Barrier protective effects of 2,4,6-trihydroxy-3-geranyl acetophenone on lipopolysaccharides-stimulated inflammatory responses in human umbilical vein endothelial cells

Article abstract of DOI:10.1016/j.jep.2016.07.032

Pharmocological relevance 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), is a phloroglucinol compound found naturally in Melicope ptelefolia. Melicope ptelefolia has been used traditionally for centuries as natural remedy for wound infections and inflammatory diseases. Aim of the study Endothelial barrier dysfunction is a pathological hallmark of many diseases and can be caused by lipopolysaccharides (LPS) stimulation. Therefore, this study aims to investigate the possible barrier protective effects of tHGA upon LPS-stimulated inflammatory responses in human umbilical vein endothelial cells (HUVECs). Materials and methods HUVECs were pretreated with tHGA prior to LPS stimulation, where inflammatory parameters including permeability, monocyte adhesion and migration, and release of pro-inflammatory mediators were examined. Additionally, the effect of tHGA on F-actin rearrangement and adhesion protein expression of LPS-stimulated HUVECs was evaluated. Results It was found that pretreatment with tHGA inhibited monocyte adhesion and transendothelial migration, reduced endothelial hyperpermeability and secretion of prostaglandin E₂ (PGE₂). Additionally, tHGA inhibited cytoskeletal rearrangement and adhesion protein expression on LPS-stimulated HUVECs. Conclusion As the regulation of endothelial barrier dysfunction can be one of the therapeutic strategies to improve the outcome of inflammation, tHGA may be able to preserve vascular barrier integrity of endothelial cells following LPS-stimulated dysfunction, thereby endorsing its potential usefulness in vascular inflammatory diseases.

Full text of DOI:10.1016/j.jep.2016.07.032

Author’s Accepted Manuscript
Barrier Protective Effects of 2,4,6-Trihydroxy-3-
Geranyl Acetophenone on Lipopolysaccharides-
Stimulated Inflammatory Responses in Human
Umbilical Vein Endothelial Cells
Yi Joong Chong, Musa Nazmi Firdaus, Alicia Ng
Chean Hui, Khozirah Shaari, Daud Ahmad Israf,
Chau Ling Tham
www.elsevier.com/locate/jep
PII:
S0378-8741(16)30455-X
http://dx.doi.org/10.1016/j.jep.2016.07.032
JEP10297
DOI:
Reference:
To appear in:
Journal of Ethnopharmacology
Received date: 18 February 2016
Revised date: 6 June 2016
Accepted date: 9 July 2016
Cite this article as: Yi Joong Chong, Musa Nazmi Firdaus, Alicia Ng Chean Hui
Khozirah Shaari, Daud Ahmad Israf and Chau Ling Tham, Barrier Protective
Effects of 2,4,6-Trihydroxy-3-Geranyl Acetophenone on Lipopolysaccharides-
Stimulated Inflammatory Responses in Human Umbilical Vein Endothelial Cells
Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.07.032
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Barrier Protective Effects of 2,4,6-Trihydroxy-3-Geranyl Acetophenone on Lipopolysaccharides-
Stimulated Inflammatory Responses in Human Umbilical Vein Endothelial Cells
Yi Joong Chong1, Musa Nazmi Firdaus1, Alicia Ng Chean Hui2, Khozirah Shaari2, Daud Ahmad Israf1,
Chau Ling Tham1*
1Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia, Serdang 43300, Malaysia.
2Faculty of Science, Universiti Putra Malaysia, Serdang 43300, Malaysia.
* Corresponding Author’s Email: chauling@upm.edu.my
Abstract
Pharmocological relevance:
2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), is a phloroglucinol compound found naturally in
Melicope ptelefolia. Melicope ptelefolia has been used traditionally for centuries as natural remedy
for wound infections and inflammatory diseases.
Aim of the study:
Endothelial barrier dysfunction is a pathological hallmark of many diseases and can be caused by
lipopolysaccharides (LPS) stimulation. Therefore, this study aims to investigate the possible barrier
protective effects of tHGA upon LPS-stimulated inflammatory responses in human umbilical vein
endothelial cells (HUVECs).
Materials and methods:
1

