Dihydrohomoplantagin and Homoplantaginin, Major Flavonoid Glycosides from Salvia plebeia R. Br. Inhibit oxLDL-Induced Endothelial Cell Injury and Restrict Atherosclerosis via Activating Nrf2 Anti-Oxidation Signal Pathway

Abstract

Oxidized low-density lipoprotein (oxLDL)-induced endothelium injury promotes the development of atherosclerosis. It has been reported that homoplantaginin, a flavonoid glycoside from the traditional Chinese medicine Salvia plebeia R. Br., protected vascular endothelial cells by inhibiting inflammation. However, it is undetermined whether homoplantaginin affects atherosclerosis. In this study, we evaluated the effect of homoplantaginin and its derivative dihydrohomoplantagin on oxLDL-induced endothelial cell injury and atherosclerosis in apoE-/- mice. Our results showedthat both dihydrohomoplantagin and homoplantaginin inhibited apoptosis and the increased level of ICAM-1 and VCAM-1 in oxLDL-stimulated HUVECs and the plaque endothelium of apoE-/- mice. Additionally, both of them restricted atherosclerosis development of apoE-/- mice. Mechanistic studies showed that oxLDL-induced the increase in ROS production, phosphorylation of ERK and nuclear translocation of NF-κB in HUVECs was significantly inhibited by the compounds. Meanwhile, these two compounds promoted Nrf2 nuclear translocation and increased the anti-oxidation downstream HO-1 protein level in HUVECs and plaque endothelium. Notably, knockdown of Nrf2 by siRNA abolished the cell protective effects of compounds and antagonized the inhibition effects of them on ROS production and NF-κB activation in oxLDL-stimulated HUVECs. Collectively, dihydrohomoplantagin and homoplantaginin protected VECs by activating Nrf2 and thus inhibited atherosclerosis in apoE-/- mice.

1. Introduction

Atherosclerosis, a chronic inflammatory vascular disease, is a major cause of morbidity and mortality in the modern society. Currently, the prevention and treatment of atherosclerosis are all important areas of medical research. Although the pathological features of atherosclerosis are complex, injury of the endothelial lining of lesion-prone areas from the arterial vasculature is one of the main pathological processes of atherosclerosis [1]. Accumulating evidence has also demonstrated that oxidized low-density lipoprotein (oxLDL), an important dangerous factor in the initiation and progression of atherosclerosis, induces endothelial cell injury. In the initial stage of atherosclerosis, oxLDL activates endothelial cells by inducing the expression of several cell surface adhesion molecules including ICAM-1 and VCAM-1 which recruit circulating monocytes from the blood into intima, where monocytes differentiate into macrophages and become foam cells by internalizing modified lipoproteins [2,3]. Additionally, oxLDL could induce endothelial cell apoptosis, which results in increased vascular permeability to cells, promoting lipids deposition and the formation of necrotic core, thus contributing to the instability and rupture of atherosclerotic lesions. Therefore, attenuating oxLDL-induced endothelial cell injury may be a promising strategy to prevent the development of atherosclerosis [4].

In recent years, the importance of traditional Chinese herbal medicine in the treatment of cardiovascular diseases has attracted extensive attention from scientists [5,6]. Homoplantaginin was identified to be the main flavonoid component of traditional Chinese herbal medicine Salvia plebeia R. BR. (Labiatae), used for the treatment of a variety of diseases including hepatitis, cough, diarrhea, gonorrhea, menorrhagia, tumors, and hemorrhoids [7,8]. More and more research has revealed the biological activity of homoplantaginin by using of various cell and mice models [9,10,11,12]. It is reported that homoplantaginin exhibited protective effects on H₂O₂-injured hepatocyte cells and lipopolysaccharide-induced inflammatory liver injury [9]. In addition, homoplantaginin antagonizes oxLDL-induced foam cell formation in murine macrophages [10]. Attractively, homoplantaginin could ameliorate free fatty acid-induced endothelial insulin resistance and endothelial inflammation, suggesting homoplantaginin may be a potential candidate compound for the prevention and treatment of vascular diseases [11,12]. However, whether homoplantaginin could abolish oxLDL-induced endothelial cell injury and inhibit atherosclerosis needs further research. Therefore, in this study, we investigated the protection role and molecular mechanisms of homoplantaginin and its derivative dihydrohomoplantagin in the process of atherosclerosis, using a model of endothelial cell injury induced by oxLDL in vitro and apoE-/- atherosclerotic mice model in vivo.

