A novel anti-inflammatory natural product from Sphaeranthus indicus inhibits expression of VCAM1 and ICAM1, and slows atherosclerosis progression independent of lipid changes
© Srivastava et al. 2015
Received: 9 February 2015
Accepted: 29 May 2015
Published: 5 June 2015
A large body of evidence suggests that atherosclerosis is an inflammatory disease, in which cytokines and growth factors play a major role in disease progression. The methanolic extracts of Sphaeranthus indicus as well as its active ingredient, 7-hydroxy frullanoide (7-HF), are shown to suppress LPS-induced cytokine production from mononuclear cells, and inhibit the expression of VCAM1, ICAM1 and E-selectin by TNF-α- stimulated HUVECs in a concentration-dependent manner. We tested the hypothesis that the inhibition of cytokines and adhesion molecules should attenuate the progression of atherosclerosis, independent of changes in the lipid profile. Studies were carried out in two animal models: a high fat-fed LDLr-/- mouse and a high fat-fed hyperlipidemic hamster. Methanolic extract of S. indicus was dosed to hyperlipidemic LDLr-/- at 100 and 300 mg (equivalent to 20 and 60 mg 7-HF)/kg body weight/ day for 8 weeks, and plasma lipids as well as aortic lesion area were quantitated. Hyperlipidemic hamsters were treated with one dose of 200 mg/kg/day. S. indicus extract treatment did not alter the lipid profile in both animal models, but reduced aortic lesion area in LDLr-/- mice and hyperlipidemic hamsters by 22 % and 45 %, respectively. Fenofibrate, included as a reference agent, decreased aortic lesions by 26 % in LDLr -/- mice and 84 % in hyperlipidemic hamsters, respectively, which was driven by massive reductions in proatherogenic lipoproteins. The lipid-independent anti-atherosclerotic activity of S. indicus was associated with the reductions in the circulating levels of MCP-1, TNF-α, and IL-6 via phosphorylation and degradation of IkB-α that prevents translocation of NF-kB in the nucleus to induce proinflammatory cytokines. Our findings demonstrate that anti-inflammatory agents that lower pro-inflammatory proteins inhibit the progression of atherosclerosis. The methanolic extract of S. inducus, currently being used to treat psoriasis, offer promise to benefit individuals who have high circulating pro-inflammatory cytokines, and predisposed to coronary artery disease.
A number of studies for the past 15 years suggest that atherosclerosis, the main cause of coronary artery disease (CAD), is an inflammatory disease in which inflammation plays a key role in setting the stage as well as causing the progression of atherosclerosis (Reviewed in [1–4]. Immune cells are predominantly present in the early atherosclerotic lesions, and their effector molecules have been shown to accelerate progression of the lesions leading to acute coronary syndrome [3, 5]. Thus, immune mechanisms interact with metabolic risk factors to initiate, propagate, and activate lesions in the arteries. In addition to vascular endothelial and smooth muscle cells, blood borne inflammatory and immune cells constitute an important part of an atheroma, which is preceded by an accumulation of lipid-laden cells in the subendothelium [3, 6–9]. Endothelial cells recruit leukocytes by selectively expressing major adhesion molecule on the surface. Examples of specific adhesion molecules involved in initiation of atherosclerotic plaques include vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM1), and endothelial cell selectin (E-selectin) [8, 9]. The chemoattractant cytokine, monocyte chemoattractant protein-1 (MCP-1), interacts with the monocyte chemokine receptor CCR2, recruiting the monocytes to the arterial endothelium and facilitating their entry in the subendothelial space [9, 10].
During the past one decade, inflammatory nature of atherosclerosis has attracted basic, translational, and clinical researchers to find scientific basis leading to a robust link between inflammatory biomarkers and cardiovascular disease (CVD) in outcome studies. Towards this end, high sensitivity c-reactive protein (hsCRP), an acute phase reactant released during inflammatory processes [11, 12], has been recognized as a powerful predictor of traditional markers of cardiovascular risk [13–15]. Basic and clinical research data suggest that treatment with statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) to lower low-density lipoprotein (LDL) cholesterol levels also reduces hsCRP . Additional support for the anti-inflammatory and immunimodulatory actions of statins came from clinical research. Thus, the magnitude of risk reduction associated with statin therapy may exceed that expected on the basis of the LDL-C lowering alone. Prospective evidence provided by the JUPITER trial (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) demonstrated that patients with normal LDL-C levels but elevated hsCRP levels showed highly significant (-44 %) reduction in adverse cardiovascular events , suggesting additional benefit of hsCRP reduction and demonstrating an inflammatory component in the CVD risk.
Immune-mediated inflammatory disease, including atherosclerosis, psoriatric arthritis (PsA) [18–21], and rheumatoid arthritis (RA) [22–26], are characterized by common inflammatory morbidity and mortality. Chronic activation of innate and adaptive inflammatory pathways that provide an essential defense against “foreign” substances ranging from bacterial products to endogenous oxidized lipids, may contribute to atherosclerotic plaque progression, destabilization, and ultimately rupture with subsequent clinical sequelae such as myocardial infarction or stroke [20, 27].
Recently, it was demonstrated that the extract of Sphaeranthus indicus as well as its active ingredient, 7-HF, a sesquiterpene lactone, inhibits the LPS-induced release/synthesis of several pro-inflammatory mediators such as TNF-α, IL-1β and IL-6 in freshly isolated human peripheral blood monocytes . Moreover, both of these prevented constitutive proinflammatory cytokine production in primary cultures of rheumatoid synovial cells, and oral administration of 7-HF effectively suppressed the clinical signs of established arthritis in DBA/1 collagen-induced arthritis model . However, the antiatherosclerotic activities of S. indicus extract and 7-HF have not been evaluated. We hypothesized that the anti-inflammatory activities of S. indicus extract and 7-HF, shown to cause lowering of VCAM1, ICAM1, and E-selectin, may inhibit the progression of arterial lesion formation. To test this hypothesis, we employed two widely studied animal models, LDLr -/-  and hyperlipidemic hamsters , and evaluated antiatherosclerotic activities of S. indicus extract. Our results show that the antiatherosclerotic efficacy of S. indicus methanolic extract occurs via attenuation of proinflammatory cytokines and adhesion molecules, and is independent of changes in the plasma lipid profiles.
