gene expression levels of selected factors in hacat...

Gene expression levels of selected factors in HaCaT

cells upon treatment with plant extracts against hair

loss

H. Aydın1, B. Akyıldırım1, N. Çelik1, H. Sevinç1, M. Kartal2 and M. Türkoğlu1

1Biota Laboratories, Sancaktepe, 34785 Istanbul, Turkey

2Bezmialem Vakıf University, Bezmialem Center for Education, Research and Practice in Phytotherapy

Abstract

Background: Hair loss (alopecia) is a serious problem due to many different reasons. It is of

great interest; therefore, to develop new therapies for treating alopecia and for enhancing hair

growth. Treatments that are utilizing natural products such as plants and their derivatives are

attracting more attention especially studying the mechanism at the cellular level.

Objective: The objective of this study was to examine the hair-growth effect of plant extracts

that have been either traditionally used for treating hair loss or selected through a literature

review. This collection includes the extracts of Myrtus communis L., Urtica dioica L.,

Rosmarinus officinalis L., Raphanus sativus L., Lepidium sativum L., and Ficus carica L..

Our investigation was based on the changes in the gene expression levels of selected factors

such as VEGF, SRD5α (5α-reductase), and IL-1α in HaCaT cells treated with the plants

extracts.

Methods and Results: Plant extracts were prepared with different methods such as Soxhlet

and decoction in different solvents such as water, water-ethanol or oil. Non-cytotoxic

concentrations of the extracts were determined by Cell Proliferation Assay using XTT

reagent. Human keratinocyte (HaCaT) cells were then incubated with the extracts in the

determined concentrations. RNA isolations were carried out for both untreated and treated

cells by using Tri-reagent. Gene expression levels of the factors that have been implicated in

hair growth control were measured by RT-qPCR analysis. Extracts were tested on HaCaT

cells and the effects of these extracts on the VEGF, SRD5α, TNF-α, and IL-1α gene

expression levels were monitored. Among these extracts, Urtica dioica, Ficus carica and

Lepidium sativum led to significant inhibition of SRD5a gene expression (p <0.05). Myrtus

communis, on the other hand, significantly increased the expression level of VEGF.

Discussion and Conclusion: SRD5α gene expression was found to be significantly

downregulated by Urtica dioica, Lepidium sativum and Ficus carica extracts implying the

potential inhibitory effects of the extracts on androgenic activity in HaCaT cells. Upregulation

of VEGF expression by Myrtus communis extract represented the potential hair-growth effect

of the extract. According to these preliminary results, these plant extracts are promising

candidates for enhancing hair growth hence may be used as therapeutic agents against hair

loss. As a result, this study will give an insight about potential hair-growth, anti-inflammatory

and anti-androgenic effects of the selected plants extracts.

Introduction

The presence of hair is characteristic for mammals since it exerts a wide range of functions

including physical protection, thermal insulation, sensory functions and social interactions.

For humans, hair is of greater importance and there are many human diseases associated with

hair loss. Hair loss, also known as alopecia, is a common and distressing problem both for

men and women. The effect of hair disorders on patients’ psychological wellbeing and on

social interactions cannot be ignored. Therefore, treatments for hair loss attract considerable

attention and it is a growing research area which is heavily invested in (Cotsarelis and Millar,

2001). In order to treat hair loss, scientists try to have a molecular understanding of hair loss.

Hair follicle is responsible for the production of hair shaft. The formation of hair follicle takes

place during embryogenesis: hair follicle develops from the embryonic epidermis. Then the

keratinocyte cells of hair follicle differentiate into three enclosed cylindrical parts: the central

most layer forms the hair shaft (fiber), the middle layer forms the inner root sheath (IRS) and

the outermost cylinder forms the outer root sheath (ORS) (Stenn and Paus, 2001; Rogers,

2004). The IRS forms the hair shaft and directs its outward movement whereas the ORS

separates the whole structure from the dermis (Stenn and Paus, 2001). The hair follicle is

remodeled under the control of repetitive cycles of growth (anagen), regression (catagen), rest