2. Results

2.1. Dihydrohomoplantagin and Homoplantaginin Inhibited oxLDL-Induced Apoptosis and the Increase in ICAM-1 and VCAM-1 Protein Levels in HUVECs

The chemical structures of dihydrohomoplantagin and homoplantaginin (named compound S1 and compound 3, respectively) are shown in Figure 1. To elucidate the effect of S1 and S3 on vascular endothelial cell (VEC) injury under proatherogenic conditions, we first performed in vitro experiments in HUVECs with oxLDL treatment. In oxLDL-treated groups, about 80% HUVECs displayed typical characteristics of apoptosis including nuclear condensation and fragmentation using Hoechst 33258 staining, while after S1 or S3 treatment, the number of apoptotic cells significantly decreased (Figure 1). Results from TUNEL assay demonstrated that S1 and S3 significantly reduced the number of TUNEL-positive cells, respectively (Figure 2A,C). Moreover, the decreased protein level of Bcl2/Bax, a hallmark of apoptosis, was observed in oxLDL-treated HUVECs, the effects of which were reversed by S1 or S3 (Figure 2D,E).

OxLDL exposure enhanced intercellular adhesion molecule-1 (ICAM-1) and VCAM-1 expression on the endothelium, which is responsible for the oxLDL-induced endothelial injury. Therefore, we further explored the effect of dihydrohomoplantagin and homoplantaginin on the expression of ICAM-1 and VCAM-1. The results of Western blot revealed that S1 or S3 decreased oxLDL-induced up-regulation of ICAM-1 and VCAM-1 protein level in HUVECs, respectively (Figure 2D,E).

2.2. Dihydrohomoplantagin and Homoplantaginin Inhibited Plaque Endothelium Apoptosis and the Adhesion Factor Protein Level in apoE-/- Mice

To further investigate the effect of dihydrohomoplantagin and homoplantaginin on VEC injury under proatherogenic conditions in vivo, 6 weeks of male apoE-/- mice were fed with high-fat diet for 12 weeks, and were injected intraperitoneally with compound S1 or S3 daily for 8 weeks. The body weight of mice was measured every week during compounds injection and there was no significant difference between control and compounds-treated groups (Supplemental Figure S1). Moreover, compounds S1 and S3 also had no effect on organ weights (Supplemental Table S1), suggesting they had no obvious toxicity to mice. Then, we assessed cell apoptosis by TUNEL staining (Figure 3A) and the protein level of ICAM-1 and VCAM-1 by immunofluorescence analysis of plaque endothelium from apoE-/- mice. It is observed that, in comparison with the control group, the endothelium of apoE-/- mice with S1 or S3 treatment showed less TUNEL positive area and decreased ICAM-1 and VCAM-1 protein level, suggesting S1 and S3 inhibited endothelium injury during atherosclerosis development (Figure 3B–E).

2.3. Dihydrohomoplantagin and Homoplantaginin Restricted Atherosclerosis Development in apoE-/- Mice

Because it is believed that the injury of endothelial cell mediate the atherosclerosis progression, we further determined the anti-atherosclerotic activity of dihydrohomoplantagin or homoplantaginin in apoE-/- mice. Oil-red O staining of whole aortas and H&E staining of aortic roots demonstrated less atherosclerotic plaque areas and necrotic areas with S1 or S3 than control groups (Figure 4A,C). Moreover, these two compounds promoted the stability of plaque, displaying with the decreased lipid deposition, increased collagen content, reduced macrophage cell area and increased the number of SMCs (Figure 4B). The results of vulnerability index also demonstrated dihydrohomoplantagin or homoplantaginin enhanced atherosclerotic plaque stability (supplemental Figure S2)