Materials and methods
RPMI 1640 medium, anti-β-actin, anti-histone, phenazine methosulfate (PMS), Dulbecco’s phosphate buffered saline (DPBS) and fetal bovine serum (FBS) were purchased from Sigma. Calpain Inhibitor I, and antibodies against IκBα phosphorylated IκBα p65 were obtained from Calbiochem (Merck Biosciences). Anti-ICAM-1 (clone BBIG-I1) anti-VCAM-1 (clone BBIG-V1) and anti-E-selectin (clone BBIG-E4), isotype control mouse IgG1 (clone 11711.11), the secondary antibody (anti-mouse IgG-HRP antibody) and bacteria derived recombinant human TNF-α were products of R&D Systems (Minneapolis, MN). Protease inhibitor cocktail was procured from Roche. The CellTiter 96® Aqueous One Solution Cell Proliferation Assay were purchased from Promega (Madison, WI).
Source of Sphaeranthus Indicus and 7-hydroxy frullanolide
Methanolic extract of the fruits of Sphaeranthus Indicus was prepared in-house and dissolved in DMSO as a 20 mg/ml stock as described . 7-hydroxy frullanolide (7 HF), isolated in-house from the above plant was dissolved in DMSO as a 20 mM stock. It was purified and identified with the use of ESI-MS and 1H- and 13C-NMR analyses .
Human peripheral blood mononuclear cells assay
Human peripheral blood mononuclear cells (PBMC) were harvested using Ficoll- Hypaque density gradient centrifugation (1.077 g/ml; Sigma Aldrich; St. Louis, MO) from healthy volunteers and suspended in assay medium [RPMI 1640 culture medium (Sigma Aldrich) containing 10 % heat inactivated fetal bovine serum (FBS; JRH Biosciences; Lenexa, KA), 100U/ml penicillin (Sigma Aldrich) and 100 μg/ml streptomycin (Sigma Aldrich)]. PBMC (2 × 105) per well were transferred into a 96-well plate. The cells were pre-treated with varying concentrations of 7HF, S. indicus extract, or 0.5 % dimethyl sulfoxide (DMSO) or 10 μM 4- (4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole [SB203580; a p38 MAPK inhibitor which is known to suppress induced production of TNF-α and IL-6; Sigma Aldrich] for 1 h at 37 °C, 5 % CO2 and stimulated with 1 μg/ml lipopolysaccharide (LPS; Escherichia coli serotype 0127:B8; Sigma Aldrich). The cells were incubated for 6 h at 37 °C, 5 % CO2 followed with collection of supernatants and assayed for TNF-α, IL-6, IL-8, and IL-1β by Enzyme-Linked Immunosorbent Assay (ELISA; OptiEIA ELISA sets; BD Biosciences). The 50 % inhibitory concentration (IC50) values were calculated by a nonlinear regression method using GraphPad software (Prism 3.03). In all experiments, a parallel plate was run to ascertain the toxicity of 7HF. The toxicity was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega; Madison, WI). In every experiment, each condition was run in triplicate wells.
Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs) and the complete medium were obtained from Cascade Biologics (Portland, Oregon). Cells were grown in endothelial cell growth medium M200 supplemented with 2 % low serum growth supplements as per the manufacturer’s recommended protocol. The growth medium was changed every other day until confluence. Cells under passage 8 were used for this study. The cells used for the experiments had a viability >98 % as determined by trypan blue exclusion test.
Evaluation for the viability of endothelial cells
The CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) was used to assess cell viability. The assay is composed of the tetrazolium compound MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent (PMS). The soluble product in the medium was measured with a spectrophotometer at 490-nm absorbance. Background absorbance from the control wells (same media, no cells) was subtracted. Cells incubated in control media were considered 100 % viable.
Cell enzyme-linked immunosorbent assay
The surface expression of endothelial cell adhesion molecules was quantified using cell enzyme-linked immunosorbent assay. Briefly, confluent HUVECs in 96-well fibronectin–coated plates were pretreated with various concentrations of S. indicus or 7-HF for 30 min before being stimulated with 1 ng/mL TNF-α for the indicated time. The expressions of ICAM-1 and E-selectin were evaluated after TNF-α stimulation for 4 h, and expression of VCAM-1 was evaluated after 6 h of stimulation. The cells were fixed with 1 % paraformaldehyde and the non-specific binding of antibody was blocked using bovine serum albumin (2 % in DPBS). The cells were then washed and incubated with monoclonal mouse anti-human ICAM-1, VCAM-1, E-selectin or the isotype control mouse IgG1 overnight at 4 °C. Subsequently, cells were washed and incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG for 90 min. Binding of the secondary antibody was determined by incubating with 3,3′,5,5′ tetramethylbenzidine (TMB) substrate from BD Biosciences (SD California) and then terminating the reaction by 2 N sulphuric acid. Surface expression of adhesion molecules was quantified by measuring absorbance at 490 nm in an automated microtitre plate reader (Spectramax, Molecular Devices, USA).
To assay IκBα, cytoplasmic extracts were prepared from cells pretreated with either S. indicus extract or 7 HF for 2 h and then stimulated with TNF-α either in the presence or absence of ALLN, a calpain inhibitor, resolved on 10 % sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, the proteins were transferred to nitrocellulose membrane, probed with antibodies against either IκBα or phosphorylated IκBα at serine 32, and detected by chemiluminescence (ECL; Sigma).
NF-κB protein localization
For the determination of NF-κB localization, Western blot analysis was carried out with cytoplasmic and nuclear extracts using anti-human NF-κB primary antibody. These extracts were prepared as per manufacturers protocol (Chemicon). Briefly, treated cells were lysed in 300 μl of hypotonic lysis buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5-5 mM DTT, 0.1 % Triton X-100, sodium orthovanadate, 1 mM and protease inhibitor cocktail. The residual pellet after cytosolic fraction collection was treated with extraction buffer containing 20 mM HEPES (pH7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 0.5-5 mM DTT, 1.0 % NP-40, 25 % (v/v) glycerol, sodium orthovanadate, 1 mM, and protease inhibitor cocktail. Twenty microgram protein were taken for Western Blot analysis as described above.
