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. 2021 Apr 27;16(4):e0250663. doi: 10.1371/journal.pone.0250663

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

Yasuko Yoshioka 1,2,#, Yayoi Kamata 2,#, Mitsutoshi Tominaga 2,#, Yoshie Umehara 2, Ikuyo Yoshida 1, Nobuya Matsuoka 1, Kenji Takamori 2,3,*
Editor: Yi Cao4
PMCID: PMC8078742  PMID: 33905439

Abstract

In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes.

Introduction

Itching is a sensation of discomfort that leads to scratching. Histamine is a well-known pruritogen. Clinically, histamine H1 receptor antagonists are commonly used to treat pruritus, but they are often ineffective against pruritic skin diseases, such as atopic dermatitis (AD) and xerosis [1, 2].

Histologically, cutaneous sensory nerve fibers are localized in the vicinity of the boundary between the epidermis and the dermis in normal skin. On the other hand, in the AD or xerosis skin in the disease-state, a large numbers of sensory nerve fibers penetrated and sprouted through the basal cell layer, resulting in epidermal hyperinnervation. The nerve fibers extending just under the stratum corneum with the skin barrier disruption are thought to cause itch hypersensitivity against external stimuli [1–6].

Our previous studies demonstrated that epidermal innervation is regulated by the balance between nerve elongation factors (e.g. nerve growth factor [NGF]) and nerve repulsion factors (e.g. semaphorin 3A [Sema3A]) [4]. NGF produced by keratinocytes is one of the major growth factors that determines skin innervations, with higher local NGF concentrations in the lesional skin of patients with AD or xerosis than in normal skin [1]. Decreased levels of Sema3A expression in the epidermis have been reported in patients with AD [7] and in an experimental model [8], concomitant with an increase in epidermal nerve density. Sema3A has been shown to inhibit NGF-induced sprouting of sensory afferents in the adult mammalian spinal cord [9], and elevated levels of NGF reduced the Sema3A-induced collapse of sensory growth cones [10]. These findings suggest that decreasing Sema3A levels in the epidermis can accelerate epidermal nerve growth in dry-skin-based diseases.

Experimentally, it has been reported that replacement of recombinant Sema3A in the lesional skin of NC/Nga mice normalized epidermal hyperinnervation, resulting in suppression of itch-related scratching behavior and improved dermatitis [11, 12]. This raises the possibility that enhancement of epidermal Sema3A levels may be a promising therapeutic approach for improving pruritus associated with intra-epidermal nerve density. It is generally thought that small molecules (<500 Da) pass through the normal skin barrier [13], whereas larger molecules, such as recombinant Sema3A with a molecular weight of 113.5 kDa, would be less likely to penetrate the stratum corneum barrier. Even if it is absorbed into the epidermis from the cracked stratum corneum of lesional skin, a protein molecule also risks side effects such as contact dermatitis. In addition, recombinant Sema3A is an unstable substance in ointment base [11]. It is likely that there may be many problems in the clinical application of Sema3A direct replacement therapy. Therefore, we considered that topical application of a low-molecular-weight compound with the potential to induce endogenous Sema3A production from human epidermal keratinocytes, rather than Sema3A itself, is a useful approach to improve itch caused by epidermal hyperinnervation.

In this study, we screened a library of herbal plant extracts, which have been confirmed to be safe or use in external preparations, to discover compounds with potential to induce endogenous Sema3A production from normal human epidermal keratinocytes (NHEKs). Here, we describe effect of Scutellaria baicalensis extract on Sema3A production in human epidermal keratinocytes.

Materials and methods

Preparation of test samples

The ninety-one herbal plants used for screening in this study are shown in Table 1. Ethanol extracts of the herbal plants were obtained from Maruzen Pharmaceuticals Co., Ltd. (Hiroshima, Japan) and Koei Kogyo Co., Ltd. (Tokyo, Japan). These ethanol extracts were evaporated under reduced pressure and lyophilized as test samples. Lyophilized samples were reconstituted at 100 mg/mL with DMSO, and each test sample was diluted with basal medium at a concentration in the range of 0.02–500 μg/mL and used in each experiment.

Table 1. A list of herbal plants used in screening of Sema3A inducers.

