Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 985345, 7 pages
Keratinocytes Using aComposition ContainingCarnitine,
ThiocticAcid and SawPalmetto Extract
1State University of New York (SUNY), Albany, NY, USA
2Advanced Restoration Technologies, Inc., 9035 North 15th Place, Phoenix, AZ 85020, USA
Correspondence should be addressed to Geno Marcovici, email@example.com
Received 29 December 2008; Accepted 8 July 2009
Copyright © 2011 Sridar Chittur et al. This is an open access article distributed under the Creative CommonsAttribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia
(AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, β-sitosterol, in subjects with
AGA and showeda highlypositive response to treatment. In this study, we soughtto determine whether blockade of inflammation
using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the
expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative
ofHFkeratinocytes,specifically, stimulationofcultured humankeratinocytecellsin vitro, wemeasuredchangesingeneexpression
of a spectrum of well-known inflammatorymarkers. Lipopolysaccharide (LPS) provided an inflammatorystimulus. In particular,
we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and
LTB(4)associatedwith pathwaysinvolvedininflammationandapoptosis.Ourdatasupport thehypothesisthatthetestcompound
exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These
findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel
two-pronged approach in the treatment of AGA with improved efficacy over current modalities.
The pathogeneses of benign prostatic hyperplasia (BPH)
and androgenetic alopecia (AGA) are mediated in part
by the transcriptional pathways downstream of the steroid
hormone androgen receptor (AR). The predominant ligand
in these tissues is dihydrotestosterone (DHT), which is
formed by theconversionofthe inactive formoftestosterone
(T)andiscatalyzedbytheenzyme 5-alphareductase (5-AR).
Anti-androgens and inhibitors of 5-AR have proven
effective in the treatment of BPH as well as AGA, attesting to
theircommon disease mechanisms. Boththe pharmaceutical
compound, finasteride (Proscar or Propecia) and the lipo-
sterolicextract ofSerenoa repens (LSESr)haveshown efficacy
in the treatment of BPH and AGA. Notably, in a direct
comparison of LSESragainst finasteride, it has been reported
that LSESr exhibited a 3-fold greater inhibition of 5-AR in in
vitro assays .
Finasteride (at a dose of 5mg, as in Proscar) is used as
the treatment of choice for BPH, particularly in the USA.
A number of well-controlled studies point to its efficacy in
ameliorating the signs and symptoms of BPH . In large,
double-blind, placebo-controlled clinical studies recruiting
over 1600 patients, it was shown that the administration of
finasteride reduced the size ofthe prostate by a mean of22%,
following 6 months of therapy . Likewise, multiple well-
controlled clinical trials reinforce the utility of LSESr in the
setting of BPH, predominantly in Europe . Investigators
have found that LSESr is well tolerated and has greater
efficacy than placebo and similar efficacy to finasteride in
improving symptoms in men with BPH .
During the course of a clinical trial of Proscar for BPH,
it was noted serendipitously that there was a cessation of
hair loss in study subjects receiving drug . Therefore,
finasteride (at a dose of 1mg; as in Propecia) was subse-
quently investigated in clinical trials for the treatment of
men with AGA. In affected individuals, long-term treatment
with finasteride 1 mg/day over 5 years was well-tolerated,
led to visible improvements in scalp hair growth and slowed
the further progression of hair loss that occurred without
2Evidence-Based Complementary and Alternative Medicine
treatment . LSESris well known for its role in BPH as a 5-
AR inhibitor, leading us to postulate a similar effect in AGA.
Previously,we tested LSESranditsglycoside,β-sitosterol,
in subjects with AGA and showed a highly positive response
to treatment. The blinded investigative staff assessment
reported that 60% of study subjects dosed with the active
study formulation were rated as improved at the conclusion
of the trial and established the effectiveness of naturally
occurring 5-AR inhibitors against AGA for the first time .
Notwithstanding, the common mechanism of androgens
in their pathogenesis, several lines of emerging evidence
suggest that both BPH and AGA are also associated with
significant dysregulation in the expression of inflammatory
cytokines . For example, gene expression profiling of
prostate tissue from BPH patients revealed molecular signa-
tures containing genes associated with inflammation .
