Evaluation of Ginsenoside Rg1 as a Potential Antioxidant for Preventing or Ameliorating Progression of Atherosclerosis

Purpose: To determine whether Rg1 inhibits H 2 O 2 -induced injury in human umbilical vein endothelial cells (HUVECs), an injury often regarded as a key early event in the development of atherosclerosis. Methods: Cell viability of HUVECs treated with Rg1 and/or H 2 O 2 was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide ( MTT) assay. Lactate dehydrogenase (LDH) release, lipid peroxidation, and reserved oxidase were detected using different available kits. The apoptosis pathway involved in the effect of Rg1 was also evaluated. Results: Exposing HUVECs to 100 μmol/L H 2 O 2 significantly decreased cell viability (78.12 ± 1.78 %), nitric oxide production, and nitric oxide synthase, superoxide dismutase, and glutathione activities, but markedly increased malondialdehyde content (from 26.87 ± 3.97 to 45.84 ± 3.50 nmol/mg of protein) and LDH release (from 8.63 to 31.42 %) (p < 0.05). These results were accompanied by a decrease in mitochondrial membrane potential and up-regulation of Bid and caspase-3, -8, and -9 mRNA expressions. However, pretreatment with different Rg1 concentrations (4, 8, and 16 µmol/L) markedly attenuated these changes (p < 0.05). Conclusion: Rg1 may protect HUVECs against H 2 O 2 -induced injury via the anti-oxidative and anti-apoptosis mechanisms, which could be applied potentially for the prevention of endothelial cell dysfunctions associated with atherosclerosis.


INTRODUCTION
The endothelium is a thin layer of cells that line the interior surface of blood vessels, forming an interface between the circulating blood in the lumen and the rest of the vessel wall [1].Endothelial injury or the loss of proper endothelial function is a hallmark for vascular diseases and is often regarded as a key early event in the development of atherosclerosis (AS) [2].
Numerous studies have shown that oxidative stress, which is defined as excessive production of reactive oxygen species (ROS), is one of the main causes of endothelial cell injury [3].ROS are continuously generated in cells as products of cellular oxidation-reduction processes and as the mechanisms of biophylaxis [4].Hydrogen peroxide (H 2 O 2 ) is one of the most important ROS that can easily penetrate the plasma membrane and can affect neighboring cells and H 2 O 2 -producing cells.It plays a central role in vascular pathophysiology and has been extensively used in studying the oxidative injury model of endothelial cell in vitro [5].
Panax ginseng is a medicinal plant cultivated in many oriental countries.P. ginseng has been used for thousands of years as a traditional medicine and a daily supplement in China.Numerous active ingredients of P. ginseng have been isolated and characterized.Among these, ginsenoside Rg1 is one of the most active and abundant steroid saponins that shares structural similarity with many steroid hormones [6].Gingenoside Rg1 can attenuate oxidative damage in the liver of thioacetamide-treated rats [7].In addition, several studies have shown that Rg1 has protective effects on the central nervous and endocrine systems [8].However, the effects of ginsenoside Rg1 on regulating the free-radical scavenging system and protecting endothelial cells against H 2 O 2 -induced oxidative damage are still unknown.Therefore, the present study aims to investigate the protective effect of Rg1 on H 2 O 2 -induced injury in human umbilical vein endothelial cells (HUVECs).

EXPERIMENTAL Cell culture
HUVECs were obtained from the College of Medicine of Nanchang University.Fetal calf serum (FCS), Dullbecco's modified Eagle's medium (DMEM), and serum-free medium were purchased from Gibco (Invitrogen, USA).Ginsenoside Rg1 was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
The HUVECs were incubated in DMEM medium supplemented with 10% FCS, 100 IU/mL penicillin, 100 IU/mL streptomycin, and 0.29g/L L-glutamine and then maintained at 37 °C in a humidified atmosphere with 5% CO 2 .The culture media were changed every 2 days.Upon reaching confluence, the cells were harvested by treatment with 0.25% trypsin and 0.02% EDTA for 1 min and then replanted.

Determination of cytotoxicity of ginsenoside Rg1 on HUVECs
When cells were 80 % confluent, the media were removed and the fresh serum-free media with different concentrations of Rg1 (1, 4, 16, 64, and 250 µmol/L) were added and incubated for 24h.Thereafter, the media containing Rg1 were discarded and the cells were washed twice with PBS.Then, cells were incubated with 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma, USA) (final concentration 0.5 mg/ml) for 4 h at 37 °C.The media were removed from each well and 200 μL of DMSO was added.The absorbances were measured at 490 nm using a Thermo Scientific Multiskan MK3 Microplate Reader (Thermo Fisher, USA).