No Scientific name Part No. Scientific name Part
1 Uncaria gambir Leaf, burgeon 47 Ziziphus jujuba Fruit
2 Arnica montana Flower 48 Thymus vulgaris Aerial part
3 Aloe ferox, Aloe africana Leaf 49 Syzygium aromaticum Bud
4 Aloe arborescens Leaf 50 Citrus unshiu Pericarp
5 Ginkgo biloba Leaf 51 Capsicum annuum Fruit
6 Urtica thunbergiana Leaf 52 Angelica acutiloba Root
7 Artemisia capillaris Flower head 53 Calendula officinalis Flower
8 Foeniculum vulgare Fruit 54 Panax ginseng Root
9 Thea sinensis Leaf 55 Lonicera japonica Leaf, stem
10 Malva sylvestris L. Flower 56 Eriobotrya japonica Leaf
11 Rosa multiflora Fruit 57 Tussilago farfata Flower, leaf
12 Rabdosia japonica Aerial part 58 Poria cocos Sclerotium
13 Scutellaria baicalensis Root 59 Ruscus aculeatus Rootstock
14 Phellodendron amurense Bark 60 Carthamus tinctorius Flower
15 Fucus vesiculosus Whole alga 61 Mentha piperita Leaf
16 Glycyrrhiza glabra, Glycyrrhiza uralensis Root, rootstock 62 Paeonia suffruticosa Root bark
17 Cinchona succirubra Bark 63 Humulus lupulus Female Flower
18 Prunus armeniaca Seed 64 Aesculus hippocastanum Seed
19 Mallotus philippinensis Bark 65 Melissa officinalis Leaf
20 Sasa veitchii Leaf 66 Prunus persica Leaf
21 Matricaria chamomilla Flower 67 Eucalyptus globulus Leaf
22 Sophora flavescens Root 68 Saxifraga stolonifera Whole plant
23 Citrus paradisi Fruit 69 Citrus junos Fruit
24 Cinnamomum cassia Bark 70 Lavandula angustifolia Flower
25 Gentiana lutea Root, rootstock 71 Thea sinensis Leaf
26 Geranium thunbergii Aerial part 72 Ganoderma lucidum Fruit body
27 Saccharomyces cerevisiae Whole 73 Rosa canina Fruit
28 Arctium lappa Root 74 Rosmarinus officinalis Leaf
29 Oryza sativa Seed coat 75 Thymus serpyllum Aerial part
30 Asiasarum sieboldii Root, rootstock 76 Hydrangea serrata var. thunbergii Leaf
31 Salvia officinalis Leaf 77 Coptis japonica Rootstock
32 Crataegus cuneata Fruit 78 Hypericum perforatum Aerial part
33 Gardenia jasminoides Fruit 79 Artemisia princeps Leaf
34 Zanthoxylum piperitum Pericarp 80 Lonicera japonica Flower
35 Rehmannia glutinosa Root 81 Thea sinensis var. assamica Leaf
36 Perilla frutescens var. Crispa Leaf 82 Acorus calamus Rootstock
37 Tilia cordata Flower 83 Achillea millefolium Whole plant
38 Paeonia lactiflora Root 84 Morus alba Root bark
39 Houttuynia cordata Aerial part 85 Luffa cylindrica Aerial part
40 Sanguisorba officinalis Root, rootstock 86 Lilium candidum Bulb
41 Zingiber officinale Rootstock 87 Coix lachryma-jobi var. ma-yuen stapf Seed
42 Betula pendula Bark 88 Cordyceps sinensis Whole
43 Pinctada fucate Nacre 89 Machilus odoratissima Bark
44 Equisetum arvense Whole 90 Punica granatum Pericarp
45 Cnidium officinale Rootstock 91 Vitis vinifera Leaf
46 Swertia japonica Whole plant
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Cell culture

NHEKs derived from adult epidermis (Lonza, Basel, Switzerland) were cultured in keratinocyte basal medium (KBM-Gold; Lonza, Basel, Switzerland) supplemented with KGM-Gold SingleQuot (Lonza), containing 0.15 mM calcium, at 37°C with 5% CO2. Cells were passaged at 60–70% confluence, and the experiments were performed using sub-confluent cells within four passages.

