Echinacoside Rb1 Attenuates Age-associated Vascular Impairment By Modulating The Gas6 Pathway

ABSTRACT

Context: Echinacoside Rb1 (Rb1) exerts many beneficial effects and protects against cardiovascular disease. 

Objective: To investigate whether Rb1 could attenuate age-related vascular impairment and identify the mechanism. Materials and methods: Female C57BL/6J mice aged 2 and 18 months, randomly assigned to Young, Young þ 20 mg/kg Rb1, Oldþ vehicle, Old þ 10 mg/kg Rb1 and Old þ 20 mg/kg Rb1 groups, were daily intraperitoneal injected with vehicle or Rb1 for 3 months. The thoracic aorta segments were used to inspect the endothelium-dependent vasorelaxation. Left thoracic aorta tissues were collected for histological or molecular expression analyses, including aging-related proteins, markers relevant to calcification and fibrosis, and expression of Gas6/Axl. 

Results: We found that in the Oldþ vehicle group, the expression of senescence proteins and cellular adhesion molecules were significantly increased, with worse endothelium-dependent thoracic aorta relaxation (58.35% ± 2.50%) than in the Young group (88.84% ± 1.20%). However, Rb1 treatment significantly decreased the expression levels of these proteins and preserved endothelium-dependent relaxation in aged mice. Moreover, Rb1 treatment also reduced calcium deposition, collagen deposition, and the protein expression levels of collagen I and collagen III in aged mice. Furthermore, we found that the downregulation of Gas6 protein expression by 41.72% and mRNA expression by 52.73% in aged mice compared with young mice was abrogated by Rb1 treatment. But there was no significant difference on Axl expression among the groups. 

Conclusions: Our study confirms that Rb1 could ameliorate vascular injury, suggesting that Rb1 might be a potential anti-aging-related vascular impairment agent.

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Introduction

Over the next 30 years in China, there is an estimated increase from 12.6% to 26% of adults aged 65 and above (Zhou and Walker 2021). Although the human lifespan is increasing, age-related diseases continue to be a topic of concern. Cardiovascular diseases are the most common diseases that affect elderly individuals. Vascular aging accompanied by vascular structural and functional changes is an important independent risk factor for cardiovascular diseases (Lakatta and Levy 2003). Epidemiological surveys have shown that 82% of patients who die of cardiovascular diseases are 65 years old or older (Moslehi et al. 2012). Therefore, exploring the mechanism of age-associated vascular impairment and finding agents that can protect vascular function against age-related decline might play important roles in reducing the incidence and prevalence of age-associated cardiovascular disorders in elderly individuals. 

Arterial aging has been implicated in the pathogenesis of vascular dysfunction and various cardiovascular diseases and is a hallmark of aging (Minamino and Komuro 2007). Vascular alterations resulting from aging vary and include endothelial dysfunction, hypertrophy of vascular smooth muscle cells (VSMCs), arterial dilatation, reorganization of the extracellular matrix (ECM), vascular calcification, and an increase in the collagen-to-elastin ratio with fragmentation and vascular stiffing (Tolle et al. 2015). Brandes et al. (2005) reported that age-related endothelial dysfunction was commonly characterized by an imbalance in endothelium-derived relaxation and contractile factors. Further study by Herrera et al. (2010) also showed that endothelial dysfunction was closely associated with a progressive decline in endothelium-dependent vasodilatation. However, it is still unclear how aging affects vasodilatation or triggers imbalances in related factors and ultimately leads to vascular dysfunction. Acosta et al. (2008) showed that vascular cells undergoing senescence could produce adhesion molecules, including plasminogen activator inhibitor 1 (PAI-1), a vascular cell adhesion protein 1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1). All these adhesion molecules were thought to play important roles in monocyte recruitment to atherosclerotic sites and then initiate vascular remodeling, ultimately resulting in a reduction in compliance and an increase in stiffness (Acosta et al. 2008; Kovacic et al. 2011). Vascular stiffness is the most important manifestation of vascular aging. It is attributed to ECM remodeling and contributes to a decline in vasodilatation. Previous studies have demonstrated that vascular stiffness and VSMC senescence are attributed to medial calcification and collagen deposition (Mauriello et al. 1992; McClelland et al. 2006; Nakano-Kurimoto et al. 2009; Wendorff et al. 2015; Kim et al. 2018). Therefore, vasodilatation dysfunction might result from calcification and fibrosis. Reducing calcium or collagen deposition may delay aging-related arterial impairment.