HUVECs were pretreated with tHGA prior to LPS stimulation, where inflammatory parameters
including permeability, monocyte adhesion and migration, and release of pro-inflammatory mediators
were examined. Additionally, the effect of tHGA on F-actin rearrangement and adhesion protein
expression of LPS-stimulated HUVECs was evaluated.
Results:
It was found that pretreatment with tHGA inhibited monocyte adhesion and transendothelial migration,
reduced endothelial hyperpermeability and secretion of prostaglandin E2 (PGE2). Additionally, tHGA
inhibited cytoskeletal rearrangement and adhesion protein expression on LPS-stimulated HUVECs.
Conclusion: As the regulation of endothelial barrier dysfunction can be one of the therapeutic strategies
to improve the outcome of inflammation, tHGA may be able to preserve vascular barrier integrity of
endothelial cells following LPS-stimulated dysfunction, thereby endorsing its potential usefulness in
vascular inflammatory diseases.
Keywords
2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), Melicope ptelefolia, phloroglucinol, barrier protective
effect, endothelial dysfunction, lipopolysaccharides.
2

Graphical Abstract
1. Introduction
The endothelium is a functional barrier that lines the blood vessel, in which this barrier plays
major roles in homeostasis including coagulation, fibrinolysis and immune responses. During
inflammation, the endothelium is activated and releases a cascade of inflammatory mediators such as
MCP-1 and IL-8 for recruitment of monocytes and neutrophils (Gavard, 2009; Zhang et al., 2011).
Additionally, the endothelium also upregulates the expression of adhesion molecules and dissociates the
tight junctional proteins to facilitate diapedesis and fluid permeation (Gavard, 2009; Harmey et al.,
2002). In between the alteration of protein expression, the endothelium permeability is increased, and
the recruitment of circulating leukocytes is heightened. Therefore this initiates the vascular barrier
3

dysfunction cascade (Thiermermann et al., 1995). This is deleterious to the host as such
hyperinflammatory activity may cause systemic shock and tissue damage, both of which may be fatal.
Therefore, intervention on such severe inflammatory state is necessary.
LPS is found on the membrane of gram-negative bacteria, and it is frequently found in high
concentration in patients who are suffering from septicemia and endotoxemia (Rivers et al., 2001).
Activation of TLR4 by LPS will lead to downstream upregulation of various pro-inflammatory signaling
molecules that play important role in propagating inflammatory responses such as NF-ĸB (Hoefen &
Berk, 2002) and MAP kinases (Sawa et al., 2008). In high concentrations, LPS may lead to fatal
conditions such as multiple organ dysfunction and septic shock (Dellinger et al., 2004), both of which
are linked to vascular barrier disruption (Angus & Van der Poll, 2013; Balk, 2000). It has been
suggested that therapeutic approaches aimed at maintaining vascular barrier integrity constitute a
potential approach in the management of sepsis (Dellinger et al., 2004; Goldenberg et al., 2011).
Melicope ptelefolia (Champ. ex Benth.) is a shrub growing in many areas of the Southeast Asia
commonly used as a traditional Chinese medicine for the treatment of inflammatory reactions including
abscesses, wound infection, eczema and dermatitis (Jiangsu New Medical School, 1986). In Malaysia,
the shoots and young leaves of Melicope ptelefolia are believed to be high in nutritional and medicinal
values and amongst the popular ‘ulam’ or green salad favored by the local Malay population. Our
research group had successfully isolated a new natural product namely 2,4,6-trihydroxy-3-geranyl
acetophenone (tHGA) from Melicope ptelefolia (Shaari et al., 2006). tHGA was identified as one of the
three marker compounds that are found abundantly in young leaves but not in mature leaves of Melicope
ptelefolia (Shuib et al., 2011). tHGA is a drug-like compound containing a phloroglucinol structural-
core as the bioactive principle (Ng et al., 2006; Shaari et al., 2006) (Figure 1).
4

Our research group previously reported significant antioxidant and nitric oxide inhibitory
activities of Melicope ptelefolia, suggesting its potential anti-inflammatory properties (Abas et al., 2006).
Our recent work also demonstrated that tHGA prevents airway inflammation in an ovalbumin-induced
murine model of acute allergic asthma. The airway hyperresponsiveness, pulmonary cellular infiltration,
goblet cell metaplasia, cytokine and cysteinyl leukotriene secretion and systemic IgE concentrations
were all reduced following systemic treatment of tHGA (Ismail et al., 2012). Bioassay studies have
shown that tHGA exerts dose-dependent inhibition against 5-lipoxygenase (5-LOX) and both
cyclooxygenase (COX) isoforms (Shaari et al., 2011), both of which play an important role in mediating
LPS-stimulated inflammation. Virdis et al, revealed that inhibition of COX-2 improved the outcome of
LPS-stimulated endothelial dysfunction due to reduction of pro-inflammatory signal transduction and
reactive oxygen species (Virdis et al., 2005), therefore giving insights on the potential of tHGA on
inhibiting LPS-stimulated vascular dysfunction. There have not been any attempts to evaluate the use of
tHGA as an adjunct therapeutic in sepsis or LPS-associated endothelial dysfunction. Hence, this study
was carried to evaluate the barrier protective effect of tHGA upon LPS-stimulated endothelial
dysfunction.
Figure 1: Structure of 2,4,6-Trihydroxy-3-Geranyl Acetophenone
5

