Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells

Accepted Manuscript

Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells Lai Yen Fong , Chin Theng Ng , Zhi Li Cheok , Mohamad Aris Mohd Moklas , Muhammad Nazrul Hakim , Zuraini Ahmad PII: DOI: Reference:

S0944-7113(15)00384-0 10.1016/j.phymed.2015.11.019 PHYMED 51943

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

14 October 2015 17 November 2015 26 November 2015

Please cite this article as: Lai Yen Fong , Chin Theng Ng , Zhi Li Cheok , Mohamad Aris Mohd Moklas , Muhammad Nazrul Hakim , Zuraini Ahmad , Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells, Phytomedicine (2016), doi: 10.1016/j.phymed.2015.11.019

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Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells

Muhammad Nazrul Hakima and Zuraini Ahmada,*

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Lai Yen Fonga, Chin Theng Nga, Zhi Li Cheoka, Mohamad Aris Mohd Moklasb,

Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti

Putra Malaysia, Serdang, Selangor, Malaysia.

Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti

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Putra Malaysia, Serdang, Selangor, Malaysia.

* Corresponding author.

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Zuraini Ahmad

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Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

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Phone number : +60389472313 E-mail address: [email protected]

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ABSTRACT Background: Endothelial cell activation is characterized by increased endothelial permeability and increased expression of cell adhesion molecules (CAMs). This allows monocyte adherence and migration across the endothelium to occur and thereby initiates atherogenesis process. Asiatic acid is a major triterpene isolated from Centella asiatica

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(L.) Urban. and has been shown to possess anti-oxidant, anti-hyperlipidemia and antiinflammatory activities.

Purpose: We aimed to investigate protective effects of asiatic acid on tumor necrosis

factor-α (TNF-α)-induced endothelial cell activation using human aortic endothelial cells (HAECs).

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Study design: For cell viability assays, HAECs were treated with asiatic acid for 24 h. For other assays, HAECs were pretreated with various doses of asiatic acid (10 – 40 μM) for 6 h followed by stimulation with TNF-α (10 ng/ml) for 6 h.

Methods: Fluorescein isothiocyanate (FITC)-dextran permeability assay was performed using commercial kits. Total protein expression of CAMs such as E-selectin, ICAM-1,

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VCAM-1 and PECAM-1 as well as phosphorylation of IκB-α were determined using western blot. The levels of soluble form of CAMs were measured using flow cytometry.

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Besides, we also examined the effects of asiatic acid on U937 monocyte adhesion and monocyte migration in HAECs using fluorescent-based assays.

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Results: Asiatic acid significantly suppressed endothelial hyperpermeability, increased VCAM-1 expression and increased levels of soluble CAMs (sE-selectin, sICAM-1,

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sVCAM-1 and sPECAM-1) triggered by TNF-α. Neither TNF-α nor asiatic acid affects PECAM-1 expression. However, asiatic acid did not inhibit TNF-α-induced increased monocyte adhesion and migration. Interestingly, asiatic acid suppressed increased

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phosphorylation of IκB-α stimulated by TNF-α. Conclusion: These results suggest that asiatic acid protects against endothelial barrier disruption and this might be associated with the inhibition of NF-κB activation. We have demonstrated a novel protective role of asiatic acid on endothelial function. This reveals the possibility to further explore beneficial effects of asiatic acid on chronic inflammatory diseases that are initiated by endothelial cell activation.

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Keywords Asiatic acid; TNF-α; Human aortic endothelial cells; Cell adhesion molecules; NF-κB

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Abbreviations HAECs, human aortic endothelial cells; TNF-α, tumor necrosis factor-α; CAMs, cell adhesion molecules; sCAMs; soluble form of cell adhesion molecules; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1;

PECAM-1, platelet endothelial cell adhesion molecule-1; sE-selectin, soluble E-selectin;

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sICAM-1, soluble ICAM-1; sVCAM-1; soluble VCAM-1; sPECAM-1, soluble PECAM1; NF-κB, nuclear factor-κB; IκBα, inhibitor of NF-κB alpha; FITC-dextran, fluorescein isothiocyanate conjugated-dextran

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Introduction

Atherosclerosis is a chronic inflammatory disease initiated by endothelial dysfunction

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and ultimately develops into coronary heart disease. Hyperlipidemia, diabetes and hypertension are risk factors known to initiate atherogenesis. In early stage of

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atherosclerosis, endothelial dysfunction is accompanied by activation of endothelial cells that involves a complex interplay between leukocytes, endothelial cells and cytokines

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(Sitia et al., 2010). In response to pro-inflammatory stimuli, the surface expression of cell adhesion molecules (CAMs) such as E-selectin, intercellular adhesion molecule (ICAM)1 and vascular cell adhesion molecule (VCAM)-1 are up-regulated in endothelial cells.

