Article
Dexpanthenol Promotes Cell Growth by Preventing Cell
Senescence and Apoptosis in Cultured Human Hair
Follicle Cells
Jae Young Shin, Jaeyoon Kim






Citation: Shin, J.Y.; Kim, J.; Choi,
Y.-H.; Kang, N.-G.; Lee, S.
Dexpanthenol Promotes Cell Growth
by Preventing Cell Senescence and
Apoptosis in Cultured Human Hair
Follicle Cells. Curr. Issues Mol. Biol.
2021, 43, 1361–1373. https://doi.org/
10.3390/cimb43030097
Academic Editors: Raffaele Capasso,
Maria Grazia Ferraro and
Dongchul Kang
Received: 12 August 2021
Accepted: 24 September 2021
Published: 28 September 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Curr. Issues Mol. Biol. 2021, 43, 1361–1373. https://doi.org/10.3390/cimb43030097
, Yun-Ho Choi, Nae-Gyu Kang * and Sanghwa Lee *
LG Household & Health Care (LG H&H) R&D Center 70, Magokjoongang 10-ro, Gangseo-gu, Seoul 07795, Korea;
sjy2811@lghnh.com (J.Y.S.); kjy5281@lghnh.com (J.K.); youknow@lghnh.com (Y.-H.C.)
* Correspondence: ngkang@lghnh.com (N.-G.K.); shleek@lghnh.com (S.L.); Tel.: +82-2-6980-1533 (N.-G.K.);
+82-2-6980-1210 (S.L.)
Abstract: Dexpanthenol (D-panthenol) is a precursor of vitamin B5 (pantothenic acid) and is widely
used for dietary supplements and topical applications. D-panthenol has long been used in hair care
products for the purpose of anti-hair loss, its effects and the underlying mechanisms, however, were
barely reported. In this study, the effects of D-panthenol on human hair follicle cells, including dermal
papilla cells (hDPCs) and outer root sheath cells (hORSCs), were investigated. D-panthenol enhanced
the cell viability, increasing the cellular proliferation marker Ki67 in cultured hDPCs. The markers for
apoptosis (Caspase3/9) and cell senescence (p21/p16), reported to be expressed in aged or resting
phase follicles, were significantly reduced by D-panthenol. Anagen-inducing factors (ALP; β-catenin;
versican), which trigger or elongate the anagen phase, were stimulated by D-panthenol. On the other
hand, D-panthenol reduced TGF-β1 expressions in both mRNA and protein levels. The expression of
VEGF, which is important for peripheral blood vessel activation; was up-regulated by D-panthenol
treatment. In cultured hORSCs, cell proliferation and viability were enhanced, while the mRNA
expression of cell senescence markers (p21/p16) was significantly down-regulated. The expressions
of both VEGF and its receptor (VEGFR) were up-regulated by D-panthenol. In conclusion, our data
suggest that the hair growth stimulating activity of D-panthenol was exerted by increasing the cell
viability, suppressing the apoptotic markers, and elongating the anagen phase in hair follicles.
Keywords: D-panthenol; dermal papilla; outer root sheath; anti-hair loss; anagen; follicle aging
1. Introduction
Anagen is a specific period occupying the majority of the hair cycle. In anagen,
hair follicular cells go through vigorous cell proliferation and differentiation to make the
hair shaft [1]. During catagen and telogen, cells in the lower part of the hair follicle go
through apoptosis, which leads to hair follicle shrinkage and hair shaft loss, respectively [2].
Given the clinical importance of the anagen-catagen transformation in human hair growth
disorders, the abnormal termination of the anagen phase triggers gradual hair thinning [3].
In this context, it has long been regarded that extension of the anagen phase is a main
strategy of anti-hair loss treatment.
The dermal papilla and outer root sheath are the special compartments of the hair
follicle, playing important roles in hair follicle morphogenesis and regeneration. Previous
studies suggested that the anti-apoptotic and proliferative potencies of dermal papilla cells
are the first clue for anti-hair loss property [4]. In particular, alkaline phosphatase (ALP)
activity in dermal papilla has been regarded as an important factor for the progression of
the hair follicle cycle inducing hair follicle neogenesis and telogen to anagen transition [5].
Growth factors from the dermal papilla, including insulin-like growth factor-1 (IGF-1),
vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF) stimulate
matrix keratinocytes to proliferate and differentiate into the hair shaft during anagen [6–8].
https://www.mdpi.com/journal/cimb
Curr. Issues Mol. Biol. 2021, 43 1362

The transition of hair cycles is also mainly controlled by the dermal papilla. Secreted
proteins, such as transforming growth factor (TGF) β1, TGF β2, and dickkopf1 (DKK-1) are
known to induce the transition from anagen to telogen [9–11]. On the other hand, the outer
root sheath (ORS), which takes a mold part of the hair follicle structure, plays a connective
role between bulge stem cells and bulb dermal cells (dermal papilla and dermal sheath
cells) in the anagen phase [12]. When the telogen to anagen transition signaling begins,
bulge regional outer root sheath cells begin to proliferate and to differentiate downward
to activate the dermal papilla [13]. Once the ORS cells escape from quiescent/conserved
status, cellular proliferative and anti-apoptotic characteristics are more important than
stem cell characteristics for maintaining the anagen phase. While several markers, such
as SOX9, CD34, and Keratin 15 in the bulge region show stem cell lineage maintenance
functions, cellular activation markers, such as Ki67 and BCL2, in the lower part of ORS
are considered as proliferative and anagen-elongating factors [12,14]. This differentiated
compartment of the ORS could interact only with the basal part of the hair follicle, the
dermal papilla.
D-panthenol, the alcohol form of pantothenic acid (Vitamin B5), is a well-known
cosmetic and pharmaceutical ingredient used for anti-inflammatory, skin regenerating, and
stratum corneum hydrating effects [15–17]. Because of these skin improving properties,
D-panthenol is widely used to relieve atopic dermatitis, nappy rash symptoms, and sun-
burns [18,19]. D-panthenol was also reported to facilitate wound repair by up-regulating
wound-healing associated molecules, such as IL-6, IL-1β, CYP1B1, CXCL1, CCL18 and
KAP4-2 [20,21].
In addition, D-panthenol has long been used for hair and scalp care products, es-
pecially for the purpose of anti-hair loss. Several clinical studies reported that the oral
administration of D-panthenol improved female pattern hair loss as well as male androge-
netic alopecia [22,23]. Its effects on hair follicle cells and underlying mechanisms, however,
were barely reported.
In this study, we investigated the in vitro hair growth-promoting properties of D-
panthenol in cultured hDPCs and hORSCs. The effects of D-panthenol on the cell growth
and the expression of apoptosis/cell senescence related genes were measured to elucidate
cellular proliferative properties. The expression levels of anagen and telogen markers
were assessed to examine the effect of D-panthenol on hair follicle cycle modulation. The
effect of D-panthenol on the expression of several growth factors and receptors, including
TGF-β1, VEGF and VEGFR, were also evaluated.

2. Materials and Methods

2.1. Culture of Human Dermal Papilla Cells (hDPCs) and Outer Root Sheath Cells (hORSCs)
hDPCs were purchased from Promocell (Heidelberg, Germany) and hORSCs were
purchased from ScienCell research laboratories (Carlsbad, CA, USA). hDPCs were cultured
in a basal medium supplemented with Supplement Mix, which contains 4% fetal calf serum,
0.4% bovine pituitary extract, 1 ng/mL of basic fibroblast growth factor, and 5 µg/mL of
insulin. hORSCs were cultured in a mesenchymal stem cell medium containing 5% FBS,
1% penicillin streptomycin and 1% MSCGS (Mesenchymal Stem Cell Growth Supplement)
which is provided by the manufacturer. Cells were maintained in a humidified incubator
at 37 ◦ C with 5% CO2 . hDPCs under the passage number 5 were used for experiments.

2.2. Cell Viability Assay

D-panthenol was purchased from xinfa pharmaceutical (Shandong, China) and the
stock solution was prepared by dissolving in DMSO. The effects of D-panthenol on cell via-
bility were examined using CCK-8 assay (Dojindo, Rockville, MD, USA), following the man-
ufacturer’s protocols. Briefly, hDPCs (3 × 103 cells/well) and hORSCs (3 × 103 cells/well)
were seeded in 96-well plates and cultured for 24 h. Triplicate cultures of hDPCs and
hORSCs were treated with various concentrations of D-panthenol and cultured for another
24 h. The generation of NADH and NADPH was determined by CCK-8 assay which
Curr. Issues Mol. Biol. 2021, 43 1363

indicates the cell viability. The absorbance at 450 nm was measured using an Epoch micro
plate spectrophotometer (BioTek, Winooski, VT, USA). Minoxidil was purchased from
Sigma Aldrich (St. Louis, MO, USA) and the stock solution was prepared by dissolving
in DMSO.