Effect of ginsenoside Rg1 on viability of H 2 O 2 -induced HUVECs
Based on preliminary findings, 100 μmol/L of H 2 O 2 was selected to establish the oxidative stress model in the present study.HUVECs were randomly divided into the control, H 2 O 2 and four ginsenoside Rg1 groups.In the ginsenoside Rg1 groups, the cells were pre-treated with different final concentrations of ginsenoside Rg1 (1, 4, 16, and64 µmol/L) for 24 h, followed by co-culture with 100 μmol/L of H 2 O 2 for another 8 h.Finally, the effects of Rg1 on the H 2 O 2 -induced cell injury in HUVECs were measured through MTT assay as described above.

Determination of lactate dehydrogenase (LDH) release, nitric oxide (NO) production, malondialdehyde (MDA) content, superoxide dismutase (SOD), glutathione (GSH), and nitric oxide synthase (NOS) activities
LDH release (%), NO production, MDA concentration, and SOD, GSH, and NOS activities were determined using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, China).LDH was released from injured cells and used as an index of cell injury.LDH release (%) was calculated as in Eq 1.The MDA levels were evaluated by the thiobarbituric acid reacting substance (TRARS) methods.Briefly, MDA reacts with thiobarbituric acid at an acidic high temperature and form a stable chromophoric production.The absorbance of the production was determined at 532 nm and expressed as nmol/mg protein [9].The activities of NOS were spectrophotometrically measured with commercially-available kits based on the oxidation of oxyhaemoglobin to methaemoglobin by nitric oxide.NO production was tested by measuring the accumulation of nitrites, a stable product of NO metabolism in the supernatant of the cells [10].In brief, the supernatant of the cells was mixed with the Griess reagent (1 percent sulfanilamide, 0.1 percent N-lnaphathyletylenediamine dihydrochloride and 2.5 percent phosphoric acid) at room temperature for 10 min.Nitrite products in the supernatants were determined by measuring absorbance at 540 nm.The assay of SOD activity was based on the ability of SOD to inhibit the oxidation of hydroxylamine by O 2− produced from the xanthine-xanthine oxidase system.One enzyme unit (U) of SOD activity was defined as the amount of enzyme required to cause 50 % inhibition in the rate of nitro-blue tetrazolium (NBT) reduction at 560 nm and was expressed as U/mg protein [11].GSH was measured based on the fact that 5, 5-dithiobis 2-nitrobenzoic acid reacts with GSH to generate 2-nitro-5thiobenzoic acid which is yellow.Hence, the GSH concentration in the assay mixture can be determined by measuring absorbance at 412 nm [11].
where LDHs is the release of LDH in the supernatant and LDHc is the release of LDH from the cell lysate.

Flow cytometry analysis
The apoptosis of HUVECs was assessed by an annexin-V/propidium iodide binding assay (Biosciences, USA) according to the manufacturer's instructions.The results were interpreted as follows: cells negative for both PI and Annexin-V-FITC staining were considered as live cells; PI-negative and Annexin-V-FITCpositive stained cells were considered in early apoptosis; PI positive and Annexin-VFITCpositive-stained cells were considered in late apoptosis or secondary necrosis.

Measurement of mitochondrial membrane potential (ΔΨm)
Rhodamine 123, a cationic fluorescent dye whose mitochondrial fluorescence intensity decreases quantitatively with the dissipation of the mitochondrial membrane potential, was used to evaluate perturbations in mitochondrial membrane potential [12].The HUVECs were cultured as described above and divided into the control, H 2 O 2 , and Rg1 groups.Subsequently, the cells were harvested, washed twice with cold phosphate-buffered saline, and then stained with 1 μmol/L Rhodamine 123 for 30 min in the dark at room temperature.Fluorescence was measured via flow cytometry at an excitation wavelength of 485 nm.