In vivo cytokine production study
Group of 10 mice were treated orally with Sphaeranthus Indicus methanolic extract, 60 min prior to lipopolysaccharide injection (1 mg/kg, i.p.). Levels of TNF-α in the plasma, were measured 1.5 h after lipopolysacchride injection, and IL-1β were done 4 b after LPS treatment.
Real-time quantitative PCR analysis
Quantitation of messenger RNA (mRNA) was done in aortic total RNA by real-time quantitative RT-PCR using an ABI Prism 7700 sequence detector. All PCR reactions were performed in a total volume of 50 μl and included the following components: cDNA derived from 20 ng of total RNA, 400 nM each of forward and reverse primers, RNase-free water, and 25 μl of Power SYBR Green PCR Master Mix (ABI), an optimized buffer system containing AmpliTaq Gold DNA polymerase and dNTPs. All PCR reactions were performed in quadruplicate using pooled cDNA samples (n = 6). Cycling parameters were as follows: after an initial denaturation step for 10 min at 95 °C, 40 subsequent cycles were performed in which samples were denatured for 15 s at 95 °C followed by primer annealing and elongation at 60 °C for 1 min. Relative quantities of mRNA were calculated from CT values using the comparative CT method (ΔΔCT;  using GAPDH as an internal reference. Primer pairs for real-time PCR were designed using Primer3 software and sequence information obtained from GenBank (NCBI).
Atherosclerosis intervention study in hamsters
Animals and diet
All animal procedures were performed as per guidelines provided by the Institutional Animal Care and Use Committee. Golden Syrian (GS) hamsters (10-12 weeks old) obtained from Charles River, were housed in groups of three in appropriately sized solid-bottom cages with contact bedding. Room lighting conditions were adjusted to 12 h light and 12 h dark cycle as follows: light between the hours of 6 AM to 6 PM and dark between the hours of 6 PM to 6 AM. All animals were allowed to acclimate for 5 days in the vivarium and were fed standard rodent chow unless otherwise mentioned before initiating the study.
After the acclimation period, baseline blood chemistry was done in animals after 12-h fasting followed by blood (700 μl – 800 μl) withdrawal by retro-orbital puncture in a 3 ml Vacutainer® tube containing K3EDTA. Blood samples were centrifuged (4000 rpm, 20 min, 4 °C) in an Eppendorf tube to obtain plasma, which was transferred (250–300 μl) into a clean tube. The aliquot of plasma sample was analyzed for triglycerides, total cholesterol, LDL-C, direct-HDL-C, glucose, AST and ALT. LDL, triglycerides, cholesterol, and glucose were quantitated on automated chemistry analyzer, Hitachi 917, using Roche diagnostic kits (Indianapolis). After bleeding, animals were earmarked for identification and then were returned to their cages and feed replaced. All animals were put on a high fat diet consisting of Purina 5001 plus 10 % coconut oil, 10 % corn oil, 0.5 % cholesterol, and 5 % fructose. This diet composition accelerates hyperlipidemia in hamsters. Animals were fed high fat diet for 4 weeks. This study started with a total of 50 animals. At the end of 4 weeks of feeding high fat high cholesterol diet, the hamsters were fasted overnight and bled as described above. Plasma samples were analyzed for total cholesterol, triglycerides, LDL-C, HDL-C, glucose, AST and ALT as described above. Based on the body weight, and blood chemistry those animals that did not develop adequate hyperlipidemia (total cholesterol >1000 mg/dl and total triglycerides >1200 mg/dl) were excluded from the study before grouping . Animals were then randomly grouped into 3 groups of 10 animals in each group as follows: group 1- vehicle control CMC 0.5 %; group 2- fenofibrate 100 mg/kg/day; group 3- methanolic extract of S. indicus 200 mg/kg/day. Animals were fed high fat high cholesterol diet and concomitantly dosed by oral gavage once daily in the morning between 8 and 9 AM. At the end of 10 weeks of treatment, hamsters were euthanized under CO2 and blood withdrawn by cardiac puncture and processed as described above. The thorax was opened and vasculature perfused first with heparinized saline (40 units/ml) for 2 min and then with 10 % formalin for 5 min. The aorta attached to heart and containing aortic arch, thoracic and abdominal aorta to the femoral artery bifurcation were removed and placed in 10 % formalin for en face staining and lesion quantitation. Aortic lipid contents were quantitated by en face staining with Oil Red O and atherosclerotic lesion area coverage determined by image analysis.
Isolated aortae from the above study were placed on to a tray containing black wax. Connective tissues sticking around the artery were removed as much as possible. Abdominal aorta and renal arteries, iliac bifurcation, aortic arch and major branches were exposed. The aorta was snipped at the heart where it leaves the heart. Major branches were snipped at approximately 0.5 cm away from where it joins the aorta. Iliac arteries were snipped from the bifurcation point. The aorta was placed into 10 % formalin for lesion measurement.
Each section of the aorta was opened longitudinally and pinned to a black wax plate using stainless steel pins. The plate containing the aorta sections was then rinsed with 70 % ethanol (5 min), immersed in Oil Red O staining solution (10 min) followed by destaining with 70 % ethanol on a shaker for 5 min at room temperature. The aorta was rinsed with deionized water and then submerged with PBS. Quantitation and imaging was done as described . Atherosclerosis burden was expressed as % lesion per area.
Atherosclerosis intervention study in LDLr-/- mice
Animals and diet
Male LDLr-/- mice (C57Bl/6 J background) were procured from Jackson Laboratories (Bar Harbor, Maine) at 6 weeks of age. Mice were allowed to acclimatize on regular chow diet for one week followed by feeding a high fat high cholesterol (HF) diet with 45 % calories from butter fat plus 0.21 % cholesterol for 2 weeks to acclimatize to the high fat diet. On an average, the food consumption was around 3 g/day. One group of mice was fed rodent chow, Purina 5001 (n = 11).