2.3. mRNA Analysis

hDPCs (1 × 106 cells/well) and hORSCs (1 × 106 cells/well) were seeded in 6-well
plates and cultured for 24 h. Then D-panthenol was treated at appropriate concentrations
for 24 h. Total RNA was isolated using an RNA isolation kit (Qiagen, RNeasy mini kit),
according to the manufacturer’s guide. After RNA isolation, cDNA was synthesized by
reverse transcription using an eCube cDNA synthesis kit (philekorea, Korea) with a PCR
thermocycler (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s
protocol. cDNA obtained from control cells and D-panthenol treated cells were subjected
to real-time PCR analysis. TaqMan probes used in this study were as follows: GAPDH
assay id 4352934E; Caspase3 assay id Hs00234387_m1; Caspase 9 assay id Hs00962278_m1;
CDKN1A assay id Hs00355782_m1; CDKN2A assay id Hs00923894_m1; ALP assay id
Hs01029144_m1; Versican assay id Hs00171642_m1; TGF-β1 assay id Hs00998133_m1; Bcl2
assay id Hs00608023_m1; Bax assay id Hs00180269_m1; VEGFA assay id Hs00900055_m1;
VEGFR (KDR) assay id Hs00911700_m1. TaqMan One-Step RT-PCR Master Mix Reagents
(Life Technologies, Carlsbad, CA, USA) was used. The PCR reactions were performed on
an ABI7500 Real Time PCR system following the manufacturer’s protocol. The resulting
data were analyzed with ABI software (delta-delta Ct method).

2.4. Western Blot Analysis

hDPCs (1 × 106 cells/dish) were seeded in 100 mm culture dishes and cultured for
24 h. D-panthenol was treated at appropriate concentrations for 24 h. Cells were then
washed with ice-cold PBS and lysed on ice in an M-PER buffer (Thermo Fisher Scientific,
Waltham, MA, USA) supplemented with a Complete™ protease inhibitor cocktail and
phosphatase inhibitor (Roche, Indianapolis, IN, USA). 40 µg of protein was analyzed by
Western blotting with appropriate antibodies to evaluate protein expression; β-catenin
(1000:1 dilution, Santa Cruz, CA, USA), CDKN2A/p16INK4a (1000:1 dilution, Abcam,
Cambridge, UK), p21 (1000:1 dilution, cell signaling technology, Danvers, MA, USA),
caspase3 (1000:1 dilution, cell signaling technology, Danvers, MA, USA), GAPDH (2000:1
dilution, Santa Cruz, CA, USA). Western blot was analyzed by a chemiluminescence
detector (iBright FL1500, Thermo Fisher Scientific, Waltham, MA, USA).

2.5. Immunocytochemistry
hDPCs (5 × 104 cells/well) were seeded in 24-well plates. After 24 h, cells were treated
with appropriate concentrations of D-panthenol for 24 h. After an ice cold PBS wash, hDPCs
were fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were then
permeabilized with PBS containing 0.1% triton X-100 and blocked with PBS containing 5%
FBS and 1% BSA. After consecutive incubation with the primary antibodies (200:1 dilution,
Abcam, Cambridge, UK) at 4 ◦ C for 12 h and the alexa 488 nm or alexa 594 nm conjugated
secondary antibodies (1000:1 dilution, Thermo Fisher Scientific, Waltham, MA, USA) at
room temperature for 1 h, nucleus were stained with DAPI (2000:1 dilution, Thermo
Fisher Scientific, Waltham, MA, USA) in the dark for 10 min. High resolution fluorescence
images were taken using the EVOSTM FL Auto2 Imaging System (Thermo Fisher Scientific,
Waltham, MA, USA).

2.6. Alkaline Phosphatase (ALP) Staining and Quantification

hDPCs (5 × 103 cells/well) were plated in 96-well plates and cultured for 24 h. Cells
were treated with D-panthenol for 24 h, and then fixed in 4% paraformaldehyde for 10 min
and washed with PBS. For efficient staining, cells were permeabilized with PBS containing
0.1% triton X-100. After permeabilization, ALP was stained with Vector® Blue kit. Vector
 2.6. Alkaline Phosphatase (ALP) Staining and Quantification
hDPCs (5 × 103 cells/well) were plated in 96-well plates and cultured for 24 h. Cells
Curr. Issues Mol. Biol. 2021, 43
were treated with D-panthenol for 24 h, and then fixed in 4% paraformaldehyde for 10 1364
min and washed with PBS. For efficient staining, cells were permeabilized with PBS con-
taining 0.1% triton X-100. After permeabilization, ALP was stained with Vector® Blue kit.
Vector blue reagents 1, 2, and 3 were mixed in 200 mM of Tris-HCl (pH 8.5) solution. 100
μL ofblue reagents
mixed 1, 2, andwas
ALP solution 3 were mixed
treated in 200
to each mMand
well of Tris-HCl
incubated(pHfor 8.5) solution.
30 min in the 100
dark.µL of
Aftermixed ALP solution
treatment, the whole was treated
area to each
of each wellmeasured
well was and incubated
by theforEVOS
30 min in the
TM FL dark.
Auto2 Im-After
treatment, the whole area Scientific,
of each well was measured by the TM
aging System (Thermo Fisher Waltham, MA, USA). To EVOS
quantify FLtheAuto2
number Imaging
of
ALP positive cells and intensity, fluorescence images were obtained under the same con-ALP
System (Thermo Fisher Scientific, Waltham, MA, USA). To quantify the number of
positive
ditions usingcells
561and intensity,
nm/594 fluorescence images were
nm (Excitation/Emission) obtainedwhich
wavelength under react
the same
withconditions
ALP
usingCelleste
staining. 561 nm/594
imagenm (Excitation/Emission)
analysis software was used wavelength which react
for quantification withFisher
(Thermo ALP staining.
Sci-
Celleste
entific, image
Waltham, MA,analysis
USA).software was used for quantification (Thermo Fisher Scientific,
Waltham, MA, USA).
2.7. Statistical Analysis
2.7. Statistical Analysis
All experimental data were presented as the mean ± standard deviation (S.D.) of at
All experimental data were presented as the mean ± standard deviation (S.D.) of at
least 3 independent experiments, otherwise indicated. Experimental results were ana-
least 3 independent experiments, otherwise indicated. Experimental results were analyzed
lyzed using the SigmaPlot (Systat Software Inc., San Jose, CA, USA). The statistical signif-
using the SigmaPlot (Systat Software Inc., San Jose, CA, USA). The statistical significance of
icance of the difference was determined using Student’s t-test. The value of p < 0.05 was
the difference was determined using Student’s t-test. The value of p < 0.05 was considered
considered statistically significant.
statistically significant.
3. Results
3. Results
3.1. D-Panthenol Stimulated
3.1. D-Panthenol the Growth
Stimulated of Cultured
the Growth hDPCs
of Cultured hDPCs
To evaluate the the
To evaluate cellcell
proliferative effect
proliferative effectof of
D-panthenol,
D-panthenol,hDPCs
hDPCswereweretreated
treated with
with 5 µM
μM to 5 mMmM of ofD-panthenol.
D-panthenol.The Theviability
viabilityofofhDPCs
hDPCs waswas measured
measured bybycellcell counting
counting kit 8kit 8
(CCK8)
(CCK8)
assayassay
afterafter
24 h 24 h treatment.
treatment. Minoxidil
Minoxidil was usedwas asused as a positive
a positive control.control. D-panthenol
D-panthenol stimulated
stimulated the growth
the growth of hDPCsof hDPCs in a concentration-dependent
in a concentration-dependent mannermanner
(Figure (Figure 1A). Max-
1A). Maximal growth
imalincrement
growth increment was approximately
was approximately 30% at a 30%2.5 mMat a concentration.
2.5 mM concentration. Also, fluores-
Also, fluorescent staining
cent results
stainingshowed
results that
showed
Ki67that Ki67 positive
positive cells were cells were increased
increased by the by the D-panthenol
D-panthenol treatment
(Figure
treatment 1B). 1B).
(Figure

(A)

(B)
Figure 1. D-panthenol
Figure stimulated
1. D-panthenol the growth
stimulated of cultured
the growth human human
of cultured dermal papilla
dermal cells (hDPCs).
papilla (A)
cells (hDPCs).
The growth
(A) The growth of hDPCs was assessed by CCK-8 assay after 24 h treatment of D-panthenol. (B)aKi67,
of hDPCs was assessed by CCK-8 assay after 24 h treatment of D-panthenol. (B) Ki67,
a cell proliferation marker was immunostained after 24 h treatment of D-panthenol and the propor-
tions of Ki67 positive cells were calculated (×200 magnification). Ki67 positive cells were marked by
green fluorescence and total cells were marked by DAPI. * p < 0.05, ** p < 0.01 versus control group.
Data are expressed as mean ± SD.
 cell proliferation marker was immunostained after 24 h treatment of D-panthenol and the propor-
tions of Ki67 positive cells were calculated (×200 magnification). Ki67 positive cells were marked by
Curr. Issues Mol. Biol. 2021, green
43 fluorescence and total cells were marked by DAPI. * p < 0.05, ** p < 0.01 versus control group. 1365
Data are expressed as mean ± SD.