2. Materials and Methods
2.1 Reagents
Lipopolysaccharide (O111:B5), Evans Blue, Albumin and BCECF-AM were purchased from Sigma (St
Louis, MO, USA). Sodium Fluorescein (Na-F), dimethyl sulfoxide and 3-(4,5-Dimethylthiazol-2-yl)-2-
Diphenyltetrazolium Bromide (MTT) were purchased from Amresco (Solon, OH, USA). Water soluble
tetrazolium-1 (WST-1) was purchased from Millipore (Billerica, MA, USA). Cell culture inserts and
Collagen I was purchased from BD Biosciences (Franklin Lake, NJ, USA). Alexa Fluor-488 conjugated
phalloidin was purchased from Invitrogen (Carlsbad, CA, USA). Mouse Anti-Human ICAM-1 (sc107),
Mouse Anti-Human VCAM-1 (sc-13160) and goat anti-mouse IgG (sc-2005) was purchased fron Santa
Cruz Biotechnology (Santa Cruz, CA)
2.2 Synthesis of tHGA
tHGA was synthesized according to previously described method (Ismail et al., 2012). A well stirred
mixture of phloracetophenone (1.000 g, 6 mmol), geranyl bromide (0.876 g, 4.80 mmol), and anhydrous
potassium carbonate (0.415 g, 3.00 mmol) in dry acetone (3.5 mL) was refluxed for 6 h. The reaction
mixture was filtered and evaporated under reduced pressure to give an oily orange residue that was
purified by flash column chromatography on Si gel (petroleum ether-EtOAc, 10:1) to afford 2,4,6-
Trihydroxy-3-geranylacetophenone (tHGA) as a light yellow powder; mp 128–130 °C. 1H NMR
(CD3OD) δH1.58 (3 H, s, Me), 1.63 (3 H, s, Me), 1.76 (3 H, s, Me), 2.64, (3H, s, COMe), 1.96 (2H,
q, J = 7.5 Hz), 2.06 (2H, m), 3.21 (2H, d, J = 6.5 Hz), 5.08 (1H, t, J = 7 Hz), 5.20 (1H, t, J = 6.5 Hz),
5.92 (1H, s, ArH); IR (KBr) νmax 3405, 1627 cm-1; EIMS m/z (%) [M] + 304 (38), 289 (3), 261 (9), 235
(25), 181 (100).
2.3 Cell Culture
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Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Cascade Biologics. The cells
were maintained in Endogro-LS Complete Media Kit (Merck Milipore) at 37oC in a humidified
incubator with 5% CO2. U937, a human monocytic cell line, was purchased from ATCC. The cells were
maintained at a density of 2 105 - 1 106 cells/mL in RPMI 1640 supplemented with 10% FBS. For
this study, only HUVECs between passage 3 to passage 5 were used.
2.4 MTT Cell Viability Assay
Effect of tHGA on viability of cells were examined using MTT reagent. HUVECs were grown at a
concentration of 4 103 HUVECs/well. The seeding concentration is determined based on MTT
optimization assay whereby the optimal seeding range of HUVECs for MTT is between 1.25 103 to
5 104 cells/well in 96 well plate (data not shown). On the next day, different concentrations of tHGA
with the presence of 1 µg/mL LPS were added into each individual well and co-incubated in a
humidified CO2 incubator for 24 h at 37°C. MTT (5 mg/mL) was then added followed by incubation in
CO2 incubator at 37oC of 4 h. Finally, live cells containing formazon salt were then lysed with DMSO,
and the amount of formazon salt was determined via measuring OD at 570nm using microplate reader
(Tecan Austria GmbH, Austria).
2.5 Adhesion Assay
Monocyte adhesion to HUVECs was performed as described previously with some modification (Tham
et al., 2015). Confluent HUVECs in 96 well plates were first pretreated with tHGA or Dexamethasone
for 6 h, followed by LPS stimulation for 5 h. Meanwhile, U937 cells were prelabelled with 4 µg/mL
BCECF-AM for 1 h immediately prior to co-culture. Following stimulation, HUVECs were washed with
PBS, followed by co-cultured with 1 105 U937 in 100 µL media for 1 h to allow adhesion of the
monocytes to the endothelial monolayer. Non-adherent cells were then washed thrice with PBS, and
7