After the acclimation period of one week, baseline blood chemistry was done in animals after 4-h fasting followed by blood (150 μl) withdrawal by retro-orbital puncture tube containing K3EDTA. Each blood sample was centrifuged (4000 rpm, 20 min, 4 °C) using an Eppendorf tube, and the supernatant transferred into a new clean tube. The aliquot of plasma sample was analyzed for triglycerides and total cholesterol. After bleeding, animals were earmarked for identification and then were returned to their cages and feed replaced. All animals were put on a high fat diet consisting of Purina 5001 plus 21 % fat, and 0.21 % cholesterol.
The LDLr -/- mice on high fat diet were divided into 3 groups (n = 11/group) as follows: Group 1- Vehicle, high fat high cholesterol (HF) with 45 % calorie from butter milk and 0.21 % cholesterol; Group 2, HF diet plus Methanolic extract of S. indicus (100 mg/kg body weight/day); Group 3, HF diet plus methanolic extract of S. indicus (300 mg/kg body weight/day). Mice were fed pelleted HF diet, and test agent was administered by oral gavage in the morning once daily. New batches of dosing solution was prepared every week and stored as aliquots at 4 oC. Food was replaced with fresh food at the intervals of every 3 days. Body weights were monitored every week, and feeding continued for 8 weeks. At the end of 8 weeks of treatment, mice were bled retro-orbitally under isoflurane anesthesia, and plasma analyzed for triglycerides, cholesterol, LDL-C, HDL-C, glucose, and cytokine level. Mice were sacrificed by carbon dioxide asphyxiation. Aorta were removed, and processed for Oil Red O staining as described [34, 35]. Aortae from 4 mice in each group were isolated without formalin fixing to prepare total RNA .
Mean values of treated groups were compared to those of the vehicle treated group. Statistical significance was determined by ANOVA. Statistical comparisons across treatment groups were done. All results were presented as mean ± SD. A p value of <0.05 was considered significant.
S. indicus extract decreases TNF-α stimulated pro-inflammatory cytokines in human peripheral blood monocytic cells
S. indicus extract and 7-HF decrease the expression of VCAM-1, ICAM-1 and E-selectin by TNF-α stimulated HUVECs
The cell cytotoxicity was assessed by MTS assay. Treatment of HUVECs with 1 ng/mL of TNF-α did not result in cytotoxicity (data not shown). When incubated with 1, 3 and 10 μg/mL of S. indicus extract, cell viability was 89 ± 3 %, 89 ± 3 % and 100 ± 0 % respectively. Treatment with 0.3, 1 and 3 μM did not affect cell viability (89 ± 3 %, 89 ± 3 % and 100 ± 0 % respectively).
Effect of S. indicus extract on inhibition of TNF-α and IL-1β production in mice
Effect of S. indicus extract and 7-HF on NF-κB activation by TNF-α
Effect of S. indicus extract and 7-HF on TNF-α-induced phosphorylation and degradation of IκBα
To ascertain whether S. indicus extract inhibits IκB-α degradation by blocking its phosphorylation, the cyoplasmic extracts were examined by western blot analysis using antibodies that detect only the serine-phosphorylated form of IκB-α. The results show that the serine phosphorylated form of IκBα appears at 15 and 30 min (Fig. 5b, lanes 3 and 4), which completely disappeared when cells were pretreated with S. indicus extract (Fig. 5b, lanes 3 and 4), indicating that it completely abolished the TNF-α induced phosphorylation of IκB-α. However, the cells pretreated with 7-HF did not show similar results. While the differences in the untreated and treated groups are evident when equal amounts of protein was loaded to each lane, it should be noted that internal control was not included. Nevertheless, the results do indicate inhibition of IκBα phosphorylation.
Taken together, the cell ELISA and Western blot analysis, suggest that S. indicus extract inhibits TNF-α-induced IκBα phosphorylation and degradation, which causes a decrease in NF-κB resulting in decreased surface expression of adhesion molecules. In contrast, 7-HF, one of its bioactive principles, without affecting the phosphorylation and degradation of IκB-α, inhibits the activation of NF-κB thereby reducing the surface expression of adhesion molecules.
Effect of S. sphaericus extract on plasma lipids and atherosclerosis in hamsters
Effect of S. sphaericus extract on plasma lipids and atherosclerosis in LDLr-/- mice
Effect of S. sphaericus extract on cytokines in LDLr-/- mice
In the course of screening natural products to find novel anti-inflammatory drugs as TNF-α inhibitors we found that S. indicus extract and 7-HF, one of its active constituents, characterized as a sesquiterpene lactone, suppresses the cytokine production in a concentration-dependent manner from LPS-stimulated human peripheral blood mononuclear cells as well as from synovial cells obtained from rheumatoid arthritis patients undergoing knee replacement surgery. Most importantly, S. indicus extract was also found to arrest disease progression in an in vivo mouse model for rheumatoid arthritis . Since atherosclerosis is an inflammatory disease , we sought to investigate if anti-inflammatory activities of S. indicus inhibited atherosclerosis progression. We also looked into the mechanism of anti-inflammatory properties of S. indicus.
It has been previously documented that sesquiterpene lactones or sesquiterpene lactone-containing plant extracts exert potent anti-inflammatory effect, at least in part, through inhibition of transcription factor NF-κB , causing a decrease in adhesion molecule expression and subsequent leukocyte adhesion. Here, we show that natural product, S. indicus extract along with one of its active constituent, 7-HF mediate anti-inflammatory effects by effectively modulating the expression of cell adhesion molecules via suppression of NF-κB/IκBα signaling in TNF-α activated HUVECs.
Clearly, S. indicus extract (at the dose of 10 μg/ml) significantly inhibited the expression of the ICAM-1 (54 % inhibition), VCAM-1 (64 % inhibition), and E-selectin (88 % inhibition) in TNF-α activated HUVECs (Fig. 2). Likewise, 7-HF, a sesquiterpene lactone, (at the dose of 3 μM) dramatically inhibited ICAM-1 (75 % inhibition), VCAM-1 (90 % inhibition), and E-selectin (100 % inhibition) in TNF-α treated HUVECs (Fig. 2). Similar effects with compounds such as 1,4-Dihyroxyxanthone (65 μg/mL) , diclofenac (750 μM) , pyrrolidine dithiocarbamate (1 mM)  and N-acetyl cysteine (100 μM)  on cytokine-induced expression of cell adhesion molecules have been demonstrated by previous investigators, albeit at higher concentrations. Interestingly, S. indicus extract and one of its bioactive components, 7-HF, were found to be effective at comparatively lower concentrations and therefore, may be used in controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules such as atherosclerosis, bacterial sepsis, inflammation and tumor metastasis.