3.2. D-Panthenol Reduced Apoptotic and Senescence Markers in Cultured hDPCs

3.2. D-Panthenol Reduced Apoptotic and Senescence Markers in Cultured hDPCs
Pantothenic acid was previously reported to prevent the apoptotic process induced
Pantothenic acid was previously reported to prevent the apoptotic process induced by
by oxidative stress in several mammalian cell types [24]. Caspase 3 and caspase 9, which
oxidative stress in several mammalian cell types [24]. Caspase 3 and caspase 9, which lead
lead to cell apoptosis, are regarded as anagen termination markers [25]. During the cat-
to cell apoptosis, are regarded as anagen termination markers [25]. During the catagen and
agen and telogen phase of the hair follicle, the miniaturization of the dermal papilla is
telogen phase of the hair follicle, the miniaturization of the dermal papilla is committed by
committed by several molecules. The apoptosis signaling triggered by caspase 9 and
several molecules. The apoptosis signaling triggered by caspase 9 and ended by caspase
ended by caspase 3 results in mitochondrial dysfunction [26]. As shown in Figure 1A, D-
3 results in mitochondrial dysfunction [26]. As shown in Figure 1A, D-panthenol treatment
panthenol treatment increased the cell growth which is represented by mitochondrial
increased the cell growth which is represented by mitochondrial function (CCK8). To
functionevaluate
(CCK8). whether
To evaluate whether up-regulated
up-regulated mitochondrialmitochondrial function iswith
function is associated associated
apoptosis and
with apoptosis and of
senescence senescence
hDPCs, the of mRNA
hDPCs,expressions
the mRNAofexpressions
apoptosis—andof apoptosis—and se- genes
senescence-related
nescence-related genes were measured after the D-panthenol treatment. The
were measured after the D-panthenol treatment. The mRNA expressions of apoptosis mRNA ex-
pressionsmarkers,
of apoptosis markers,
caspase 3 and caspase
caspase 39,and
were caspase 9, werereduced
significantly significantly
by 20% reduced
comparedby to the
20% compared to the control (Figure 2A). Along with the anti-apoptotic effects, cell senes-
control (Figure 2A). Along with the anti-apoptotic effects, cell senescence markers, p21 and
cence markers,
p16, werep21also
andsignificantly
p16, were also significantly
reduced reducedinby
by D-panthenol D-panthenol in concen-manners.
concentration-dependent
tration-dependent manners. Relative mRNA expressions
Relative mRNA expressions of p21 and p16 were significantly of p21 and reduced
p16 werebysignifi-
60% and 50%,
cantly reduced
respectively (Figure 2B). In addition, protein expression levels of p21,expression
by 60% and 50%, respectively (Figure 2B). In addition, protein p16 and caspase 3
levels ofwere
p21, p16 and caspase
significantly 3 were
reduced bysignificantly
60%, 40% and reduced by 60%, 40%(Figure
50%, respectively and 50%, respec-
2C). These results
tively (Figure 2C). These results indicate that D-panthenol could prevent catagen/telogen
indicate that D-panthenol could prevent catagen/telogen entry by preventing the cell
entry bysenescence
preventingand the apoptosis
cell senescence and apoptosis
in dermal papilla. in dermal papilla.

(A)

(B)
Figure 2. Cont.
Curr.
Curr. Issues Mol.
IssuesCurr. Biol.
Biol. 2021,
Mol.Issues Mol. 1,
2021, FOR
Biol.
1, FOR PEER
2021,
PEER43 REVIEW
REVIEW 66 1366

(C)
(C)
Figure
Figure 2. D-panthenol
2. Figure
D-panthenol reduced
reduced apoptosis
2. D-panthenol apoptosis and
and senescence
senescence
reduced apoptosis markers
markers in
and senescence in cultured
in hDPCs.
cultured
markers cultured(A)
hDPCs. (A) mRNA
mRNA
hDPCs. (A) mRNA
expression levels
levels of
expressionexpressionof caspase
caspase 3,
3, caspase
caspase 99 in
in hDPCs
hDPCs after
after 24
24 h
h treatment
treatment of
of D-panthenol.
D-panthenol. (B)
(B) mRNA
mRNA
levels of caspase 3, caspase 9 in hDPCs after 24 h treatment of D-panthenol. (B) mRNA
expression levels
levels of
expressionexpressionof senescence
senescence markers
markers (p21,
(p21, p16) after
p16)(p21, 24
24 h
afterp16) treatment
hafter
treatment of
of D-panthenol.
D-panthenol. (C)
(C) Protein
Protein
levels of senescence markers 24 h treatment of D-panthenol. (C) Protein
levels
levels of
of p21,
p21, p16
p16 and
and caspase
caspase 3 were
were analyzed by
by Western blot
blot and quantitated. ** pp << 0.05, ** pp <<
0.01 versuslevels of p21,
control p16
group. and 3caspase
Data are
analyzed
3 were as
expressed mean
Western
analyzed ± by Western
SD.
and quantitated.
blot and quantitated. 0.05,
* p < **
0.05, ** p < 0.01
0.01 versus control group. Data are expressed as mean ± SD.
versus control group. Data are expressed as mean ± SD.
3.3.
3.3. D-Panthenol
D-Panthenol Significantly
Significantly
3.3. D-Panthenol Increased the
the Anagen
IncreasedIncreased
Significantly Anagen theMarkers
Anagen in
Markers in Cultured
Cultured
Markers hDPCs
hDPCs hDPCs
in Cultured
To elucidate
To elucidate the correlation
the correlation
To elucidate between D-panthenol-induced
betweenbetween
the correlation D-panthenol-induced
D-panthenol-inducedhDPC
hDPC growthhDPC and
growth and hair
hairand hair
growth
cycle
cycle modulation
modulation (anagen
(anagen and
and catagen),
catagen), hDPC
hDPC specific
specific anagen
anagen markers,
markers,
cycle modulation (anagen and catagen), hDPC specific anagen markers, such as alkaline such
such as
as alkaline
alkaline
phosphatase
phosphatase (ALP)
(ALP) and
phosphatase and β-catenin,
β-catenin,
(ALP) were
were measured
and β-catenin, measured after
after the
were measured D-panthenol
the after
D-panthenol treatment.
treatment.
the D-panthenol After
After After
treatment.
24 h treatment
24 h treatment of 50 μM to
of 50 μMofto505µM
24 h treatment 5 mM of D-panthenol,
mMtoof5 D-panthenol,
mM of D-panthenol, the number
the number and intensity
and intensity
the number of ALP
of ALP
and intensity ofposi-
posi-
ALP positive
tive
tive cells were
cellscells significantly
werewere
significantly increased
increased
significantly by
by 40%
increased 40%by(Figure
(Figure 3A).
3A). The
40% (Figure The mRNA
mRNA
3A). expression
expression
The mRNA level
level of
expression of level of
ALP
ALP was
wasALPalso significantly
alsowas up-regulated
also significantly
significantly by
by 40%
up-regulated
up-regulated 40% in by 5540%
in mM
mMinD-panthenol
5 mM D-panthenol
D-panthenol treated
treated groups
treated groups
groups
(Figure
(Figure 3B).
3B). Alkaline
(Figure phosphatase
3B). Alkaline
Alkaline phosphatase activity
activity is
phosphatase considered
activity
is as
as an
an inherent
is considered
considered marker
marker for
as an inherent
inherent hair
marker
for hair for hair
growth
growth promotion in dermal papilla [27]. Furthermore, ALP activity was reported to be
promotion
growth in dermal
promotion inpapilla
dermal [27]. Furthermore,
papilla [27]. ALP
Furthermore, activity
ALP was reported
activity was to
reported
be to be
regulated by Wnt/β-catenin
regulatedregulated pathway
by Wnt/β-catenin
by Wnt/β-catenin activation
pathway pathway [28].
activationactivation Histochemical studies
[28]. Histochemical
[28]. Histochemical revealed that
studies revealed
studies revealed that that
both
both ALP and
ALPboth
andALPβ-catenin were
were highly
and β-catenin
β-catenin wereexpressed
highly in
in the
highly expressed
expressed the early to
to mid-anagen
in the
early phase
phase of
early to mid-anagen
mid-anagen the
ofphase
the of the
hair
hair follicle
hair[27,29].
follicle follicleAs
[27,29]. As shown
[27,29].
shown Asin Figure
Figurein3C,
inshown immunocytochemical
Figure
3C, 3C, immunocytochemical
immunocytochemical staining revealed
staining staining that
revealedrevealed
that that
D-panthenol
D-panthenol concentration
D-panthenol
concentration dependently
concentration
dependently up-regulated
dependently
up-regulated the
the protein
up-regulated theexpressions
protein of
of β-catenin
protein expressions
expressions of β-catenin
β-catenin
and
and versican.
versican. Western
and versican. blot
blot and
WesternWestern RT-PCR
andblot analyses
and RT-PCR
RT-PCR analyses confirmed
analyses
confirmed the
the increased
confirmed β-catenin
the increased
increased pro-
pro- protein
β-catenin
β-catenin
tein and versican
and mRNA
versican mRNA levels, respectively
levels, (Figure
respectively
tein and versican mRNA levels, respectively (Figure 3C). 3C).
(Figure 3C).