(telogen) and shedding (exogen) (Cotsarelis and Millar, 2001). In anagen phase, follicular

keratinocyte cells proliferate rapidly and hair shaft thickens and elongates. Therefore, the

duration of anagen phase determines the length of the hair shaft (Price, 1999). During

catagen, the epithelial cells at the base of the follicle undergo programmed cell death

(apoptosis) reducing the size of the follicle as it enters the resting phase (telogen) (Cotsarelis

and Millar, 2001). At the end of telogen, hair is released and shed in exogen phase and the

next cycle is initiated (Price, 1999).

The cyclic regeneration of hair is thought to be affected and controlled by cyclic cellular

mechanisms. Besides the genetic factors and dietary changes, there are a number of

significant factors which are thought to be responsible for the cellular signaling mechanisms

underlying the hair growth cycle. The hair follicle is highly sensitive to numerous growth

factors, cytokines, neuropeptides and hormones (Schneider et al., 2009). Growth factors such

as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and

keratinocyte growth factor (KGF) are thought to be important for anagen maintenance.

VEGF, for example, is best known for its role in angiogenesis which, in turn, promotes cell

proliferation. VEGF is also identified as a major mediator of hair follicle growth and showed

to improve follicle vascularization promoting hair growth (Yano et al., 2001). KGF, as a

member of fibroblast growth factor (FGF) family, was found to be an important endogenous

mediator of normal hair growth and development (Danilenko et al., 1995). On the other hand,

cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1α/β (IL-1α/β) and interferon-

γ (IFN-γ), have been described to induce catagen either by inducing apoptosis (TNF-α) or by

increasing inflammation (IL-1α/β, IFN-γ) (Schneider et al., 2009; Konur et al., 2005). In

addition to these factors, stereoid 5α-reductase, an enzyme that catalyzes the conversion of

testosterone into 5α-dihydrotestosterone (DHT), was found to play an important role in hair

growth control. The conversion of testosterone into DHT amplifies the androgenic signal in

androgen-related target tissues. Previously, prostate was known to be one of these target

tissues which is rich in 5α-reductase; however, the studies in the 1970s and 1980s have stated

that the skin is also rich in 5α-reductase. Later, it was found that 5α-reductase activity was

associated with the androgen-dependent skin disorders such as acne vulgaris and androgenetic

alopecia (Chen et al., 1998).

Based on this knowledge about the molecular mechanisms of hair growth, treatments for hair

loss basically aim to prolong anagen, convert telogen follicles to anagen, prevent apoptosis of

follicle keratinocytes during catagen and, if possible, generate new follicles. At molecular

level, these therapeutic approaches include regulation of growth factors, cytokines and/or

other signaling molecules that are known to have roles in hair growth. Despite the ever-

increasing demand for treating hair loss, there are only two clinically proven and FDA-

approved treatments for hair loss: finasteride and minoxidil. Finasteride is a highly specific

5α-reductase (type 2) inhibitor whereas minoxidil is a nonspecific drug (Price, 1999).

However, finasteride is approved for the treatment of androgenetic alopecia only in men while

minoxidil is proven to be useful in both men and women with various conditions such as

androgenetic alopecia and alopecia areata (Price, 1999). In order to develop new therapeutic

approaches that overcome the limitations of finasteride and minoxidil current studies require

better understanding of hair biology. Besides the drugs, use of natural products, such as plants

and their derivatives, for hair loss treatment have been gathered ever-increasing attention as in

the case of many other disorders.

Plants are inseparable part of both natural ingredients of drugs and alternative, complementary

medicine which have become popular in the last 10-15 years. Throughout history, plants and

botanical extracts have been used in order to treat diseases within the various kind of

formulation by humans. New innovative hair products are also developed from natural

products, especially from plants. Botanical extracts that support the health, texture and

integrity of hair are widely used in cosmetic formulations.