2.4. Dihydrohomoplantagin and Homoplantaginin Inhibited ROS/ERK/NF-ĸB Signaling Pathway

Studies have provided compelling evidence that oxLDL stimulate excessive or sustained ROS production, which leads to vascular oxidative stress injury and thus promotes atherosclerosis [13]. Therefore, we determine whether the anti-atherosclerotic activity of dihydrohomoplantagin and homoplantaginin could be attributed to the anti-oxidative actions of these two compounds. The results revealed that incubation of HUVECs with oxLDL increased the cellular ROS level and this was strongly suppressed by S1 or S3 (Figure 5A,B). Moreover, these two compounds significantly inhibited the ROS level of atherosclerotic lesion in apoE-/- mice, respectively (Figure 5C,D).

Research from another group has shown that the increased ROS level could activate ERK/NF-κB signaling pathway, and finally promote the up-regulation of adhesion molecule expression in vascular endothelial cells [14]. Here, we further investigated the effect of dihydrohomoplantagin and homoplantaginin on ERK/NF-κB activation. Consistent with other studies, the phosphorylation of ERK increased and NF-κB translocated into the nuclei in oxLDL-treated HUVECs, and these were dramatically inhibited by S1 or S3 (Figure 6).

2.5. Dihydrohomoplantagin and Homoplantaginin Activated Nrf2 /HO-1 Anti-Oxidation Signal Pathway

Nuclear factor erythroid-related factor 2 (Nrf2) is well demonstrated to play a central role in the protection of cells against oxidative damage. When Nrf2 is activated, it quickly translocates into the nucleus and increases the expression of antioxidant genes, such as heme oxygenase 1 (HO-1). To clarify the mechanisms of dihydrohomoplantagin and homoplantaginin anti-oxidation, we examined whether dihydrohomoplantagin and homoplantaginin affect Nrf2 /HO-1 anti-oxidation signal pathway. Immunofluorescence staining results showed that both compounds increased nuclear Nrf2 protein level compared with control groups (Figure 7A). Moreover, the protein levels of HO-1 were enhanced by compounds in oxLDL-stimulated HUVECs and plaque endothelium of apoE-/- mice (Figure 7B–D). These results suggested dihydrohomoplantagin and homoplantaginin activated Nrf2 /HO-1 anti-oxidation signal pathway.

2.6. Dihydrohomoplantagin and Homoplantaginin Inhibition of oxLDL-Induced VEC Injury and ROS/NF-κB Activation Were Nrf2-Dependent

To further determine whether Nrf2 is directly involved in cell protection of dihydrohomoplantagin and homoplantaginin, cells were transfected with either scramble siRNA or siNrf2 RNA. Hoechst 33258 staining results revealed that compounds could not inhibit oxLDL-induced apoptosis in siNrf2 groups (Figure 8A). Western blot analysis demonstrated that the effects of compound S1 or S3 on the protein level of Bcl2/Bax, ICAM-1, VCAM-1 and HO-1 were abolished by knocking down of Nrf2 (Figure 8B). Moreover, S1 and S3 inhibition of oxLDL-induced ROS and NF-κB nuclear translocation were reversed by Nrf2 siRNA (Figure 9). Therefore, the above experiments suggested the Nrf2 was a key target of dihydrohomoplantagin and homoplantaginin.

3. Discussion

The present study demonstrated the novel role of homoplantaginin and dihydrohomoplantagin in protection against oxLDL-induced injury in endothelial cell and the development of atherosclerosis in apoE-/- mice. Homoplantaginin and dihydrohomoplantagin exhibited the protective effects through activating the Nrf2/HO-1 anti-oxidation signal pathway (Figure 10).