adhered U937 were lysed with 0.2% Triton-X 100. The fluorescence intensity was quantified at 485-nm
excitation and 530-nm emission using a spectrofluorometer (Tecan M200 Infinite, Mannedorf,
Switzerland).
2.6 Cell Migration Assay
Cell migration assay was performed using 8 µm PET cell culture inserts. The inserts allow formation of
an inner and outer chamber, where migration of monocytes across the chambers will be measured.
Following coating of the inner chamber of the insert with 1 µg/cm2 collagen, HUVECs were seeded
until confluent on the insert membrane. On the day of the experiment, HUVECs were pretreated with
tHGA for 6 hours and stimulated with LPS for 4 h. The insert was then washed and transferred to a
clean well containing DMEM with 20% FBS. Serum starved U937 (1x106 cells/well) were added on the
upper chamber and allowed to migrate through the HUVEC monolayer for 4 h. Migrated cells on the
bottom chamber were harvested and incubated with WST-1 dye for 4 h before measuring the absorbance
at 450-nm using a spectrophotometer (Tecan M200 Infinite, Mannedorf, Switzerland).
2.7 Permeability Assay
Permeability assay was performed using a modified protocol from previous studies using Sodium
Fluorescein (Na-F) and Evans Blue Albumin (EBA) (Takata et al., 2013; Yamada et al., 2014).
HUVECs were seeded until confluent on the membrane of the insert. Upon pretreatment with tHGA for
6 h and stimulation with LPS for 4 h, the insert was washed and transferred to a clean well containing
500 µL assay buffer (136mM NaCl, 0.9mM CaCl2, 0.5mM MgCl2, 2.7mM KCl, 1.5mM KH2PO4,
10mM NaH2PO4, 25mM glucose and 10mM HEPES, pH7.4). Then, 200 µL of assay buffer containing
10µg/mL Na-F and 165µg/mL EBA was added into the insert. Both Na-F and EBA were allowed to
permeate for 10 min. The lower chamber buffer was collected after 10 min, and permeated Na-F
8

(Fluorescence intensity: 485-nm excitation, 535-nm emission) and EBA (Optical Density: 620-nm) were
measured using dual spectrophotometer/spectrofluorometer (Tecan M200 Infinite, Mannedorf,
Switzerland). The permeability index was normalized according to a previously described method
(Maruo et al., 1992).
2.8 F-Actin Staining
Phalloidin is a compound isolated from Amanita phalloides that binds specifically to F-actin. Phalloidin
conjugated with Alexa-Fluor 488 was used for staining of F-actin. HUVECs were first seeded in 4-well
chamber slides. Then, the cells were pretreated with tHGA or Dexamethasone for 6 h, followed by
stimulation with LPS for 24 h. The cells were then washed with PBS, and fixed using 4%
paraformaldehyde for 10 mins. After two washes with PBS, the cells were permeated with 0.2% Triton
X-100 for 5 mins. Then the cells were blocked with 1% BSA for 20 mins to reduce nonspecific binding.
The cells were then stained using Alexa-Fluor 488 phalloidin diluted in 1% BSA (1:200) for 20 mins
and counterstained with 1 µg/mL Hoescht 33342. Finally, the slides were mounted using MOWIOL 4-
88 Mounting Agent. F-Actin fibers were then examined using Leica DM2500 Microscope (IL, USA)
under 400x magnification.
2.9 Western Blot Analysis
Confluent HUVECs were pretreated with tHGA for 6 h prior to induction with LPS stimulation for 24 h.
The cells were lysed in RIPA lysis buffer (150mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate,
0.1% SDS, 50mM Tris, pH8.0) to obtain whole cell lysate. The protein amount in whole cell lysate was
quantified by using BCA assay. Equal amount of protein (20 μg) was electrophoresed on 8% SDS-
polyacrylamide gel prior to blotting into PVDF membrane using Mini Trans-Blot Cell (Biorad, CA).
The membrane was then blocked with 5% BSA in Tris-Buffered Saline with 0.05% Tween-20 (TBST)
9