Likewise, chronic inflammation of the hair follicle (HF) is
also considered a contributing factor in AGA . Another
recent study reported a relationship between moderate to
extensive alopeciaand chroniclow-grade inflammation .
Histologically, it has been shown that in scalp biopsies
from AGA patients, sustained follicular inflammation with
hair loss and, thus, is described as a possible cofactor in the
complex etiology of the disorder .
On the basis of such findings, the utility of treating
BPH-affected patients with anti-inflammatory agents in
combination with 5-AR inhibitors is currently under inves-
tigation. In one study, combination therapy with alpha(1)-
adrenergic receptor antagonists [alpha(1)-ARAs] and the 5-
AR inhibitor finasteride was significantly more effective than
either component alone in reducing BPH-related symptoms
(P = .006 versus doxazosin monotherapy; P < .001 versus
finasteride monotherapy) and lowering the rate of overall
clinical progression (P < .001 versus either monotherapy
alone) . The greatest efficacy was observed in patients
with a markedly enlarged prostate, more severe symptoms
and higher PSA levels. These data suggest that the treatment
of BPH is enhanced by the use of anti-inflammatory agents
in combination with 5-AR blockade . Taken together,
these lines of evidence led us to postulate that inflammation
is a rational therapeutic target in pattern hair loss (AGA).
In this study, we sought to determine whether blockade
of inflammation using a composition containing LSESr,
carnitine and thioctic acid (TA) could alter the expression
of molecular markers of inflammation in a well-established
in vitro system. We found that our compound effectively
repressed LPS-activated expression of a number of genes
involved in skin inflammation and apoptosis. Our findings
suggest that 5-AR inhibitors combined with blockade of
approach in the treatment of AGA with improved efficacy
over current modalities.
2.1. Cell Culture and Challenge. Human keratinocyte cells
(HaCaT) were grown in low glucose DMEM supplemented
with 10% FBS, 2mM l-glutamine, 1mM sodium pyruvate
solution, 0.1mM NEAA, 100Uml−1penicillin, 100μgml−1
streptomycin at 37◦C and 5%CO2. They were subcultured
on Day 2 and grown to 80% confluency on six-well plates.
of LPS (Sigma, St Louis, MO, USA) for 2h. The media was
then replacedwith fresh LPS atthesame concentrationalong
with the LSESr, TA and carnitine test substance, hereafter
designated as TS-050508A (1:1000). Each condition was
carried out in triplicate. The cells were harvested in 1ml
trizol after 22h. Control wells were treated with 100ng or
200ng for 24h and harvested in 1ml trizol. Alternately, the
HaCat cells were treated with TS-050508A (1:1000) for 2h.
The media was then replaced with either 100ng or 200ng of
LPS along with fresh TS-050508A (1:1000). Each condition
was carried out in triplicate. The cells were harvested in 1ml
trizol after 22h. Control wells were treated with TS-050508A
(1:1000) for 24h and harvested in 1ml trizol. Cell viability
was determined by trypan blue staining.
2.2. Trypan Blue Staining. The HaCaT cells were treated
with 0.5 ml 0.05% trypsin–EDTA and placed at 37◦C in
5%CO2for 12min. The cells were neutralized with 0.5 ml
Dulbecco’s Modified Eagle’s Medium (DMEM) 10% Fetal
Bovine Serum (FBS). 30 μl of trypan blue was mixed with
30μl of cell suspension. Approximately 10μl was loaded into
a haemocytometer and the cells were counted. Counts were
takenat 3, 6, 12and 24h for the NoLPS, 100, 200 and 400ng
LPS conditions, respectively. Under our culture conditions
and over this concentration range of LPS, the effects of LPS
on cell viability, as measured by trypan blue, were variable
but there was no consistent decrease in viability as LPS
concentration increased. Therefore, we used LPS at the lower
end of this concentration range to ensure cell viability. Our
findings are consistent with those observed by others .