(A)
(A) (B)
(B)
Figure 3. Cont.
 Curr.Issues
Curr. IssuesMol.
Mol.Biol.
Biol.2021,
2021,1,43
FOR PEER REVIEW 1367
7

Curr. Issues Mol. Biol. 2021, 1, FOR PEER REVIEW 7

(C) (D)
Figure
Figure3.3.Anagen
Anagen markers
markers of of hDPCs
hDPCswere
weresignificantly
significantly increased
increased by by D-panthenol
D-panthenol treatment.
treatment. (A)
(A) Alka-
(C) (D)
Alkaline phosphatase staining (×40 magnification) and (B) mRNA expression of ALP
line phosphatase staining (×40 magnification) and (B) mRNA expression of ALP after 24 h treatment after 24 h treat-
Figure of
ment 3. Anagen markersinofcultured
D-panthenol hDPCs were significantly
hDPCs. increased
(C) Versican by D-panthenol
and β-catenin treatment. (A)
expressions were double
of D-panthenol in cultured hDPCs. (C) Versican and β-catenin expressions were double stained
Alkaline phosphatase
stained staining (×40 magnification)
by immunocytochemistry after 24 hand (B) mRNAofexpression
treatment of ALP(×200
D-panthenol after 24 h treat-
magnification). (D)
by immunocytochemistry after 24 h treatment of D-panthenol ( × 200 magnification).
ment of D-panthenol in cultured hDPCs. (C) Versican and β-catenin expressions were double (D) Western
Western blot analysis of β-catenin and RT-PCR analysis of versican mRNA were also performed. *
blot analysis
stained of β-catenin andafter
by immunocytochemistry RT-PCR
24 h analysis
treatmentof ofversican mRNA
D-panthenol (×200were also performed.
magnification). (D) * p < 0.05,
p < 0.05, ** p < 0.01 versus control group. Data are expressed as mean ± SD.
Western blot versus
** p < 0.01 analysiscontrol
of β-catenin
group.andData
RT-PCRare analysis
expressed of versican
as meanmRNA
± SD.were also performed. *
p < 0.05, ** p < 0.01 versus control group. Data are expressed as mean ± SD.
3.4.
3.4.D-Panthenol
D-PanthenolReducedReducedTGF-β1
TGF-β1Expression
ExpressionininCultured
CulturedhDPCs
hDPCs
3.4. D-Panthenol
TGF-β1 Reduced TGF-β1 Expression in Cultured hDPCs
TGF-β1was wasreported
reportedtotostimulate
stimulatethe thehair
hairfollicle
follicletotoenter
enterthethecatagen
catagenphase
phase[30].
[30].InIn
TGF-β1 was
androgenetic reported precocious,
alopecia, to stimulate the hair follicle
repeated to enter the
stimulation of catagen
the dermalphasepapilla
[30]. In by andro-
androgenetic alopecia, precocious, repeated stimulation of the dermal papilla by androgens
androgenetic
gens inhibits alopecia,
the growth precocious, repeated
of adjacent stimulation
epithelial cells ofwhich
the dermal
leads papilla
the by
toprogression andro-
progression of hor-
inhibits
gens thethe
inhibits growth
growthofofadjacent epithelial
adjacent epithelial cells
cells which
which leads
leads to progression
to the the of hor-of hormonal
monal baldness
baldness [31].
[31].[31]. To
To evaluate evaluate
whetherwhether D-panthenol possibly inhibits the catagen entry
monal baldness To evaluate whetherD-panthenol possibly
D-panthenol possibly inhibits
inhibits the the catagen
catagen entryentry of the
of the
ofdermal dermal
papilla,
the dermal papilla,
the the
papilla, the
effects effects
of of
effects of D-panthenol
D-panthenol
D-panthenolon onthe on
the mRNAthe
mRNA and mRNA
andprotein and protein
proteinexpression expression
expression levels of
levels
TGF-β1
levels ofTGF-β1
of TGF-β1 were
were assessed.
were assessed.
As shown
assessed. Asinshown
As shown Figure in4A,
in Figure Figure
5 mM
4A, 4A,
5 mM 5 mM D-panthenol
D-panthenol
D-panthenol significantly
significantly
significantly decreased
decreased
decreased
the mRNA themRNA
the mRNA
expression expression
expression of in
of TGF-β1
of TGF-β1 TGF-β1 in cultured
in cultured
cultured hDPCs by
hDPCs hDPCs
by 30% by
30%compared 30% compared
compared to the to the
to the control.
control.
control. Immunocytochemistry
Immunocytochemistry
Immunocytochemistry results results
results showed
showed showed
thatthat that D-panthenol
D-panthenol
D-panthenol also decreased
also decreased
also decreased the
theTGF-
TGF-β1 theprotein
TGF-
β1
β1 protein
level in alevel
protein levelinina concentration-dependent
a concentration-dependent
concentration-dependent mannermanner (Figure
manner
(Figure 4B).
(Figure
4B). 4B).

(A) (B)

Figure 4. D-panthenol reduced TGF-β1 expression in cultured hDPCs. Treatment of D-panthenol for
24 h decreased the (A)(A) mRNA and (B) protein expressions of TGF-β1 in cultured
(B) hDPCs (×200 mag-
nification). * p < 0.05, ** p < 0.01 versus control group. Data are expressed as mean ± SD.
rr. Issues Mol. Biol. 2021, 1, FOR PEER REVIEW 8
Curr. Issues Mol. Biol. 2021, 1, FOR PEER REVIEW 8

Figure 4. D-panthenol reduced TGF-β1 expression in cultured hDPCs. Treatment of D-panthenol

Curr. Issues Mol. Biol. 2021, 24Figure
for 43 4. D-panthenol
h decreased reduced
the (A) mRNA TGF-β1
and expression
(B) protein in cultured
expressions hDPCs.
of TGF-β1 Treatment
in cultured of D-panthenol
hDPCs (×200 1368
for 24 h decreased
magnification). the
* p < 0.05, ** (A) mRNA
p < 0.01 andcontrol
versus (B) protein expressions
group. of TGF-β1asinmean
Data are expressed cultured
± SD.hDPCs (×200
magnification). * p < 0.05, ** p < 0.01 versus control group. Data are expressed as mean ± SD.
3.5. D-Panthenol Stimulated VEGF Expression in Cultured hDPCs
3.5. D-Panthenol Stimulated
3.5. D-Panthenol VEGF Expression
Stimulated in Cultured
VEGF Expression hDPCs hDPCs
in Cultured
In the hair follicle, VEGF plays a critical role for hair growth promotion, especially in
In the hair follicle,
In the VEGF plays
hair follicle, VEGF a critical
plays arole for hair
critical role growth
for hairpromotion, especiallyespecially
growth promotion, in
anagen phase elongation [7,32,33]. VEGF from dermal papilla attracts adjacent blood ves-
anageninphase
anagenelongation [7,32,33]. VEGF
phase elongation from
[7,32,33]. dermal
VEGF frompapilla
dermal attracts adjacent
papilla blood
attracts ves- blood
adjacent
sel to supply vascular-derived growth factors to the hair follicles. VEGF also stimulates
sel to supply
vessel tovascular-derived growth factors
supply vascular-derived growthtofactors
the hair follicles.
to the VEGF also
hair follicles. VEGF stimulates
also stimulates
adjacent cells, such as outer root sheath cells, matrix progenitor cells, sebaceous glands,
adjacentadjacent cells,assuch
cells, such outerasroot
outer root sheath
sheath cells, matrix
cells, matrix progenitor
progenitor cells, sebaceous
cells, sebaceous glands,glands,
sweat glands, and epidermis to make the hair follicle fully matured and the hair shaft
sweat glands,
sweat glands, and epidermis
and epidermis to make to the
make the
hair hair follicle
follicle fully matured
fully matured and the and theshaft
hair hair shaft
differentiated [34]. As shown in Figure 5, the protein level of VEGF was up-regulated by
differentiated [34]. As shown in Figure 5, the protein level of VEGF
differentiated [34]. As shown in Figure 5, the protein level of VEGF was up-regulated by was up-regulated by
the D-panthenol treatment.
the D-panthenol
the D-panthenol treatment.
treatment.