Echinacoside Rb1 (Rb1) is one of the active components found in ginseng. Research has reported that Rb1 exerts many beneficial antisenescence and anti-apoptotic effects, particularly in protecting the myocardium and endothelium (Zheng et al. 2017; Zhou et al. 2017; Zheng et al. 2020). Recent studies have demonstrated that Rb1 can reduce chronic kidney disease-associated vascular calcification and type I collagen expression in rats (Zhou et al. 2019b). Findings have also suggested that Rb1 reduces type I collagen expression (Kwok et al. 2012). However, the effects of Rb1 on age-associated vascular impairment have not been investigated. 

It has been reported that Rb1 inhibits vascular calcification through growth arrest-specific gene 6 (Gas6) transactivation in vitro (Nanao-Hamai et al. 2019). Gas6, a member of the vitamin K-dependent protein family, is implicated in the regulation of multiple cellular functions after binding to its receptor Axl, a membrane receptor tyrosine kinase (Nakano et al. 1997; Fridell et al. 1998; Yanagita et al. 2001). Some studies have indicated that the Gas6/Axl pathway plays a pivotal role in vascular biology and diseases such as vascular calcification vascular remodelling and atherosclerosis (Korshunov et al. 2006; Hurtado et al. 2010; Son et al. 2010). We hypothesize that Rb1 can attenuate age-related vascular impairment by suppressing calcification and fibrosis, which is potentially associated with the regulation of the Gas6/Axl pathway.

Materials and methods

Experimental reagents

Rb1 (purity greater than 98%) was obtained from Victory (Sichuan, China). Antibodies against p21Cip1 (ab188224), p16INK4a (ab51243), ICAM-1 (ab179707), VCAM-1 (ab134047), PAI-1 (ab222754) and GAPDH (ab181603) were purchased from Abcam (MA, USA). Antibodies against collagen I (14695), collagen III (22734), and Axl (13196) were purchased from Proteintech Group (MA, USA). Antibodies against Gas6 (A8545) and p16INK4a (A0262) were purchased from ABclonal Technology (Hubei, China). Modified Krebs-Henseleit (K-H) buffer, phenylephrine (a potent vasoconstrictor) and acetylcholine (Ach, an endothelial-dependent NO donor) were purchased from Sigma-Aldrich (MO, USA). All other reagents used were of analytical grade. 

Animals and management

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-Sen University. Most longevity studies have shown effects on females (Hsu et al. 2018; Qin et al. 2018); hence, we performed our long-term study using female mice. Female C57BL/6J mice aged 2 and 18 months old were obtained from the Centre of Experimental Animals, Sun Yat-Sen University. The mice were fed a normal diet and housed under controlled conditions (24 ± 2  C and 50 ± 5% humidity) with 12 h light/dark photoperiods under specific pathogen-free conditions. 40 young female C57BL/6J mice at 2 months of age were randomly assigned to the Young and Young þ 20 mg/kg Rb1 (Young þ Rb1-20) groups (n ¼ 20 each). These mice were treated with a daily intraperitoneal injection of vehicle (sterile saline solution) or 20 mg/kg Rb1 dissolved in the vehicle for 3 months before euthanasia by CO2 asphyxiation at 5 months of age. 60 aged female C57BL/6J mice at 18 months of age were randomly assigned to the Old þ vehicle, Old þ 10 mg/kg Rb1 (Old þ Rb1-10) and Old þ 20 mg/kg Rb1 (Old þ Rb1-20) groups (n ¼ 20 each). The mice that survived until the end of this experiment received daily intraperitoneal injections of the vehicle alone or Rb1 (10 or 20 mg/kg) for 3 months, after which the mice were euthanized at 21 months of age. Experiments were repeated at least three times with 4-5 mice per group.