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These favor the recruitment of circulating leukocytes and hence promote firm attachment between leukocytes and endothelial cells. In addition, ICAM-1 and VCAM-1 serve as signaling molecules that promote the production of reactive oxygen species (ROS), a key player of TNF-α-induced increased permeability (van Wetering et al., 2003; Wolf et al., 2013). Following the binding of monocytes to endothelium, platelet endothelial cell adhesion molecule (PECAM)-1 regulates the transmigration of monocytes across the blood vessel wall. Enzymatic cleavage of the surface CAMs results in the secretion of 3

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soluble form of cell adhesion molecules (sCAMs) such as soluble E-selectin (sE-selectin), soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1) and soluble PECAM-1 (sPECAM-1) (Leeuwenberg et al., 1992). The level of sICAM-1 is elevated in hyperlipidemic individuals and associated with high cardiovascular risk (Karasek et al., 2005). Importantly, an increase in endothelial permeability to small molecules occurs in

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parallel with the diapedesis of monocytes, leading to disruption of endothelial barrier that subsequently ease the foam cell formation (Funk et al., 2012). Therefore, natural

compounds that protect against early atherogenic events that occur before the formation of atherosclerotic lesion might have beneficial effects in preventing atherosclerosis. Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, is found to be

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expressed in atherosclerotic lesion (Barath et al., 1990). During the progression of

atherosclerosis, TNF-α sustains and propagates the inflammatory response by increasing the surface expression of CAMs, stimulating the production of inflammatory cytokines and chemokines as well as enhancing endothelial permeability. Although the molecular pathways that lead to TNF-α-induced increased permeability are not well understood,

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accumulating evidence in recent years has suggested that the CAMs are central mechanisms in mediating endothelial hyperpermeability stimulated by TNF-α (Frank and

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Lisanti, 2008; Marcos-Ramiro et al., 2014). Nuclear factor-κB (NF-κB) family consists of a group of transcription factors that

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regulate various cellular processes such as inflammation, immune response and programmed cell death. In cytoplasm, the binding of these transcription factors with an

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inhibitory protein, inhibitor of NF-κB-alpha (IκBα), prevents them from entering the nucleus. Upon activation, IκBα phosphorylates and undergoes degradation while

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releasing the bound NF-κB dimer into the nucleus. The transcription cascades of many pro-inflammatory genes are then being initiated. Thus, inhibition of the NF-κB pathway might be a promising therapeutic strategy to prevent inflammatory diseases that are perpetuated by cytokines. Asiatic acid is one of the pentacyclic triterpenoids isolated from Centella asiatica (L.) Urban., a traditional medicinal plant that is commonly found in swampy areas of most tropical countries including India, China, Indonesia, Malaysia and other Asian countries. 4

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C. asiatica is well known for its wound healing and neuroprotective effects in Ayurvedic medicine. In Malaysia and Thailand, it is consumed as raw vegetables or blended into juice and served as tonic drinks (Hashim, 2011). The lipid lowering effects of C. asiatica extract were previously reported by other groups. C. asiatica extract was shown to reduce total cholesterol, triglyceride and plasma glucose in hyperlipidemic rats (Supkamonseni

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et al., 2014). A fraction of ethanol extract of C. asiatica was reported to improve lipid profiles in chemical-induced hyperlipidemic mice and high fat diet induced-hamster models (Zhao et al., 2014). In several clinical studies, total triterpenic fraction of C.

asiatica (TTFCA) was demonstrated to prevent the progression of atherosclerotic plaques in asymptomatic patients when administered in combination with Pycnogenol, a pine

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bark extract (Belcaro et al., 2015a; Belcaro et al., 2015b). In particular, TTFCA has been shown to improve capillary permeability in hypertensive patients, and this is associated with reduction of microcirculatory symptoms (Belcaro et al., 1990; De Sanctis et al., 2001). Anti-inflammatory, anti-angiogenesis and anti-oxidant effects of asiatic acid have also been reported previously (Huang et al., 2011; Kavitha et al., 2011; Pakdeechote et al.,

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2014). A study conducted using diabetic rats has reported that the ability of asiatic acid to reduce plasma glucose level might be associated with the hypolipidemic effect of asiatic

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acid (Ramachandran et al., 2014). Besides, asiatic acid also improves lipid profile of metabolic syndrome rats through maintaining the equilibrium between iNOS and eNOS

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expression (Pakdeechote et al., 2014). Besides, asiatic acid protects against high fat diet-induced liver injury in mice through

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inhibiting the NF-κB and mitogen-activated protein kinase (MAPK) pathways (Yan et al., 2014). These previous data suggest that asiatic acid possesses potential lipid lowering and anti-inflammatory effects. However, anti-atherosclerotic effects of asiatic acid and its

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underlying mechanisms are not well documented. Therefore, the aim of this study was to investigate the protective effects of asiatic acid on early atherogenic events, in the context of endothelial cell activation triggered by cytokines. We examined the in vitro effects of asiatic acid on TNF-α-induced increased endothelial permeability, expression of adhesion molecules, monocyte adhesion and monocyte migration. In addition, the effect of asiatic acid on NF-κB activation elicited by TNF-α was also explored in this study.

