1. Introduction
Blackcurrant (
Ribes nigrum L.) contains high levels of anthocyanins, including cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, and delphinidin-3-rutinoside [
1]. These anthocyanins are reported to have some health benefits, such as in the prevention of breast cancer and reduction of inflammation and obesity [
2,
3,
4]. Furthermore, blackcurrant contains various bioactive compounds and has been traditionally used in the treatment of various conditions, including rheumatic disease [
5].
Phytoestrogens, including isoflavones, lignans, coumestans, and flavonoids, which are found in many foods, are chemically diverse plant compounds that exert estrogenic effects in animals [
6,
7,
8,
9]. Recent studies have reported that blackcurrant extract (BCE) and anthocyanins derived from blackcurrant act as phytoestrogens by signaling through both estrogen receptor (ER)α and ERβ [
10,
11].
Estrogens affect the functions of various organs and tissues, including blood vessels, bones, brain, skin, and hair, and participate in the mechanisms underlying several diseases, such as metabolic syndrome [
12,
13,
14,
15]. The function of women’s ovaries decreases gradually beginning in their late 20s. Moreover, estrogen secretion decreases sharply at around 40 years of age; this decrease is associated with menopausal disorder, which involves symptoms such as thinning hair, hair loss, hot flashes, headaches, dizziness, palpitations, and malaise.
To alleviate menopausal symptoms, postmenopausal women may take hormone replacement therapy. However, it is necessary to consider the risk of venous thrombosis and breast cancer when using estrogen preparations. In contrast, phytoestrogens have not been reported to be associated with these risks and are therefore considered important substitutes for estrogen preparations [
16,
17]. In female pattern hair loss (FPHL), such as that occurring during menopause, the number of hairs decrease at the head top in the center, which makes the hair thin [
18]. There are few changes in the crown and hairline. Ovariectomy decreases the secretion of estrogen from the ovary, therefore ovariectomized (OVX) rodents are widely used as a menopausal model [
19]. Furthermore, OVX mice have been reported to be useful animal models of FPHL [
20,
21].
The hair follicle consists of epithelial and mesenchymal tissues. Hair follicle stem cells are involved in the production of hair and supply the necessary cells to make hair by division and differentiation. Keratin (K) 19, K15, and cluster of differentiation (CD) 34 are hair follicle stem cell markers [
22,
23,
24]. K19 is expressed in the epidermis of the skin, the bulge region of the hair follicle, and the outermost layer of the outer root sheath [
25,
26,
27]. CD34 and K15 are localized on the hair bulge region, and K15 is also localized on the hair germ [
28,
29,
30]. Hair follicle dermal papilla cells (HFDPCs) are important cells present at the tip of the hair follicle and take up nutrients from capillaries to grow hair [
31,
32].
Previous studies reported that BCE and blackcurrant anthocyanins improved dermal health and increased the production of collagen, elastin, and hyaluronic acid in OVX rats [
33]. However, the effects of phytoestrogen on hair follicles are still unknown. Accordingly, this study aimed to investigate the beneficial effects of BCE on FPHL in OVX rats as a menopausal model.
3. Materials and Methods
3.1. Materials and Cell Culture
BCE powder, CaNZac-35, was purchased from Koyo Mercantile Co. (Tokyo, Japan). BCE contained high concentrations of anthocyanins (38.0 g/100 g BCE) [
11]. HFDPCs derived from the temples of Caucasian middle-aged women were obtained from PromoCell (Heidelberg, Germany). Cells were maintained in follicle dermal papilla cell growth medium (PromoCell). All cell culture experiments were conducted at 37 °C in a humidified incubator containing 5% CO
2.
3.2. Microarray Gene Expression Profiling
HFDPCs were seeded in 21 cm
2 culture dishes and grown until reaching confluence. The medium was then replaced with phenol red-free follicle dermal papilla cell basal medium, with or without BCE (1.0 μg/mL) for 24 h. After washing cells twice with phosphate-buffered saline (PBS), total RNA was extracted using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. RNA labeling and hybridization were performed using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (version 6.5, 2010; Agilent Technologies, Santa Clara, CA, USA). Briefly, 100 ng total RNA from each sample was linearly amplified and labeled with Cy3-dCTP. The resulting labeled cRNAs were purified using an RNAeasy mini kit (Qiagen). Labeled and fragmented cRNA was hybridized to a SurePrint G3 Human Gene Expression microarray (8 × 60 K version 3; Agilent Technologies). Labeling, hybridization, image scanning, and data analysis were performed at Macrogen Japan Corp. (Tokyo, Japan). The microarray dataset is accessible at
http://www.ncbi.nlm.nih.gov/geo under accession code GSE 117360.
3.3. IPA
Genes showing a greater than 2.0-fold upregulation following exposure of HFDPCs to 1.0 μg/mL BCE were analyzed using IPA software (version 36601845) (Qiagen, Germantown, MD, USA). The z-score algorithm was utilized to reduce the possibility of false-positive results, where z scores greater than 2.0 indicated that transcript expression was significantly increased, and z scores less than −2.0 indicated that expression was significantly decreased.