Figure 5. D-panthenol stimulated VEGF expression in cultured hDPCs. The protein level of VEGF
Figure 5.
in cultured D-panthenol
Figure
hDPCs 5.
was stimulated
D-panthenol
evaluated VEGF expression
stimulated
by immunocytochemistry in cultured
VEGF expression after 24hDPCs.
in cultured The protein
hDPCs.
h treatment ofThe level oflevel
protein
D-panthenol VEGF of VEGF
in cultured hDPCs
(×200 magnification). was
* phDPCsevaluated
< 0.05, ** by immunocytochemistry
p <evaluated
0.01 versusbycontrol after 24 h treatment
group. Data are expressed of D-panthenol
in cultured was immunocytochemistry after 24 hastreatment
mean ± SD.
of D-panthenol
(×200 magnification). * p < 0.05, ** p < 0.01 versus control group. Data are expressed as mean ± SD.
(×200 magnification). * p < 0.05 versus control group. Data are expressed as mean ± SD.
3.6. D-Panthenol Stimulated the Growth of Cultured hORSCs and Reduced the Senescence
3.6. D-Panthenol Stimulated
3.6. D-Panthenol the Growth
Stimulated of Cultured
the Growth hORSCs
of Cultured and Reduced
hORSCs the Senescence
and Reduced the
Markers
MarkersSenescence Markers
The hORSCs are keratinocytes which originated from epidermal bulge stem cells and
The hORSCs
The hORSCs are keratinocytes
are keratinocytes which originated
which originated from epidermalfrom epidermal
bulge stembulge stem cells
cells and
comprise the outermost part of the hair follicle. Hair follicle hORSCs go through repeating
compriseandthe
comprise
outermost thepart
outermost
of the hair part of theHair
follicle. hairfollicle
follicle. Hair follicle
hORSCs hORSCs
go through go through
repeating
proliferation and apoptosis during hair follicle elongation and regression cycles, respec-
repeating
proliferation andproliferation
apoptosis during and apoptosis during
hair follicle hair follicle
elongation and elongation
regression and regression
cycles, respec- cycles,
tively [35]. During late telogen and early anagen, bulge stem cells migrate along the ORS
respectively
tively [35]. During late[35].telogen
Duringand late early
telogen and early
anagen, bulgeanagen, bulge
stem cells stem cells
migrate along migrate
the ORS along the
to reach theORShairtobulb
reach [36].
the Because
hair bulbthe proliferative
[36]. Because capacity
the of hORSCs
proliferative determines
capacity of hORSCs thedetermines
to reach the hair bulb [36]. Because the proliferative capacity of hORSCs determines the
anagen, catagen or telogen phasetelogen
of the hair follicle
thestructure, thestructure,
effects oftheD-panthenol
anagen,the anagen,
catagen orcatagen
telogenorphase phase
of the of follicle
hair hairstructure,
follicle the effects of effects of D-panthenol
D-panthenol
on the cell viability were investigated in several parameters [37]. The treatment of D-pan-
on the cell viability were investigated in several parameters [37]. The treatment of D-pan- of D-
on the cell viability were investigated in several parameters [37]. The treatment
thenol significantly stimulated the growth of cultured hORSCs in a concentration-depend-
thenol panthenol
significantly significantly
stimulated the stimulated
growth of the growth
cultured of cultured
hORSCs hORSCs in a concentration-
in a concentration-depend-
ent mannerdependent
(Figure 6A). NADH
manner and
(FigureandNADPH
6A).NADPH generation,
NADH generation, presented
and NADPHpresented as cell
generation, viability,
presented were
as cell viability,
ent manner (Figure 6A). NADH as cell viability, were
increased by 15%
were and 20%
increased in
by the
15% presence
and 20% of 0.15
in themM and
presence 0.3ofmM0.15 ofmMD-panthenol,
and 0.3 mM respec-
of D-panthenol,
increased by 15% and 20% in the presence of 0.15 mM and 0.3 mM of D-panthenol, respec-
tively. D-panthenol significantly increased the Bcl2/Bax ratio toBcl2/Bax
1.71 and ratio
1.88 folds, sug-
tively. respectively.
D-panthenol D-panthenol
significantly significantly
increased theincreased
Bcl2/Baxthe ratio to 1.71 and 1.88to 1.71 andsug-
folds, 1.88 folds,
gesting a possible anti-apoptotic
suggesting a possible effect of
anti-apoptoticD-panthenol
effect of in cultured
D-panthenol hORSCs
in cultured(Figure
hORSCs 6B).(Figure 6B).
gesting a possible anti-apoptotic effect of D-panthenol in cultured hORSCs (Figure 6B).
Furthermore, mRNA expressions
Furthermore, of p21 and p16 wereand significantly
were reduced by 50% in 2.5
Furthermore, mRNAmRNA expressions
expressions of p21 and of p21
p16 werep16 significantlysignificantly
reduced by reduced
50% inby 2.550% in
mM of D-panthenol
2.5 mM oftreatment,
D-panthenol indicating
treatment,thatindicating
cell senescence
that could
cell be prevented
senescence could by
be D-
prevented by
mM of D-panthenol treatment, indicating that cell senescence could be prevented by D-
panthenol (Figure 6C). (Figure 6C).
D-panthenol
panthenol (Figure 6C).

(A) (B)
(A) (B)
Figure 6. Cont.
 Curr.Issues
Curr. IssuesMol.
Mol.Biol.
Biol.2021,
2021,1,43FOR PEER REVIEW 1369
9
Curr. Issues Mol. Biol. 2021, 1, FOR PEER REVIEW 9

(C) (C)
Figure 6.Figure
Figure6.6.D-panthenol
D-panthenol stimulated
D-panthenol stimulated
the growth
stimulated the
theofgrowth
humanofof
growth human
outer outer
root
human root
sheath
outer sheath
cells
root cells
cells(hORSCs).
(hORSCs).
sheath (A) The (A)
(hORSCs). (A)The
The
growth ofgrowth
hORSCs of hORSCs
was was
assessed assessed
after 24 hafter 24
treatment h treatment
of of D-panthenol.
D-panthenol. (B) (B)
Relative Relative
mRNA mRNA expression
expression
growth of hORSCs was assessed after 24 h treatment of D-panthenol. (B) Relative mRNA expression
ratiostoofBax
ratios of Bcl2 Bcl2 to Bax
were were calculated
calculated after 24 hafter 24 h treatment
treatment of D-panthenol.
of D-panthenol. (C) mRNA (C)expression
mRNA expression
of of
ratios of Bcl2 to Bax were calculated after 24 h treatment of D-panthenol. (C) mRNA expression of
senescence markers (p21 and p16) in cultured hORSCs after 24 h treatment
senescence markers (p21 and p16) in cultured hORSCs after 24 h treatment of D-panthenol. * p < of D-panthenol. * p <
senescence markers (p21 and p16) in cultured hORSCs after 24 h treatment of D-panthenol. * p < 0.05,
0.05, ** p 0.05,
< 0.01**versus
p < 0.01 versusgroup.
control controlData
group.
are Data are expressed
expressed as mean ±asSD.mean ± SD.
** p < 0.01 versus control group. Data are expressed as mean ± SD.
3.7.
3.7.D-Panthenol
3.7. D-Panthenol Induced
Induced the
D-Panthenol the
theExpression
Expression
Induced of VEGFofand
Expression ofVEGF
VEGFR
VEGF and VEGFR
andin ininCultured
Cultured
VEGFR CulturedhORSCs
hORSCs hORSCs
Several Several
reports reports demonstrated
demonstrated that VEGF that VEGF
mediated mediated
the
Several reports demonstrated that VEGF mediated the proliferation the proliferation
proliferation of of
hORSCsof hORSCs
hORSCs
through
through the the ERK
ERK pathway
through pathway
the ERK [12]. [12].
To clarify
pathway To
[12]. theclarify the
To underlying underlying mechanism
mechanism for
clarify the underlying for the proliferative
the proliferative
mechanism for the prolifer-
effect of effect
ative of D-panthenol
D-panthenol
effect on hORSCs,
ofonD-panthenol
hORSCs, changes changes
in mRNA
on hORSCs, in levels
mRNA
changes oflevels
in several
mRNA of growth
severaloffactors
levels growth
severalfactors
were
growth were
fac-
evaluated.evaluated.
tors note,Of
Of were note, VEGFA
VEGFA
evaluated. expressionexpression
Of note, was was
prominently
VEGFA prominently
expression wasincreased
increased by 70%, by
prominently 70%,
comparablecomparable
increased to
to by 70%,
that
that of the of the
minoxidil minoxidil
treatment,treatment,
a positivea positive
control control
(Figure (Figure
7A). The 7A). The expression
expression
comparable to that of the minoxidil treatment, a positive control (Figure 7A). The ex- of VEGF of VEGF
receptorreceptor
2 (KDR)2was
pression (KDR)
of alsowas
VEGF also significantly
significantly
receptor 2 (KDR) wasincreased
increased by D-panthenol
by D-panthenol
also significantly in in by
a concentration-de-
a concentration-de-
increased D-panthenol in a
pendent
pendent manner manner (Figure 7B).
(Figure 7B). Our data
concentration-dependent Our data
indicate
manner indicate
(Figurethat7B). that
D-panthenolD-panthenol
inducedthat
Our data indicate induced the proliferation
the proliferation
D-panthenol induced
of
of cultured cultured
the hORSCs hORSCs
through
proliferation through VEGF signaling.
VEGF signaling.
of cultured hORSCs through VEGF signaling.