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Materials and methods Chemicals and reagents Asiatic acid was purchased from ChromaDex (CA, USA) with the purity of 93.7% (Supplementary material S1), 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-

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acetoxymethyl ester (BCECF-AM) was purchased from Sigma (MO, USA). Rabbit polyclonal anti-ICAM-1 antibody, mouse monoclonal anti-E-selectin, anti-VCAM-1 and anti-PECAM-1 antibodies and anti-mouse IgG HRP-conjugated were purchased from Santa Cruz Biotechnology (Texas, USA). Rabbit monoclonal anti-phospho-IκBα

antibody and anti-rabbit HRP-conjugated secondary antibody were purchased from Cell

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Signaling Technology (MA, USA). TNF-α was purchased from Peprotech (NJ, USA).

Simvastatin and methyl thiazoyltetrazolium (MTT) were purchased from Calbiochem (NJ, USA). Cell culture

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Human aortic endothelial cells (HAECs) were purchased from American Type Cell Culture (ATCC) and maintained in endothelial cell medium (Sciencell, CA, USA)

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supplemented with 5% fetal bovine serum, endothelial cell growth supplement, penicillin (100 U/ml) and streptomycin (100 µg/ml). The medium was changed every two days until the cells were 80 - 90% confluent. All the experiments were performed using cells

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between passage 3 to 5. U937 cells, a suspension human leukemic monocyte lymphoma cell line, were purchased from ATCC and maintained in RPMI-1640 medium containing

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10% Hyclone fetal bovine serum (Thermo Fisher Scientific, IL, USA). The cell density was maintained at 1 x 105 to 2 x 106 cells per ml.

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For all the experiments except the cell viability assays, HAECs were pretreated with asiatic acid (10, 20, 30 and 40 μM) for 6 h before stimulated with TNF-α (10 ng/ml) for another 6 h. Cell viability assays Cell viability was assessed using MTT assay as described previously (Ng et al., 2015). HAECs were treated with various concentrations of asiatic acid (10 - 200 µM) for 24 h. 6

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After 24 h, we added 10 µl of MTT solution (5 mg/ml in PBS) into each well and incubated for 4 h. Then, all the solution was removed and 100 µl of DMSO was added to dissolve the purple formazan salt formed. The absorbance was read at 570 nm with a reference wavelength of 650 nm. In addition, cell viability was also assessed by a fluorometric-based assay. ATP fluorometric assay kit (Biovision, CA, USA) was used to

excitation/emission wavelengths of 535 nm/587 nm. Permeability assay in vitro

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quantify the ATP level in viable cells. Fluorescence intensity was read at

In vitro Vascular Permeability Assay kit (Milipore, MA, USA) was used to measure the

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passage of fluorescein isothiocyanate (FITC) conjugated-dextran across HAECs

monolayer according to manufacturer’s protocol. HAECs were grown in collagen-coated cell culture inserts for 3 days to allow the formation of cell monolayers. The inserts were placed in a 24-well plate where the bottom wells were filled with 500 µl of endothelial cell medium. After treatment, 150 µl of FITC- dextran was added to the inserts and

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incubated for 20 min. At the end of experiment, 100 µl of media was collected from the bottom wells and transferred to a black 96-well plate. Fluorescence intensity was

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measured at wavelengths of 485 nm excitation and 535 nm emission using a fluorescence microplate reader (Infinite M200, TECAN, Männedorf, Switzerland). Results are expressed as a percentage compared to control (Relative fluorescence unit fluorescence unitcontrol x 100%).

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treatment/Relative

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Measurement of soluble CAMs in cell culture supernatant The concentrations of sE-selectin, sP-selectin, sICAM-1, sVCAM-1 and s-PECAM-1 in

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cell culture supernatant were measured by using human adhesion 6plex kit (eBioscience, Vienna, Austria) according to the manufacturer’s protocol. The fluorescent intensity of samples was acquired using BD FACS Calibur flow cytometer (BD Biosciences, NJ, USA). Standards were run in parallel with samples for each independent experiment and the concentrations of samples were obtained from the standard curve.