before overnight incubation with polyclonal antibody specific for ICAM-1 (1:1000), VCAM-1 (1:1000)
or β-Actin (1:10000). After washing, the membrane was hybridized with HRP-conjugated antibody
specific to the primary antibody (1: 2000). The membrane was incubated with Western Bright Sirius
HRP Substrate (Advansta, CA) and viewed under chemiluminescence by a CCD camera imaging system
(Vilber Lourmet, Marne-la-Vallee, France). Band intensity was quantified by Image J software and
normalized to β-Actin.
2.10 ELISA for MCP-1 and PGE2
The secretion of MCP-1 (R&D Systems, Catalog no: DCP00) and PGE2 (R&D Systems, KGE004B)
was determined by enzyme immunoassay according to the manufacturer’s protocol (Minneapolis, MN,
USA). Briefly, confluent HUVECs in 96 well plate were pretreated with tHGA or Dexamethasone for 6
h, followed by induction with LPS for 24 h in 100 µL of complete media. The cell culture supernatant
were collected upon completion of LPS stimualtion for analysis using the ELISA kit, based on the
manufacturer’s protocol.
2.11 Statistical Analysis
All statistical data are expressed as means ± S.E.M of three independent experiments in triplicate. All
analyses were performed using SPSS 22.0. One-way analysis of variance (ANOVA) followed by post
hoc Dunnett’s test were used for all statistical analysis. Values of P≤0.05 were considered to be
statistically significant.
3. Results
3.1. tHGA does not affect HUVECs viability
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The results from MTT assay as per summarized in Figure 2 was used to determine the concentrations of
tHGA which are non-cytotoxic to HUVECs. The results indicated that tHGA in presence or absence
(data not shown) of LPS is non-cytotoxic at concentrations of ≤20 µM. Subsequently, assays were
performed using concentrations of 20 µM, 5 µM and 1.25 µM. Dexamethasone (10 µM) or Quercetin
(100 µM) were used as drug controls.
Figure 2: tHGA is non-cytotoxic at 20µM and below. HUVECs were seeded at 96-well plate
overnight before treatment with LPS and tHGA. tHGA is non-cytotoxic at 20 µM and below, and is
cytotoxic at 40 µM and above. Results were expressed with mean ± S.E.M. of three independent
experiments performed in triplicate. *** P ≤ 0.001 significantly different from LPS-stimulated control
without any treatment.
3.2 tHGA reduces monocyte adhesion and transendothelial migration
11

LPS is known to stimulate the expression of adhesion molecules on endothelial cells to enhance the
adhesion and transendothelial migration of leukocytes. We have examined the effect of tHGA on the
adhesion of U937 monocytes to HUVECs and subsequent migration through the HUVEC monolayer.
Figure 3A shows that upon LPS stimulation, the adhesion of U937 on HUVECs is significantly
increased in contrast to unstimulated cells. Pretreatment with tHGA or Dexamethasone caused
significant decrease in fluorescence intensity, indicating a lowered adhesion of BCECF-AM labelled
U937 to the HUVECs.
Additionally, we examined the capability of tHGA in inhibiting migration of monocytes across activated
endothelial cells. HUVECs were first pretreated with tHGA or Dexamethasone prior to LPS stimulation.
Figure 3B shows increased monocyte migration following LPS stimulation and 20 µM and 5 µM tHGA
effectively inhibited migration of U937 cells across the HUVEC monolayer.
12

Figure 3: tHGA attenuates barrier dysfunction of LPS-stimulated HUVECs. tHGA significantly
reduced monocyte adhesion (A) and transendothelial migration (B) of and through LPS-stimulated
HUVEC monolayer at 5 and 20 µM. Additionally, tHGA also significantly reduced leakage of large and
small molecules through LPS-stimulated HUVECs, at 5 and 20 µM, as per indicated by Evans Blue
Albumin leakage (C) and Sodium Fluorescein leakage (D). Results were expressed with mean ± S.E.M.
of three independent experiments performed in triplicate. *P≤0.05, **P≤0.01 and ***P≤0.001
significantly different from LPS-stimulated control without any treatment.
13