(A) (A) (B) (B)

Figure 7.Figure
Figure 7.7. The
The mRNA The mRNA
mRNAexpression
expression levels of levels
expression levelsof
VEGF ofVEGF
andVEGF
VEGFRand VEGFR
andwere
VEGFR were
were up-regulated
up-regulated up-regulated by
by D-panthenol
by D-panthenolD-panthenol
treatmenttreatment
in cultured
treatment inincultured
culturedhORSCs.
hORSCs. The mRNA
hORSCs. The
The mRNA
mRNAexpression
expression ofof(A)
of (A) VEGFA
expression (A)andVEGFA and
and(B)
(B) VEGFR
VEGFA VEGFR
(B)(KDR)
VEGFR (KDR)
was(KDR)was
was
evaluatedevaluated
by RT-PCR
evaluated by RT-PCR
byafter 24 hafter
RT-PCR 24
24hhtreatment
treatment
after of
ofD-panthenol.
of D-panthenol.
treatment * p < 0.05,****
D-panthenol. pp<p<<0.05,
0.01****
0.05, pp<<0.01
versus versus
control
0.01 control
controlgroup.
group.
versus group.
Data are Data are expressed
expressed as mean ±as
Data are expressed
mean ± SD.
SD.
as mean ± SD.

4.4.Discussion
4. Discussion Discussion
In
Inthe
In the present present
thestudy,
presentstudy,
westudy, we
weinvestigated
investigated
investigated the ininvitro
vitroanti-hair
theanti-hair
the in vitro anti-hairlossofeffects
loss
loss effects effectsofofD-panthenol,
D-panthenol,D-panthenol,
focusing
focusingfocusing onthe
on themodulation
on the modulation modulation ofthe
of
of the hair thehair
haircycle
follicle follicle
follicle cyclein
cycle
in cultured incultured
cultured humanpapilla
human
human dermal dermaland
dermal papillaand
papilla and
outer rootouter
outer rootcells.
root
sheath sheath
sheath cells. D-panthenol
D-panthenol is widely is widely
widely
used asused
used as
a skinasapharmaceutical
askin
skinpharmaceutical
pharmaceutical
and cos- and cosme-
and cos-
meceuticalceutical
meceutical ingredient.
Most ofMost
ingredient.
ingredient. Most ofofthe
theresearch
the research research on
onthethe pharmacological
pharmacological
on the pharmacological action ofaction of D-panthenol
of
D-panthenolD-panthenol
is
is mainly
mainly focused
focused onon skin
skin hydration,
hydration, anti-irritation
anti-irritation andand wound
wound
is mainly focused on skin hydration, anti-irritation and wound healing. Little was done healing.
healing. Little
Little wasinwas done
done in
Curr. Issues Mol. Biol. 2021, 43 1370

in the field of anti-hair loss research, especially concerning the cell-based mechanisms,
despite its wide use in hair and scalp care products.
We have found that D-panthenol enhanced the growth of hDPCs and hORSCs
(Figures 1A and 6D), inducing the expression of cell proliferation marker (Ki67, Figure 1B)
in cultured hDPCs. Cellular growth in hair follicles has been reported as a significant
characteristic of the anagen phase [38–41]. The dermal papilla goes through miniaturiza-
tion when hair follicles proceed to catagen and telogen [42,43], after which it recovers its
original size in the course of anagen initiation. The recovering growth force of the dermal
papilla could grant anagen persistency against several insults, such as DHT, TGF- β, and
oxidative stress. Unlike the dermal papilla, the outer root sheath establishes the outer
structure of the hair follicle from the upper bulge to lower bulb through the most vigorous
proliferation during the anagen phase [44]. The outer root sheath, on the other hand, does
not go through miniaturization, but instead total extinction during the regression, catagen
phase [45]. In this context, we also have found that apoptosis signals were significantly re-
duced by D-panthenol treatment in cultured hair follicle cells (Figures 2A and 6B). Dermal
papilla is considered to resist apoptosis, associated with high levels of Bcl-2 and a lack of
death receptors during the entire hair cycle in mice and humans [45–48]. However, they
may be susceptible to apoptosis under certain experimental conditions [49,50] and it has
been reported that minoxidil prevented cellular apoptosis in dermal papilla cells [46]. Our
data suggest that the reduction of the apoptotic markers by D-panthenol treatment could
explain at least in part the cellular mechanism of its clinical anti-hair loss efficacy.
D-panthenol treatment also significantly reduced the mRNA expression of cell senes-
cence markers, p21 and p16 (Figures 2B and 6C). p16 and p21 are well-known senescence
markers which inhibit cyclin-dependent kinase activity. Senescence-related cell prolifer-
ation markers are decreased by the increased expression of p21 and p16 [51,52]. Indeed,
balding DPCs and ORSCs, expressing high levels of p16, showed a much slower growth
rate and immature hair follicle differentiation [53]. Taken together, our study suggests
that D-panthenol could possibly support hair growth by stimulating the cell proliferation,
suppressing the apoptosis and preventing the senescence in hair follicle DPCs and ORSCs.
Alkaline phosphatase activity is a well-known anagen marker of dermal papilla. We
have found that D-panthenol treatment increased ALP activity and also up-regulated
the mRNA expression of ALP (Figure 3A,B). The expression of versican, another dermal
papilla anagen marker, was also elevated by D-panthenol treatment both in protein and
mRNA levels (Figure 3C,D). Since Wnt/β-catenin pathway activation plays a crucial role
for maintaining anagen in the dermal papilla, we investigated the effect of D-panthenol
on Wnt/β-catenin signaling. Up-regulated β-catenin protein level was confirmed by both
immunostaining and Western blotting (Figure 3C,D). The precise mechanisms, however,
need to be elucidated. Our results demonstrate that D-panthenol could assist anagen phase
establishment and maintenance by stimulating ALP, versican expression, and Wnt/β-
catenin signaling.
Not only anagen maintenance, but also catagen inhibition could be a main strategy
for anti-hair loss therapies. TGF-β1 expressed in bald DPC is known to stimulate catagen
entry of the hair follicle. We have found that D-panthenol significantly reduced TGF-
β1 expressions in both mRNA and protein levels (Figure 4A,B). Taken together, our data
suggest that D-panthenol could support hair growth by inhibiting catagen entry, elongating
anagen phase.
Among growth factors, VEGF is reported to stimulate cell proliferation both in the
dermal papilla and outer root sheath. VEGF exerts its effects through VEGF receptor-2,
a receptor tyrosine kinase expressed in both the dermal papilla and outer root sheath. It
was revealed that the expression of VEGF was up-regulated by D-panthenol treatment
not only in hDPCs but also in hORSCs (Figures 5 and 7B). The enhanced expression of
VEGF in ORSCs, even though only in mRNA levels, is not a commonly reported event
whose implication should be elusive. In addition, the expression of VEGF-R2 (KDR) was
also up-regulated by D-panthenol in cultured hORSCs. Our results demonstrate that D-
Curr.
Curr.Issues
IssuesMol.
Mol.Biol. 2021,43
Biol.2021, 1, FOR PEER REVIEW 1371
11

could support
panthenol couldhair growth
support hairbygrowth
stimulating the VEGFthe
by stimulating signals,
VEGFwhich is which
signals, important to deter-
is important
mine
to hair follicle
determine size by size
hair follicle angiogenesis in both in
by angiogenesis hDPCs and hORSCs.
both hDPCs and hORSCs.
In the
In the present
present study,
study,we wedemonstrated
demonstratedthe thein invitro
vitrohair
hairgrowth-supporting
growth-supportingactivities
activities
of D-panthenol
of D-panthenol and elucidated
elucidated several
severalunderlying
underlyingcellular
cellularand
andmolecular
molecularmechanisms
mechanisms of
D-panthenol.
of D-panthenol. In spite of the
In spite of in vitro
the evidence,
in vitro our study
evidence, has major
our study limitations
has major since neither
limitations since
in vivo in
neither mimicking systems,
vivo mimicking such assuch
systems, human hair follicle
as human organ culture,
hair follicle nor an nor
organ culture, animal
an
model have
animal model been
haveadopted for further
been adopted for elucidation. Despite these
further elucidation. limitations
Despite our data our
these limitations still
havestill
data meaningful implications
have meaningful that our findings
implications that ourdo provide
findings dosome cellular
provide some and molecular
cellular and
clues of D-panthenol
molecular for its hair
clues of D-panthenol growth
for its stimulating
hair growth activities
stimulating whichwhich
activities have have
not been
not
demonstrated
been demonstrated yet. yet.
In
In conclusion, findings demonstrate
conclusion, our findings demonstratethat thatD-panthenol
D-panthenolstimulated
stimulatedthe thecell
cellprolif-
pro-
liferation and decreased the expression of cell senescence markers
eration and decreased the expression of cell senescence markers such as p16 and p21 both such as p16 and p21
both in cultured
in cultured hDPCshDPCsand and hORSCs.
hORSCs. D-panthenol
D-panthenol reduced
reduced the expression
the expression of apoptosis
of apoptosis and
and senescence
senescence markers,
markers, increasing
increasing the the expression
expression of anagen
of anagen markers
markers andand decreasing
decreasing thethe
ex-
expression
pression of of catagen-inducing
catagen-inducing factors
factors in cultured
in cultured hDPCs.
hDPCs. Furthermore,
Furthermore, D-panthenol
D-panthenol stim-
stimulated
ulated the the expression
expression of of VEGF
VEGF inin bothhDPCs
both hDPCsand andhORSCs,
hORSCs, additionally
additionally up-regulating
up-regulating
VEGF-R
VEGF-R expression in cultured hORSCs. Our data demonstrate that D-panthenolcould
expression in cultured hORSCs. Our data demonstrate that D-panthenol could
promote
promote hairhair growth
growth by by prolonging
prolonging the the anagen
anagen phase
phase and
and preventing
preventing catagen
catagen entry
entry by by
stimulating
stimulating the the dermal
dermal papilla
papilla and
and outer
outer root
rootsheath
sheathcells.
cells. The
Theeffects
effectsofofD-panthenol
D-panthenolon on
hair
hair follicle
follicle cells
cellsare
aresummarized
summarizedin inFigure
Figure8.8.