2.3. Nucleic Acids Preparation and PCR-Based Expression
Array. Total RNA was isolated from all samples using
standard protocols and further cleaned using RNeasy mini
columns according to the manufacturer’s recommendations
(Qiagen, Gaithersburg, MD, USA). The RNA was deemed
of good quality for the 260/280, 260/230 and the 28s/18s
ribosomal ratios were close to 2. The RNA (500ng) was then
reverse transcribed to cDNAusing the protocolsas suggested
in the RT2First strand kit (SA Biosciences, Frederick, MD,
USA). The cDNA was then used to prepare a master mix
as per the RT2qPCR kit protocol (SA Biosciences). A 10μl
aliquot of this mix was loaded on to the wells in a custom
384-well plate (designed to include most of the genes in
the inflammation and cytokineresponse pathway). The cycle
time (Ct)valuesfromthese reactionswere analyzed using the
PCRarray data analysis template to calculate the ΔΔCt and
the fold change [fold change = 2(−ΔΔCt)].
2.4. RT–PCR Validation of Results. Equal amount of RNA
(1μg) was taken for all samples and reverse transcription
was performed using RT2First Strand kit (Cat# C-03) from
SA Biosciences. The total volume of the reaction was 20μl,
and the final RT product was diluted to a volume of 100
Evidence-Based Complementary and Alternative Medicine3
μl. Individual PCR assays were performed with the off-the-
shelf pre-validated primer pairs from SA Biosciences on the
Mix (Cat#PA-011, SABiosciences).PCR assays foreach gene
target were performed in triplicates for each sample. Each
PCR reaction contained cDNA synthesized from 10 ng total
RNA. Relative changes in gene expression were calculated
using theΔΔCt(threshold cycle)method.Foldchange values
are calculated using the formula 2−ΔΔCt. There were three
biological replicates in each treatment group. Comparisons
between groups were made using unpaired Student’s t-tests
with the level of significance set at P < .05.
3.1. Test Compound Suppressed LPS-activated Gene Expres-
sion. In this study, we used a well-validated in vitro assay
representative of HF keratinocytes, specifically, stimulation
of cultured HaCaT cells. We measured changes in gene
expression of a spectrum of well-known inflammatory
markers, using lipopolysaccharide (LPS) as an inflam-
matory stimulus. Using untreated cells as a baseline for
gene expression, we tested our compound under seven
conditions: (i) 100ng LPS alone (low dose); (ii) 200ng
LPS alone (high dose); (iii) compound alone (1:1000);
(iv) compound followed by low-dose LPS; (v) compound
followed by high-dose LPS; (vi) low-dose LPS followed
by the compound and lastly (vii) high-dose LPS followed
by the compound. Overall, we found that treating HaCaT
cell cultures with our compound resulted in reduced LPS-
mediated inflammatory gene expression without inducing a
negative effect on cell viability. In particular, we found that
the composition effectively suppressed LPS-activated gene
expression of chemokines associated with pathways involved
in inflammation and apoptosis.
3.2. Global Expression Analysis Reveals Inhibition of Inflam-
matory Pathways. The RT2Profiler PCR Array from Super-
Array was used to interrogate the effect of the novel
composition on inflammation. This array contains 384 well-
validated genes known to be involved in stromal, endothelial
and epithelial inflammatory pathways. The classes of genes
representedonthearray includedtheCCligand chemokines,
toll-like receptor genes and their ligands, epithelial cell
adhesion molecules, and members of the CXC chemokine
family and leukotriene lipid mediators derived from the 5-
lipoxygenase pathway of arachidonic acid metabolism.
When measured against theuntreated cells, we noted sta-
tistically significant (P ≤ .05) fold changes in the following
genes: CCL3, CCL8, CCL17, CCL24, CCR7, CXCL2, CXCL6,
IL18RAP, IL1A, IL1B, IL1F10 LTB(4) and TLR4, (Table 1).
Below we will focus on three genes, CCL17, CXCL6 and
LTB(4) (Figure 1).