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Fluorescence labeling of U937 monocytes U937 monocytes at a concentration of 3 x 106 cells per ml were fluorescently labeled with 2 µM of BCECF-AM in RPMI-1640 medium for 45 min at 37 oC and 5% CO2. The

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monocytes were washed twice with PBS containing 0.5% BSA to remove unbound dye before resuspended in endothelial cell medium. BCECF-AM-labeled U937 monocytes were then used for monocyte adhesion and migration assays. U937 monocyte adhesion assay

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U937 monocyte adhesion assay was carried out according to the procedures previously described with some modifications (Ang et al., 2011). BCECF-AM-labeled monocytes with a density of 1 x 105 cells per well were added onto the monolayers of HAECs and incubated for 30 min at 37 oC and 5% CO2. The monolayers were washed three times

with PBS to remove non-adhering U937 monocytes. The attached monocytes were then

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lysed with 0.1% Triton X-100 in 0.1M NaOH. The fluorescence intensities were measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

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The fluorescence intensities of known numbers of labeled monocytes were also measured and used to construct a standard curve for each set of experiment. The number of attached monocytes for each group was calculated from the standard curve. Results are expressed

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as a percentage compared to normal control. For qualitative analysis, HAECs were grown onto 22 mm collagen-coated coverslips (BD Bioscience, NJ, USA). At the end of

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treatment, BCECF-AM-labeled monocytes were added to the HAECs and non-binding monocytes were removed by gently washing the cell monolayer with PBS. Then, the cells

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were fixed in 3.7% paraformaldehyde and mounted with ProLong Gold antifade agent (Molecular Probes, OR, USA). 5 random fields were captured for each independent experiment using a Leica DM2500 fluorescence microscope (Leica Microsystems Vertrieb GmbH, Wetzlar, Germany). Migration assay in vitro

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A fluorescent-based transendothelial migration assay was performed as described (Ramirez et al., 2008) with some modifications. Cell culture inserts with the pore size of 3 µm (BD Biosciences, NJ, USA) were coated with rat tail collagen type I (BD Biosciences, NJ, USA). HAECs were grown onto the collagen-coated inserts at a density of 2 x 105 cells per insert for three days to allow confluent monolayers to be formed.

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After treatment, the BCECF-AM-labeled U937 monocytes were added to the upper chamber and incubated at 37 oC for 2 h to allow transmigration of monocytes to the lower chamber. Media from the lower chamber was collected and transferred to a black opaque 96-well plate for the measurement of fluorescence intensity using a fluorescence

microplate reader (Infinite M200, TECAN, Männedorf, Switzerland) at excitation and

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emission wavelengths of 485 nm and 535 nm, respectively. The fluorescence intensities for known numbers of labeled monocytes were used to plot standard curves and the number of migrated monocytes was calculated from the curve. Western blot analysis

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The cells were lysed in RIPA buffer. The supernatant was collected and the protein concentrations were quantified using bicinchoninic acid (BCA) protein assay reagent kit

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(Pierce, Rockford, USA). Equal amount of proteins were loaded onto 10-15% polyacrylamide gel and resolved by SDS-PAGE. The proteins were transferred onto a PVDF membrane, blocked in 5% BSA for 1 h and incubated with the following primary

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antibodies; ICAM-1 (1:3000 dilution), VCAM-1 (1:1000 dilution), E-selectin (1:2000 dilution), PECAM-1 (1 : 3000 dilution), phospho-IκBα (1:1000 dilution). The membranes

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were washed three times, 5 min each, and incubated with HRP-linked secondary antibody (1:5000 dilution). Chemiluminescent signal was developed using LuminataTM Forte

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Western HRP substrate (Milipore, MA, USA). Densitometry analysis was performed using Image J software. Statistical analysis The results are expressed as the mean ± standard mean of error (SEM). All the data were analyzed with one way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was considered as statistically significant. 9

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Results

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Asiatic acid does not decrease viability of HAECs In order to ensure all the concentrations of asiatic acid used in this study does not affect the viability of HAECs, MTT assay was performed by incubating the cells with various concentrations of asiatic acid for 24 h. Asiatic acid, when applied up to 40 µM, did not cause significant cell death (Fig. 1A). These results are further confirmed with a more

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sensitive fluorometric assay that measures the ATP level in viable cells. We obtained

similar results as MTT assay where 40 µM of asiatic acid did not significantly reduce the amount of ATP compared to control cells (Fig. 1B). Therefore, 10 - 40 µM were chosen as the treatment dosages in this study. Taken together, the inhibitory effects of asiatic acid in all the subsequent experiments were not due to cytotoxic effect in HAECs.