Turkey is rich in flora and approximately 12.000 varieties of plants are growing naturally

(Davis et al., 1965-85; 1988; Güner et al., 2000; Özhatay and Kültür 2006; Özhatay et al.

2009). Local people are using the plants that are growing naturally in proximity for many

different purposes. “People living in the vicinity of a region to exploit information to meet the

various needs of plants and the effect on plants’ can be used as the definition of the term of

ethnobotany. Recently, the studies performed utilization of the plants for different purposes in

Turkey. Studies stated that nearly 500 plants are used for medicinal purposes.

In the present study we examined the hair-growth effect of plant extracts that have been either

traditionally used for treating hair loss or selected through a literature. This collection

includes the extracts of Myrtus communis, Urtica dioica, Rosmarinus officinalis, Raphanus

sativus, Ficus carica and Lepidium sativum. Although some of the selected plant has proven

effects in different areas, insufficient scientific data on the hair. Our investigation was based

on the changes in the gene expression levels of selected factors (VEGF, SRD5α (5α-

reductase), IL-1α and TNF-α in HaCaT cells treated with the plants extracts.

Materials & Methods Preparation of Plant Extracts

The different parts of plants were collected from Turkey. These plants species listed below in

the Table 1. These plants were identified using the stereomicroscope and Flora of Turkey and

the Aegean Islands.

Latin name of plant The part of used

Raphanus sativus Rhizomes

Lepidium sativum Leaf

Ficus carica Leaf

Urtica dioica Leaf

Myrtus communis Leaf

Rosmarinus officinalis Leaf

Table 1: Plants species and their parts of used

- Raphanus sativus fresh rhizomes juice was prepared.

- Lepidium sativum fresh leaf was fine-cut and extracted with 50% distilled water and

50% ethanol with pressure extractor for 120 minutes at 15°C.

- Ficus carica fresh leaf was fine-cut and extracted with 100% distilled water for 3

hours at 50°C.

- Urtica dioica dried leaf was fine-cut and extracted with 100% distilled water for 3

hours at 50°C.

- Myrtus communis dried leaf was fine-cut and extracted with 100% distilled water for 3

hours at 50°C.

- Rosmarinus officinalis dried leaf was fine-cut and extracted with 30% distilled water

and 70% ethanol with pressure extractor for 120 minutes at 15°C.

The extracts were filtered through a filter paper into a sterile bottle.

Cell Culture

The human keratinocyte cell line (HaCaT) was cultured in Dulbecco’s modified Eagle’s

medium with high glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS)

and 100U/ml gentamicin. Cells were maintained at 37℃ in a humidified atmosphere at 5%

CO2 in Newbrunswick incubator. All supplements and media were purchased from Sigma

Aldrich (Germany).

XTT Cell Proliferation Assay

Roche XTT Cell Proliferation kit was used for cytotoxicity assay. HaCaT cells were seeded

into 96 well plates (1x104 cells/well) and were subjected to different concentrations of plant

extracts. XTT and activator reagents were added to the plates after 72 hour incubation period

according to the manufacturer’s instructions. Then, cells were incubated at 37℃ for 4 hours in

order that XTT reagent was reduced to formazan compound. The optical density of soluble

formazan compound was measured at 495 nm with a BIO-RAD microplate reader (Japan)

(650 nm as reference wavelength).

RNA Isolation

Total RNA was extracted from cells treated with plant extracts and non-treated cells by using

TRI-reagent according to manufacturer’s instructions (Sigma Aldrich, USA). The

concentration and purity of isolated RNA samples were determined by measuring optical

densities at 260 nm and 280 nm using Bio-spect nano (Japan).

Reverse Transcription

Roche Transcriptor First Strand cDNA Synthesis Kit was used for reverse transcription.

cDNA synthesis was performed with 500 ng total RNA, 2 μM final concentration of gene

specific primers of VEGF, IL-1α, 5α- reductase, TNF-α and GAPDH (Integrated DNA

Technologies), 10 U of Transcriptor Reverse Transcriptase, 20 U of Protector RNase

Inhibitor, 1mM each of dNTP mix and Transcriptor Reverse Transcription Buffer (5X)

according to the manufacturer’s instructions (Roche).