3.3 tHGA reduces permeability of LPS-stimulated HUVEC monolayer
Stimulation of endothelial cells with LPS disrupts monolayer integrity. Therefore it paved ways for
blood content, such as proteins, to infiltrate to surrounding tissues (Thiermermann et al., 1995). We
examined the effect of tHGA in mediating endothelial monolayer permeability by examining flux of
EBA and Na-F across the monolayer. There are two mechanisms for solute permeation through the
endothelium: transcellular for large molecules, and paracellular for small molecules (<30µm). In order
to mimic both mechanisms of permeation, EBA (MW=67kDa) and Na-F (MW=376 Da) were used to
trace permeation via transcellular and paracellular routes, respectively (Maruo et al., 1992). Figures 3C
and 3D show that LPS is able to increase the permeability EBA and Na-F, and tHGA is able to suppress
the permeability of both tracers at 20 µM and 5 µM. tHGA at 1.25 µM exhibited no significant effect in
inhibiting influx of both tracers. This indicated that tHGA may reduce both transcellular and paracellular
permeation.
3.4 tHGA preserves F-actin arrangement of LPS-stimulated HUVECs
Stress fibers are contractile actomyosin bundles in endothelial cells that play a role in contraction and
increase in intracellular gaps (Bogatcheva & Verin, 2008). Under normal conditions, F-actin is
distributed throughout cells with localization on the cellular periphery (Figure 4A). Upon LPS
stimulation, cytoskeleton rearrangement occurs with formation of stress fibers (4B), which are
characterized as F-actins scattered over the cells as shown. HUVECs pretreated with tHGA (Figure 4C)
and dexamethasone (Figure 4D) were shown to have less amount of stress fibers and maintained strong
localization of peripheral F-actin in comparison to untreated cells.
14

Figure 4: tHGA reduces stress fibers in LPS-stimulated HUVECs. Treated HUVECs were stained
with Alexa-Fluor 488 Phalloidin and Hoescht 33258 prior to viewing under 400x magnification. F-actin
is normally localized at the peripherals of the cells, forming dense peripheral bands (indicated with
yellow arrow) (A). The bands were disrupted and rearranged into stress fibers upon stimulation with
LPS (B). Cells with high amount of stress fibers are marked with white arrow. Pretreatment with 20 µM
tHGA (C) or 10 µM Dexamethasone elicited strong preventive effect on F-actin rearrangement, where
dense peripheral bands were still abundantly found in the cells.
3.5 tHGA inhibits expression of ICAM-1 and VCAM-1 of LPS-stimulated HUVECs
15

Cell adhesion molecules (CAMs) play major role in mediating the adhesion of monocytes towards
HUVECs. The expression of the proteins are upregulated with stimulation of LPS. Pretreatment of
tHGA at 20µM and 5µM was found to be effective in suppressing the expression of the proteins (Figure
5). Such inhibitory effect of tHGA may be one of the many factors that causes decreased monocyte
adhesion and migration, ultimately exhibiting barrier protective effect on LPS-stimulated HUVECs.
Figure 5: tHGA suppresses protein expression of ICAM-1 and VCAM-1. Pretreatment of 20 µM
and 5 µM tHGA significantly suppressed the expression of ICAM-1 (A) and VCAM-1 (B). Results
were expressed with mean ± S.E.M. of three independent experiments performed in triplicate. *P≤0.05,
**P≤0.01 and ***P≤0.001 significantly different from LPS-stimulated control without any treatment.
3.6 tHGA inhibits PGE2, but does not inhibit MCP-1 release in LPS-stimulated HUVECs
16

Prostaglandin E2 (PGE2) and monocyte chemoattractant protein-1 (MCP-1) play important roles in LPS-
stimulated inflammation. Upon stimulation by LPS, PGE2 released by endothelial cells will upregulate
inflammation (Engström et al., 2012) and enhance vascular dysfunction (Omori et al., 2014).
Meanwhile, MCP-1 is an important chemoattractant protein which recruits circulating monocytes to the
inflammatory region.
Figure 6 summarizes the effect of tHGA on PGE2 and MCP-1 production by LPS-stimulated HUVECs.
Both PGE2 and MCP-1 concentration were significantly increased by 1.6 fold and 13.5 fold respectively
upon stimulation with LPS. tHGA pretreatment effectively inhibited PGE2 release. In particular, 20µM
tHGA inhibited PGE2 secretion to near non-stimulated levels. However, tHGA is ineffective in
inhibiting MCP-1 secretion at all tested concentrations.
Figure 6: tHGA inhibits PGE2 secretion but not MCP-1. tHGA significantly inhibited the secretion
of PGE2 secretion (A) at 5 and 20 µM, but not MCP-1 secretion (B). Results were expressed with mean
± S.E.M. of three independent experiments performed in triplicate. *** P ≤ 0.001 significantly different
from LPS-stimulated control without any treatment.
17