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Asiatic acid suppresses TNF-α-induced endothelial barrier disruption in HAECs

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The amount of FITC-labeled dextran that passed through the HAECs monolayer was used to indicate the integrity of endothelial barrier. As shown in Fig. 2, TNF-α significantly impaired the barrier integrity and caused an increase in permeability to

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156.8 ± 4.8 % of control. Asiatic acid significantly abolished the TNF-α-induced increased permeability in a dose-dependent manner (P < 0.05). However, asiatic acid

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alone did not alter the baseline permeability. We used simvastatin, a drug that is commonly used in clinical practice to prevent atherosclerosis, as a positive control for our

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assays. Simvastatin significantly decreased the hyperpermeability stimulated by TNF-α to 104.9 ± 3.45 % of control (Fig. 2). These results indicate that asiatic acid maintains the barrier integrity and prevents the barrier disruption induced by TNF-α. Asiatic acid decreases TNF-α-induced increased release of soluble CAMs Non-activated HAECs released sVCAM-1 and sICAM-1 at 2.67 ± 0.01 ng/ml and 9.30 ± 1.78 ng/ml, respectively; while sPECAM-1 was secreted at a relatively higher level, 21.9 10

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± 0.96 ng/ml (Fig. 3). Non-activated HAECs did not secrete sE-selectin. Upon stimulation with 10 ng/ml of TNF-α, the concentrations of sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1 in the cell culture supernatant were significantly increased to 176.6 ± 31.94, 191.5 ± 7.16, 39.78 ± 0.34 and 272 ± 16.62 ng/ml, respectively (P < 0.05). Asiatic acid significantly reduced the sE-selectin, sICAM-1 and sPECAM-1 levels at all

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doses tested (P < 0.05) (Fig. 3). The suppressive effects were not dose dependent. Asiatic acid also significantly suppressed increased sVCAM-1 level at 20 - 40 µM in a dose

dependent manner (P < 0.05). Furthermore, the concentrations of all sCAMs were not

significantly different from control group when cells were treated with asiatic acid alone (40 µM). As shown in Fig. 3, simvastatin reduced TNF-α-induced protein secretion of

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sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1 (P < 0.05). In summary, asiatic acid

inhibits the secretion of sCAMs, which are biological markers used in the risk prediction of cardiovascular diseases.

Asiatic acid inhibits increased total protein expression of VCAM-1 triggered by TNF-α

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In order to investigate whether asiatic acid could also suppress total protein expression of CAMs, total expression of E-selectin, ICAM-1, VCAM-1 and PECAM-1 were detected

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using western blot. Non-stimulated HAECs did not express E-selectin and VCAM-1; while ICAM-1 and PECAM-1 were expressed (Fig. 4A). When cells were induced with TNF-α, E-selectin, ICAM-1 and VCAM-1 expression were significantly increased (P <

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0.05) (Fig. 4A). However, TNF-α did not alter PECAM-1 expression. Asiatic acid significantly reduced TNF-α-enhanced VCAM-1 protein expression by 46% when

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applied at 40 µM (P < 0.05) (Fig. 4B). 10, 20 and 30 µM of asiatic acid also decreased up-regulated VCAM-1 by 11, 13 and 20 %, respectively, but this reduction did not reach

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statistical significance. Asiatic acid also slightly decreased the up-regulated ICAM-1 expression (between 15- 21%) at all doses tested but these did not differ significantly from TNF-α- induced group (Fig. 4B). Besides, asiatic acid did not suppress TNF-αincreased E-selectin at all doses. Neither TNF-α nor asiatic acid change the total expression of PECAM-1 in HAECs. Asiatic acid alone at 40 µM did not affect the total expressions of all CAMs in non-stimulated HAECs. On the other hand, simvastatin showed diverse responses on the expression of CAMs. Simvastatin lowered the increased 11

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ICAM-1 expression; however, it further augmented the TNF-α-increased VCAM-1 and E-selectin (Fig. 4B). PECAM-1 expression was not altered by simvastatin. Collectively, asiatic acid decreases VCAM-1 expression but does not alter the increased expression of E-selectin and ICAM-1 evoked by TNF-α. In addition, asiatic acid does not affect the total PECAM-1 expression in HAECs.

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Asiatic acid does not reduce adhesion and migration of monocytes elicited by TNF-α

To evaluate whether asiatic acid could inhibit TNF-α-induced adhesion and migration of monocytes due to its suppressive effects on the increased total VCAM-1 expression,

monocyte adhesion assay and migration assay were performed under static condition.

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Qualitative analysis showed that there were only a few monocytes adhered to the control HAECs (Fig. 5A). Upon exposure to TNF-α, the number of adherent monocytes dramatically increased. Neither asiatic acid nor simvastatin displayed fewer number of adherent monocytes, compared to TNF-α-induced group (Fig. 5A). The quantitative measurement of fluorescent intensity revealed that TNF-α caused a threefold increase in

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monocyte adhesion to HAECs compared to non-stimulated cells (P < 0.05) (Fig. 5B). Pretreatment of asiatic acid at all doses did not inhibit the increased monocyte adhesion

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elicited by TNF-α (Fig. 5B). When treated with asiatic acid alone, HAECs showed no significant difference with normal cells (Fig. 5A). Consistent with qualitative analysis,

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simvastatin also failed to inhibit TNF-α-induced increased monocyte adhesion to HAECs. In monocyte migration assay, TNF-α enhanced the migration of monocytes through