Real-Time Quantitative Polymerase Chain Reaction

Real-time PCR (qPCR) reaction was carried out in Light Cycler 96 (Roche). And Fast Start

DNA Green Master Kit (Roche) was used. Briefly, total volume of reaction mix was 20 ul

containing 10 μl SYBR Green Master Mix (2X), 0.5 μM of reverse and forward primers, 2.5

ng cDNA and appropriate amount of nuclease free water. All samples were run as triplicates

in each run including a non-template control and four standards (1:1, 1:10, 1:100, 1:1000).

The PCR parameters were determined separately for each target according to melting and

annealing temperatures of primers. Each parameter includes a pre-incubation step for 10 min

at 95°C and followed by 45 cycles 3-step Amplification and melting step. Melting curve

analysis was performed to verify specificity. For each factor upon treatment with each extract,

five repeats of gene expression analysis was done (n=5). For quantitation of RT-qPCR results,

ΔΔCt method was used (2-ΔΔCt

).

Statistical Analysis

All data are representative of five repeats (n=5) and expressed as mean ±standard error of the

means (SEM). Statistical evaluation was performed by Unpaired t-test using Graph Pad Prism

5 Software (USA) and the results with p value less than 0.05 were accepted as significant.

Results

Cytotoxicity Analysis

Based on cell proliferation ratios of extract-treated cells with respect to the control cells,

cytotoxicity levels of the extracts were determined. Among all six extracts, Ficus carica

extract was found to have the minimum cytotoxic effect on HaCaT cells. For Myrtus

communis, Urtica dioica and Rosmarinus officinalis extracts, 1% concentration was chosen as

non-cytotoxic dose. 3% concentration was chosen for Raphanus sativus and 5% concentration

was chosen for Lepidium sativum and Ficus carica (Figure 1).

3%

80% 57%

46%

87% 95% 100%

0.00%

50.00%

100.00%

Ce

ll P

rolif

era

tio

n

Concentration

Myrtus communis

0%

61% 42% 33%

71% 87%

100%

0.00%

50.00%

100.00%

Ce

ll P

rolif

era

tio

n

Concentration

Rosmarinus officinalis

51%

97% 94% 92% 93% 95% 100%

0.00%

50.00%

100.00%

Ce

ll P

rolif

era

tio

n

Concentration

Ficus carica

14% 34%

77% 83% 94% 87% 100%

0.00%

50.00%

100.00%

Ce

ll P

rolif

era

tio

n

Concentration

Lepidium sativum

15% 19%

62%

91% 98% 100%

0.00%

50.00%

100.00%

Ce

ll P

rolif

era

tio

n

Concentration

Raphanus sativus

Figure 1: Cytotoxicity analysis results of Myrtus communis (A), Urtica dioica (B), Rosmarinus officinalis (C),

Ficus carica (D), Lepidium sativum, and Raphanus sativus (F) extracts. (Dashed bars represent the extract

concentrations chosen for incubation).

Gene Expression Analysis (Real Time qPCR)

According to the cytotoxicity analysis results, HaCaT cells were treated with the non-

cytotoxic concentrations of the extracts. From both treated and untreated cells, total RNA

isolation was carried out. Gene-specific primers were used for reverse transcription and

VEGF, SRD5α, IL-1α, TNF-α and GAPDH cDNAs were prepared. Gene expression analysis

of these factors was then performed via RT-qPCR. Results were represented as “Target /

GAPDH Fold Change”.

Based on our RT-qPCR data, Myrtus communis extract significantly increases the gene

expression levels of VEGF, SRD5α and TNF-α (8 fold, 11 fold and 2.5 fold, respectively)

3%

56% 63% 72% 74% 80% 100%

0.00%

50.00%

100.00%

Ce

ll P

rolif

era

tio

n

Concentration

Urtica dioica

105%

A B

C D

E F

whereas it causes a significant reduction in IL-1α expression level, compared to untreated

control cells. (Figure 2, Table 2).