3.3. Chemokine Ligand CCL17. In this study, our com-
pound showed modest but statistically significant anti-
inflammatory activity against CCL17. The HaCaT cells
treated with 100ng LPS displayed a 1.76 up-regulated fold
1 : 1000 drug only
100ng LPS only
100ng LPS + drug
drug + 100ng LPS
200ng LPS only
200ng LPS + drug
drug + 200ng LPS
Figure1: Noteworthy foldchangeoccurring inCCL17, CXCL6 and
change as compared to untreated controls. Dosing the cells
with 200ng LPS resulted in a down-regulation of CCL17
by a factor of −1.43-fold. The cells treated with the test
compound alone down-regulated expression of CCL17 by
a factor of −2.17 from baseline, whereas cells treated with
compound followed by low-dose LPS resulted in a down-
regulation of −1.71-fold. Overall, the results showed that
our compound down-regulated the inflammatory marker
CCL17. The test composition also blocked the up-regulation
of message, both prior to and subsequent to LPS-mediated
3.4. CXC Chemokine Ligand, CXCL6. We show statistically
significant anti-inflammatory activity in an LPS-mediated
model of skin inflammation via the marker CXCL6. The
low-dose LPS-only (100ng) stimulated cells resulted in
CXCL6 being up-regulated by 1.57-fold. When measured
against baseline, the test compound alone down-regulated
expression of CXCL6 by a factor of −3.09. After incubating
the cells with the test compound followed by exposure
to low-dose LPS, CXCL6 expression showed a −3.28-fold
change as measured against baseline. These results show
a blockade of CXCL6 inflammatory message in the LPS-
stimulated HaCaT cells by the test compound.
3.5. Leukotriene B(4). In this study, our test compound
showed a noteworthy effect in the leukotriene [LTB(4)]
marker. The HaCaT cells treated with 100ng LPS inflam-
matory agonist displayed an up-regulated fold change for
4Evidence-Based Complementary and Alternative Medicine
Table 1: Gene expression profile of inflammatory markers expressing statistically significant fold change.
1 : 1 000 drug
only Fold upor
1 : 1000 drug
drug no LPS
100 ng LPS only
100 ng LPS
drug no LPS
100 ng LPs+
drug Fold upor
drug no LPS
LPS Fold up- or
drug no LPS
200 ng LPS only
200 ng LPS
drug no LPS
200 ng LPS+
drug Fold upor
drug no LPS
LPS Fold upor
drug no LPS
Dark gray shadings indicates only test sample shows weak amplification. Bold text indicates stasticallysignificant fold changes.
LTB(4) of 1.33 when measured against baseline. Gene
expression of LTB(4) showed a down-regulation of −2.64-
fold in the cells treated with the test compound alone. A
100ng LPS incubated cells followed by the test compound
showed marked change in LTB(4) expression demonstrating
a −4.59-fold change as measured against baseline. These
results show that our compound blockedthe LPS-stimulated
up-regulation of the inflammatory marker LTB(4).
In summary, we found that our test compound atten-
uated expression for markers of inflammation, and specif-
ically for CCL17, CXCL6 and LTB(4). Our data support
the hypothesis that the test compound exhibits anti-
inflammatory characteristics in a well-established in vitro
assay representing HF keratinocyte gene expression.
Inthisstudy,we sought todemonstratethata testcompound
containing LSESr, carnitine and TA represses LPS-activated
expression of inflammatory genes using a well-validated in
LPS-stimulated HaCaT cells [16, 17]. We assayed for changes
in gene expression across a spectrum of well-characterized
inflammatory markers and found that our test compound
demonstrates anti-inflammatory properties in vitro. Plant
extracts or mixtures comprise a repertoire of chemical
entities that have pleiotropic effects on cellular physiology.
As such, they have great potential in the multi-target
approach to diseases [18, 19]. Microarray analysis of gene
expression following exposure of cells to plant extracts can
be useful for elucidating the molecular networks impacted
by herbal extracts and mixtures . In particular, plant-
based therapies have proven efficacious in the treatment of
inflammatory skin disorders, such as atopic dermatitis .