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HAECs monolayer (P < 0.05) (Fig. 6). However, neither asiatic acid nor simvastatin reduced the enhanced monocyte transmigration (Fig. 6). These results demonstrate that asiatic acid does not inhibit TNF-α-induced increased adherence of monocytes to the

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endothelium and the subsequent migration of monocytes across the cell monolayers. Asiatic acid decreases phosphorylation of IκB-α triggered by TNF-α Activation of the NF-κB pathway leads to up-regulation of the expression of cell adhesion molecules and endothelial barrier disruption in response to inflammatory cytokines such as TNF-α. Thus, the effect of asiatic acid on phosphorylation of IκB induced by TNF-α was investigated to determine whether barrier protective effects of 12

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asiatic acid involve NF-κB pathway. Administration of TNF-α to HAECs significantly increased phosphorylation level of IκB to approximately two folds higher, compared to non-activated HAECs (P < 0.05) (Fig. 7A). Asiatic acid significantly decreased the TNFα-stimulated increased phosphorylation of IκBα at 20, 30 and 40 µM by 46, 53, 50 %, respectively (P < 0.05) (Fig. 7B). HAECs treated with asiatic acid alone did not affect

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basal phosphorylation level of IκBα in non-stimulated cells. Simvastatin also suppressed increased phosphorylation of IκBα by 95 % (P < 0.05) (Fig. 7B). In summary, asiatic acid might work by inhibiting NF-κB activation in HAECs.

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Discussion

Anti-inflammatory and anti-hyperlipidemia effects of asiatic acid, a pentacyclic triterpenoid derived from C. asiatica, have been demonstrated previously using animal models (Huang et al., 2011; Ramachandran et al., 2014). Yet, whether asiatic acid possesses anti-atherogenic effect remains unknown. In particular, the effects of asiatic

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acid on endothelial cell activation have not been demonstrated yet. In the present study, we showed that asiatic acid inhibited endothelial hyperpermeability, the increased levels

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of all the sCAMs (sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1) and the upregulated protein expression of VCAM-1 in TNF-α-activated HAECs. However, asiatic acid failed to inhibit the increased E-selectin and ICAM-1 protein expression while PECAM-1

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expression remained unchanged. Furthermore, asiatic acid did not decrease monocyte adhesion and migration in HAECs. Importantly, asiatic acid suppressed increased

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phosphorylation of IκBα stimulated by TNF-α. The concentrations of asiatic acid used in the present study were ranged from 10 - 40 µM. These concentrations are in agreement

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with previous in vitro studies using various cell types. For example, 10 - 30 µM of asiatic acid was shown to inhibit collagen expression in human keloid fibroblast, acting through the PPAR-γ pathway (Bian et al., 2013) while 5 – 20 µM of asiatic acid inhibited VEGFinduced angiogenesis in human umbilical vein endothelial cells and human brain microvascular endothelial cells (Kavitha et al., 2011). Besides, 70 – 120 μM of asiatic acid was demonstrated to inhibit NFκB pathway in RAW 264.7 macrophage cell line

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(Patil et al., 2015) and 30 – 120 μM of asiatic acid was shown to possess antiinflammatory effects in RAW 264.7 macrophage cell line (Yun et al., 2008). Our data clearly showed that asiatic acid enhances endothelial barrier function by suppressing the passage of FITC-dextran. Improvement of endothelial barrier function may serves as a therapeutic strategy in chronic inflammatory diseases, such as

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atherosclerosis, as endothelial hyperpermeability initiates atherogenesis by allowing

influx of low density lipoprotein into the subendothelial space. A recent study conducted by our group has demonstrated that asiaticoside, the glycoside of asiatic acid, inhibits

endothelial hyperpermeability and prevents redistribution of filamentous (F)-actin elicited by TNF-α in HAECs (Fong et al., 2015). Based on our observations, asiatic acid exerts

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stronger suppressive effect on TNF-α-induced endothelial hyperpermeability, compared to asiaticoside. Indeed, aglycones such as asiatic acid have been shown to be biologically more active than their respective glycosides (Caballero-George et al., 2004). In the view to elucidate the protective effects of asiaticoside against early atherogenic

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events, we extended our observation to the roles of CAMs. Asiatic acid improves TNF-αinduced endothelial activation by selectively inhibited VCAM-1 expression. Surprisingly,

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the increased productions of all sCAMs were significantly decreased by asiatic acid. Plasma levels of sCAMs are considered as important biomarkers of the endothelial function and elevated levels of sCAMs are greatly correlated with the occurrence of

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future cardiovascular diseases or death (Ribeiro et al., 2009). Our data showed that asiatic acid might be beneficial in lowering the risk of cardiovascular events. Interestingly, a

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recent study reported that sVCAM-1 disrupts the integrity of blood-brain barrier through binding with its receptor, integrin α-4, on brain microvascular endothelial cells, while