Figure 8. Summarized
Figure Summarizedininvitro
vitroeffects of of
effects D-panthenol in hair
D-panthenol follicle
in hair cells.cells.
follicle All these
All effects in combi-
these effects in
nation couldcould
combination lead to hairtogrowth
lead promotion.
hair growth ↑:upregulation,
promotion. ↓:downregulation
↑:upregulation, ↓:downregulation.

AuthorContributions:
Author Contributions: Conceptualization
Conceptualization and andmethodology:
methodology:J.Y.S.
J.Y.S.and S.L.;
and Experiment
S.L.; Experiment processing:
process-
J.Y.S., J.K. and Y.-H.C.; Formal analysis, data curation and statistical analysis: J.Y.S.; Writing-original
ing: J.Y.S., J.K. and Y.-H.C.; Formal analysis, data curation and statistical analysis: J.Y.S.; Writing-
draft preparation:
original J.Y.S.; writing-review
draft preparation: and editing:
J.Y.S.; writing-review andN.-G.K. and
editing: S.L.; Supervision
N.-G.K. and project man-
and S.L.; Supervision and
agement: N.-G.K. and S.L. All authors have read and agreed to the published
project management: N.-G.K. and S.L. All authors have read and agreed to the published version of version
the manu-
of
script.
the manuscript.
Funding: This
Funding: This research
researchreceived
receivedno noexternal
externalfunding.
funding.
Data Availability Statement: The datasets used and/or analyzed during the current study are avail-
Data Availability Statement: The datasets used and/or analyzed during the current study are
able from the corresponding author on reasonable request. Some data may not be available because
available from the corresponding author on reasonable request. Some data may not be available
of the policy of company and ethical restrictions.
because of the policy of company and ethical restrictions.
Conflicts of Interest: The authors declare no conflict of interest.
Conflicts of Interest: The authors declare no conflict of interest.

References
Curr. Issues Mol. Biol. 2021, 43 1372

References
1. Schneider, M.R.; Schmidt-Ullrich, R.; Paus, R. The hair follicle as a dynamic miniorgan. Curr. Biol. 2009, 19, R132–R142. [CrossRef]
2. Whiting, D.A. Possible mechanisms of miniaturization during androgenetic alopecia or pattern hair loss. J. Am. Acad. Dermatol.
2001, 45, S81–S86. [CrossRef]
3. Giacomini, F.; Starace, M.; Tosti, A. Short anagen syndrome. Pediatr. Dermatol. 2011, 28, 133–134. [CrossRef]
4. Kim, J.; Shin, J.Y.; Choi, Y.H.; Jang, M.; Nam, Y.J.; Lee, S.Y.; Jeon, J.; Jin, M.H.; Lee, S. Hair Growth Promoting Effect of Hottuynia
cordata Extract in Cultured Human Hair Follicle Dermal Papilla Cells. Biol. Pharm. Bull. 2019, 42, 1665–1673. [CrossRef] [PubMed]
5. Lee, S.H.; Yoon, J.; Shin, S.H.; Zahoor, M.; Kim, H.J.; Park, P.J.; Park, W.S.; Min do, S.; Kim, H.Y.; Choi, K.Y. Valproic acid induces
hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells. PLoS ONE 2012,
7, e34152. [CrossRef] [PubMed]
6. Itami, S.; Kurata, S.; Takayasu, S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth
factor-I from dermal papilla cells. Biochem. Biophys. Res. Commun. 1995, 212, 988–994. [CrossRef] [PubMed]
7. Lachgar, S.; Charveron, M.; Gall, Y.; Bonafe, J.L. Minoxidil upregulates the expression of vascular endothelial growth factor in
human hair dermal papilla cells. Br. J. Dermatol. 1998, 138, 407–411. [CrossRef]
8. Li, W.; Man, X.Y.; Li, C.M.; Chen, J.Q.; Zhou, J.; Cai, S.Q.; Lu, Z.F.; Zheng, M. VEGF induces proliferation of human hair follicle
dermal papilla cells through VEGFR-2-mediated activation of ERK. Exp. Cell Res. 2012, 318, 1633–1640. [CrossRef]
9. Inoue, K.; Aoi, N.; Yamauchi, Y.; Sato, T.; Suga, H.; Eto, H.; Kato, H.; Tabata, Y.; Yoshimura, K. TGF-beta is specifically expressed
in human dermal papilla cells and modulates hair folliculogenesis. J. Cell. Mol. Med. 2009, 13, 4643–4656. [CrossRef]
10. Shin, H.; Yoo, H.G.; Inui, S.; Itami, S.; Kim, I.G.; Cho, A.R.; Lee, D.H.; Park, W.S.; Kwon, O.; Cho, K.H.; et al. Induction of
transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells.
BMB Rep. 2013, 46, 460–464. [CrossRef]
11. Kwack, M.H.; Sung, Y.K.; Chung, E.J.; Im, S.U.; Ahn, J.S.; Kim, M.K.; Kim, J.C. Dihydrotestosterone-inducible dickkopf 1 from
balding dermal papilla cells causes apoptosis in follicular keratinocytes. J. Invest. Dermatol. 2008, 128, 262–269. [CrossRef]
[PubMed]
12. Vidal, V.P.; Chaboissier, M.C.; Lutzkendorf, S.; Cotsarelis, G.; Mill, P.; Hui, C.C.; Ortonne, N.; Ortonne, J.P.; Schedl, A. Sox9 is
essential for outer root sheath differentiation and the formation of the hair stem cell compartment. Curr. Biol. 2005, 15, 1340–1351.
[CrossRef] [PubMed]
13. Rompolas, P.; Deschene, E.R.; Zito, G.; Gonzalez, D.G.; Saotome, I.; Haberman, A.M.; Greco, V. Live imaging of stem cell and
progeny behaviour in physiological hair-follicle regeneration. Nature 2012, 487, 496–499. [CrossRef] [PubMed]
14. Zhang, H.; Nan, W.; Wang, S.; Zhang, T.; Si, H.; Yang, F.; Li, G. Epidermal Growth Factor Promotes Proliferation and Migration of
Follicular Outer Root Sheath Cells via Wnt/beta-Catenin Signaling. Cell Physiol. Biochem. 2016, 39, 360–370. [CrossRef] [PubMed]
15. Biro, K.; Thaci, D.; Ochsendorf, F.R.; Kaufmann, R.; Boehncke, W.H. Efficacy of dexpanthenol in skin protection against irritation:
A double-blind, placebo-controlled study. Contact Dermat. 2003, 49, 80–84. [CrossRef]
16. Gehring, W.; Gloor, M. Effect of topically applied dexpanthenol on epidermal barrier function and stratum corneum hydration.
Results of a human in vivo study. Arzneimittelforschung 2000, 50, 659–663. [CrossRef]
17. Stettler, H.; Kurka, P.; Wagner, C.; Sznurkowska, K.; Czernicka, O.; Bohling, A.; Bielfeldt, S.; Wilhelm, K.P.; Lenz, H. A new topical
panthenol-containing emollient: Skin-moisturizing effect following single and prolonged usage in healthy adults, and tolerability
in healthy infants. J. Dermatolog. Treat 2017, 28, 251–257. [CrossRef]
18. Stamatas, G.N.; Tierney, N.K. Diaper dermatitis: Etiology, manifestations, prevention, and management. Pediatr. Dermatol. 2014,
31, 1–7. [CrossRef]
19. Adam, R. Skin care of the diaper area. Pediatr. Dermatol. 2008, 25, 427–433. [CrossRef] [PubMed]
20. Heise, R.; Skazik, C.; Marquardt, Y.; Czaja, K.; Sebastian, K.; Kurschat, P.; Gan, L.; Denecke, B.; Ekanayake-Bohlig, S.;
Wilhelm, K.P.; et al. Dexpanthenol modulates gene expression in skin wound healing in vivo. Skin Pharmacol. Physiol. 2012, 25,
241–248. [CrossRef]
21. Marquardt, Y.; Amann, P.M.; Heise, R.; Czaja, K.; Steiner, T.; Merk, H.F.; Skazik-Voogt, C.; Baron, J.M. Characterization of a
novel standardized human three-dimensional skin wound healing model using non-sequential fractional ultrapulsed CO2 laser
treatments. Lasers Surg. Med. 2015, 47, 257–265. [CrossRef]
22. Kutlu, O.; Metin, A. Systemic dexpanthenol as a novel treatment for female pattern hair loss. J. Cosmet Dermatol. 2021, 20,
1325–1330. [CrossRef]
23. Kutlu, O. Dexpanthenol may be a novel treatment for male androgenetic alopecia: Analysis of nine cases. Dermatol. Ther. 2020,
33, e13381. [CrossRef]
24. Wojtczak, L.; Slyshenkov, V.S. Protection by pantothenic acid against apoptosis and cell damage by oxygen free radicals—The
role of glutathione. Biofactors 2003, 17, 61–73. [CrossRef]
25. Kim, M.H.; Kim, S.H.; Yang, W.M. Beneficial effects of Astragaloside IV for hair loss via inhibition of Fas/Fas L-mediated
apoptotic signaling. PLoS ONE 2014, 9, e92984. [CrossRef]
26. Fujita, E.; Egashira, J.; Urase, K.; Kuida, K.; Momoi, T. Caspase-9 processing by caspase-3 via a feedback amplification loop
in vivo. Cell Death Differ. 2001, 8, 335–344. [CrossRef] [PubMed]
27. Iida, M.; Ihara, S.; Matsuzaki, T. Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium
in mouse vibrissal follicles. Dev. Growth Differ. 2007, 49, 185–195. [CrossRef]
Curr. Issues Mol. Biol. 2021, 43 1373