Here, we found that treating HaCaT cell cultures with
our compound resulted in a statistically significant (P <
.05) reduction of LPS-mediated inflammatory gene expres-
sion without inducing a negative effect on cell viabil-
ity. Specifically, we noted that the composition effectively
suppressed LPS-activated gene expression of chemokines
CCL17, CXCL6 and LTB(4). The over-expression of each of
these markers has previously been observed in the setting
of inflammation and apoptosis. We focused here on short
term and immediate changes in gene expression, using
array hybridization, followed by qPCR. The gene expression
fold changes we observed were statistically significant. Our
approach is supported in the literature by many other
to stimuli such as LPS, in addition to UV irradiation and
wounding in the HaCaT cells and keratinocytes [22–25].
Our rationale for selecting CXCL6, CCL17 and LTB(4)
for further study relates to their role in inflammatory
disorders of the skin, mucosae and epithelia. CXCL6 is a
CXC chemokine expressed by macrophages, epithelial and
mesenchymal cells during inflammation. Recently, it was
shown that the transcriptome of the aging prostate stroma
is characterized by the up-regulation of several genes that
encode secreted inflammatory mediators, including CXCL6
. Similarly, in the skin, CCL17 (a CC chemokine ligand)
is up-regulated under stress, injury or inflammation .
Evidence-Based Complementary and Alternative Medicine5
Skin and hair
Figure 2: Many organ systems are affected by pro-inflammatory
CCL17 is also over-expressed in stromal, endothelial and
epithelial tissues in autoimmune disorders including the
inflammatory bowel diseases, Crohn’s disease and ulcerative
colitis (UC) . Finally, LTB(4) is a lipid inflammatory
mediator derived from membrane phospholipids by the
sequential actions of cytosolic phospholipase A2 (PLA2), 5-
lipoxygenase (5-LO) and leukotriene A(4) [LTA(4)] hydro-
lase. LTB(4), best known as a neutrophil chemoattractant, is
now recognized to exert other important effects contribut-
ing to inflammatory and immune responses. Reinforcing
the connection between androgen-mediated disorders and
inflammatory processes, LTB(4)expression hasbeendemon-
strated in acne vulgaris .
While LPS caused upregulation of CXCL6 and LTB(4)
at both doses as expected, we observed a modest downreg-
ulation of CCL17 at the higher dose of LPS. Likewise, in a
recent study using the LPS challenge cell culture model, the
authors noted that their responses might have also differed
from predicted expectation based on the presence orabsence
of specific cell-surface receptors in 2D culture versus the
3D environment . Thus, the observation that high-dose
LPS itself partially lowers CCL17 expression does not change
our interpretation, since the treatment represses expression
beyond the level of high-dose LPS. The overall result of
our experiment is that LPS challenge (at low dose) induces
the expected increase in CCL17, and the treatment group
demonstrates potent downregulation of CCL17.
A large body of evidence supports the involvement
of pro-inflammatory processes in the development and
progression of numerous disorders, including inflammatory
bowel disease, ischemic heart disease and asthma (Figure 2)
. Chronic inflammation is recognized at the molecular
and cellular levels as the final common pathway of many sys-
temicand degenerativediseases, includingthoseaffectingthe
skin . It has also been observed that inflammation, even
that induced by emotional stress, plays a role in hair loss,
and recent studies point to inflammation as a contributing
factor to AGA . In a murine model, the authors demon-
strated that psychoemotional stress indeed alters actual HF
cycling in vivo, that is, prematurely terminates the normal
duration of active hair growth (anagen) . Furthermore,
inflammatory events deleterious to the HF are present in the
HF environment of stressed mice (perifollicular macrophage
cluster, excessive mast cell activation). It has also been shown
experimentally that high-dose pro-inflammatory cytokines
induce apoptosis of HF keratinocytes in vivo .
Histologically, sustained microscopic follicular inflam-
mation with connective tissue remodeling is a noted cofactor
in the complex etiology of AGA . Disruption of HF
homeostasis, via injury, insult or inflammation results in
a shift from proliferation to apoptosis. Cumulatively, these
findings underline the susceptibility HFs display when
subjected to inflammatory insult.