Transfection and measurement of promoter activity

NHEKs were seeded in 24-well flat bottom plates for 24 hours. The culture medium was replaced with antibiotic-free medium and the cells were co-transfected with 500 ng/well pGL3-Sema3A vector containing the firefly luciferase gene and 50 ng/well pRL3-TK vector containing the Renilla luciferase gene (Promega, Madison, WI, USA) using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer’s instructions. pGL3-Sema3A vector was produced in accordance with the methods reported by Kamata et al. [14]. At 24 hours after the start of transfection, the medium was replaced with fresh medium containing the test samples shown in Table 1, apigenin, baicalein, baicalin, chrysin, or wogonin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at various concentrations, and NHEKs were further incubated for 24 or 48 hours at 37°C. The cells were washed and lysed with passive lysis buffer, and luciferase activities were analyzed using a dual-luciferase reporter assay system with a 2030 ARVO-X4 Multi label Plate Reader (Perkin Elmer, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Relative luciferase activity was expressed as a relative ratio of experimental data to untreated control data after normalization with firefly luminescence/Renilla luminescence ratio for each sample well. Cultured cells were observed using a microscopic and checked for morphologically damaged cells by test sample treatment. Data obtained from damaged cells were excluded from the results.

Measurement of Sema3A at mRNA and protein levels in cultured NHEKs

NHEKs were incubated with five plant extracts, Arnica montana (sample No. 2), Artemisia capillaris (No. 7), Malva sylvestris L. (No. 10), Scutellaria baicalensis (No. 13), or Hydrangea serrata var. thunbergii (No. 76), at a final concentration of 50 μg/mL for 3 or 6 hours at 37°C. The cells were subsequently harvested and transcription levels of the human Sema3A gene were quantified using quantitative real-time PCR. Quantification was performed using an Applied Biosystems Fast Real-Time PCR system (Applied Biosystems) using PrimeScript™ One Step RT-PCR Kit (Takara Bio Inc., Shiga, Japan) and the primers 5’-ACCCAACTATCAATGGGTGCCTTA-3’ (forward) and 5’-AACACTGGATTGTACATGGCTGGA-3’ (reverse). As an internal control, ribosome protein S18 (RPS18) mRNA was amplified using the primers 5′-TTTGCGAGTACTCAACACC AACATC-3′ (forward) and 5′-GAGCATA TCTTCGGCCCACAC-3′ (reverse). The amounts of mRNA were normalized relative to those of RPS18 and expressed relative to ratios of the untreated controls.

The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol. Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Cell counting assay

A Cell Counting Kit (CCK)- 8 (Dojindo, Kumamoto, Japan) was used to assess the cell toxicity of each compound on NHEKs. Cells were seeded at 1 × 104 cells per well in 96-well culture plates. After incubation overnight at 37°C, an extract of S. baicalensis or A. montana (50 μg/mL) was added to each well, then incubated for 6 or 24 hours at 37°C. At each time point, CCK-8 solution was added to each well and incubated for 3 hours at 37°C. Cell viability was estimated by measuring the absorbance at 450 nm using a 2030 ARVO-X4 Multi label Plate Reader (Perkin Elmer).

Reconstructed human epidermis (RHE) models

RHE model (EPISKIN, SA, Lyon, France) is an in vitro reconstructed human epidermis from normal human keratinocytes cultured on an inert polycarbonate filter at the air-liquid interface, in a chemically defined medium, featuring normal ultrastructure and functionality similar to human tissue in vivo [15]. RHE models were cultured in accordance with the manufacturer’s protocol. Briefly, inserts containing the RHE models were shipped at room temperature in a multi-well plate filled with an agarose-nutrient solution in which they were embedded. The inserts were carefully taken out of the multi-well plate, any remaining agarose adhering to the outer sides of the insert was removed, and the inserts were placed in a plate in which each well has been filled with Maintenance Medium (EPISKIN, Lyon, France) overnight at 37°C, 5% CO2. Next, 100 μL of the extract of A. montana or S. baicalensis (100 or 500 μg/mL) was applied from the stratum corneum side of the reconstructed epidermis samples and cultured for 24 hours. The concentration of Sema3A protein in the culture supernatants was measured by ELISA as described above.