Endothelial cell injury has been confirmed to be the initial step in the atherosclerosis development [15]. It is believed that oxLDL induces endothelium injury and enhances atherosclerosis by increasing endothelial cell oxidative stress [16]. Thus, inhibiting oxLDL-induced endothelial cell injury has becoming a promising strategy to prevent the development of atherosclerosis [4]. Recently, homoplantaginin and dihydrohomoplantagin were isolated from the traditional Chinese herbal medicine Salvia plebeia R. BR. (Labiatae) by our research group. Homoplantaginin and dihydrohomoplantagin belong to flavone glucopyranosides compounds. It is known that flavone, such as Salvia plebeia extract, hesperetin, naringenin, eriodictyol, isosakuranetin, and their respective glycosides, support and enhance the body’s defenses against oxidative stress and help the organism in the prevention of cardiovascular diseases [10,17]. Moreover, eight flavone compounds including luteolin 4’ methyl ether 7-O-β-d-4C1-glucopyranoside 8-methoxyapigenin 7-O-β-d-4C1-galactopyranoside, isovitexin, 8-methoxyluteolin 7-O-β-d-4C1-glucopyranoside, diosmetin, cirsimaritin, luteolin, and apigenin have been found to exhibit anti-inflammatory, antidiabetic, antioxidant activities, and anti-atherosclerosis in a dose-dependent manner [18]. Previous studies also confirmed that natural flavonoid Vitexin, which was identified as apigenin-8-C-b-d-glucopyranoside, exhibited anti-oxidative and anti-inflammatory properties in PC12 cells [19]. Although homoplantaginin has displayed various biological properties including inhibition of free fatty acids-induced endothelial insulin resistance and endothelial inflammation [9,10,11,12], whether homoplantaginin alleviate vascular endothelial cells injury induced by oxLDL and inhibit atherosclerosis is unclear. Here, we found that homoplantaginin and dihydrohomoplantagin significantly inhibited apoptosis and the increased ICMA-1 and VCAM-1protein level in oxLDL-stimulated HUVECs and endothelium of apoE-/- mice. Importantly, dihydrohomoplantagin and homoplantaginin enhanced atherosclerotic plaque stability of apoE-/- mice.

It is reported that increased ROS production induced by oxLDL promotes NF-κB translocation into the nuclei and thus facilitates vascular endothelial cell injury [20]. Moreover, numerous studies have shown that ROS accumulation can activate ERK/NF-κB pathways [21,22]. Here, the results demonstrated that dihydrohomoplantagin and homo-plantaginin significantly inhibited the increased ROS level in oxLDL-stimulated HUVECs and atherosclerotic lesion of apoE-/- mice, respectively. Moreover, the increased p-ERK level and the NF-κB nuclear translocation induced by oxLDL was significantly inhibited by dihydrohomoplantagin and homoplantaginin, indicating dihydrohomoplantagin and homoplantaginin inhibited ROS/ERK/NF-ĸB signaling pathway.

Nrf2 has been confirmed to be involved in several detoxifying and antioxidant defense processes by inducing anti-oxidative gene expression. As the Nrf2 targeted gene, HO-1 expression was demonstrated to play an important role against oxidative stress [23]. It is reported that 7,8-Dihydroxyflavone activated Nrf2/HO-1 signaling pathways and protected against osteoarthritis [24]. Additionally, methylnis-solin-3-O-β-d-glucopyranoside protected VECs against oxidative damage through the Nrf2/HO-1 pathways [25]. Meanwhile, Apigenin-7-O-β-d-(-6″-p-coumaroyl)-glucopyranoside treatment elicited a neuro-protective effect through Nrf2 activation [26]. However, whether dihydrohomoplantagin and homoplantaginin exert antioxidant effect by activating Nrf2 remains unclear. Here, we found that dihydrohomoplantagin and homoplantaginin promoted Nrf2 nuclear translocation and increased the anti-oxidation downstream HO-1 protein level in HUVECs and plaque endothelium. Notably, the down-regulation of Nrf2 by small siRNA of Nrf2 abolished the cell protective effects of compounds and antagonized the inhibition effects of them on ROS production and NF-κB activation in oxLDL-stimulated HUVECs. These results suggested dihydrohomoplantagin and homoplantaginin protected VECs against oxLDL-induced oxidative injury by activating Nrf2.

