Cell-Based Screening Identifies the Active Ingredients from Traditional Chinese Medicine Formula Shixiao San as the Inhibitors of Atherosclerotic Endothelial Dysfunction

Abstract

In this study, we performed a phenotypic screening in human endothelial cells exposed to oxidized reduced density lipoprotein (an in vitro standard of atherosclerotic endothelial dysfunction) to make identical the effective compounds in Shixiao San. After investigating the suitability and reliableness of the cell-based screening course using atorvastatin as the positive superintend drug, this method was applied in screening Shixiao San and its extracts. The management of n-butanol fraction on endothelial cells exhibited stronger comforting effects against oxidized low density lipoprotein-induced indignity when compared with other fractions. Cell viability, the of the same rank of nitric oxide, endothelial nitric oxide synthase and endothelin-1 were moderate, respectively. The assays revealed n-butanol bit significantly elevated the survival ratio of impaired cells in culture. In parallel, n-butanol fraction exhibited the highest interdiction of inflammation. The generation of prostaglandin-2 and tendency to adhere molecule (soluble intercellular adhesion molecule-1) was obviously declined. Furthermore, n-butanol fragment suppressed the production of reactive oxygen form and malondialdehyde, and restored the nimbleness of superoxide dismutase. Compounds identification of the n-butanol fragment was carried out by ultra recondite liquid chromatography coupled to quadrupole time-of-hasty departure tandem mass spectrometry. The active ingredients including quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside revealed the efficacy of anti-atherosclerosis after exposing ~ward endothelial cells. The current work illustrated the pharmacology reality of Shixiao San and clearly indicated the greater active components in Shixiao San. More importantly, the proposed confined apartment-based screening method might be individually suitable for fast evaluating the anti-atherosclerosis efficiency of Traditional Chinese Medicines and screening in a puzzle the interesting ingredients of Traditional Chinese Medicines.

Citation: Wang X, Zhang R, Gu L, Zhang Y, Zhao X, et al. (2015) Cell-Based Screening Identifies the Active Ingredients from Traditional Chinese Medicine Formula Shixiao San for example the Inhibitors of Atherosclerotic Endothelial Dysfunction. PLoS ONE 10(2): e0116601. doi:10.1371/magazine.pone.0116601

Academic Editor: Pratibha V. Nerurkar, College of Tropical Agriculture and Human Resources, University of Hawaii, UNITED STATES

Received: August 23, 2014; Accepted: December 12, 2014; Published: February 20, 2015

Copyright: © 2015 Wang et al. This is each open access article distributed under the stipulations of the Creative Commons Attribution License, that permits unrestricted use, distribution, and representation in any medium, provided the prototype author and source are credited

Data Availability: All suitable data are within the paper.

Funding: The authors current no specific funding for this work.

Competing interests: The authors have declared that ~t any competing interests exist.

Introduction

Atherosclerosis (AS) is a complicated vascular tumult involving lipid accumulation, cell death, oxidative detriment and inflammatory responses in the arterial wall, resulting in heart disease and stroke. Endothelial dysfunction is considered to subsist an initial step in the pathogenesis of AS [1]. High plasma horizontal of low-density lipoprotein (LDL) could be oxidatively modified to be oxidized LDL (Ox-LDL), what one. is closely correlated with accelerated AS [2].

When salubrious endothelial cell function is impaired ~ dint of. atherogenic risk factor (Ox-LDL) [3], the pleasant balance between proliferation and apoptosis would subsist disordered firstly, which is widely evaluated through cell viability assay (Roche, Sweden) [4]. The imbalance betwixt vasoconstriction and vasodilatation is also triggered ~ the agency of Ox-LDL. It is well-known that nitric oxide (NO) is single in kind of the vasodilators, and endothelin-1 (ET-1) is the force vasoconstrictor, both of which oppositely put in order the enothelial-dependent vasomotion. Thus, the hale condition condition of vasomotion attributed to the unvarying release of NO and ET-1 [5, 6].

Besides, increasing evidence shows a relationship between oxidative significance and endothelial function, and oxidative inclemency has been recognized as a solution mechanism in the development of vascular harm, particularly AS [7]. There are diverse possible mechanisms for causing the oxidative stress of endothelial function in AS, including enhanced prolongation of reactive oxygen species (ROS) and decreased remit of NO, as well as one attenuated antioxidant system (Superoxide dismutase, SOD) [8]. Moreover, oxidation of membrane lipids, human being of the primary events in oxidative honey-combed damage, can be assessed by area of malondialdehyde (MDA), a breakdown issue of lipid peroxides [9].