Materials and methods Experimental reagents

Rb1 (purity greater than 98%) was obtained from Victory (Sichuan, China). Antibodies against p21Cip1 (ab188224), p16INK4a (ab51243), ICAM-1 (ab179707), VCAM-1 (ab134047), PAI-1 (ab222754) and GAPDH (ab181603) were purchased from Abcam (MA, USA). Antibodies against collagen I (14695), collagen III (22734) and Axl (13196) were purchased from Proteintech Group (MA, USA). Antibodies against Gas6 (A8545) and p16INK4a (A0262) were purchased from ABclonal Technology (Hubei, China). Modified Krebs-Henseleit (K-H) buffer, phenylephrine (a potent vasoconstrictor) and acetylcholine (Ach, an endothelial-dependent NO donor) were purchased from Sigma-Aldrich (MO, USA). All other reagents used were of analytical grade. 

Animals and management

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-Sen University. Most longevity studies have shown effects on females (Hsu et al. 2018; Qin et al. 2018); hence, we performed our long-term study using female mice. Female C57BL/6J mice aged 2 and 18 months old were obtained from the Centre of Experimental Animals, Sun Yat-Sen University. The mice were fed a normal diet and housed under controlled conditions (24 ± 2  C and 50 ± 5% humidity) with 12 h light/dark photoperiods under specific pathogen-free conditions. 40 young female C57BL/6J mice at 2 months of age were randomly assigned to the Young and Young þ 20 mg/kg Rb1 (Young þ Rb1-20) groups (n ¼ 20 each). These mice were treated with a daily intraperitoneal injection of vehicle (sterile saline solution) or 20 mg/kg Rb1 dissolved in the vehicle for 3 months before euthanasia by CO2 asphyxiation at 5 months of age. 60 aged female C57BL/6J mice at 18 months of age were randomly assigned to the Old þ vehicle, Old þ 10 mg/kg Rb1 (Old þ Rb1-10) and Old þ 20 mg/kg Rb1 (Old þ Rb1-20) groups (n ¼ 20 each). The mice that survived until the end of this experiment received daily intraperitoneal injections of the vehicle alone or Rb1 (10 or 20 mg/kg) for 3 months, after which the mice were euthanized at 21 months of age. Experiments were repeated at least three times with 4-5 mice per group.

Determination of vasorelaxation in thoracic aortic rings

After the animals were euthanized, thoracic aortic rings were rapidly removed and placed in cold K-H buffer solution (118 mmol/L NaCl, 4.75 mmol/L KCl, 25 mmol/L NaHCO3, 1.18, mmol/L MgSO4 1.18 mmol/L KH2PO4, 2.54 mmol/L CaCl2, and 11.1 mmol/L glucose), and the surrounding fat and tissue were carefully debrided. The length of the aortic rings was approximately 3 mm. The aortic segments were suspended on stainless steel hooks, aerated in K-H buffer, and continuously oxygenated with 95% O2 at 37  C. The aortic rings were then connected to FORT-10 force transducers for MacLab data acquisition. An initial passive tension in the aortic rings was set as 3 mN for 30 min to achieve vessel ring stability before further experimentation. The K-H buffer was replaced every 10 min. Phenylephrine was added to the tissue bath at a concentration of 10 5 mol/L to induce vasoconstriction. After achieving vessel ring stability, the vasorelaxant Ach (10 9 –10 5 mol/L) was added to the tissue bath in immediate succession to obtain the dose-response curve. The relaxation at each concentration was measured and is expressed as the percentage of force generated in response to phenylephrine.

Immunohistochemistry

Thoracic aorta tissues were fixed in formalin, dehydrated, embedded in paraffin, and cut into 4 lm cross sections. These sections were incubated in citrate buffer (pH 6.0) and microwaved for 10 min twice after undergoing deparaffinization and rehydration. Goat serum (10%) (Gibco, BRL, NY, USA) was used to block non-specific binding for 1 h at 37  C, followed by overnight incubation with primary antibodies against p21Cip1 (1:1000) and p16INK4a (1:200) at 4  C in a humid box. After three washes in PBS, horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) were added and incubated for 60 min at 37  C, followed by diaminobenzidine (1:100, Abcam) staining. Haematoxylin was applied to counterstain the cell nuclei. The sections were covered with neutral gum and photographed under a Zeiss microscope.

Haematoxylin-eosin (HE) staining

Paraffin-embedded thoracic aorta sections were stained by hematoxylin for 2 min. After washing and treating with 1% acidic alcohol, the sections were treated with eosin staining for 3 min. Afterward, the sections were washed, dehydrated, and treated by xylene before microscopic observation. The thoracic aorta sections were photographed under a Zeiss microscope.