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sICAM-1 did not affect the permeability of the blood-brain barrier (Haarmann et al., 2015). Upon binding with lymphocytes, VCAM-1 increases the production of ROS and destabilizes adherens junctions (Vockel and Vestweber, 2013). Based on these data, we suggest that the barrier protective effect of asiatic acid might be associated with its inhibitory effects on increased sVCAM-1 level and VCAM-1 expression stimulated by TNF-α. Moreover, our findings also showed that the degree of suppression of asiatic acid on the secretions of sCAMs are much higher than its inhibitory effect on total expression 14

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of CAMs. This implies that asiatic acid may interfere with the process of proteolytic cleavage that releases sCAMs from the cell surface. Future studies are needed to investigate the effect of asiatic acid on proteolytic cleavage process. Asiatic acid has been shown to inhibit NF-κB pathway in various animal models (Yan et al., 2014). A previous study reported that anti-inflammatory effect of asiatic acid may act

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via NF-κB pathway by decreasing phosphorylation of IκBα in LPS-induced RAW 264.7 macrophage at 30, 60 and 120 µM by 30 %, 60 % and 90 %, respectively (Yun et al.,

2008). In agreement to these previous studies, we showed that asiatic acid inhibited TNFα-induced phosphorylation of IκBα in HAECs at 20-40 µM by inhibitory percentages of 45-53 %. These data suggest that the protective effect of asiatic acid against endothelial

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cell activation might involve reduction of NF-κB activation. It is well established that

NF-κB regulates the transcription of ICAM-1 and VCAM-1 genes. Some triterpenoids have been shown to act selectively by suppressing VCAM-1 expression without affecting ICAM-1 expression in cytokine-induced endothelial cells (Moon et al., 2011). Besides, the transcription of ICAM-1 gene is regulated by several transcription factors as ICAM-1

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promoter possesses different binding sites for NF-κB, activator protein 1 (AP-1) and signal transducers and activators of transcription (STAT). Hence, it is possible that asiatic

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acid does not inhibit transcription factors other than NF-κB, leading to its inability to decrease ICAM-1 protein expression in TNF-α- stimulated HAECs. As a consequence,

assays.

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asiatic acid also failed to exert suppressive effects in monocyte adhesion and migration

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Statins, inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, are widely used in clinical practice to reduce plasma cholesterol level. In addition to their

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cholesterol-lowering effect, simvastatin has been shown to possess pleiotropic effects including improvement of endothelial function, suppression of vascular inflammation, inhibition of platelet activation and smooth muscle cell proliferation. However, experimental data on how simvastatin regulates the expression of CAMs are controversial. Previous studies reported that simvastatin down regulates both the mRNA and protein expression of ICAM-1 and VCAM-1 as well as suppresses the increased leukocyte adhesion stimulated by cytokines in endothelial cells (Zapolska-Downar et al., 2004). On 15

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the contrary, simvastatin potentiates the expression of ICAM-1 and VCAM-1 in cytokine-treated endothelial cells (Pozo et al., 2006). We reported that simvastatin suppressed ICAM-1 protein expression while augmented the expression of VCAM-1 and E-selectin in TNF-α-stimulated HAECs. PECAM-1 expression was not affected by simvastatin. Furthermore, simvastatin also decreased the levels of sICAM-1, sVCAM-1,

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sE-selectin and sPECAM-1. Unexpectedly, simvastatin did not inhibit the increased monocyte adhesion and migration induced by TNF-α under static condition. The vascular protective effects of simvastatin observed in vivo could not be demonstrated in the

present study and this may relate to the static condition of the assays performed. A

previous study reported that the potentiating effects of simvastatin on the expression of

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CAMs observed in static condition are prevented by application of a unidirectional

laminar shear stress to the endothelial cells (Rossi et al., 2010). Nevertheless, we showed that simvastatin suppressed TNF-α-induced phosphorylation of IκB-α, which indicates that the barrier protective effect of simvastatin might be partly mediated through

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suppression of the NF-κB pathway.

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Conclusions

In conclusion, the protective effects of asiatic acid on early atherogenic events triggered

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by TNF-α were demonstrated through the suppression of endothelial hyperpermeability, secretions of sCAMs (sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1) and total VCAM-1 protein expression. The barrier protective effect of asiatic acid might correlate

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with its inhibitory effects on NF-κB activation. However, asiatic acid does not reduce the interaction between monocytes and endothelial cells. This study also provides new

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insights into the roles of TNF-α on activation of HAECs particularly in barrier function and expression of CAMs. Our findings suggest a novel protective role of asiatic acid on endothelial function and enrich the existing data on the biological activities of asiatic acid.

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Acknowledgment This study was funded by Research University Grant Scheme (RUGS) from Universiti

Conflict of interest The authors declared that there is no conflict of interest.