Furthermore, endothelial dysfunction accompanied by inflammatory process leads to increased clinging molecule to the activated endothelium [10]. For prototype, soluble intercellular adhesion molecule-1 (sICAM-1), simulation as a leukocyte adhesion molecule, openly contributes to inflammatory responses within the hot spark vessel wall by increasing endothelial cell activation and augmenting atherosclerotic plaque making up [11]. Prostaglandin E2 (PGE2) is ~y important mediator of active inflammation onward with activation and recruitment of macrophages and pannage cells [12]. Therefore, it is decisive to supervise these parameters associated through endothelial cell function at the seasonably stage of AS discovery process.

Shixiao San, originally recorded in the Complete Collection of Prescriptions (Taiping Huimin Heji Ju Fang), is getting increasing attention worldwide for explicating pharmacological machinery [13, 14]. As a well-known TCM ~ry, Shixiao San has been widely used as far as concerns the prevention and treatment of cardiovascular distemper in modern clinical therapy. Our foregoing studies have demonstrated that Shixiao San effectively diminish the degree of LDL particles [14, 15]. Indeed, tyrannical level of plasma LDL results in endothelium-conditioned dysfunction, so it is probably that Shixiao San could revive endothelial function, in part, by murky serum LDL levels. Nevertheless, there are with reference to something else few reports regarding the reliable pharmacological exercise of Shixiao San on treating endothelial dysfunction. Besides, compound chemical compositions of Shixiao San constrain it extremely difficult to evaluate and hide the bioactive ingredients. Hence, a sincere and rapid method is urgently needed to inspire the efficacy and discover the bioactive compounds of Shixiao San in compensation for endothelial dysfunction.

A variety of approaches since screening and analysis of the bioactive fractions or compounds in TCM esteem been developed and improved during the out of the reach of decades [16]. Researchers tend to segregate chemical compounds from Traditional Chinese Medicines (TCMs) and test them individually on animal models in accustomed ways [17]. However, these approaches are time consuming, toilsome, and unsuitable for the rapid screening of bioactive compounds of TCMs. Cell-based screening typically refers to a continuous experiment in which abundant drug candidates are efficiently tested to identify their biological activity end cell assay in additional biological or pharmacological experiments. Compared by the whole animal models, the cellular models based on different diseases and mechanisms are in greater numbers adaptive to large-scale candidates screening in TCMs, since they have overcome the shortcomings of denizen of the deep models, such as time consuming, technical complexity, poor repeatability, expensive cost, low throughput and kind difference [18, 19]. Therefore, this unbiased screening is expanding expeditiously in drug discovery recently [20]. Human endothelial lonely dwelling line EA.hy926 is commonly accepted considered in the state of a tool in exploring the pathogenesis of cardiovascular diseases [21]. To perform this study, Ox-LDL exposing put ~ EA.hy926 was carried out as an in vitro pathological model on account of high throughput screening. Through evaluating the parameters over, interesting fraction and compounds from Shixiao San that rule endothelial cell functions would be fast tested in vitro in preclinical models.

Herein, we constructed a unusual and rapid cell-based screening strategetics to systematically explore compounds of Shixiao San respecting their efficacy against atherosclerotic endothelial dysfunction. Compounds identification was carried lacking by ultra high liquid chromatography coupled to quadrupole time-of-flock tandem mass spectrometry (UHPLC/Q-TOF MS). This rule not only offered evidence-based facts about the therapeutic mechanism of Shixiao San in corpuscular level, but also screened out the anti-atherosclerotic aspirant compounds. More importantly, for the ~ and foremost time, a standard operating procedure relating to a cell-based screening method that be possible to rapidly detect the target component from a intricate web sample of anti-atherosclerotic candidate drugs has been recommended.

Experimental

Chemicals, reagents and materials

Ox-LDL, human (Yiyuan Biotechnologies, China) was stored at 4°C, and the hoard solution was freshly prepared in phosphate-buffered salt-spring before applied to the cultures by a final concentration of 100 μg/mL. Dulbecco’s modified eagle’s pervading substance (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), penicillin and streptomycin were obtained from Sigma (St. Louis, MO, USA). The trading kits used in biochemical assays of MDA, SOD and NO were purchased from Nanjing Jiancheng Bioengineering Institute, (Nanjing, China). Human enzyme-linked immusorbent make an ~ of (ELISA) Kits for measurement of endothelial nitric oxide synthase (eNOS), ET-1, PGE2 and sICAM-1 topics were from Senxiong Biological Limited Corporation (Shanghai, China). The damp materials of Typhae Pollen and Faeces Trogopterori were purchased from Tong-Ren-Tang TCM abundance (Shenyang, China). Atorvastatin (Atv) was with the understanding by Pfizer. The reference standard of isorhamnetin-3-O-(2G-α-L-rhamnosyl)-rutinoside was obtained from National Institute in opposition to Food and Drug Control (Beijing, China). Isorhamnetin-3-O-neohesperidoside was purchased from Aladdin Reagent Inc. Isorhamnetin-3-O-rutinoside was from Chengdu Pufei De Biotech Co., Ltd. Quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, kaempferol-3-O-(2G-α-L-rhamnosyl)-rutinoside and kaempferol-3-O-neohesperidoside were separate in our laboratory. Distilled water prepared with demineralized water was employed throughout the examination. Acetonitrile of HPLC grade was from Fisher Scientific (Fair Lawn, NJ, USA). Formic tart of HPLC grade was provided ~ the agency of Shandong Yuwang Industrial Co., Ltd. (Yucheng, China).

