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Short communication
Arborvitae (Thuja plicata) essential oil significantly inhibited critical
inflammation- and tissue remodeling-related proteins and genes in human
dermal fibroblasts
Xuesheng Han*, Tory L. Parker
d
oTERRA International, LLC, 389 S. 1300 W., Pleasant Grove, UT 84062, USA
Received 21 December 2016; revised 6 February 2017
Available online 20 February 2017
Abstract
Arborvitae (Thuja plicata) essential oil (AEO) is becoming increasingly popular in skincare, although its biological activity in human skin
cells has not been investigated. Therefore, we sought to study AEO's effect on 17 important protein biomarkers that are closely related to
inflammation and tissue remodeling by using a pre-inflamed human dermal fibroblast culture model. AEO significantly inhibited the expression
of vascular cell adhesion molecule 1 (VCAM-1), intracellular cell adhesion molecule 1 (ICAM-1), interferon gamma-induced protein 10 (IP-10),
interferon-inducible T-cell chemoattractant (I-TAC), monokine induced by interferon gamma (MIG), and macrophage colony-stimulating factor
(M-CSF). It also showed significant antiproliferative activity and robustly inhibited collagen-I, collagen-III, plasminogen activator inhibitor-1
(PAI-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2). The inhibitory effect of AEO on increased production of
these protein biomarkers suggests it has anti-inflammatory property. We then studied the effect of AEO on the genome-wide expression of
21,224 genes in the same cell culture. AEO significantly and diversely modulated global gene expression. Ingenuity pathway analysis (IPA)
showed that AEO robustly affected numerous critical genes and signaling pathways closely involved in inflammatory and tissue remodeling
processes. The findings of this study provide the first evidence of the biological activity and beneficial action of AEO in human skin cells.
©2017 The Authors. Published by Elsevier B.V. on behalf of Socie
´te
´Franc¸aise de Biochimie et Biologie Mole
´culaire (SFBBM). This is an open
access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Arborvitae essential oil; Vascular cell adhesion molecule 1; Intracellular cell adhesion molecule 1; Interferon gamma-induced protein 10; Interferon-
inducible T-cell chemoattractant; Collagen-III
1. Introduction
Arborvitae (Thuja plicata), also known as western red
cedar, and its essential oils have been traditionally used as a
natural insect repellent and wood preservative, primarily
because of its insecticidal and antimicrobial property [1e3].
Recently, the topical use of Arborvitae essential oil (AEO) for
skincare has gained popularity. However, a literature search
revealed no existing studies of the biological activities of AEO
in human cells. Therefore, we evaluated the biological activ-
ities of a commercially available AEO in a pre-inflamed
human dermal fibroblast culture model, which was designed
to model the disease biology of chronic skin inflammation.
First, we analyzed the effect of AEO on 17 important protein
biomarkers that are closely related to inflammation and tissue
remodeling. Then, we studied its effect on genome-wide gene
expression in the same cell culture.
2. Materials and methods
All experiments were conducted using a BioMAP system
HDF3CGF, which was designed to model the pathology of
chronic inflammation in a robust and reproducible manner.
Nonstandard abbreviations: AEO, Arborvitae (Thuja plicata) essential oil.
*Corresponding author.
E-mail address: lhan@doterra.com (X. Han).
Available online at www.sciencedirect.com
ScienceDirect
Biochimie Open 4 (2017) 56e60
http://www.journals.elsevier.com/biochimie-open
http://dx.doi.org/10.1016/j.biopen.2017.02.003
2214-0085/©2017 The Authors. Published by Elsevier B.V. on behalf of Socie
´te
´Franc¸aise de Biochimie et Biologie Mole
´culaire (SFBBM). This is an open access
article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
The system comprises three components: a cell type, stimuli to
create the disease environment, and a set of biomarker (pro-
tein) readouts to examine how the treatments affected the
disease environment [4].
2.1. Cell culture
Primary human neonatal fibroblasts (HDFs) were prepared
as previously described [5] and were plated under low serum
conditions for 24 h before stimulation with a mixture of
interleukin (IL)-1b, tumor necrosis factor (TNF)-a, interferon
(IFN)-Y, basic fibroblast growth factor (bFGF), epidermal
growth factor (EGF), and platelet-derived growth factor
(PDGF). The cell culture and stimulation conditions for the
HDF3CGF assays have been described in detail elsewhere and
were performed in a 96-well plate [5,6].
