miR-513a-5p eliminated the anti-oxidative, anti-inflammatory and anti-endothelial dysfunction effects of Rb3 pretreatment in ox-LDL-treated HAECs, while the effects of miR-513a-5p overexpression were reversed by ZBTB20 siRNA (Figure 5B–G). Collectively, Rb3 reduced oxidative stress, inflammation and endothelial damage caused by ox-LDL through the miR-513a-5p/ZBTB20 axis.
Discussion
Endothelial dysfunction and apoptosis are thought to be the initial elements in the development of AS, contributing to the pathophysiology of the disease.3 Inhibiting ox-LDL-induced endothelial dysfunction and endothelial cell death is thought to be a possible therapeutic treatment for AS.17 The management of the inflammatory response is critical for the treatment of AS, and inhibiting pro-inflammatory cytokines is a promising technique for preventing AS.18 Except for the inflammatory response, which has an impact on lipid metabolism in AS and vice versa,19 endothelial dysfunction also appears to be a frequent hallmark of AS, and the endothelial dysfunction could be induced by ox-LDL via the acceleration of oxidative stress.20 A previous study reported that tofacitinib has shown vascular protective properties in HAECs by inhibiting ox-LDL-induced inflammation and oxidative stress.21 In addition, saxagliptin-mediated endothelial protection is associated with the inhibition of the inflammatory response induced by ox-LDL.22 In the current investigation, we discovered that Rb3 pretreatment reduced MDA formation and SOD caused by ox-LDL, as well as inhibited the release of IL-6 and TNF-α, indicating that Rb3 had anti-inflammatory and anti-oxidative effects in cellular models of AS. Furthermore, we discovered that Rb3 boosted the viability and eNOS expression of ox-LDL-treated HAECs while inhibiting the generation of sICAM-1, indicating that Rb3 prevented endothelial dysfunction in cellular models of AS. Our results revealed an anti-AS effect of Rb3 in vitro.
The significance of miRNA in vascular disorders has recently gained a lot of interest.23 The miRNA is associated with various pathophysiological processes, including growth, proliferation, apoptosis, and autophagy. Recent research has found that aberrant miRNA expression occurs at various phases of AS.24 Increased plasma levels of miR-216a were found in elderly CVD patients, and were responsible for triggering endothelial cell inflammation via the Smad3 pathway.25 In addition, miR-513a-5p was downregulated in human aortic VSMCs stimulated by IκBα,26 as well as HAECs treated with ox-LDL, while the expression of miR-513a-5p could be increased with Rb3 treatment. Furthermore, it has been revealed that miR-513a-5p plays a role in TNF-α/endotoxin-induced apoptosis by suppressing the production of endothelial cell apoptosis protein X-linked inhibitors,27 thus being a vital miRNA in regulating cell damage caused by an inflammatory response. In this study, we not only found that Rb3 treatment increased the expression of miR-513a-5p, which was inhibited by ox-LDL stimulation, but also determined that the beneficial effects of Rb3 were eliminated by miR-513a-5p inhibitors. This indicates that miR-513a-5p had a novel role in regulating Rb3-mediated anti-oxidative, anti-inflammatory and anti-endothelial damage in AS.
The ZBTB20, as a member of the large complex tramtrack bric-a-brac (BTB)/poxvirus and zinc finger (POZ) family,28 has the ability to suppress IκBα gene transcription while also promoting nuclear factor-κB (NF-κB) activation.29 In addition, ZBTB20 levels are elevated in human AS lesions and ox-LDL-stimulated macrophages. The ZBTB20 knockdown has been shown to decrease the oxidative stress level and inflammatory response of ox-LDL-stimulated macrophages.30 Therefore, we hypothesize that miR-513a-5p could play a role by inhibiting ZBTB20. Our results showed that miR-513a-5p targeted ZBTB20 in ox-LDL-treated HAECs. Additionally, the enhanced ZBTB20 expression caused by ox-LDL was blocked by Rb3 treatment. Moreover, the inhibition of miR-513a-5p eliminated the anti-oxidative, anti-inflammatory and anti-endothelial dysfunction effects of Rb3 in ox-LDL-treated HAECs, while ZBTB20 knockdown could have enhanced the anti-oxidative, anti-inflammatory and anti-endothelial dysfunction effects. Therefore, Rb3 might exert its anti-AS effect through the miR-513a-5p/ZBTB20 axis.
Limitations
The intracellular signal transduction pathway is very complicated. In the present study, miR-513a-5p was selected as a candidate miRNA based only on the relevant literature. In future studies, we should select more miRNAs or miRNA expression profiles to explore the protective role of Rb3 in AS. Furthermore, the role of Rb3 in animal models and clinical research needs to be further investigated.
Conclusions
In conclusion, we proved that Rb3 reduced oxidative stress, inflammation and endothelial damage caused by ox-LDL through the miR-513a-5p/ZBTB20 axis. These results supported the beneficial role of Rb3 as an anti-AS agent.