Preparation of Shixiao San fractions

The method with respect to preparing Shixiao San has been described beforehand [14]. Then, The crude extract of unfinished medicinal materials was suspended in sprinkle and calender and successively partitioned with petroleum ether (PE), methylene chloride (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (BuOH) to afford PE, CH2Cl2, EtOAc and BuOH fractions considered in the state of well as an H2O residue, followed ~ dint of. freeze-drying procedure. The freeze-dried powders were dissolved by DMEM separately and filtrated with 0.22μm cellulose acetate membrane.

Experimental design

EA.hy926 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in DMEM containing 1 g/L grape-sugar supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in 10% CO2. The cells were sown into plates and randomly assigned into nine groups in the manner that described below containing six parallel samples by group. In group III-VII;, decisive concentrations of Shixiao San and other fractions in whole the assays were equivalent to 100 mg/L crude extract of raw medicinal materials. In collection IX, final concentration of Atv in the analyze was 5 mg/L.

Control cluster Cells were first incubated with struck dumb. medium for 12 h, then incubated through the replaced blank medium for a different 12 h.

Model group Cells were primeval incubated with the medium containing Ox-LDL conducive to 12 h, then incubated with the replaced mean average containing Ox-LDL for another 12 h.

Shixiao San arrange Cells were first incubated with the pervading substance containing Ox-LDL for 12 h, afterwards incubated with the replaced medium containing Ox-LDL and Shixiao San beneficial to another 12 h.

PE group Cells were highest incubated with the medium containing Ox-LDL conducive to 12 h, then incubated with the replaced means containing Ox-LDL and PE part for another 12 h.

CH2Cl2 arrange Cells were first incubated with the mean average containing Ox-LDL for 12 h, sooner or later incubated with the replaced medium containing Ox-LDL and CH2Cl2 piece for another 12 h.

EtOAc form into ~s Cells were first incubated with the mean containing Ox-LDL for 12 h, hereafter incubated with the replaced medium containing Ox-LDL and EtOAc section for another 12 h.

BuOH arrange Cells were first incubated with the moderation containing Ox-LDL for 12 h, sooner or later incubated with the replaced medium containing Ox-LDL and BuOH part of a unit for another 12 h.

H2O collection Cells were first incubated with the agency containing Ox-LDL for 12 h, then incubated with the replaced medium containing Ox-LDL and H2O part for another 12 h.

Atv assemblage (positive control) Cells were first incubated with the medium containing Ox-LDL in favor of 12 h, then incubated with the replaced medium containing Ox-LDL and Atv part for another 12 h.

After handling, the cells were assigned to calculus of cell viability assay, level of intracellular ROS, MDA and SOD assays and extracellular NO, eNOS, ET-1, PGE2 and sICAM-1 assays. After investigating the suitability and reliability of the method using Atv during the time that positive control, each group was examined using the assays as being its anti-atherosclerotic activities, then the fragment with high activity was selected because further identification using an UHPLC/Q-TOF MS connected view. In order to test whether the identified compounds be the subject of biological activity, the compounds which acquire reference standards in candidate fraction were given individually ordeal with in the Ox-LDL mediated examination. Therefore, any interesting compounds from Shixiao San that rule endothelial cell functions would be in posse to test in this method.

Cell viability trial and intracellular ROS measurement

After mix with ~s treatment as described in section 2.3, the fund MTT solution was added to the whole of wells of the assay, and plates were incubated 4 h. Afterwards the supernatant was discarded and 150 μL of DMSO per well was added to solubilize formazan crystals during 10 min on a shaker. The optical closeness was measured by a microplate reader (3001, Thermo Scientific, Finland) at a wavelength of 490 nm.

The measurement of intracellular ROS was based put ~ ROS-mediated conversion of non-fluorescent 2, 7-dichlorofuorescin diacetate (DCFH-DA) into DCFH [22]. The cells were washed by PBS and then incubated with DCFH-DA (10 μM) and DNA contaminate Hoechst 33342 (10 mmol/L) at 37°C ~ the sake of 30 min. At the end of incubation, the DCFH fluorescence of the cells from each well was measured at an issuance wavelength of 530 nm and an excitation wavelength of 485 nm by a FLX 800 microplate fluorescence reader (Biotech Instruments Inc., USA). The background was obtained from cell-free conditions and the results were expressed in the manner that the percentage of control (non-stimulated cells) fluorescence power. The representative pictures were evaluated through TCS NT Sp5 LSCM instrument (Leica, Germany).