To further delineate the mechanism by which S. indicus extract and 7-HF may be acting, we studied its effect on NF-κB activation. Using Western blot analysis, we show that S. indicus extract and 7-HF both appear to block TNF-α induced p65 translocation from the cytoplasm to the nucleus. In this respect, 7-HF possibly behaves differently from other known sesquiterpene lactones  such as helenalin , parthenolide  and isodeoxyelephantopin , which exert their anti-inflammatory effect by either targeting the IκB kinase complex or inhibiting the DNA binding of p65 homodimer. The mode of action of 7-HF appears to be similar to compounds like caffeic acid phenethyl ester (CAPE)  and DHMEQ , which block NFκB by inhibiting its binding to promoter DNA without affecting the degradation of IκB-α. Because NF-κB is responsible for the induced expression of ICAM-1, VCAM-1 and E-selectin , these results suggest that both these agents inhibit NF-κB dependent transcription of cell adhesion molecule genes. However, it is quite possible that S. indicus extract and 7 HF may also inhibit other transcription factors.
In addition, we also showed that pretreatment of cells with proteasome inhibitor ALLN prevents the destruction of IκB-α and results in accumulation of phosphorylated IκB-α. On the other hand, treatment of cells with S. indicus extract did not result in the accumulation of phosphorylated IκB-α in the cytoplasm. This led us to conclude that S. indicus extract possibly prevents NF-κB by preventing phosphorylation of IκB-α rather than proteolysis per se . It is well known that the pathway leading to activation of NF-κB is common for these activators, implying that S. indicus extract acts at a molecular step common to both the LPS- and TNF pathways, which could be at the level of IKK or upstream of IKK. The effect of S. indicus extract on the cascade upstream of IKK remains to be clarified, although it is quite possible that S. indicus extract may suppress different steps in the NF-κB activation pathway.
The standardized S. indicus extract (10 μg/ml) contains approximately 0.6 μg/ml or 3 μM of 7-HF, and since we observed similar effects of S. indicus extract at 10 μg/ml and 7-HF at 3 μM (data not shown), we postulated that the effects on the expression of cell adhesion molecules and NF-κB activation seen with the crude extract of S. indicus extract may be related to its content of 7-HF. However, there are as yet uncharacterized active components besides 7-HF, which may be responsible for the additional potency of S. indicus extract that is reflected in the inhibition of IκB-α phosphorylation and degradation.
Thus, S. indicus extract and 7-HF a) significantly reduced ICAM-1, VCAM-1 and E-selectin expression; b) markedly inhibits the translocation of p65 from cytoplasm to the nucleus; and c) inhibits IκB-α phosphorylation and degradation. These activities of S. indicus extract and 7-HF suggest its potential to treat a wide variety of NF-κB-linked pro-inflammatory diseases such as rheumatoid arthritis, atherosclerosis and inflammatory bowel disease.
We, therefore, evaluated anti-atherosclerotic activities of S. indicus preparation in two widely studied animal models of atherosclerosis. Based on our in vitro results, we chose a dose equivalent to 120 mg/kg/day of active ingredient, 7-HF to examine the potential of S. indicus preparation to treat atherosclerosis. Both hamster and the LDLr-/- mice showed efficacy in terms of slowing down atherosclerosis progression without modulating circulating proatherogenic lipoproteins, suggesting that the reductions in lesion area was not due to proatherogenic lipoproteins, but other mechanism(s). This effect was very different than those observed with fenofibrate, where reductions in lesion area were associated with reductions in proatherogenic lipoproteins in hamsters  as well as in LDLr-/- mice . Reductions in IL-6, TNF-α, and MCP-1 do suggest that S. indicus preparation decreased aortic lipid deposition by reducing circulating pro-inflammatory cytokines. These data are consistent with in vitro cell-based findings that showed NF-kB-mediated down-regulation of pro-inflammatory cytokines and adhesion molecules. Natural products that inhibits NF-kB pathway lower cell adhesion molecules and pro-inflammatory cytokines, leading to reduced atherosclerotic burden [56–58] without changes in circulating lipoprotein levels.
We hypothesized that anti-inflammaory agents showing efficacy in inflammatory diseases like psoriasis, colitis and rheumatoid arthritis will inhibit atherosclerosis progression. Indeed, individuals with psoriasis [18, 20, 59–62], inflammatory bowel disease , and rheumatoid arthritis [22, 25, 64–67] have greater risks of developing coronary artery disease. These findings together with chronic inflammation in atherosclerosis [5, 68] and end stage kidney disease in diabetics  have led to increasing efforts to discover and develop anti-inflammatory agents to treat coronary artery disease . NFkB inhibitors are among anti-inflammatory agents showing anti-atherosclerotic activities [57, 58, 71]. High-density lipoproteins shown to regress atherosclerosis is believed to have an aniinflammatory component , in addition to promoting reverse cholesterol transport . The anti-atherosclerotic activities of many of the natural products like alicin , catechins , abscisic acid , marine-derived wax esters , dried plums , and a number of plant-derived flavonoids , are attributed to their anti-inflammatory activities. The anti-atherosclerosis efficacy of S. indicus preparation in the present study is not surprising, as a number of anti-inflammatory agents possess anti-atherosclerosis activities via NF-kB mediated pathways [57, 58, 71]. Sesquiterpene lactones have been shown in apoE-/- mice and guinea pigs to have anti-atherosclerosis efficacy via their anti-inflammatory activities [80, 81]. Here we showed that S. indicus preparation with major ingredient 7-HF, a sesquiterpene lactone, inhibited atherosclerosis progression in two diet-induced atherosclerosis models, LDLr knockout and hamsters.