Statistical analyses

Unless otherwise indicated, values were presented as the standard deviation (S.D.) of the mean. Statistical analyses were performed using one-way or two-way ANOVA followed by Dunnett’s multiple comparison tests with GraphPad Prism 8 (GraphPad Software, Inc., CA, USA), with p < 0.05 defined as statistically significant.

Results

Screening of Sema3A inducers from herbal plant extracts using a reporter gene assay

Cultured NHEKs were transfected with luciferase reporter gene constructs containing -1444 bp of the human Sema3A promoter as described previously [14]. Following transfection, cells were treated with various types of herbal plant extracts at a final concentration of 50 μg/mL for 24 hours and the cell lysates were subjected to luciferase assays. The results are shown in Table 2. From 91 different types of herbal plant extracts, 48 extracts exhibited significantly increased relative luciferase activity compared with the untreated controls, with no cell damage morphologically by the addition thereof. The dose–response relationship was examined with the lower concentration range for the top 5 samples (sample Nos. 2, 7, 10, 13, and 76) among the 48 herbal plants extracts showing significantly higher activity compared with the control. The samples of No. 7, 10, and 76 increased activity in a dose-dependent manner (Fig 1). No dose-dependent increases in activity were observed with samples No. 2 and 13, although in sample No. 13, it seemed that its activity probably reached a plateau, because it was the most active at a low concentration of 5 μg/mL.

Table 2. A summary of Sema3A reporter gene activity by herbal plant extracts.

No. Mean S.D. Significant difference P value No. Mean S.D. Significant difference P value
1 1.60 0.07 *** 0.0007 47 1.05 0.06 n.s. 0.9994
2 2.21 0.20 **** < 0.0001 48 1.24 0.14 n.s. 0.367
3 1.62 0.18 *** 0.000 49 1.43 0.12 * 0.0108
4 1.53 0.07 ** 0.004 50 1.02 0.01 n.s. 0.9998
5 1.60 0.06 *** 0.001 51 1.62 0.10 *** 0.0002
6 1.67 0.13 *** 0.000 52 1.08 0.05 n.s. 0.999
7 2.81 0.21 **** < 0.0001 53 1.29 0.27 n.s. 0.1762
8 1.73 0.07 **** < 0.0001 54 1.23 0.09 n.s. 0.3833
9 1.55 0.30 ** 0.002 55 1.33 0.03 n.s. 0.0841
10 1.97 0.16 **** < 0.0001 56 1.38 0.30 * 0.0348
11 1.44 0.17 * 0.02 57 1.41 0.13 * 0.019
12 1.39 0.17 n.s. 0.05 58 1.29 0.12 n.s. 0.996
13 1.95 0.09 **** < 0.0001 59 1.49 0.14 n.s. 0.9042
14 1.03 0.06 n.s. 1.00 60 1.38 0.08 n.s. 0.9829
15 1.32 0.06 n.s. 0.20 61 1.40 0.21 n.s. 0.9741
16 1.69 0.16 **** < 0.0001 62 1.55 0.14 n.s. 0.8322
17 1.29 0.23 n.s. 0.31 63 0.67 0.05 n.s. 0.992
18 1.19 0.06 n.s. 0.81 64 No data No data - -
19 No data No data - - 65 1.62 0.10 n.s. 0.7277
20 1.40 0.07 * 0.05 66 1.25 0.13 n.s. 0.9991
21 1.25 0.03 **** < 0.0001 67 1.57 0.18 n.s. 0.7978
22 1.20 0.03 **** < 0.0001 68 1.03 0.15 n.s. 0.9999
23 1.03 0.02 n.s. 0.8836 69 1.13 0.04 n.s. 0.9996
24 1.24 0.05 **** < 0.0001 70 1.36 0.26 n.s. 0.9893
25 1.19 0.04 **** < 0.0001 71 1.31 0.11 ** 0.0062
26 1.25 0.05 **** < 0.0001 72 1.35 0.01 *** 0.0006
27 1.20 0.02 **** < 0.0001 73 1.16 0.04 n.s. 0.2914
28 1.23 0.01 **** < 0.0001 74 1.38 0.07 *** 0.0006
29 1.19 0.02 **** < 0.0001 75 1.42 0.13 **** < 0.0001
30 1.23 0.02 **** < 0.0001 76 2.27 0.17 **** < 0.0001
31 1.34 0.02 **** < 0.0001 77 1.29 0.03 ** 0.0062
32 1.27 0.00 **** < 0.0001 78 1.38 0.06 *** 0.0006
33 1.28 0.05 **** < 0.0001 79 1.54 0.08 **** < 0.0001
34 1.30 0.01 **** < 0.0001 80 1.33 0.04 *** 0.0006
35 0.96 0.26 n.s. 0.9993 81 1.32 0.02 ** 0.0062
36 1.17 0.03 n.s. 0.2271 82 1.18 0.09 n.s. 0.2914
37 1.10 0.03 n.s. 0.7929 83 1.38 0.13 *** 0.0002
38 1.19 0.03 n.s. 0.1783 84 0.02 0.01 **** < 0.0001
39 1.18 0.03 n.s. 0.2098 85 0.99 0.08 n.s. 0.9997
40 1.26 0.03 * 0.0244 86 0.96 0.07 n.s. 0.9534
41 1.26 0.24 * 0.0197 87 0.99 0.08 n.s. 0.9998
42 1.10 0.01 n.s. 0.82 88 0.87 0.04 n.s. 0.8227
43 1.24 0.01 * 0.0368 89 1.01 0.22 n.s. > 0.9999
44 1.31 0.01 ** 0.0037 90 0.85 0.13 n.s. 0.398
45 1.19 0.01 n.s. 0.1383 91 0.83 0.04 n.s. 0.3129
46 1.27 0.07 * 0.0143
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NHEKs were transfected with pGL3-Sema3A. After transfection, the cells were incubated with the herbal plant extracts shown in Table 1 at a final concentration of 50 μg/mL for 24 hours at 37°C. Cell lysates were subjected to luciferase activity measurement as described in the Materials and methods. All results were expressed as mean ± S.D. of triplicate samples.