4. Discussion
The endothelium respond to physical and chemical signals through regulation of vascular tone,
cellular adhesion, thrombo-resistance, smooth muscle cell proliferation, and vessel wall inflammation
(Deanfield et al., 2007). When activation becomes exaggerated and sustained, the endothelial responses
are detrimental to host and may lead to loss of selective permeability, resulting in vascular leakage that
may eventually lead to shock (Lee and Slutsky, 2010). LPS from gram-negative bacteria can initiate the
septic cascade through altered regulation of proadhesive properties and increased permeability (Aird,
2003). Its effect on the endothelium frequently leads to shock due to excessive fluid leakage. Therefore
prevention of vascular leakage can be used as a method in managing LPS-stimulated systemic
inflammation such as endotoxemia and sepsis (Goldenberg et al., 2011).
Phloroglucinol is a class of compound that is commonly found in plant extracts. Prior study
reported that phloroglucinol alone was able to inhibit LPS-induced barrier disruption in human
endothelial cells (Bae, 2012). Moreover, phlorotannin, which is a compound that contains
phloroglucinols as common monomer unit, was found to possess barrier protective effect on HMGB1-
mediated proinflammatory responses (Kim et al., 2012). The compound of interest in this study, 2,4,6-
trihydroxy-3-geranyl acetophenone (tHGA), is one of the marker compounds that is originally found in
the young leaves of Melicope ptelefolia. It has been reported to exhibit several anti-inflammatory effects,
including anti-asthmatic effect (Ismail et al., 2012) and dual LOX/COX inhibitory effect (Shaari et al.,
2011). Inhibition on COX-2 has been found to be effective in improving the outcome of LPS-stimulated
endothelial dysfunction. Therefore, the barrier protective effect of tHGA on LPS-stimulated endothelial
barrier dysfunction was explored in this study.
18

As upregulation of COX-2 derived PGE2 secretion has been observed to promote alteration of
barrier integrity in LPS-stimulated endothelial cells (Engström et al., 2012), we examined the effect of
tHGA on PGE2 secretion released by LPS-stimulated HUVEC monolayers. The results demonstrated
that PGE2 secretion was strongly inhibited by tHGA. One possible explanation for this inhibition could
be the dual inhibitory effect of tHGA on 5-LOX and COX expression (Shaari et al., 2011). LPS
stimulates cells via the TLR4 receptor, which subsequently initiates the arachidonic acid cascade. In
order to synthesize PGE2, COX-2 plays a major role in converting arachidonic acid to prostaglandin H2
(PGH2), which is the precursor of PGE2. Therefore, it is hypothesized that tHGA may have inhibited
COX-2 activity, which subsequently reduced production of PGH2 and lastly, PGE2.
In LPS-stimulated endothelial barrier dysfunction, inflamed endothelia are known to express
high levels of MCP-1 and cell adhesion molecules (CAMs) to facilitate recruitment and binding of
monocytes to the endothelium to initiate the extravasation process (Sprague & Khalil, 2009). As
adhesion events often happen early in the inflammatory process and are essential in the regulation of
extravasation, thus blocking the adhesion events may disrupt the leukocyte extravasation and all
downstream events to prevent leukocytes from entering into the affected tissue (Simmons, 2006). In this
study, tHGA was shown to reduce adhesion of activated U937 on LPS-stimulated HUVECs, as well as
the subsequent transendothelial migration of U937 through LPS-stimulated HUVECs. Such results
suggest that tHGA may have effects on molecules that play important roles in mediating monocyte
adhesion and transendothelial migration, namely MCP-1 and CAMs.
Monocyte chemoattractant protein-1 (MCP-1) is one of the key chemokines that regulate
migration and infiltration of monocytes/macrophages (Deshmane et al., 2009). It is interesting to know
the above barrier protective effects exerted by tHGA is solely due to inhibition on the adhesion events
that further disrupts subsequent leukocyte transendothelial migration, or also partly due to inhibition on
19