Urtica dioica extract, did not significantly affect the expression levels of VEGF, IL-1α and

TNF-α. However, it was found to downregulate 5α-reductase since it reduced gene expression

level of SRD5α almost by half, compared to untreated control cells (Figure 3, Table 2).

Myrtus

communis

VEGF 8,384 ± 1,04 (P=0,0021)

SRD-5α 11,66 ± 0,692 (P=0,0001)

TNF-α 2,623 ± 0,6035 (P=0,0361)

IL-1α 0,4500 ± 0,09891 (P=0,0014)

Urtica dioica

VEGF 0,6102 ± 0,1415 (P=0,0511)

SRD-5α 0,5873 ± 0,05858 (P=0,0021)

TNF-α 492,5 ± 314,2 (P=0,1688)

IL-1α 176,4 ± 86,10 (P=0,0878)

Control Myrtus communis

Control Myrtus communis Control Myrtus communis

Control Myrtus communis

Figure 2: Gene expression levels of VEGF (A), TNF-α (B), SRD5α (C) and

IL-1α (D) in Myrtus communis extract-treated HaCaT cells

Table 2: Fold changes in the

expression levels of VEGF, SRD5α,

TNF-α and IL-1α genes

Control Urtica dioica Control Urtica dioica

Control Urtica dioica Control Urtica dioica


IL-1α (D) in Urtica dioica extract-treated HaCaT cells




Cells treated with Rosmarinus officinalis extract showed 31-fold increased expression

of TNF-a gene together with 7.5-fold increase in VEGF gene expression level compared to

untreated cells (Figure 4, Table 4).

Ficus carica extract, which was found to be the least cytotoxic extract of all studied, caused

significant reduction in expression levels of all genes of interest with the most reduction in

SRD5α gene expression (0.07-fold, p<0.0001) (Figure 5, Table 5).

Rosmarinus

officinalis

VEGF 7,450 ± 1,865 (P=0,0258)

SRD-5α 1,292 ± 0,06272 (P=0,0096)

TNF-α 31,01 ± 10,56 (P=0,0295)

IL-1α 0,8000 ± 0,1693 (P=0,2821)

Ficus carica

VEGF 0,2300 ± 0,04041 (p<0.0001)

SRD-5α 0,0700 ± 0,0200

(p<0.0001)

TNF-α 0,2433 ± 0,1033

(p=0.0019)

IL-1α 0,1867 ± 0,03844

(p<0.0001)

Control Control

Control Control






IL-1α (D) in Rosmarinus officinalis extract-treated HaCaT cells





IL-1α (D) in Ficus carica extract-treated HaCaT cells




Control Ficus carica Control Ficus carica

Control Ficus carica Control Ficus carica

A

C D

B

Compared to untreated cells, cells treated with Lepidium sativum extract showed significantly

reduced expression of VEGF, SRD5α and TNF-α genes with no significant effect on IL-1α

gene expression (Figure 6, Table 6).

Similar to Lepidium sativum, Raphanus sativus extract significantly reduced expression levels

of VEGF, SRD5α and TNF-α while not affecting IL-1α significantly (Figure7, Table 7).

Lepidium

sativum

VEGF 0,2167 ± 0,04372 (p<0.0001)

SRD-5α 0,05667 ± 0,0033

(p<0.0001)

TNF-α 0,4133 ± 0,1703 (p=0.0262)

IL-1α 0,6900 ± 0,1756 (p=0.1522)

Raphanus sativus

VEGF 0,1400 ± 0,0361

(p<0.0001)

SRD-5α 0,08667 ± 0,0240 (p<0.0001)

TNF-α 0,4733 ± 0,1559 (p=0.0278)

IL-1α 1,697 ± 0,4817 (p=0.2216)

A

C D

B

Control Lepidium sativum





IL-1α (D) in Lepidium sativum extract-treated HaCaT cells




A

C D

B

Control Raphanus sativus





IL-1α (D) in Raphanus sativus extract-treated HaCaT cells




Discussion

The demand for treating hair loss with natural products and the botanical wealth of Turkey are

the principal motivations of the present study. Additionally, the lack of studies about the

molecular effects of already-used plants on hair cells paved the way for this study.