* p < 0.05,

** p < 0.01,

*** p < 0.001,

**** p < 0.0001, n.s: no significant difference (one-way ANOVA followed by Dunnett’s test). No data: data obtained from cells in which morphological damage was observed by the addition of sample was not used.

Fig 1. Effects of five positive hits of plant extracts on Sema3A reporter gene expression.

Fig 1

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NHEKs were transfected with pGL3-Sema3A. After transfection, the cells were incubated with five plant extracts, namely No. 2, 7, 10, 13, and 76 at a final concentration of 5, 15, and 50 μg/mL for 48 hours at 37°C. Cell lysates were subjected to luciferase activity measurement as described in Materials and methods. All results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Effects of the herbal plant extracts Sema3A induction at mRNA and protein levels in NHEKs

We next examined the induction of Sema3A mRNA and protein levels with the five positive plants extracts (sample No. 2, 7, 10, 13, and 76) by quantitative real-time PCR and ELISA, respectively. Expression of Sema3A mRNA in the NHEKs treated with No. 2, 7, and 13 was markedly increased at both 3 and 6 hours compared with untreated controls (Fig 2). In samples No. 10 and 76, they were slightly increased at 3 and 6 hours, respectively.

Fig 2. Effects of five positive plant extracts on the expression of Sema3A mRNA in cultured NHEKs.

Fig 2

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NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3 and 6 hours at 37°C. Expression of Sema3A mRNA was examined using quantitative real-time PCR analysis. The levels of Sema3A mRNA were normalized relative to those of RPS18. All results are expressed as mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Sema3A protein levels were measured in the culture supernatants of NHEKs treated with five tested samples using ELISA (Fig 3A). At 3 hours after sample treatment, differences in Sema3A protein levels were hardly noted in the culture supernatants compared with the control group. However, from 6 hours onward, sample treatment led to an overall increase in Sema3A expression, and Sema3A levels were significantly increased with sample No. 13 compared with the controls at 24 hours. In sample No. 2, we noted a tendency to increase, although no statistically significant difference was observed. Increased expression at the protein level was also observed using ELISA with cell lysates of cultured NHEKs with sample No. 13 but not sample No. 2 (Fig 3B). We further tested the effects of samples No. 2 and No. 13 on cell viability of cultured NHEKs using a cell counting assay. In the assay, the cell viabilities of NHEKs treated with these compounds was significantly increased at 6 hours compared with untreated controls, whereas samples No. 2 and No. 13 reduced cell viabilities to 89.8% and 78.2% at 24 hours, respectively (Fig 3C).