In summary, our results revealed that dihydrohomoplantagin and homoplantaginin significantly inhibited oxLDL-induced VEC apoptosis and the expression of ICAM-1 and VCAM-1. The research elucidated that dihydrohomoplantagin and homoplantaginin inhibited oxLDL-induced ROS overproduction, the phosphorylation of ERK and NF-κB activation through activating Nrf2/HO-1 anti-oxidative signaling pathway. Collectively, dihydrohomoplantagin and homoplantaginin protected VECs by activating Nrf2 and thus restricted atherosclerosis in apoE-/- mice. Therefore, our study provides a lead compound for the treatment of atherosclerosis.

4. Materials and Methods

4.1. Preparation of Dihydrohomoplantagin and Homoplantaginin

The dry herbs of Salvia plebeia R. BR. were purchased from Qilu hospital (Jinan, China), then powdered, refluxed with 60% ethanol two times and 95% ethanol two times. The evaporated extract was suspended in water and successively partitioned with petroleum ether and n-butanol. The ethyl acetate fraction was subjected to polyamide column chromatography (60~80 mesh) eluted by with a gradient of CH₃OH/H₂O (1:5→100:0). Dihydrohomoplantagin was obtained from the 20% CH₃OH elution using silica gel column chromatography CH₂Cl₂/H₂O (9:1) and high-performance liquid chromatography (HPLC), homoplantaginin was obtained from the 30% CH₃OH elution using silica gel column chromatography, then eluted by CH₂Cl₂/H2O (10:1 and 100:13) and HPLC. Two chemical structures were identified through spectroscopic analysis as reported previously [7,27].

4.2. Cell Culture and Treatment

HUVECs were obtained from ScienCell (San Diego, CA, USA) and cultured on gelatincoated plastic dishes in M199 medium with 20% (v/v) bovine serum and 10 IU/mL fibroblast growth factor 2 (FGF2). The cells were maintained at 37 °C under humidified conditions and 5% CO2. HUVECs at no more than passage 25 were used in the experiments. When HUVECs were grown to 80% confluency, the cells were stimulated with oxLDL or oxLDL and compounds.

4.3. Antibodies

The primary antibodies used for Western blot and immunofluorescence staining including Bcl-2, Bax, HO-1, ICAM-1, VCAM-1, phospho-ERK, and ERK were purchased from Proteintech Group, Inc. (Chicago, IL, USA). The corresponding peroxidase conjugated secondary antibodies used for Western blot were acquired from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China).

4.4. Western Blot Analysis

Protein was isolated from HUVECs using radio immune precipitation assay (RIPA) lysis buffer (Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). The total protein concentration was determined using a quantitative BCA protein kit (Beyotime, Shenzhen, Guangdong, China). Proteins from different groups were mixed with 5× gel loading buffer and boiled for 5 min. Equal amounts of protein (20 μg) were separated by SDS-PAGE. Separated proteins were transferred to PVDF membranes and then the membranes were incubated in 5% non-fat milk for 1 h at 37 °C to block the non-specific binding. After that, incubation with specific primary antibodies (1:500) for targeted proteins was performed for 18 h at 4 °C. After three washes in Tris-buffered saline/Tween 20 for 10 min for each time, membranes were incubated with corresponding HRP-conjugated secondary antibody (1:2000) for 1.5 h at room temperature. The protein bands were detected by enhanced chemiluminescence (Millipore Corporation, Billerica, MA, USA). The β-actin was used as the internal control.

4.5. Hoechst 33258 Staining

Nuclear fragmentation of apoptotic cells was determined by Hoechst 33258 staining. After treatment, HUVECs were incubated with Hoechst 33258 solution (10 mg/mL, Dingguo Changsheng Biotechnology Co., Ltd., DH163) for 20 min at 37 °C, and then were detected using a fluorescence microscopy (LEICA DMi8, Wiesbaden, Germany).