Assessment of intracellular MDA and SOD

The cells were washed through ice-cold PBS and centrifuged at 1000 r at 4°C because of 10 min. The pellets were resuspended by 1000 μL of PBS, freeze-thawed two times at −20°C and centrifuged at 10,000r at 4°C because of 15min. The supernatant was collected because MDA and SOD assays, according to the instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and the activities of enzymes were expressed similar to units per milligram protein. Protein compression into a small compass was determined by the bicinchoninic tart (BCA) method, using BSA as a concern standard.

Measurement of extracellular NO, eNOS, ET-1, PGE2 and sICAM-1

The cultured intervening substance was collected and centrifuged for 15 min at 4000 r 4°C, and on that account the supernatants were used for the following analyses. The sum ~ amount of NO was assessed through using a colorimetric assay kit. Human ELISA kits from Senxiong Biological Limited Corporation (Shanghai, China) were employed against the measurement of eNOS, ET-1, PGE2 and sICAM-1 separately in cultured medium.

Determination of piece by UHPLC/Q-TOF MS

Each portion was examined using the assays in opposition to its anti-atherosclerotic activities and hereafter the fraction with high activity was selected according to further characterization using UHPLC/Q-TOF MS scheme. UHPLC/Q-TOF MS analytical procedures were performed ~ward an Accurate-Mass Q/TOF 6520 mass spectrometer by an Agilent 1290 LC system (Agilent, USA). The software Mass Hunter workstation was applied to arrangement operation and data collection. The LC disunion was achieved on a Zorbax Eclipse Plus C18 line (100 mm × 2.1mm, 1.8 μm). The mobile phase consisted of A (0.01% formic sour in water) and B (0.01% formic sour in methanol). The following gradient program was used: 0–7min, 28% B; 7–20 min, 28–45% B; 20–40 min, 45–80% B. The solvent flow rate was 0.2 mL/min. For total UHPLC/Q-TOF MS experiments, the Q-TOF mass spectrometer was operated in the negative ion style with an electropray ionization source (ESI-). The look into range was set at m/z 100–900. Instrument calibration was performed through a sodium formate solution consisting of 10 mM sodium hydroxide in isopropanol/0.2% formic acid (1:1, v/v).

Constituents were identified by comparing their retention time, mass value and MS/MS fragmentation with the corresponding parameters of the reference standards. The constituents, insufficiency of reference standards, were identified ~ the agency of comparing the accurately measured mass importance and MS/MS fragments with the relative length reported in references. Finally, the compounds which have reference standards in candidate bit were given individually in the Ox-LDL mediated test.

Statistical analysis

All data was expressed viewed like mean ± standard deviations (SD) and statistical analysis was performed using SPSS software bundle (version 16.0, SPSS Inc., Chicago, IL, USA). The premises following a normal distribution was evaluated by Shapiro-Wilk test. The differences were analyzed through one-way analysis of variance (ANOVA), followed through post hoc analysis with Tukey-Kramer example [23]. Statistical significance was considered whenever the value of *p < 0.05, and **p < 0.01 indicated very much significant.

Results and Discussion

Concentration-sustained by viability losses in EA.hy926 cells induced through Ox-LDL

We first carried away the concentration-dependent study of viability losses in EA.hy926 cells induced through Ox-LDL. Medium containing different concentrations of Ox-LDL (0, 25, 50, 100, 200, 300 and 400 μg/mL) was added to catch the most appropriate concentration in the pattern group. The cells were incubated towards 24 h followed by observation beneath an inverted microscope and measurement ~ the agency of MTT assay. The variation of cell viability and the morphological evaluation displayed an Ox-LDL dose-dependent viability failure to win in EA.hy926 cells. At the condensation of 0, 25, 50, 100, 200, 300 and 400 μg/mL, the simplest organism viability was 100 ± 0.00%, 85.36 ± 3.63%, 79.61 ± 2.18%, 53.89 ± 3.81%, 35.20 ± 1.94%, 15.54 ± 0.54% and 13.78 ± 0.31%, particularly. In summary, 100 μg/mL of Ox-LDL was determined to have ~ing the most appropriate concentration which would have ~ing applied in the model groups.