Fig 3. Effects of five positive plant extracts on the production of Sema3A proteins in cultured NHEKs.

Fig 3

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(A) NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3, 6, and 24 hours at 37°C. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. *p < 0.05, (one-way ANOVA followed by Dunnett’s test). (B) NHEKs were incubated with sample No. 2 (A. montana) or No. 13 (S. baicalensis) at 50 μg/mL for 6 and 24 hours. After that, the cell lysates were prepared with lysis buffer, and then the levels of Sema3A protein in the cell lysates were measured using ELISA. (C) Cell viability of cultured NHEKs treated with the extract of A. montana or S. baicalensis was measured using a cell counting assay. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA followed by Dunnett’s test).

Effect of the plant extracts on Sema3A protein production in RHE models

The Sema3A-inducing activity of the extracts of S. baicalensis and A. montana was evaluated using RHE models. These plant extracts at concentrations of 100 or 500 μg/mL were applied to the stratum corneum side of RHE models. They were cultured for 24 hours, and then Sema3A protein levels in the culture supernatant were measured. In the S. baicalensis-treated group, the concentration of Sema3A increased in a dose-dependent manner, with significantly higher values noted in the 500 μg/mL S. baicalensis-treated group compared with the untreated control (Fig 4). Meanwhile, application of A. montana extract did not affect Sema3A concentration in the culture supernatant.

Fig 4. Effects of S. baicalensis and A. montana extracts on Sema3A production in RHE models.

Fig 4

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A 100 μL sample of the extract of S. baicalensis, A. montana (100 or 500 μg/mL) or PBS (vehicle) was applied from the stratum corneum side of cultured RHE samples, and the RHE samples were cultured for a further 24 hours. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. All results are expressed as means ± S.D. of three independent experiments. *p < 0.05, (one-way ANOVA followed by Dunnett’s test).

Bioactive components in S. baicalensis contributing to Sema3A induction

We examined which components in the extract of S. baicalensis contributed to Sema3A induction (Fig 5A). Of the known chemical components in the extract of S. baicalensis, we conducted Sema3A reporter gene assays in NHEKs treated with the five major bioactive flavones, baicalin, baicalein, wogonin, chrysin, and apigenin. The experiment was performed with a maximum concentration of 5 μg/mL and a 3-fold dilution series. The molar concentration of each compound is shown in S1 Table. The extract of S. baicalensis, baicalin, and baicalein all significantly increased the relative luciferase activity at a concentration of 5 μg/mL, and the increase in activity of these samples was dose dependent. Notably, baicalein markedly increased its activity at a lower concentration of 0.56 μg/mL compared with the extract of S. baicalensis and baicalin. Such significant alterations were observed with chrysin and apigenin but were not dose dependent. Meanwhile, wogonin significantly decreased the luciferase activity at a concentration of 5 μg/mL

Fig 5. Effects of S. baicalensis–derived compounds on Sema3A reporter gene expression.

Fig 5

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(A) NHEKs were transfected with pGL3-Sema3A vector. After transfection, the cells were incubated with S. baicalensis extract and S. baicalensis-derived compounds: apigenin, baicalein, baicalin, chrysin, and wogonin, at the indicated concentrations, a 3-fold dilution with a maximum concentration of 5 μg/mL, for 24 hours at 37°C. Cell lysates were subjected to the measurement of luciferase activities as described in the Materials and methods. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test). (B) Chemical formulae of major bioactive flavones from S. baicalensis.