There have been many plants commonly used by people for hair care. On the other hand,

there are only two clinically-approved treatments for hair disorders, each having limitations to

some extent. Taking these into account, developing new treatments that utilize plants and

demonstrating their effects on the molecular mechanism of hair growth become a remarkable

research area. In this study, extracts of Myrtus communis, Urtica dioica, Rosmarinus

officinalis, Raphanus sativus, Ficus carica and Lepidium sativum were chosen and tested for

their potential hair-growth effects. In order to demonstrate their effects, the changes in the

expression levels of VEGF, SRD5α, TNF-α, and IL-1α genes upon treatment were monitored.

Myrtus communis extract was found to increase the expression levels of all factors of interest

to some extent. By taking this overall increase into consideration, Myrtus communis extract

may be said to enhance HaCaT cell proliferation hence to upregulate synthesis of all factors.

Especially, the 8-fold increase in VEGF gene expression is remarkable since VEGF is a

growth factor that facilitates hair-growth. Taken together, these results demonstrated potential

hair-growth effect of Myrtus communis extract.

According to our results, Urtica dioica extract affected the gene expression level of only

SRD5α gene and it reduced SRD5α gene expression almost by half. This selective

downregulation of SRD5α gene by Urtica dioica extract represents the potential anti-

androgenic effect of Urtica dioica extract since SRD5α gene encodes for 5α-reductase

enzyme that converts testosterone into dihydrotestosteron which results with the shortening of

anagen phase. Urtica dioica, therefore, is a promising candidate plant to use in hair-loss

treatment, especially in men.

Upon treatment with Rosmarinus officinalis extract, HaCaT cells showed highly increased

levels of both VEGF (7.5-fold) and TNF-α (31-fold) genes. Even if it increases VEGF gene

expression, the 31-fold increase in the expression level of TNF-α gene, which is an apoptosis-

inducer, pointed out the anti-proliferative effect of Rosmarinus officinalis extract on HaCaT

cells. Therefore, Rosmarinus officinalis extract was not included into the list of plants that can

be used for hair-loss treatment.

In contrast to Myrtus communis, Ficus carica extract decreased to gene expression levels of

all factors of interest to some extent. In this regard, it can be said that Ficus carica extract

may have a negative effect on HaCaT cell proliferation hence decreased the synthesis of all

factors. However, the reduction in the expression level of SRD5α gene was the most among

all : its expression was reduced by almost one-fifteenth whereas the expression level of other

factors were reduced by one-fifth. Despite its overall negative effect on HaCaT cell

proliferation, Ficus carica extract, with its significant SRD5α downregulation, can have

potential anti-androgenic effect.

Lepidium sativum extract and Raphanus sativus extract treatments ended up with results

similar to those of Ficus carica just with the difference of no significant effect on IL-1α.

Therefore, Lepidium sativum and Raphanus sativus may also have anti-androgenic effects.

None of all plant extracts tested caused upregulation of VEGF and downregulation of other

factors at the same time. Such extract could be the ideal to use for hair-loss treatment.

Nevertheless, combination of two or more extracts with different but complementary effects

can give results like such an ideal extract can do. For example, Myrtus communis and Urtica

dioica could be a good combination.

This study gave an insight about potential hair-growth, anti-inflammatory and anti-androgenic

effects of the selected plants extracts. Further studies with different factors together with

clinical studies can pave way to use of this plant extracts as therapeutic agents against hair

loss.

Acknowledgements

This study was supported by TUBİTAK in the context of “Industrial R&D Projects Grant

Programme” with the project ID 3130970.

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