In summary, we showed that the main component of S. indicus preparation, 7-HF, is an anti-inflammatory agent that works through NF-kB-mediated pathway, and inhibits atherosclerosis progression. These findings offer promise for further clinical benefits to patients with inflammatory and cardiovascular diseases.
The authors would like to thank Roda Dalal and Aurellio Lobo for their excellent technical support, and to Piramal Life Sciences, Mumbai for research facility.
# Part of the studies reported here was presented at the International Atherosclerosis Society, Boston, June, 2009.
- Ross R. Atherosclerosis: current understanding of mechanisms and future strategies in therapy. Transplant Proc. 1993;25:2041–3.Google Scholar
- Ross R. Atherosclerosis is an inflammatory disease. Am Heart J. 1999;138:S419–20.View ArticleGoogle Scholar
- Libby P. Inflammation in atherosclerosis. Nature. 2002;420:868–74.View ArticleGoogle Scholar
- Libby P. Inflammation and cardiovascular disease mechanisms. Am J Clin Nutr. 2006;83:456S–60.Google Scholar
- Libby P, Crea F. Clinical implications of inflammation for cardiovascular primary prevention. Eur Heart J. 2010;31:777–83.View ArticleGoogle Scholar
- Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature. 2008;451:904–13.View ArticleGoogle Scholar
- Cybulsky MI, Iiyama K, Li H, et al. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001;107:1255–62.View ArticleGoogle Scholar
- Li H, Cybulsky MI, Gimbrone Jr MA, Libby P. Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma. Am J Pathol. 1993;143:1551–9.Google Scholar
- Gu L, Okada Y, Clinton SK, et al. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998;2:275–81.View ArticleGoogle Scholar
- Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998;394:894–7.View ArticleGoogle Scholar
- Libby P, Ridker PM. Inflammation and atherosclerosis: role of C-reactive protein in risk assessment. Am J Med. 2004;116(Suppl 6A):9S–16.View ArticleGoogle Scholar
- Ridker PM, Danielson E, Fonseca FA, et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med. 2008;359:2195–207.View ArticleGoogle Scholar
- Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000;342:836–43.View ArticleGoogle Scholar
- Lemieux I, Pascot A, Prud’homme D, et al. Elevated C-reactive protein: another component of the atherothrombotic profile of abdominal obesity. Arterioscler Thromb Vasc Biol. 2001;21:961–7.View ArticleGoogle Scholar
- Saito M, Ishimitsu T, Minami J, Ono H, Ohrui M, Matsuoka H. Relations of plasma high-sensitivity C-reactive protein to traditional cardiovascular risk factors. Atherosclerosis. 2003;167:73–9.View ArticleGoogle Scholar
- Ridker PM, Rifai N, Clearfield M, et al. Measurement of C-reactive protein for the targeting of statin therapy in the primary prevention of acute coronary events. N Engl J Med. 2001;344:1959–65.View ArticleGoogle Scholar
- Ridker PM, Silvertown JD. Inflammation, C-reactive protein, and atherothrombosis. J Periodontol. 2008;79:1544–51.View ArticleGoogle Scholar
- Gisondi P, Fantin F, Del Giglio M, et al. Chronic plaque psoriasis is associated with increased arterial stiffness. Dermatology. 2009;218:110–3.View ArticleGoogle Scholar
- Prodanovich S, Kirsner RS, Kravetz JD, Ma F, Martinez L, Federman DG. Association of psoriasis with coronary artery, cerebrovascular, and peripheral vascular diseases and mortality. Arch Dermatol. 2009;145:700–3.View ArticleGoogle Scholar
- Kaye JA, Li L, Jick SS. Incidence of risk factors for myocardial infarction and other vascular diseases in patients with psoriasis. Br J Dermatol. 2008;159:895–902.View ArticleGoogle Scholar
- Yiu KH, Yeung CK, Zhao CT, Chan JC, Siu CW, Tam S, et al. Prevalence and extent of subclinical atherosclerosis in patients with psoriasis. J Intern Med. 2013;273(3):273–82. Epub 2012 Dec 28.Google Scholar
- Libby P. Role of inflammation in atherosclerosis associated with rheumatoid arthritis. Am J Med. 2008;121:S21–31.View ArticleGoogle Scholar
- Sarzi-Puttini P, Atzeni F, Gerli R, et al. Cardiac involvement in systemic rheumatic diseases: An update. Autoimmun Rev. 2010;9:849–52.View ArticleGoogle Scholar
- Rho YH, Solus J, Raggi P, et al. Macrophage activation and coronary atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Care Res. 2011;63:535–41.View ArticleGoogle Scholar
- Garcia-Bermudez M, Lopez-Mejias R, Gonzalez-Juanatey C, et al. Analysis of the Interferon Gamma (rs2430561, +874 T/A) Functional Gene Variant in Relation to the Presence of Cardiovascular Events in Rheumatoid Arthritis. PLoS One. 2012;7, e47166.View ArticleGoogle Scholar
- Deo SS, Chogle AR, Mistry KJ, Shetty RR, Nadkar UL. Increased prevalence of subclinical atherosclerosis in rheumatoid arthritis patients of Indian descent. Expn Clin Cardiol. 2012;17:20–5.Google Scholar
- Packard RR, Lichtman AH, Libby P. Innate and adaptive immunity in atherosclerosis. Semin Immunopathol. 2009;31:5–22.View ArticleGoogle Scholar
- Srivastava RAK, Dalal R, Lobo A, Parikh S, Sharma S. A Novel Anti-inflammatory Natural Product and its Active Ingredient, 7-Hydroxy Frullanolide Inhibits Expression of VCAM1 and ICAM1, and Slows Progression of Athersoclerosis. Boston: International Atherosclerosis Society Meeting; 2009.Google Scholar