The confined apartment viability change after treatment of Shixiao San and its extracts

The simplest organism viability carried out by MTT was significantly decreased ~wards Ox-LDL exposure. As shown in Fig. 1A, the betokening increased cell viability (compared with original group) indicated that Shixiao San effectively protected cells from Ox-LDL induced damage. The result was similar to that of Atv group. Following further treatment of different extracts, the BuOH group showed a significant improvement over the other scrap groups in cell growth. The small cavity viability reached up to 73.57 ± 7.34% whereas cells treated with BuOH fraction, during the time that the cell viability of cells treated by PE, CH2Cl2, EtOAc and H2O fragment were 52.70 ± 4.32%, 52.98 ± 5.16%, 63.21 ± 5.46% and 56.22 ± 3.88%, respectively.

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Fig 1. Effect of Shixiao San and its exracts steady OX-LDL-induced endothelial cell viability (A) and the flat of MDA (B), SOD (C) and NO (D).

EA.hy926 cells were exposed to Ox-LDL of 100 μg/mL, and treated by different samples for another 12 hours. All of the data are expressed as the means ± S.D. (n = 6). *P < 0.05, ** p < 0.01, compared through the model group.

doi:10.1371/magazine.pone.0116601.g001

Intracellular ROS of Shixiao San and its extracts

The up-rule of ROS in vascular lesions command exert detrimental effects including peroxidation of membranes lipids, endothelium-derived enzymes inactivation and apoptotic occurrences, etc. We be able to evaluate the intracellular ROS concentration end observing the intensity of fluorescence [8]. Fig. 2 shows the typical picture of the fluorescence in EAhy.926 cells for the time of the various treatments. When incubated in the instrumentality for 30 min, a sudden enlargement in fluorescence intensity of cells through Ox-LDL indicated the increasing of intracellular radicals. Whereas the increased fluorescence was significantly reduced whereas cells incubated with Shixiao San and Atv. The drift of BuOH group was similar to that of Shixiao San. However, there is no significant difference in other groups compared by model groups.

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Fig 2. Inhibitory effects of Shixiao San and its exracts up~ the production of intracellular ROS.

EA.hy926 cells were exposed to Ox-LDL of 100 μg/mL, and hereafter treated with different samples for one more 12 hours. All of the premises are expressed as the means ± S.D (n = 6). * P < 0.01, compared with the model group.

doi:10.1371/periodical.pone.0116601.g002

Effect of Shixiao San and its extracts put ~ SOD activity and MDA level

MDA is as a common thing used as an indicator of chain damage. The radical formation evaluated of the same kind with MDA, is shown in Fig. 1B. Shixiao San and Atv significantly attenuated Ox-LDL induced changes of MDA. At the corresponding; of like kind time, the increased level of MDA induced ~ means of Ox-LDL was inhibited by EtOAc and BuOH group. Specifically, the protective effect of BuOH assign places to was better than those of other extracts. As shown in Fig. 1C, the incubation of the cells with Ox-LDL for 24 h caused a weighty decrease in SOD activity compared through control group. In contrast, the incubation through Shixiao San, BuOH extract, EtOAc take out and Atv significantly decreased SOD nimbleness. The results suggested that the mechanical construction of the therapeutical effect of Shixiao San is partly due to antioxidant activity.

eNOS suitable feeling and execution and NO, ET-1 release from endothelial cells

The eNOS is physiologically turning for vascular homeostasis, keeping the vasculature dilated, protecting the intima from platelet aggregates and leukocyte adhesion, and preventing smooth muscle proliferation. eNOS-derived NO is produced ~ dint of. the vascular endothelium under basal provisions. Its production is stimulated by a multiplicity of receptor agonists as well during the time that the shear stress produced by the smooth blood. NO released by endothelial cells is a greater endogenous vasodilator system counterbalancing the vasoconstriction produced ~ the agency of the sympathetic nervous system and the rennin-angiotensin universe. Thus, eNOS and NO play significative roles in the development of AS [24]. Exposure of EA.hy926 cells to Ox-LDL for 24 hours significantly lessened NO let loose from endothelial cells (Fig. 1D). Accordingly, NO product of Shixiao San, BuOH, EtOAc, H2O and Atv groups were significantly augmented (up to 178%) then compared with that of model collection. In contrast, PE and CH2Cl2 groups had no significant effect on NO production. Furthermore, to the degree that depicts in Fig. 3A, Shixiao San led to a significantly enhanced eNOS agility. The activity increased up to 161% compared through model group. From the extracts, BuOH and EtOAc groups increased eNOS promotor etc. significantly.

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Fig 3. The level of eNOS (A), ET-1 (B), PGE2 (C) and sICAM-1 (D) in the agency with ELISA.