3.4. qPCR
HFDPCs were seeded in 21 cm
2 culture dishes and grown until reaching confluence. The medium was then replaced with phenol red-free follicle dermal papilla cell basal medium, with or without BCE (1.0 μg/mL). Cells were incubated for 24 h and washed twice with PBS. Total RNA was prepared using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was reverse-transcribed using PrimeScript RT Master Mix (TaKaRa, Tokyo, Japan). Levels of
K19 mRNA were quantified by qPCR using TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa). The PCR amplification protocol consisted of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C for 40 cycles. Transcript levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (
GAPDH) cDNA. The primers were as follows (5′→3′):
K19, forward CGGGACAAGATTCTTGGT and reverse CGTTGATGTCGGCCTCCA [
44];
GAPDH, forward TGAGAACGGGAAGTCTGTCA and reverse TCTCCATGGTGGTGAAGACG. PCR specificity was checked using s melting curve analysis. All samples were analyzed in duplicate, and relative gene expression was calculated according to the 2
−ΔΔCt method.
3.5. Animals and Treatments
OVX female Sprague–Dawley and sham surgery rats (12 weeks of age) were purchased from CLEA Japan Inc. (Tokyo, Japan). The rats were housed in air-conditioned rooms with a 12 h light/dark cycle and free access to water and food at the Institute for Animal Experiments of Hirosaki University Graduate School of Medicine. Previous studies showed that 3% BCE elicited phytoestrogen effects in the rat uterus and skin [
11,
33]. All rats received an AIN-93M diet, with or without 3% BCE, as indicated (CLEA Japan, Inc.), and were divided into three groups (
n = 6–7/group) as follows: (1) OVX rats treated with 3% BCE for three months (OVX BCE group), (2) OVX rats without BCE treatment (OVX control group), and (3) sham surgery rats without BCE treatment (sham group). At the end of the experiment, the animals were euthanized and the back skin was removed. The skin tissues were fixed in 10% formaldehyde and embedded in paraffin for histological examination. This experiment was approved by the Animal Research Committee of Hirosaki University (permission number: G16004) and was conducted in accordance with the rules for Animal Experimentation of Hirosaki University.
3.6. Enumeration of Hair Shafts per Follicular Unit and Analysis of Immunohistochemical Staining
Skin tissue sections (3 μm thick) were mounted onto silane-coated slides. The sections were deparaffinized by passing through xylene and a graded alcohol series before being stained with hematoxylin and eosin. The number of hair shafts per follicular unit was counted using a fluorescence microscope (FSX100; Olympus, Tokyo, Japan). In immunohistochemistry, tissue sections were heated in a microwave oven to 95 °C in citric acid buffer (pH 6.0) for 15 min for antigen retrieval, and then incubated with anti-Ki67 antibodies (cat. no. ab15580; Abcam, Cambridge, MA, USA; 1:100 dilution, v/v) at 4 °C overnight. The tissue sections were then incubated at room temperature for 60 min with a universal secondary antibody (Roche, Basel, Switzerland). The site for peroxidase binding was determined using DISCOVERY DAB Map Detection Kit (Roche). Sections were then counterstained with Hematoxylin II (Roche) for microscopic examination. As a negative control, nonimmune γ-globulin was used instead of the antibody. Images were captured using a fluorescence microscope (FSX100). Ki67-positive cells were counted, and the percentage was calculated relative to nuclear staining.
3.7. Immunofluorescence Staining
Deparaffinization and antigen retrieval was carried out as described above. Subsequently, the slides were incubated with anti-cytokeratin 19 (A-3) antibodies (cat. no. sc-376126; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50 dilution, v/v), anti-cytokeratin 15 (LHK15) antibodies (cat. no. sc-47697; Santa Cruz Biotechnology; 1:50 dilution, v/v), or anti-CD34 (B-6) antibodies (cat. no. sc-74499; Santa Cruz Biotechnology; 1:50 dilution, v/v) at 4 °C overnight. This was followed by incubation with Alexa Fluor 546 anti-mouse IgG secondary antibodies (cat. no. A11060; Life Technologies; dilution 1:500) for 30 min at room temperature. Nuclear staining and mounting were performed using Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). As a negative control, nonimmune γ-globulin was used instead of the antibody. Images were captured using a fluorescence microscope (FSX100).
3.8. Statistical Analysis
Results are expressed as the means ± standard deviations. Graphs were generated using Graph Pad Prism 7.0 ver. 7.03 software (Graph Pad Prism, San Diego, CA, USA). All statistical analyses were performed using BellCurve for Excel ver. 3.0 software (Social Survey Research Information, Tokyo, Japan). The qPCR results were evaluated using Student’s t-tests. Comparisons of the three groups were analyzed using one-way analysis of variance with Tukey–Kramer post-hoc tests, whereas comparisons of the two groups were evaluated using t-tests. Differences with p values of less than 0.05 were considered statistically significant.
4. Conclusions
Because BCE has phytoestrogen activity, this study investigated the effects of BCE on hair loss in OVX rats. The results showed that BCE affected the expression of many genes downstream of beta-estradiol and estrogen receptor in human HFDPCs, most notably K19. BCE also increased the number of hairs per hair follicle and the expression of the cell proliferation marker Ki67 in OVX rats. In addition, BCE enhanced the expression of hair follicle stem cell markers, such as K19, K15, and CD34, in OVX rats, similar to that in sham rats. These results suggest that BCE has phytoestrogen effects and enhanced stemness in the hair follicles of OVX rats. Further studies are necessary to determine whether BCE is an effective treatment for FPHL in menopausal women.