4.6. Intracellular ROS Assay

HUVECs were incubated with the carboxy-2′,7′-dichlorodihydrofluorescein (DCHF, Invitrogen, Carlsbad, CA, USA) probe for 30 min. After incubation, samples were washed with medium M199 3 times and then the cellular fluorescence which reflected the intracellular ROS level was detected using a fluorescence microscopy at the 488 nm examination wavelength.

4.7. Cells for Immunofluorescence Staining

After treatment, HUVECs were fixed in 4% paraformaldehyde (w/v) for 15 min at room temperature and blocked with normal donkey serum (Solarbio, Beijing, China) for 30 min, and incubated with anti-Nrf2 or NF-κB antibody for 12 h at 4 °C. Then, the samples were washed with PBST (0.05% Tween-20 in PBS) 3 times and were incubated with corresponding fluorescence labeled secondary antibody (1:200). After washing with PBS, the cellular immunofluorescence was evaluated by a fluorescent microscope.

4.8. TUNEL Staining

A terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay was performed according to the manufacturer’s instructions (Roche, Switzerland, France). Briefly, the HUVECs were fixed with 4% paraformaldehyde for 30 min and subsequently permeabilized with 0.25% Triton X-100 in 0.1% sodium citrate for 5 min at room temperature. Finally, the HUVECs were incubated with TUNEL Enzyme-TMR label mixture solution. The TUNEL-positive HUVECs were monitored by a fluorescence microscope.

4.9. Animal Model

6 weeks of male apoE-/-mice on a C57BL/6J background were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). ApoE-/- mice were fed an atherogenic diet (containing 21% fat and 0.15% cholesterol) for 12 weeks. Then, the mice were randomly divided into five groups (n = 8). The mice of each different group were intravenously injected with saline solution (control), S1 (5 mg/kg), S1 (10 mg/kg), S3 (5 mg/kg) or S3 (10 mg/kg) for six weeks, respectively. Mice were sacrificed after injection and the aortic roots were dissected and embedded in optimal cutting temperature (OCT) embedding medium (Tissue-Tek, Sakura Finetek USA, Torrance, CA) for histology and immunofluorescence assay.

All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and were approved by the ethics committee for laboratory animals of University of Jinan.

4.10. Histological and Immunofluorescence

Oil red O (Sigma-Aldrich, Zwijndrecht, The Netherlands) staining was used to assess atherosclerotic plaque area and lipid accumulation in the aorta or aortic root following the manufacturer’s protocol. Image-Pro Plus 6.0 software (Media Cybernetics, CA, United States) was applied to analyze the positive staining within the aortas. The red or orange areas were considered positive regions. The area of an aortic atherosclerosis lesion was represented by the percentage of the oil red O positively stained area relative to the whole aortic intima area.

Immunofluorescence staining was performed to detect the target protein levels in endothelium of plaque. The sections (7 μm thick) were fixed in cold acetone for 10 min, blocked with 5% BSA containing 0.2% Triton X-100 for 2 min, after which they were incubated with primary antibody (1:200) overnight at 4 °C. After incubation with the appropriate secondary antibodies (1:500) at 37 °C for 1.5 h, sections were observed by confocal laser scanning microscopy (Zeiss LSM800, Carl Zeiss, Jena, Germany).

H&E staining and Masson staining were performed according to the instructions provided by the manufacturer (Solarbio Biotechnology, Beijing, China). The microscopic images of lesions in the aortic sinus were captured by microscope. Image-Pro Plus 6.0 was used to measure the percentage of lesion area and collagen area.

4.11. Statistical Analysis

All the quantifications are expressed as mean ± SEM. Images were processed by the use of Graphpad Prism 5 (GraphPad Software, La Jolla, CA, USA) and Adobe Photoshop (Adobe, San Jose, CA, USA). In vitro experiments were routinely repeated with at least 3 independent biological replicates. Statistical evaluations were performed with the Student’s t-test or one-way ANOVA with Bonferroni correction for multiple comparisons. p ≤ 0.05 was considered statistically significant.