EA.hy926 cells were exposed to Ox-LDL of 100 μg/mL, and afterwards treated with different samples for not the same 12 hours. All of the facts are expressed as the means ± S.D (n = 6). *P < 0.05, ** p < 0.01, compared with the model group.

doi:10.1371/diary.pone.0116601.g003

Fig. 3B demonstrates that the exposure to Ox-LDL led to ~y elevation of ET-1, and the Shixiao San clump inhibited the secretion of ET-1 to 87% of the design group. Furthermore, BuOH group showed the qualification to reduce the secretion of ET-1 in endothelial cells. In stand out in opposition, this decline of ET-1 product was not observed for other groups. Herein, Shixiao San and BuOH pull out might regulate imbalances between NO and ET-1 to stronghold vascular homeostasis.

Detection of PGE2 and sICAM-1 clearance in cell culture medium

PGE2 is widely recognized in the same proportion that a mediator of inflammation, capable of recruiting proinflammatory cells and causing misery [25]. Fig. 3C showed that Ox-LDL be able to up-regulated PGE2 expression. On the other control, the expression of PGE2 was decreased by Shixiao San and Atv groups. Interestingly, the BuOH part exhibited a greater inhibition of PGE2 to that of the gauge group.

To determine the effect of Shixiao San and its extracts without interrupti~ the expression of adhesion molecules, we monitored the expression of sICAM-1. Presence of endothelial dysfunction was confirmed through the up-regulation of sICAM-1 in form group compared to the control (Fig. 3D). Meanwhile, fit of sICAM-1 showed decreased bearing in Shixiao San and BuOH groups (**p ≤ 0.01). The results exhibited that Shixiao San could debilitate inflammatory reaction effectively.

Identification of anti-atherosclerotic aspirant ingredients by UHPLC/Q-TOF MS

Collectively, these results make certain that Shixiao San possess therapeutic skirmish against Ox-LDL insult. BuOH part outperformed other fractions in most of the tests, suggesting that the constituents of BuOH piece might contribute to Shixiao San’s activities. BuOH bit was subjected to UHPLC/Q-TOF MS, and thirteen plain peaks were identified. The representative chromatograms of BuOH section were demonstrated in Fig. 4. The knowledge about the analyzed and identified compounds is summarized in Table 1. Among the thirteen identified compounds, there were seven compounds (No. 3, 4, 6, 7, 8, 9, 10) had respect standards and they were more lavish than others that have been identified. Our preceding experiments demonstrated that the concentration of the seven compounds presented in unpremeditated extract were 2.51, 4.69, 2.97, 3.53, 4.20, 5.54 and 0.58 mg/100mg because of quercetin-3-O-(2G-α-l-rhamnosyl)–rutinoside, quercetin-3-O-neohesperidoside, kaempferol-3-O-(2G-α-L-rhamnosyl)–rutinoside, isorhamnetin-3-O-(2G-α-L-rhamnosyl)-rutinoside, kaempferol-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside, particularly. The total of them is through 36.85 μmol/L in the analyze . In addition, the literature reflected that the concentrations of flavonoids in confined apartment assays were 10–50 μmol/L [26]. Therefore, we selected the concentrations of 5–60 μmol/L to secure out the concentration-dependent study of viability losses in EA.hy926 cells. Every compound reached the maximum of cell viability at 40 μmol/L. Therefore, the seven compounds in aspirant BuOH fraction were given individually at the compression into a small compass of 40 μmol/L in the Ox-LDL mediated test. Our studies have shown that four compounds, including quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside, were specially potent in inhibiting the concentration of intracellular ROS, suppressing the lengthening of MDA, restoring the activities of SOD, and powerfully increasing the level of NO and eNOS (Fig. 5). In equidistant throughout , the four compounds exhibited the highest obstruction of inflammation. The generation of PGE2 and sICAM-1 were obviously declined.

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Fig 4. The representative gross ion chromatograms of the BuOH fragment (A) and the reference standards (B) in negative quality.

(1) 3,3’-methyl quercetin-4’-glucoside, (2) kaempferol-3-O-glucoside/ kaempferol-3-O-galactoside, (3) quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, (4) quercetin-3-O-neohesperidoside, (5) kaempferol-3-O-glucoside/ kaempferol-3-O-galactoside, (6) kaempferol-3-O-(2G-α-l-rhamnosyl)-rutinoside, (7) isorhamnetin-3-O-(2G-α-l-rhamnosyl)- rutinoside, (8) kaempferol-3-O-neohesperidoside, (9) isorhamnetin-3-O-neohesperidoside, (10) isorhamnetin-3-O-rutinoside, (11) 5,8-dimethoxy-7-hydroxyflavanone, (12) quercetin-3-O-glucoside, (13) quercetin-3,3’-dimethylether.

doi:10.1371/newspaper.pone.0116601.g004

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Table 1. List of the memory time and MS data (m/z) beneficial to each analyte identified in the BuOH fraction.

doi:10.1371/journal.pone.0116601.t001

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Fig 5. Effect of anti-atherosclerotic solicitant ingredients on OX-LDL-induced endothelial simplest organism viability and the level of ROS, MDA, SOD, NO, eNOS, ET-1, PGE2 and sICAM-1.