- Fonseca LC, Dadarkar SS, Lobo AS, et al. 7-hydroxyfrullanolide, a sesquiterpene lactone, inhibits pro-inflammatory cytokine production from immune cells and is orally efficacious in animal models of inflammation. Eur J Pharmacol. 2010;644:220–9.View ArticleGoogle Scholar
- Srivastava RA, Jahagirdar R, Azhar S, Sharma S, Bisgaier CL. Peroxisome proliferator-activated receptor-alpha selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice. Mol Cell Biochem. 2006;285:35–50.View ArticleGoogle Scholar
- Srivastava RA, He S. Anti-hyperlipidemic and insulin sensitizing activities of fenofibrate reduces aortic lipid deposition in hyperlipidemic Golden Syrian hamster. Mol Cell Biochem. 2010;345:197–206.View ArticleGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.View ArticleGoogle Scholar
- Srivastava RA. Evaluation of anti-atherosclerotic activities of PPAR-alpha, PPAR-gamma, and LXR agonists in hyperlipidemic atherosclerosis-susceptible F(1)B hamsters. Atherosclerosis. 2011;214:86–93.View ArticleGoogle Scholar
- Paszty C, Maeda N, Verstuyft J, Rubin EM. Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest. 1994;94:899–903.View ArticleGoogle Scholar
- Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb. 1994;14:141–7.View ArticleGoogle Scholar
- Baker RG, Hayden MS, Ghosh S. NF-kappaB, inflammation, and metabolic disease. Cell Metab. 2011;13:11–22.View ArticleGoogle Scholar
- Binion DG, Heidemann J, Li MS, Nelson VM, Otterson MF, Rafiee P. Vascular cell adhesion molecule-1 expression in human intestinal microvascular endothelial cells is regulated by PI 3-kinase/Akt/MAPK/NF-kappaB: inhibitory role of curcumin. Am J Physiol Gastrointest Liver Physiol. 2009;297:G259–68.View ArticleGoogle Scholar
- Roman-Blas JA, Jimenez SA. Targeting NF-kappaB: a promising molecular therapy in inflammatory arthritis. Int Rev Immunol. 2008;27:351–74.View ArticleGoogle Scholar
- Butt C, Sun S, Peddle L, et al. Association of nuclear factor-kappaB in psoriatic arthritis. J Rheumatol. 2005;32:1742–4.Google Scholar
- Steffan NM, Bren GD, Frantz B, Tocci MJ, O’Neill EA, Paya CV. Regulation of IkB alpha phosphorylation by PKC- and Ca(2+)-dependent signal transduction pathways. J Immunol. 1995;155:4685–91.Google Scholar
- Inoguchi T, Li P, Umeda F, et al. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C–dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes. 2000;49:1939–45.View ArticleGoogle Scholar
- Charles P, Elliott MJ, Davis D, et al. Regulation of cytokines, cytokine inhibitors, and acute-phase proteins following anti-TNF-alpha therapy in rheumatoid arthritis. J Immunol. 1999;163:1521–8.Google Scholar
- Ohman MK, Wright AP, Wickenheiser KJ, Luo W, Russo HM, Eitzman DT. Monocyte chemoattractant protein-1 deficiency protects against visceral fat-induced atherosclerosis. Arterioscler Thromb Vasc Biol. 2010;30:1151–8.View ArticleGoogle Scholar
- Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999;340:115–26.View ArticleGoogle Scholar
- Hehner SP, Heinrich M, Bork PM, et al. Sesquiterpene lactones specifically inhibit activation of NF-kappa B by preventing the degradation of I kappa B-alpha and I kappa B-beta. J Biol Chem. 1998;273:1288–97.View ArticleGoogle Scholar
- Madan B, Prasad AK, Parmar VS, Ghosh B. 1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Bioorg Med Chem. 2004;12:1431–7.View ArticleGoogle Scholar
- Sakai A. Diclofenac inhibits endothelial cell adhesion molecule expression induced with lipopolysaccharide. Life Sci. 1996;58:2377–87.View ArticleGoogle Scholar
- Liu SF, Ye X, Malik AB. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate prevents In vivo expression of proinflammatory genes. Circulation. 1999;100:1330–7.View ArticleGoogle Scholar
- Nandate K, Ogata M, Tamura H, Kawasaki T, Sata T, Shigematsu A. N-acetyl-cysteine attenuates endotoxin-induced adhesion molecule expression in human whole blood. Anesth Analg. 2005;100:1453–7. Table of contents.Google Scholar
- Auld CA, Hopkins RG, Fernandes KM, Morrison RF. Novel effect of helenalin on Akt signaling and Skp2 expression in 3 T3-L1 preadipocytes. Biochem Biophys Res Commun. 2006;346:314–20.View ArticleGoogle Scholar
- Garcia-Pineres AJ, Lindenmeyer MT, Merfort I. Role of cysteine residues of p65/NF-kappaB on the inhibition by the sesquiterpene lactone parthenolide and N-ethyl maleimide, and on its transactivating potential. Life Sci. 2004;75:841–56.View ArticleGoogle Scholar
- Ichikawa H, Nair MS, Takada Y, et al. Isodeoxyelephantopin, a novel sesquiterpene lactone, potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis through suppression of nuclear factor-kappaB (nf-kappaB) activation and nf-kappaB-regulated gene expression. Clin Cancer Res. 2006;12:5910–8.View ArticleGoogle Scholar
- Fitzpatrick LR, Wang J, Le T. Caffeic acid phenethyl ester, an inhibitor of nuclear factor-kappaB, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats. J Pharmacol Exp Ther. 2001;299:915–20.Google Scholar
- Watanabe M, Dewan MZ, Okamura T, et al. A novel NF-kappaB inhibitor DHMEQ selectively targets constitutive NF-kappaB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo. Int J Cancer. 2005;114:32–8.View ArticleGoogle Scholar
- Zhu YP, Shen T, Lin YJ, Chen BD, Ruan Y, Cao Y, et al. Astragalus polysaccharides suppress ICAM-1 and VCAM-1 expression in TNF-α-treated human vascular endothelial cells by blocking NF-κB activation. Acta Pharmacol Sin. 2013;34(8):1036–42. 2013.46 Epub 2013 Jun 3.Google Scholar