Discussion

In the present study, we found five positive samples from 91 herbal plant extracts as potent inducers for human Sema3A gene in NHEKs using our Sema3A reporter gene assay. Among these, samples No. 2, 7, and 13 markedly increased the expression levels of Sema3A mRNA in cultured NHEKs at 3 and 6 hours (Fig 2). In addition, the extract of S. baicalensis significantly increased the level of Sema3A protein in the supernatants of NHEKs cultured for 24 hours (Fig 3A) and in cell lysates from NHEKs cultured for 6 and 24 hours (Fig 3B). Meanwhile, no significant increases in the level of Sema3A protein in culture supernatants and cell lysates from NHEKs treated with the extract of A. montana were found in this study. These findings suggested that the extract of S. baicalensis consistently has Sema3A-inducing activity in cultured NHEKs on both gene and protein expression levels. In addition, our cell counting assay showed that extracts of S. baicalensis and A. montana significantly increased cell viability at 6 hours, whereas the cell viabilities with S. baicalensis and A. montana treatments at 24 hours reduced to 89.8% and 78.2%, respectively (Fig 3C), although no morphological findings related to cytotoxicity were observed in microscopic observations up to 24 hours. Thus, these findings raise the possibility that extracts of S. baicalensis and A. montana are at least partly involved in cell proliferation and/or differentiation rather than cytotoxic events. This idea might also be supported by a previous study that baicalein, one of the components in S. baicalensis, induced keratinocyte differentiation [16].

Notably, Sema3A concentration in the culture supernatants was significantly increased in the S. baicalensis extract-treated group in RHE model, whereas no increase was observed in the A. montana extract-treated group (Fig 4). This model was reconstructed by culturing human epidermal keratinocytes, featuring normal ultrastructure and functionality similar to human tissue in vivo [15]. Each sample was applied to the stratum corneum side of the three-dimensional skin model. It is generally thought that small molecules (<500 Da) pass through the normal skin barrier [13]. The result of RHE models indicated that at least some of the components that contribute to the induction of Sema3A in the extract from S. baicalensis can pass through the stratum corneum and act on keratinocytes. From these findings, it was suggested that topical application of the S. baicalensis extract can alleviate itch caused by hyperinnervation through induction of endogenous Sema3A in epidermal keratinocytes. In fact, our preliminary study using a pruritic dry skin model mouse also showed that repeated application of S. baicalensis extract tended to relieve the itch-related scratching behavior and epidermal hyperinnervation (Yoshioka et al. unpublished observations).

S. baicalensis has long been used as the medicinal plant in Eastern traditional herbal formulations. More than 40 kinds of flavonoids, including baicalin, baicalein, wogonin, chrysin, and apigenin, have been identified from the extract of S. baicalensis, which has various pharmacological activities including anti-inflammatory, anti-allergic, anti-oxidant, anti-tumor, anti-bacterial, and neuroprotective actions [17–19]. We investigated which components in the extract of S. baicalensis contributed to Sema3A induction. Among the major bioactive flavones in S. baicalensis subjected to the assay, baicalin, baicalein, and wogonin are components specific to S. baicalensis [17, 20, 21], whereas chrysin and apigenin are present in a variety of plants [22, 23]. Our reporter gene assay showed that relative luciferase activities in cultured NHEKs were significantly increased in a dose-dependent manner by the application of an extract of S. baicalensis, baicalin and baicalein compared with the control (Fig 5A). Notably, baicalein showed significantly higher activity at a low concentration (0.56 μg/mL) among the major bioactive flavones in S. baicalensis, suggesting that it is the most active compound. Baicalein is a glucuronide conjugate (Fig 5B) and is known to be converted to its aglycone, baicalein, by β-glucuronidase derived from bacteria but not that from humans [24]. It has been previously reported that an extract of S. baicalensis contains approximately 10% baicalin and 1% baicalein [25–27], so baicalin is a major component of S. baicalensis extract. Taken together, these findings suggested that baicalin and its aglycone, baicalein, are the main active components in S. baicalensis contributing to Sema3A induction in keratinocytes.

Among the four flavone aglycones tested, baicalein showed significantly higher activity in a dose-dependent manner, but no other aglycones, wogonin, chrysin, and apigenin. Baicalein (5,6,7-trihydroxyflavone), chrysin (5,7-dihydroxyflavone), and wogonin (5,7-dihydroxy-8-methoxyflavone) have substituents in different patterns only in ring A (Fig 5B). Thus, the different structure in ring A might affect activity on the Sema3A promoter in epidermal keratinocytes, although the precise mechanisms remain unclear in this study. Recently, we reported that Sema3A mRNA expression in NHEKs is regulated by calcium via the mitogen-activated protein kinase and activator protein (AP)-1 signaling axis [14]. Meanwhile, baicalein has been shown to suppress the transcription activity of AP-1 [28]. Therefore, the extract of S. baicalensis may promote Sema3A production via different signaling pathways.