Quantitative real-time (qRT)-PCR) for mRNA expression
Total RNAs were isolated from HUVECs using Trizol reagent (Invitrogen, Carlsbad, USA) and were determined by using spectrophotometer (Beckman Instrument, California, USA).One µg of RNA was reverse-transcribed with PrimerScript™ RT-PCR kit (TaKaRa code: DRR014A) according to the manufacturer's protocol.Quantitative real-time PCR was performed to determine the expression levels of genes such as Bid, as well as caspase-3, -8, -9 using SYBR Premix Ex Taq TM (TaKaRa Code: DRR041A) in the ABI 7900HT Real-Time PCR system (Applied Biosystems, USA).The following PCR conditions were used: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 1 min.The florescence signals were detected with an ABI 7900HT Version 2.3 sequence detection system (Applied Biosystems, USA).The cycle threshold (Ct) values for each gene were determined and the gene expression data were normalized to the endogenous control β-actin.The relative mRNA expression levels were calculated via the comparative threshold cycle (∆Ct) method, where ∆Ct is the difference in Ct value between the target gene and β-actin.Primers used for qRT-PCR are listed in Table 1.

Statistical analysis
Data are expressed as mean ± SD (n ≥ 3).Statistical comparisons were performed with SPSS 17.0.One-way ANOVA and Student's ttest were carried out to determine statistical significance.P < 0.05 was considered statistically significant.

Cytotoxicity of ginsenoside Rg1 on HUVEC
The cytotoxicity of Rg1 on HUVEC was excluded by analyzing its effects on the proliferation of HUVEC.No significant difference was found in the cell viability between cells treated with 1 and 4 µmol/L of Rg1 and the control (p < 0.05).By As shown in Fig. 1 (C and D), the NO level and NOS activity in HUVECs (25.71 ± 5.00 µmol/L and 1.58 ± 0.36 U/mg of protein, respectively) significantly decreased in the H 2 O 2 group compared with those in the control group (41.99 ± 5.01 µmol/L and 2.49 ± 0.54 U/mg protein, p < 0.05).However, pre-treatment with Rg1 triggered a distinct, dose-dependent increase in NO level and NOS activity than that in H 2 O 2 group (p < 0.05).

Effect of Rg1 on H 2 O 2 -induced apoptosis in HUVECs
The amount of cells that stepped into early apoptosis increased from 0.1% in the control group to 34.0% in the H 2 O 2 group (Fig. 2)..However, the percentages of apoptosis decreased to 20.0% and 10.9% in the groups treated with 4 and 64 μmol/L Rg1, respectively.These results suggest that Rg1 elicits a protective effect on H 2 O 2 -induced apoptosis in HUVECs.This effect is particularly important in maintaining normal physiological functions and preventing the formation of atherosclerotic plaques.

Effect of Rg1 on ΔΨm
Apoptosis may be initiated through mitochondrial-induced pathways.The changes in ΔΨm reflect the initial cell apoptotic phenomenon.In the present study, ΔΨm was measured using flow cytometry.As shown in Figs.3A and 3B, a more substantial decrease in ΔΨm was observed in the H 2 O 2 group than in the control group.However, the two peaks shown in Figs.3C and 3D suggest that a high concentration of Rg1 could attenuate the decline in ΔΨm.

Effect of Rg1 on mRNA expression
Depolarization of mitochondrial membrane induces the expression of several anti-apoptotic and pro-apoptotic proteins, most notably Bid, that translocate from the mitochondria into the cytosol, thereby activating caspase-3, 8, 9 kinase.In the current study, the mRNA expressions of Bid and caspase-3, -8, and -9 were significantly up-regulated in the H 2 O 2 group compared with those in the control group.However, different concentrations of Rg1 suppressed H 2 O 2 -induced up-regulations.This result indicates that the protective mechanism of Rg1 on H 2 O 2 -induced injury in HUVECs may be involved in the anti-apopotic system (Fig. 4).