the secretion of MCP-1 by activated endothelium. Therefore, we investigated the effect of tHGA on
MCP-1 secretion through ELISA. Interestingly, our findings demonstrated no effects of tHGA upon
MCP-1 secretion by LPS-stimulated HUVECs. As leukocyte extravasation can be resulted from
morphological changes of the EC layer itself or/and inhibition of chemoattractants that target the
transmigration of leukocytes (Schenkel et al., 2002), the mode of action of tHGA in endothelial
dysfunstion may not MCP-1-dependent. Instead, tHGA might protect the EC layer itself through
attenuation of LPS-stimulated CAMs expression and endothelial hyperpermeability.
We therefore examined the effects of tHGA on another critical factor that affects adhesiveness of
endothelial cells: CAMs. Two types of CAMs have been found to play major role in mediating firm
adhesion of circulating leukocytes towards the endothelial monolayer: ICAM-1 and VCAM-1. Both the
CAMs are known to bind specifically to the integrins that are expressed in circulating monocytes, which
would lead to firm adhesion of monocytes to endothelium (Muller, 2009). Under normal condition, both
CAMs are expressed at low level by endothelial cells, and external stimulation such as TNF-α and LPS
significantly increase the expression of these proteins. Therefore, we examined the effects of tHGA on
the protein expression of ICAM-1 and VCAM-1 in LPS-stimulated HUVECs in this study. Our results
from Western Blot analysis showed that tHGA strongly decreased the protein expression of both LPS-
stimulated ICAM-1 and VCAM-1. As explained above, lowered expression of CAMs leads to reduction
of the endothelium-monocyte interaction and prevention of extravasation. This therefore leads to our
conclusion that the endothelial barrier protective effect of tHGA is primarily due to suppression of
CAMs expression, but not through MCP-1-dependent chemoattraction.
During endotoxemia, vascular permeability is significantly increased in order to facilitate
movement of protein and cells through the blood vessels. It is one of the hallmarks of endothelial barrier
dysfunction. Our results have demonstrated that tHGA at 5 and 20 µM are effective in inhibiting both
20

EBA permeation and Na-F permeation. In particular, vascular dysfunction for HUVEC treated with 20
µM tHGA is near to basal level. These results suggested that tHGA is potentially effective in lowering
endothelial vascular permeability via both transcellular and paracellular routes.
This finding was further confirmed by another experiment which demonstrated decreased stress
fiber formation in LPS-stimulated HUVECs following pretreatment with tHGA. Vascular integrity is
directly related to the cytoskeletal elements and cell-cell contact protein complexes. LPS activates
several cytoskeletal rearranging pathways, such as the GEF-H1/Rho pathway which may lead to
cytoskeletal rearrangement and pave ways to increase intercellular gaps and promote vascular
permeability (Guo et al., 2012; Wolfson et al., 2011). This is most easily observed in stress fiber
formation, which are actomyosin complexes that assist in the contraction of cells upon disruption of
tight junctions and adherens junctions. The downregulation of stress fibers may be partly responsible for
the barrier protective effect of tHGA. Collectively, inhibition on CAMs expression, endothelial
permeability and F-actin cytoskeleton rearrangement indicates that tHGA acts directly on the HUVECS
to elicit barrier protective function.
5. Conclusion
The inhibition of vascular endothelial inflammatory responses is considered a promising target for the
treatment of many vascular diseases such as atherosclerosis, shock, heart attack and sepsis (Lee et al.,
2012). Herein we described the effect of tHGA on LPS-stimulated HUVECs, in which tHGA is able to
reduce the expression of adhesion proteins, formation of stress fibers, permeability, adhesion and
leukocyte transmigration. Therefore as a whole, it is safe to conclude that tHGA is a potent anti-
inflammatory compound with barrier protective potential. With such, the compound may have potential
in treating diseases with LPS as the etiology. The mechanisms behind the endothelial barrier protective
21

properties of tHGA including its effect on mediators and the specific molecular target of tHGA that
leads to preservation of the barrier integrity should also be studied. In additionally, the compound should
be studied for its effect on in vivo disease models to maximize the compound’s potential as a potential
therapy for treating inflammatory diseases.
Conflict of Interests
The authors do not have any conflict of interests regarding the publication of this paper.
Acknowledgments
The authors thank Zulkhairi Zainol and Norasyikin Salim for excellent technical assistance. This
investigation was financially supported by Ministry of Education Malaysia under Fundamental Research
Grant Scheme (FRGS) no. 04-01-13-1195FR and Universiti Putra Malaysia under Putra Grant (GP-IPS)
no. GP-IPS/2014/9433948.
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