Masson’s trichrome staining

Paraffin-embedded thoracic aorta sections were stained in Weigert’s iron hematoxylin for 10 min after undergoing deparaffinization and rehydration and was then washed with PBS for 5 min. The tissues were then stained in Biebrich scarlet-acid fuchsin solution for 5 min and washed in distilled water. The sections then underwent differentiation and dehydration in 75% and 90% alcohol a few times, followed by being rinsed in tap water. Finally, the sections were cleared in xylene and mounted with a mounting medium. The sections were visualized with a microscope. Interstitial collagen deposition was indicated by blue-green staining.

Alizarin red S staining

For aortic calcification staining, thoracic aorta sections were stained with 2% alizarin red S solution for 10 min and washed with PBS. Then, the sections were soaked in anhydrous acetone for 30 s, a mixed solution of anhydrous acetone and xylene (volume ratio ¼ 1:1) for 15 s, and anhydrous xylene for 1 min. Interstitial calcium phosphate salts were indicated by red staining. 

Western blotting

Thoracic aortas were placed in RIPA lysis buffer (HaiGene, Haerbin, China) supplemented with a protease inhibitor (MedChemExpress, Monmouth Junction, NJ, USA) and then homogenized by ultrasonication on ice. The tissue lysate was centrifuged at 12,000  g at 4  C for 15 min, and the supernatant was collected in a new tube. Afterward, the protein concentration in the supernatant was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). SDSPAGE was performed with equal amounts of protein from each sample as previously described (Zheng et al. 2020), and the proteins were then transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated at 4  C overnight with one of the following primary antibodies after being blocked with 5% bovine serum albumin (Gibco) for 1 h at room temperature: p21Cip1 (1:1000), p16INK4a (1:1000), ICAM-1 (1:1000), VCAM-1 (1:5000), PAI-1 (1:1000), collagen I (1:1000), collagen III (1:300), Gas6 (1:1000), Axl (1:500) and GAPDH (1:1000). After being washed, the membranes were incubated with an HRP-conjugated secondary antibody (Boster, Wuhan, China) for 1 h at room temperature. Secondary antibody binding was assayed using an ECL kit (EMD Millipore), and the intensities of the bands were analyzed using ImageJ software (version 1.41; National Institutes of Health, MD, USA). GAPDH expression was used as an internal control. 

Real-time quantitative RT-PCR (qPCR)

First, total RNA was isolated from the thoracic aortas using a TaKaRa MiniBEST Universal RNA Extraction Kit (9767, TaKaRa) in accordance with the manufacturer’s protocols. The RNA concentration was then measured with a DS-11 FX Spectrophotometer (DeNovix). Next, the extracted RNA (1000 ng each for a 20 mL reaction system) was reverse transcribed into cDNA using PrimeScriptTM RT Master Mix (RR360A, TaKaRa) according to the manufacturer’s instructions in a ProFlex PCR System (Thermo Scientific). Finally, the reverse transcription products were used as templates for further PCR amplification with SYBR Premix Ex Taq II (TaKaRa). The following primer sequences were used: Gapdh: forward 50 -CAGCAACTCCCA CTCTTCCAC-30 and reverse 50 - TGGTCCAGGGTTTCTTAC TC-30; and Axl: forward 50 -GGAACCCAGGGAATATCACAGG- 30 and reverse 50 -AGTTCTAGGATCTGTCCATCTCG-30; and Gas6 forward 50 -TGCTGGCTTCCGAGTCTTC-30 and reverse 50 -CGGGGTCGTTCTCGAACAC-30. Each reaction well in a 96-well clear plate (Thermo Scientific) which contained 0.4 pmol/lL forward and reverse primers, 1 SYBR Premix Ex Taq II (RR820A, TaKaRa) and 10 ng of cDNA. The final reaction volume was 20 mL, and all samples were analyzed in triplicate. cDNA amplification was performed as follows: pre-denaturation at 95  C for 30 s, followed by 40 cycles of denaturation at 95  C for 5 s, and annealing at 60  C for 35 s in QuantStudio5 (Thermo Scientific). The 2–DDCT method was used for relative quantitative analysis of the collected data. The relative mRNA expression level of target genes was obtained by comparing data from the experimental group with those of the control group with reference to Gapdh.

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