28. Jin, Y.H.; Kim, H.; Ki, H.; Yang, I.; Yang, N.; Lee, K.Y.; Kim, N.; Park, H.S.; Kim, K. Beta-catenin modulates the level and
transcriptional activity of Notch1/NICD through its direct interaction. Biochim. Biophys. Acta 2009, 1793, 290–299. [CrossRef]
29. Handjiski, B.K.; Eichmuller, S.; Hofmann, U.; Czarnetzki, B.M.; Paus, R. Alkaline phosphatase activity and localization during the
murine hair cycle. Br. J. Dermatol. 1994, 131, 303–310. [CrossRef] [PubMed]
30. Foitzik, K.; Lindner, G.; Mueller-Roever, S.; Maurer, M.; Botchkareva, N.; Botchkarev, V.; Handjiski, B.; Metz, M.; Hibino, T.;
Soma, T.; et al. Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo. FASEB J. 2000, 14, 752–760. [CrossRef]
[PubMed]
31. Hodgins, M.B.; Choudhry, R.; Parker, G.; Oliver, R.F.; Jahoda, C.A.; Withers, A.P.; Brinkmann, A.O.; van der Kwast, T.H.;
Boersma, W.J.; Lammers, K.M.; et al. Androgen receptors in dermal papilla cells of scalp hair follicles in male pattern baldness.
Ann. N. Y. Acad. Sci. 1991, 642, 448–451. [CrossRef]
32. Yano, K.; Brown, L.F.; Detmar, M. Control of hair growth and follicle size by VEGF-mediated angiogenesis. J. Clin. Invest. 2001,
107, 409–417. [CrossRef]
33. Park, P.J.; Moon, B.S.; Lee, S.H.; Kim, S.N.; Kim, A.R.; Kim, H.J.; Park, W.S.; Choi, K.Y.; Cho, E.G.; Lee, T.R. Hair growth-promoting
effect of Aconiti Ciliare Tuber extract mediated by the activation of Wnt/beta-catenin signaling. Life Sci. 2012, 91, 935–943.
[CrossRef]
34. Wu, X.J.; Jing, J.; Lu, Z.F.; Zheng, M. VEGFR-2 Is in a State of Activation in Hair Follicles, Sebaceous Glands, Eccrine Sweat
Glands, and Epidermis from Human Scalp: An In Situ Immunohistochemistry Study of Phosphorylated VEGFR-2. Med. Sci.
Monit. Basic Res. 2019, 25, 107–112. [CrossRef]
35. Tanaka, T.; Narisawa, Y.; Misago, N.; Hashimoto, K. The innermost cells of the outer root sheath in human anagen hair follicles
undergo specialized keratinization mediated by apoptosis. J. Cutan Pathol. 1998, 25, 316–321. [CrossRef]
36. Li, H.; Masieri, F.F.; Schneider, M.; Bartella, A.; Gaus, S.; Hahnel, S.; Zimmerer, R.; Sack, U.; Maksimovic-Ivanic, D.;
Mijatovic, S.; et al. The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-
Specific Stem Cell Markers. Biomolecules 2021, 11, 154. [CrossRef] [PubMed]
37. Shi, G.; Sohn, K.C.; Kim, S.Y.; Ryu, E.K.; Park, Y.S.; Lee, Y.; Seo, Y.J.; Lee, J.H.; Kim, C.D. Sox9 Increases the Proliferation and
Colony-forming Activity of Outer Root Sheath Cells Cultured In Vitro. Ann. Dermatol. 2011, 23, 138–143. [CrossRef]
38. Li, J.; Yang, Z.; Li, Z.; Gu, L.; Wang, Y.; Sung, C. Exogenous IGF-1 promotes hair growth by stimulating cell proliferation and
down regulating TGF-beta1 in C57BL/6 mice in vivo. Growth Horm. IGF Res. 2014, 24, 89–94. [CrossRef] [PubMed]
39. Namekata, M.; Yamamoto, M.; Goitsuka, R. Nuclear localization of Meis1 in dermal papilla promotes hair matrix cell proliferation
in the anagen phase of hair cycle. Biochem. Biophys. Res. Commun. 2019, 519, 727–733. [CrossRef]
40. Oh, H.S.; Smart, R.C. An estrogen receptor pathway regulates the telogen-anagen hair follicle transition and influences epidermal
cell proliferation. Proc. Natl. Acad. Sci. USA 1996, 93, 12525–12530. [CrossRef]
41. Qiu, W.; Lei, M.; Zhou, L.; Bai, X.; Lai, X.; Yu, Y.; Yang, T.; Lian, X. Hair follicle stem cell proliferation, Akt and Wnt signaling
activation in TPA-induced hair regeneration. Histochem. Cell Biol. 2017, 147, 749–758. [CrossRef]
42. Kelly, Y.; Blanco, A.; Tosti, A. Androgenetic Alopecia: An Update of Treatment Options. Drugs 2016, 76, 1349–1364. [CrossRef]
43. Matsumura, H.; Mohri, Y.; Binh, N.T.; Morinaga, H.; Fukuda, M.; Ito, M.; Kurata, S.; Hoeijmakers, J.; Nishimura, E.K. Hair follicle
aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis. Science 2016, 351, aad4395. [CrossRef]
44. Fernandez-Flores, A.; Saeb-Lima, M.; Cassarino, D.S. Histopathology of aging of the hair follicle. J. Cutan Pathol. 2019, 46, 508–519.
[CrossRef]
45. Soma, T.; Ogo, M.; Suzuki, J.; Takahashi, T.; Hibino, T. Analysis of apoptotic cell death in human hair follicles in vivo and in vitro.
J. Invest. Dermatol. 1998, 111, 948–954. [CrossRef]
46. Han, J.H.; Kwon, O.S.; Chung, J.H.; Cho, K.H.; Eun, H.C.; Kim, K.H. Effect of minoxidil on proliferation and apoptosis in dermal
papilla cells of human hair follicle. J. Dermatol. Sci. 2004, 34, 91–98. [CrossRef]
47. Lindner, G.; Botchkarev, V.A.; Botchkareva, N.V.; Ling, G.; van der Veen, C.; Paus, R. Analysis of apoptosis during hair follicle
regression (catagen). Am. J. Pathol. 1997, 151, 1601–1617. [PubMed]
48. Matsuo, K.; Mori, O.; Hashimoto, T. Apoptosis in murine hair follicles during catagen regression. Arch. Dermatol. Res 1998, 290,
133–136. [CrossRef] [PubMed]
49. Ferraris, C.; Cooklis, M.; Polakowska, R.R.; Haake, A.R. Induction of apoptosis through the PKC pathway in cultured dermal
papilla fibroblasts. Exp. Cell. Res. 1997, 234, 37–46. [CrossRef] [PubMed]
50. Seiberg, M.; Wisniewski, S.; Cauwenbergh, G.; Shapiro, S.S. Trypsin-induced follicular papilla apoptosis results in delayed hair
growth and pigmentation. Dev. Dyn. 1997, 208, 553–564. [CrossRef]
51. Narita, M.; Nunez, S.; Heard, E.; Narita, M.; Lin, A.W.; Hearn, S.A.; Spector, D.L.; Hannon, G.J.; Lowe, S.W. Rb-mediated
heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003, 113, 703–716. [CrossRef]
52. Collado, M.; Blasco, M.A.; Serrano, M. Cellular senescence in cancer and aging. Cell 2007, 130, 223–233. [CrossRef] [PubMed]
53. Garza, L.A.; Yang, C.C.; Zhao, T.; Blatt, H.B.; Lee, M.; He, H.; Stanton, D.C.; Carrasco, L.; Spiegel, J.H.; Tobias, J.W.; et al. Bald
scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle
progenitor cells. J. Clin. Invest. 2011, 121, 613–622. [CrossRef] [PubMed]