DISCUSSION
Ginsenoside Rg1, one of the major active molecules from P. ginseng, may attenuate the oxidative damage in rat liver cells [7].However, the effects of ginsenoside Rg1 on regulating the free-radical scavenging system and protecting endothelial cells against H 2 O 2 -induced oxidative damage are still unknown.To investigate the protective effect of Rg1 on endothelial cells against H 2 O 2 -induced oxidative damage, indicators of oxidative stress-related cell injury (LDH, MDA, NO, NOS, SOD, and GSH) were measured in the present study.Lipid peroxidation is one of the main phenomena in free-radical-mediated cell damage.MDA is a byproduct of lipid peroxidation induced by excessive ROS, which has been widely used as a biomarker of oxidative stress.A significant increase in MDA production is accompanied by increased LDH release [13].NO, a diatomic free radical with a half-life of a few seconds, is one of the most important mediators in regulating endothelial functions.It is synthesized by NOS enzymes in which NO and L-citrulline are two products, and L-arginine is the substrate [14].Within the vasculature, NO induces vasodilatation, prevents platelet adhesion to endothelial cells, inhibits platelet aggregation, controls smooth muscle cell proliferation and migration, maintains an endothelial cell barrier function, and regulates apoptosis [15].At higher levels, O 2− reacts with NO to form a cytotoxic peroxynitrite (ONOO−) and decrease NO production, which plays a critical role in endothelial cell damage [16].Several studies have reported that the down-regulation of NO production is implicated in the pathogenesis and clinical course of all known cardiovascular diseases and is also related to future risks of adverse cardiovascular events [17].In the present study, the NO production, NOS activity, and mRNA expression of eNOS were significantly decreased in the H 2 O 2 group (p < 0.05).However, these H 2 O 2 -induced decreases were significantly inhibited in a dosedependent manner after pretreatment with different Rg1 concentrations prior to H 2 O 2 addition.SOD and GSH play a pivotal role in preventing ROS-induced cellular damage.Therefore, intracellular ROS can be effectively eliminated by the combined action of SOD and GSH, providing a repairing mechanism for oxidized membrane components [18].In the present study, the activities of SOD and GSH in HUVECs significantly decreased after exposure to H 2 O 2 , indicating impairment of antioxidant defenses.Nonetheless, pre-incubating HUVECs with Rg1 significantly attenuated these H 2 O 2induced cellular events and enhanced endogenous antioxidant preservation.These results suggest that Rg1 protects HUVECs against oxidative stress through the antioxidation system, which is beneficial for the prevention and treatment of AS.
Apoptosis is a highly ordered cell death process that may be initiated by mitochondrial inducing pathways [19].In other words, the mitochondria play a major role during apoptosis induction, leading to the organized degradation of cellular structure and results in the formation of apoptotic bodies [20].Recent evidence suggests that the opening of the mitochondrial permeability transition (MPT) pore is a critical event in the process leading to apoptosis.The opening of the MPT pore can cause the dissipation of the inner ΔΨm and culminate in the disruption of outer membrane integrity, which leads to the liberation of inter-membrane proteins from the mitochondrion [21].In addition, Bid is a proapoptotic Bcl-2 protein containing the only BH3 domain that can induce the opening of the mitochondrial voltage-dependent anion channel.This opening also results in the release of some pro-apoptotic factors from the mitochondria.Once in the cytosol, these apoptogenic proteins activate caspase proteases, amplifying the apoptotic process [22].
Although many potential stimuli may initiate apoptosis, these signals seem to converge on the caspase pathway to execute the final phases of the apoptotic process [23].Caspase-8 and caspase-9 have been linked to the mitochondrial death pathway and are initiator caspases that act upon caspase-3.In some cell lines, caspase-3 is the effector caspase that plays a direct role in the proteolytic cleavage of cellular proteins responsible for the progression to apoptosis.The sequential activation of caspase-3, -8, and -9 finally induce the occurrence of apoptosis [24].
In the current study, upon exposure to 100 µmol/L H 2 O 2 , a decrease in ΔΨm and an increase in the mRNA expression of Bid and caspase-3, -8, and -9 were clearly observed compared with the control.High concentrations of Rg1 ameliorated the decrease in ΔΨm (Fig. 3) and the upregulation of H 2 O 2 -induced mRNA expression .These results suggest that the protective mechanism of Rg1 on H 2 O 2 -injured HUVECs may be involved in the anti-apoptosis system initiated by mitochondrial-induced pathways.

CONCLUSION
In conclusion, ginsenoside Rg1 can protect HUVECs against H 2 O 2 -induced injury by regulating the indicators related to oxidative stress, enhancing anti-oxidant enzyme activity, and suppressing the over-expression of Bid and caspase-3, -8, and -9.The protective mechanism of Rg1 on H 2 O 2 -induced injury in HUVECs may be a caspase-dependent, anti-apoptotic mechanism involved in mitochondrial-induced pathways.The results of the present study provide important information for the treatment and prevention of oxidative stress-induced atherosclerosis.

Fig 1 :F
Fig 1: Effect of Rg1 on mediators related to oxidative stress in HUVECs.Asterisks (*) indicate statistically significant differences between control and H2O2 groups (p < 0.05), while letters a, b, and c indicate statistically significant differences between H2O2 and Rg1 groups (p < 0.05).

Table 1 :
Sequences of primers used in qRT-PCR

Effect of Rg1 on representative indicators of H 2 O 2 -induced cell injury
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