2.2. Protein-based readouts
Direct enzyme-linked immunosorbent assay (ELISA) was
used to measure the biomarker levels of cell-associated and
cell membrane targets. Soluble factors in the supernatants
were quantified using either homogeneous time-resolved
fluorescence (HTRF) detection, bead-based multiplex immu-
noassay, or capture ELISA. The adverse effects of the test
agents on cell proliferation and viability (cytotoxicity) were
measured using the sulforhodamine B (SRB) assay. For pro-
liferation assays, the cells were cultured and measured after
72 h, which is optimal for the HDF3CGF system, and the
detailed procedure has been described in a previous study [5].
Measurements were performed in triplicate wells, and a
glossary of the biomarkers used in this study is provided in
Supplementary Table S1.
2.3. RNA isolation
Total RNA was isolated from cell lysates using the Zymo
Quick-RNA MiniPrep kit (Zymo Research Corp., Irvine, CA,
USA) according to the manufacturer's instructions. RNA con-
centration was determined using a NanoDrop ND-2000 system
(Thermo Fisher Scientific). RNA quality was assessed using a
Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA,
USA) and an Agilent RNA 6000 Nano kit. All samples had an
A260/A280 ratio between 1.9 and 2.1 and a RIN score >8.0.
2.4. Microarray analysis of genome-wide gene
expression
The effect of 0.011% AEO on the expression of 21,224
genes was evaluated in the HDF3CGF system after a 24-h
treatment. Samples for microarray analysis were processed by
Asuragen, Inc. (Austin, TX, USA) according to the company's
standard operating procedures. Biotin-labeled cRNA was
prepared from 200 ng of total RNA using an Illumina Total-
Prep RNA Amplification kit (Thermo Fisher Scientific) and
one round of amplification. The cRNA yields were quantified
using ultraviolet (UV) spectrophotometry, and the distribution
of the transcript sizes was assessed using the Agilent Bio-
analyzer 2100. Labeled cRNA (750 ng) was used to probe
Illumina human HT-12 v4 expression bead chips (Illumina,
Inc., San Diego, CA, USA). Hybridization, washing, staining
with streptavidin-conjugated cyanine-3, and scanning of the
Illumina arrays were carried out according to the manufac-
turer's instructions. The Illumina BeadScan software was used
to produce the data files for each array; the raw data were
extracted using Illumina BeadStudio software.
The raw data were uploaded into R [6] and analyzed for
quality-control metrics using the beadarray package [7]. The
data were normalized using quantile normalization [8], and
then re-annotated and filtered to remove probes that were non-
specific or mapped to intronic or intragenic regions [9]. The
remaining probe sets comprised the data set for the remainder
of the analysis. The fold-change expression for each set was
calculated as the log
2
ratio of AEO to the vehicle control.
These fold-change values were uploaded onto Ingenuity
Pathway Analysis (IPA, QIAGEN, Redwood City, CA, USA,
www.qiagen.com/ingenuity) to generate the networks and
pathway analyses.
2.5. Reagents
AEO (d
oTERRA Intl., UT, USA) was diluted in dimethyl
sulfoxide (DMSO) to 8the specified concentrations (final
DMSO concentration in culture media was no more than 0.1%
[v/v]). Then, 25 mL of each 8solution was added to the cell
culture to obtain a final volume of 200 mL, and DMSO (0.1%)
served as the vehicle control. The gas chromatography-mass
spectrometry (GCeMS) analysis of AEO indicated that it
mainly contained methyl thujate (53%) and smaller amounts
of numerous other aromatic molecules.
3. Results and discussion
3.1. Bioactivity profile of AEO in pre-inflamed HDFs
We analyzed the biological activity of AEO by using an
HDF3CGF cell system, which simulated the microenvironment
of inflamed human skin cells with already boosted immune
responses and inflammatory levels. None of the four studied
concentrations (0.011, 0.0037, 0.0012, and 0.00041%, v/v) was
overtly cytotoxic, and therefore, the activity of 0.011% con-
centration was included for analysis. Key activities of bio-
markers were designated if biomarker values were significantly
different (p <0.05) from those of vehicle controls, outside of
the significance envelope, with an effect size of at least 10%
(>0.05 log ratio units, Fig. 1) and are discussed below.
The expressions of several inflammatory biomarkers, such
as vascular cell adhesion molecule 1 (VCAM-1), intracellular
cell adhesion molecule 1 (ICAM-1), interferon gamma-
induced protein 10 (IP-10), interferon-inducible T-cell che-
moattractant (I-TAC), and monokine induced by interferon
gamma (MIG), significantly decreased in response to AEO
(Fig. 1). Specifically, the levels of these protein biomarkers
were already highly elevated in the pre-stimulated inflamed
57X. Han, T.L. Parker / Biochimie Open 4 (2017) 56e60
dermal fibroblasts. The inhibitory effects of AEO on the
increased production of proinflammatory biomarkers suggest
that it might possess anti-inflammatory properties.