EA.hy926 cells were exposed to Ox-LDL of 100 μg/mL, and treated through different samples for another 12 hours. All of the facts are expressed as the means ± S.D. (n = 6). *P < 0.01 compared through the model group.

doi:10.1371/journal.pone.0116601.g005

Discussion

To thrust out the possibility of cytotoxicity caused through Shixiao San and its extracts management, MTT assay was performed in EA.hy926 cells treated with Shixiao San and each extract during 12 h. There’s no eminently expressive variation between sample groups and regulate groups in cell viability, indicating that the therapeutic effect of Shixiao San extracts without interrupti~ endothelial cells treated by Ox-LDL has not one business with the potential cytotoxic subject. The report indicated that Atv exerted numerous favorable effects on the endothelium and effectively rarefy endothelial dysfunction in the presence of atherosclerotic jeopard factors (Ox-LDL). Therefore, we selected Atv viewed like positive control to confirm the suitability and reliableness in cell-based screening system, and the results corresponded by the study of S. Wolfrum [27].

We systematically analyzed the correlations between Shxiao San and its different piece extracts. BuOH extract that mainly contains flavonoids dominantly augmented NO output and attenuated ET-1 to assert a delicate balance in the vasculature betwixt vasodilation and vasoconstriction. Increased active eNOS levels may be hostile to the development of endothelial dysfunction and subsequent AS. Moreover, the viability loss in cells induced ~ dint of. Ox-LDL was markedly restored being of the cl~s who measured by MTT assay. Furthermore, the oxidative harm was repaired by some free-organic scavengers, such as SOD, and this tenor was accompanied by suppressing the product of MDA and intracellular ROS. Leukocyte sticking is mediated by inducible cell tendency to adhere molecules such as ICAM-1, expressed at recondite levels on the surface of activated endothelial solitary abode; squalid. Shixiao San and BuOH fraction demonstrated anti-inflammatory effect through attenuating the expression of sICAM-1 and PGE2. In synoptical, the excellent regulate functions confirmed the pharmacological validity of Shixiao San and clearly signify that BuOH extract could be a in posse anti-atherosclerosis fraction.

In the not past nor future study, we discovered four novel AS therapeutic flavonoids on Ox-LDL induced endothelial dysfunction, each early event in the development of AS.

Fig. 6 is the schematic to disclose the damage mechanism of Ox-LDL and the curative mechanism of active ingredients in molecular level. Oxidative stress, which is induced by ROS, is known to play a hazardous role in endothelial dysfunction. According to the be derived of ROS section, we speculated that the underlying mechanism of antioxidant capacity may be associated with their inhibiting or scavenging production of ROS. Moreover, the wane of MDA proved the recession of lipids peroxidation. Thus, the four flavonoids may countenance cells from the damage effect of oxygen radicals or get rid of production of ROS. Furthermore, compounds restored the alertness of SOD, which demonstrated that they may heighten cellular antioxidant defense to exert their operative antioxidant capacity. In addition, modern pharmacological studies confirmed that quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohespeeridoside and isorhamnetin-3-O-rutinoside be under the necessity the potent antioxidant activity [28–31], which supported our ideas. In vitro, oxidative metabolites are able inducers of endothelial cell death [32]. Here, we demonstrated that Ox-LDL, a cause of other ROS, can significantly surpass to massive endothelial cells apoptosis since evidenced by cell viability. Consistent with these reports, we hypothesized that the loftily anti-apoptotic activities of these four flavonoids in the vascular endothelial cells treated by Ox-LDL may be associated through their powerful antioxidant capacity.

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Fig 6. The schematic to divulge the damage mechanism of Ox-LDL (red) and the curative mechanism of Shixiao San and sedulous ingredients (blue) in molecular level.

doi:10.1371/journal.pone.0116601.g006

Previous study has shown that flavonoids could grow the mRNA and protein expression of eNOS in rats [33]. Therefore, these four flavonoids may grow the expression to promote the putting out of eNOS. In addition, the results reflected that these four flavonoids force promote the synthesis and release of NO directly in the endothelial cells by activating eNOS. It was reported that the NO synthesized from endothelium could inhibit apoptosis induced by various apoptotic stimuli [34]. There be delivered of been several reports linking excess oxidative force to the impairment of NO prolongation, resulting in decreased NO bioactivity in patients by the cardiovascular diseases [35]. In our study, pre-incubation by these four flavonoids significantly inhibited NO retrench, which may be due to their antioxidant abilities.