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Figure legends Fig. 1. Cell viability assays for HAECs treated with various concentrations of asiatic acid (200 – 10 µM). (A) MTT assay was carried out to determine the cell viability in HAECs after incubation with asiatic acid for 24 h. (B) ATP level in HAECs was measured using a fluorometric method to further confirm the cell viability in HAECs treated with asiatic

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acid for 24 h. Control refers to cells treated with media only. Data are expressed as a percentage compared to control and shown as mean ± SEM from three independent experiments. AA, asiatic acid. * P < 0.05 compared to control.

Fig. 2. Effect of asiatic acid on TNF-induced increased permeability of HAECs. HAECs grown on collagen-coated cell culture inserts were pretreated with asiatic acid for 6 h or

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simvastatin for 24 h and followed by stimulation with TNF-α (10 ng/ml) for 6 h. The fluorescent intensities of FITC-dextran in bottom receiver wells were quantified at

excitation/emission wavelengths of 485 and 535 nm, respectively. Data are expressed as a percentage compared to control and the values shown are mean ± SEM from three independent experiments. AA, asiatic acid; Sim, simvastatin. # P < 0.05 compared to

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control; * P < 0.05 compared to TNF-α.

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Fig. 3. Effect of asiatic acid on release of sCAMs in TNF-α-stimulated HAECs. HAECs were pretreated with asiatic acid for 6 h or simvastatin for 24 h before induced with TNFα (10 ng/ml) for 6 h. The concentrations of sE-selectin, sICAM-1, sVCAM-1 and

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sPECAM-1 in cell culture supernatant were measured using a flow cytometer. The values shown are mean ± SEM for three independent experiments. AA, asiatic acid; Sim,

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simvastatin . # P < 0.05 compared to control; * P < 0.05 compared to TNF-α. Fig. 4. Effect of asiatic acid on increased total protein expressions of E-selectin, ICAM-1,

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VCAM-1 and PECAM-1 in HAECs stimulated by TNF-α. HAECs were pretreated with asiatic acid for 6 h or simvastatin for 24 h before induced with TNF-α (10 ng/ml) for 6 h. Western blot analysis was used to determine the total expressions of CAMs. (A) A representative blot from three independent experiments was shown. (B) Densitometry analysis of the protein expressions of E-selectin, ICAM-1, VCAM-1 and PECAM-1 was performed using Image J. Data are expressed as fold change relative to TNF-α group. The

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values shown are mean ± SEM for three independent experiments. AA, asiatic acid; Sim, simvastatin . # P < 0.05 compared to control; * P < 0.05 compared to TNF-α. Fig. 5. Effect of asiatic acid on TNF-α-induced increased monocyte adhesion in HAECs. HAECs were pretreated with asiatic acid for 6 h or simvastatin for 24 h before induced with TNF-α (10 ng/ml) for 6 h. BCECF-AM-labeled U937 monocytes were co-incubated

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with HAECs for 45 min. Qualitative and quantitative analysis were performed. (A)

Fluorescent images for adhered U937 monocytes were captured at 10x. (a) control (b)

TNF-α (c) AA 40 µM + TNF-α (d) AA 30 µM + TNF-α (e) AA 20µM + TNF-α (f) AA 10 µM + TNF-α (g) AA 40 µM alone (h) simvastatin + TNF-α. (B) Relative

fluorescence unit for each well was measured and results are expressed as a percentage

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compared to control. The values shown are mean ± SEM for three independent

experiments. AA, asiatic acid; Sim, simvastatin . # P < 0.05 compared to control. Fig. 6. Effect of asiatic acid on monocyte migration stimulated by TNF-α in HAECs. HAECs were pretreated with asiatic acid for 6 h or simvastatin for 24 h before induced

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with TNF-α (10 ng/ml) for 6 h. U937 monocytes labeled with BCECF-AM were added to the cell culture inserts and incubated for 2 h. The bottom wells were filled with

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endothelial cell media containing 30 ng/ml of monocyte chemoattractant protein-1 (MCP1) to create chemokine gradient. The fluorescent intensity of the bottom well represents the number of migrated monocytes. Results are expressed as a percentage compared to

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control. The values shown are mean ± SEM for three independent experiments. AA, asiatic acid; Sim, simvastatin . # P < 0.05 compared to control.

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Fig. 7. Effect of asiatic acid on TNF-α-induced phosphorylation of IκBα in HAECs. HAECs were pretreated with asiatic acid for 6 h or simvastatin for 24 h before induced

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with TNF-α (10 ng/ml) for 6 h. Western blot analysis was performed using total cell lysate. (A) A representative blot from three independent experiments was shown. (B) Densitometry analysis was carried out using Image J. Data are expressed as fold change relative to TNF-α group. The values shown are mean ± SEM for three independent experiments. AA, asiatic acid; Sim, simvastatin . # P < 0.05 compared to control; * P <

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Graphical Abstract

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