In order to clarify the rationale leading to this study,
l-carnitine, TA and LSESr operate through distinct, but
potentially interrelated, biochemical and molecular mech-
anisms, with each representing an important component
in the design of the test formula. Carnitine, a potent anti-
inflammatory agent, biosynthesized from the amino acids
lysine and methionine is required for the transport of fatty
acids from the cytosol into the mitochondria during the
breakdown of lipids for the generation of metabolic energy
. Carnitine exists in two stereoisomers: its biologically
active form is l-carnitine, while its enantiomer, d-carnitine,
is biologically inactive .
Accumulating evidence suggests that l-carnitine may
play a significant role in prevention and treatment ofnumer-
ous diseases as well as protection from accelerated aging
that result from oxygen free-radical damage, inflammation
and glycation (non-enzymatic glycosylation). Documented
benefits from l-carnitine include clinical improvement in
patients diagnosed with diabetes, cardiovascular disease,
hypertension, congestive heart failure, age-related deterio-
ration of brain function and vision and immune function
. One line ofresearch suggeststhat carnitine may possess
the ability to promote hair growth in vitro by increasing
energy supply to the rapidly prolife rating and the energy-
consuming anagen hair matrix .
The naturally occurring antioxidant TA was first
described as an essential cofactor for the conversion of
pyruvate to acetyl-CoA, a critical step in respiration . TA
isnowrecognized asacompoundwith manybiologicalfunc-
tions, such as the modulation of pathogenic inflammatory
events, including those in the skin .
In addition to the two constituents described above, the
ofsaw palmetto.LSESrishighlyenriched withfattyacidsand
phytosterols and has been used historically to treat urinary
tract symptoms, including BPH . LSESr has been shown
to block both isoforms of 5-alpha reductase (types I and
II) with the added benefit that, in contrast to finasteride,
LSESrdoesnot interfere with PSA(prostate-specific antigen)
levels . As previously noted, we were the first to clinically
demonstrate the utility of LSESr against AGA .
6Evidence-Based Complementary and Alternative Medicine
While the genetic architecture of AGA has not yet been
determined, it is clearly a polygenic disorder in which mul-
tiple genes, hormonal pathways and environmental factors
contribute to phenotype. To date, DHT modulation via
5-AR blockade remains the only druggable target shown
to ameliorate the progression of AGA . An important
feature of our hypothesis hinges on the enhanced efficacy of
combining anti-inflammatory agents with 5-AR inhibitors.
A paradigm for enhanced performance via concomitant
therapy already exists in the treatment of skin disease. For
example, studies show that the addition of a topical corti-
costeroid to imidazole therapy increases the bioavailability
and prolongs the activity of the antimycotic, while rapidly
reducing inflammatory symptoms .
In several studies, a synergy between TA and vitamin E
has been described, and potent antioxidant effects can be
obtained when both antioxidants are simultaneously used.
Specifically, recent findings showed that the combination of
TA plus vitamin E effectively reduces oxidative damage in
brain and cardiac ischemia as well as in other pathological
events associated with tissue inflammation and secondary to
the formation of reactive oxygen species (ROS) . In in
vitro and animal studies, l-carnitine, particularly when used
in combination with TA, has been shown to reverse age-
related changes in numerous tissues, systems and pathways
Since the clinical success rate for treatment of AGA with
androgen blockade is limited, there is enormous unmet
medical need for patients who are refractory to current
therapy. Recognizing that inflammation contributes to a
wide range of diseases, including those affecting the skin and
hair, it is our hypothesis that the blockade of inflammation
represents a new and potentially viable therapeutic avenue.
We suggest that our data may offer new insight into the
potentialfor combininganti-inflammatory compoundswith
5-AR inhibitors for the treatment of AGA.
The authors are grateful for the technical support and advice
from SuperArray Biosciences, Inc. In particular, we thank
Dr Timothy Steppe for valuable and stimulating discussions.
TheythankTiffany Devine,Marcy Kuentzeland DavidFrank
at the Center for Functional Genomics, State University of
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