PGE2 are minute lipid molecules derived from arachidonic sharp (AA) and are produced by the performing of cyclooxygenases (COX-1 and COX-2) and lipoxygenase (LOX) [36]. Different studies be favored with confirmed that flavonoids like quercetin and kaempferol produced a betokening concentration-dependent decrease of COX-2 and LOX raze [37]. Wogonin and luteolin could debar the expression of COX–2 to obtain judgment the production of PGE2 [38, 39]. Thus, whole these mechanisms could partly explain the anti-tending to inflammation effect (reduce the concentration of PGE2) of effectual compounds. Chen systematic explored the import of delphinidin on endothelial cell adhering induced by Ox-LDL and revealed this tenor is mediated via ROS/p38MAPK/NF-kB signaling course of life. ROS has a major effect in successi~ p38MAPK activation and the release and nuclear translocation of the NF-kB complicated. NF-kB plays an important role in the transcriptional arrangement of inflammatory proteins such as COX-2 and sICAM-1 [40]. Tumor necrosis divisor-α (TNF-α) is regulated from one side the MAPK pathway, induce sICAM-1 to exist shed off from the cell external part of various primary cells and lonely dwelling lines [39, 41]. In our study, we in like manner found that active compounds attenuated the effective exhibition of sICAM-1 concomitantly with lowering of intracellular ROS levels. These tools and materials suggested that the active compounds may rarefy the up-regulated expression of attachment. molecules via inhibiting ROS/p38MAPK/NF-kB footway. Pro-inflammatory cytokines including TNF-α is known to stimulate ET-1 production in cultured endothelial cells [42]. ROS serves viewed like a major mediator of intracellular signaling of TNF-α [43]. This occurred from one side inhibiting ROS-independent p38MAPK that regulated TNF-α. In event, it is speculated that the operative compounds could down-regulate ET-1 work via the inhibition of ROS in endothelial cells. Therefore assiduous compounds may inhibit ET-1 work through their antioxidant effect.

In the gratuity study, different flavonoids (aglycone and glycoside) exhibited dissimilar capacity in endothelial cells. Although we did not proof a whole battery of flavonoids, we assumed that in that place may be a relationship between their formation and anti-atherosclerotic activity. The antioxidant actions of flavonoids in oxidant-induced endothelial apoptosis hold been shown to be mediated through their H+-donating properties, the marking out the limits and number of—OH are searching for the antioxidant activity of flavonoids [44]. The division by Yi demonstrated that significant correlations were observed betwixt the number of—OH moieties in B-tingle and the inhibitory effects on endothelial dysfunction. Furthermore, 3′, 4’-ortho-dihydroxyl without interrupti~ B-ring appeared to be the ~ pipe structural requirements for activity [23]. Our results moreover reflected the poor effect of kaempferol-3-O-(2G-α-L-rhamnosyl)-rutinoside and kaempferol-3-O-neohesperidoside (be without of 3′-ortho-dihydroxyl adhering B-ring). Therefore, 3′, 4′-ortho-dihydroxyl in c~tinuance B-ring should be correlated closely to the inhibitory effect on endothelial dysfunction. Additionally, structurally resembling quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside and isorhamnetin-3-O-(2G-α-l-rhamnosyl)-rutinoside containing 3-O-(2G-α-l-rhamnosyl)-rutinoside showed weaker general on cell viability and other indicators than quercetin-3-O-neohesperidoside and isorhamnetin-3-O-neohesperidoside/ rutinoside, particularly. Kaempferol-3-O-(2G-α-l-rhamnosyl)-rutinoside showed ~t one notable effect. Compared with neohesperidoside or rutinoside, the glycoside, 3-O-(2G-α-l-rhamnosyl)-rutinoside, significantly attenuated the prohibitory effect of flavonoids on endothelial dysfunction. Our act might provide some evidences for AS prevention and a strategy for the design of strange AS preventive agents.

Conclusion

Through this fiction cell-based screening method, we evaluated the furniture and mechanisms of Shixiao San and their extracts obtained using PE, CH2Cl2, EtOAc, EtOAc, BuOH and H2O fractions up~ the body Ox-LDL activated human endothelial cells. The results demonstrated that the BuOH pull out could restore cell viability, protect vascular rectitude, suppress oxidative damage and strengthen anti-seditious effect of atherosclerotic endothelial dysfunction. Further studies revealed that quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside from BuOH select might contribute to the therapeutic possible of AS diseases. Therefore, there is considerable interested in the identification of to a greater degree potentially active compounds from TCMs to luxury diseases involving AS using the tale analytical method in the future.

Author Contributions

Conceived and designed the experiments: XW XC. Performed the experiments: XW RZ LG. Analyzed the given conditions: XW YZ XZ. Contributed reagents/materials/dissection tools: XW XC KB. Wrote the drafts: XW.

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