- Ying Z, Kherada N, Farrar B, et al. Lipoic acid effects on established atherosclerosis. Life Sci. 2010;86:95–102.View ArticleGoogle Scholar
- Ying Z, Kherada N, Kampfrath T, et al. A modified sesamol derivative inhibits progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 2011;31:536–42.View ArticleGoogle Scholar
- Chen H, Yang J, Zhang Q, Chen LH, Wang Q. Corosolic acid ameliorates atherosclerosis in apolipoprotein E-deficient mice by regulating the nuclear factor-kappaB signaling pathway and inhibiting monocyte chemoattractant protein-1 expression. Circ J. 2012;76:995–1003.View ArticleGoogle Scholar
- Gonzalez-Gay MA, Vazquez-Rodriguez TR, Gonzalez-Juanatey C, Llorca J. Subclinical atherosclerosis in patients with psoriatic arthritis. J Rheumatol. 2008;35:2070–1. author reply 2071.Google Scholar
- Gonzalez-Gay MA, Gonzalez-Vela C, Gonzalez-Juanatey C. Psoriasis: a skin disease associated with increased cardiovascular risk. Actas Dermosifiliogr. 2012;103:595–8.View ArticleGoogle Scholar
- Balci DD, Balci A, Karazincir S, et al. Increased carotid artery intima-media thickness and impaired endothelial function in psoriasis. J Eur Acad Dermatol Venereol. 2009;23:1–6.View ArticleGoogle Scholar
- Jamnitski A, Symmons D, Peters MJ, Sattar N, McInnes I, Nurmohamed MT. Cardiovascular comorbidities in patients with psoriatic arthritis: a systematic review. Ann Rheum Dis. 2013;72(2):211–6. Epub 2012 Apr 24.View ArticleGoogle Scholar
- Gandhi S, Narula N, Marshall JK, Farkouh M. Are patients with inflammatory bowel disease at increased risk of coronary artery disease? Am J Med. 2012;125:956–62.View ArticleGoogle Scholar
- Han C, Robinson Jr DW, Hackett MV, Paramore LC, Fraeman KH, Bala MV. Cardiovascular disease and risk factors in patients with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. J Rheumatol. 2006;33:2167–72.Google Scholar
- Giles JT, Szklo M, Post W, et al. Coronary arterial calcification in rheumatoid arthritis: comparison with the Multi-Ethnic Study of Atherosclerosis. Arthritis Res Ther. 2009;11:R36.View ArticleGoogle Scholar
- Di Micco P, Ferrazzi P, Libre L, et al. Intima-media thickness evolution after treatment with infliximab in patients with rheumatoid arthritis. Int J Gen Med. 2009;2:141–4.View ArticleGoogle Scholar
- Cavagna L, Boffini N, Cagnotto G, Inverardi F, Grosso V, Caporali R. Atherosclerosis and rheumatoid arthritis: more than a simple association. Mediators Inflamm. 2012;2012:147354.View ArticleGoogle Scholar
- Libby P, Okamoto Y, Rocha VZ, Folco E. Inflammation in atherosclerosis: transition from theory to practice. Circ J. 2010;74:213–20.View ArticleGoogle Scholar
- Nusair MB, Rajpurohit N, Alpert MA. Chronic Inflammation and Coronary Atherosclerosis in Patients with End-Stage Renal Disease. Cardiorenal Med. 2012;2:117–24.View ArticleGoogle Scholar
- Imanishi T, Akasaka T. Novel strategies to target inflammatory processes in atherosclerosis. Curr Pharm Des. 2013;19(9):1616–25.Google Scholar
- Cuaz-Perolin C, Billiet L, Bauge E, et al. Antiinflammatory and antiatherogenic effects of the NF-kappaB inhibitor acetyl-11-keto-beta-boswellic acid in LPS-challenged ApoE-/- mice. Arterioscler Thromb Vasc Biol. 2008;28:272–7.View ArticleGoogle Scholar
- Bursill CA, Castro ML, Beattie DT, et al. High-density lipoproteins suppress chemokines and chemokine receptors in vitro and in vivo. Arterioscler Thromb Vasc Biol. 2010;30:1773–8.View ArticleGoogle Scholar
- Srivastava RA, Srivastava N. High density lipoprotein, apolipoprotein A-I, and coronary artery disease. Mol Cell Biochem. 2000;209:131–44.View ArticleGoogle Scholar
- Gonen A, Harats D, Rabinkov A, et al. The antiatherogenic effect of allicin: possible mode of action. Pathobiology. 2005;72:325–34.View ArticleGoogle Scholar
- Auclair S, Milenkovic D, Besson C, et al. Catechin reduces atherosclerotic lesion development in apo E-deficient mice: a transcriptomic study. Atherosclerosis. 2009;204:e21–7.View ArticleGoogle Scholar
- Guri AJ, Misyak SA, Hontecillas R, et al. Abscisic acid ameliorates atherosclerosis by suppressing macrophage and CD4+ T cell recruitment into the aortic wall. J Nutr Biochem. 2010;21:1178–85.View ArticleGoogle Scholar
- Eilertsen KE, Maehre HK, Jensen IJ, et al. A wax ester and astaxanthin-rich extract from the marine copepod Calanus finmarchicus attenuates atherogenesis in female apolipoprotein E-deficient mice. J Nutr. 2012;142:508–12.View ArticleGoogle Scholar
- Gallaher CM, Gallaher DD. Dried plums (prunes) reduce atherosclerosis lesion area in apolipoprotein E-deficient mice. Br J Nutr. 2009;101:233–9.View ArticleGoogle Scholar
- Hishikawa K, Nakaki T, Fujita T. Oral flavonoid supplementation attenuates atherosclerosis development in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2005;25:442–6.View ArticleGoogle Scholar
- Lopez-Franco O, Hernandez-Vargas P, Ortiz-Munoz G, et al. Parthenolide modulates the NF-kappaB-mediated inflammatory responses in experimental atherosclerosis. Arterioscler Thromb Vasc Biol. 2006;26:1864–70.View ArticleGoogle Scholar
- Mangathayaru K, Kuruvilla S, Balakrishna K, Venkhatesh J. Modulatory effect of Inula racemosa Hook. f. (Asteraceae) on experimental atherosclerosis in guinea-pigs. J Pharm Pharmacol. 2009;61:1111–8.View ArticleGoogle Scholar
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