AEO also showed significant antiproliferative activity in
dermal fibroblasts, as measured using the SRB proliferation assay
72 h after treatment. The levels of five tissue remodeling mole-
culesdcollagen-I, collagen-III, plasminogen activator inhibitor-1
(PAI-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-
1 and TIMP-2)dsignificantly decreased in response to AEO
treatment. AEO also significantly inhibited the level of macro-
phage colony-stimulating factor (M-CSF), a cytokine that me-
diates macrophage differentiation and thus, immunomodulation.
It is noteworthy that the inhibitory effects of AEO on the
increased production of these protein biomarkers were
concentration-dependent. AEO inhibited all these factors, which
suggests that it might play important roles in tissue remodeling
and immunomodulation, and thus, the wound healing processes.
These effects of AEO are presumably mediated by slowing down
the tissue repair process, which reduces the chance of scar for-
mation or improper chronic wound healing [10,11].
Recent studies on the essential oils of T. plicata-related
species, their major active components, or both have shown
preliminary evidence of their therapeutic efficacy and safety in
disease models [12e14]. We conducted a literature search and
found that no study has been conducted on the effects of AEO
or its major component methyl thujate in human cells or
similar models. Therefore, to the best of our knowledge, the
current study is the first evidence of the biological activities of
AEO in a human skin disease model, which suggests their
anti-inflammatory, immunomodulatory, and tissue-remodeling
properties in the human skin.
3.2. Effects of AEO on genome-wide gene expression
We then analyzed the effect of 0.011% AEO (the highest
studied non-cytotoxic concentration in these cells) on the RNA
expression of 21,224 genes in the same cells. The results
showed the significantly diverse regulatory effect of AEO on
human genes, with numerous genes being either upregulated
or downregulated. Among the 200 most-regulated genes (with
a fold-change ratio of expression over the vehicle control of
j1.5j) by AEO, the majority (121 out of 200 genes) were
significantly downregulated (Table S2). A cross-comparison of
the protein and gene expression data revealed that AEO
significantly inhibited both the protein and gene expression
levels of VCAM-1,IP-10, and I-TAC. This suggests that AEO
might play a profound role in regulating these three important
players.
IPA showed that the bioactivity of AEO significantly over-
lapped with numerous canonical pathways from the literature-
validated database analysis (Fig. 2). Many of these signaling
pathways are closely related to inflammation, immunomodu-
lation, and tissue remodeling. Overall, AEO appeared to inhibit
these signaling pathways in the highly inflamed human skin
cells, suggesting it has potential anti-inflammatory and
immunomodulatory effects (see Supplementary Materials for
more information).
4. Conclusions
To the best of our knowledge, this study provides the first
evidence of the biological activities of AEO in highly
Fig. 1. The bioactivity profile of Arborvitae (Thuja plicata) essential oil (AEO, 0.011%, v/v in dimethyl sulfoxide, DMSO) in human dermal fibroblast
culture (HDF3CGF). X-axis denotes protein-based biomarker readouts. Y-axis denotes the relative expression levels of biomarkers compared with those of
vehicle controls, in log form. Vehicle control values are shaded gray, with 95% significance envelope. * indicates a biomarker designated with “key activity”:
biomarker value was significantly different (p <0.05) from that of vehicle controls at the studied concentration, outside of the significance envelope, with an effect
size of at least 10% (>0.05 log ratio units). MCP-1, monocyte chemoattractant protein; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intracellular cell
adhesion molecule 1; IP-10, interferon gamma-induced protein 10; I-TAC, interferon-inducible T-cell alpha chemoattractant; IL-8, interleukin-8; MIG, monokine
induced by gamma interferon; EGFR, epidermal growth factor; M-CSF, macrophage colony-stimulating factor; MMP-1, matrix metalloproteinase 1; PAI-1,
plasminogen activator inhibitor 1; TIMP, tissue inhibitor of metalloproteinase.
58 X. Han, T.L. Parker / Biochimie Open 4 (2017) 56e60
inflamed human skin cells. The findings show that AEO
significantly inhibited numerous protein and genes involved
in inflammation, immune responses, and tissue remodeling.
In addition, AEO diversely and significantly modulated
global gene expression. Furthermore, AEO robustly affected
various important signaling pathways in human cells. These
findings provide the first evidence for the therapeutic po-
tential of AEO in human skin cell inflammation. Further
studies on the mechanism of action and clinical efficacy of
AEO are required before drawing definite conclusions about
its therapeutic properties.
Conflict of interest
X.H. and T.P. are employees of d
oTERRA, where the study
agent AEO was manufactured.
Acknowledgment
This study was funded by d
oTERRA (Pleasant Grove,
UT, USA), and conducted at DiscoverX (Freemont, CA,
USA).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.biopen.2017.02.003.
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