In conclusion, the results of present study showed that the extract of S. baicalensis and its active compounds, baicalin and baicalein, induced Sema3A production in normal human epidermal keratinocytes. Thus, an S. baicalensis extract or its active compounds may be promising therapeutic candidates for improving histamine-resistant itch caused by epidermal hyperinnervation.

Supporting information

S1 Table. List of concentration units in μg/mL and μM for each compound tested.

(TIF)

Click here for additional data file. (395KB, tif)

Acknowledgments

The authors thank M. Takagi for technical assistance and J. Akaki for helpful comments on the analysis of herbal plants. We also thank H. Nakayama, A. Kamo, H. Matsuda, and K. Ishii for technical advice.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology (Grant number: S1311011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This research was also funded by Kobayashi Pharmaceutical Co., Ltd. (URL: KOBAYASHI Pharmaceutical Co.,Ltd.). Three of the authors (YY, IY, NM), who are employees of Kobayashi Pharmaceutical Co., Ltd, had a partial role in data collection and analysis, and preparation of the manuscript.

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Decision Letter 0

Yi Cao

18 Nov 2020

PONE-D-20-28160

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

PLOS ONE

Dear Dr. Takamori,

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Reviewer #1: This is an interesting article on the development of a topical solution from plant extracts that utilizes small molecules that can penetrate the skin barrier to induce the production of the nerve repulsion factor Sema3A. The article is well written and the experimental protocol as well as the statistical analysis are sound.

Reviewer #2: Some skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. And Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Therefore the paper of Yasuko and co-workers find that S.baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes. Thus, the study may provide a new therapeutic candidates for improving histamine-resistant itch caused by epidermal hyperinnervation.

As such, the matter is of interest, however the paper suffers for some limits:

1)In Fig 3, It is unconvincing that simply use ELISA to measure the Sema3A protein levels in the culture supernatants of NHEKs treated with five tested samples. Sema3A is not only the secreted protein, but also abundant in intracellular of NHEKs. It is recommended to use WB or immunofluorescence to observe the protein levels in different treatment groups.

2)In Fig 3, samples No. 2, and 13 can increase the expression levels of Sema3A in cultured NHEKs at 24 hours. Do these drugs have any effect on cell viability?

3)In Fig5a, the results showed that the luciferase activities in cultured NHEKs were increased dose dependently by the application of baicalin orbaicalein. Are there statistically significant differences as compared to the control ?

Once the above concerns are fully addressed, the manuscript could be accepted for publication in this journal.

Reviewer #3: PONE-D-20-28160

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocyte

Reviewer’s comment　

Authors are interested in an importance of epidermal Sema3A level to prevent sensory nerve fiber elongation leading to serious scratching in pruritic skin diseases such as AD and xerosis. This manuscript describes herbal plant extracts were tested for an in vitro Sema3A induction activity in normal human keratinocytes (NHEKs). The major findings were obtained from gene promoter assay by Sema3A promoter that authors characterized, qPCR of Sema3A mRNA, ELISA of secretory Sema3A protein, and human skin equivalent model. The authors showed results of the primary screening of gene promoter activity for 91 herbal plant extracts. In further screening by qPCR of mRNA, the remaining was five herbal extracts No.2, 7, 10, 13 and 76. Finally, S. baicalensis extract was selected as the best one in screened samples. In addition, baicalein and its glucuronide are proved to be main active component among 5 flavonoids included in S. baicalensis.

Although the data are interesting and authors are expected to progress this research in future, I have the following concerns regarding the experiment and interpretation of the data.

1. Page 2 line 22: “Previous studies” is “Our previous studies”.

2. Page 3 line 36-39: Do authors think whether or not the active extract-induced Sema3A may lead to keratinocyte differentiation or migration?

3. Page 5 line 74: Authors mentioned Sema3A ointment in Abstract. Please add the reference here.

4. Page 6 line 87: Sample preparation is mentioned more in detail in the text.

5. Page 8 line 105, Page 11 line 160-161: According to pGL3-Sema3A vector containing luciferase gene, the sentence “Cultured NHEKs were……with luciferase reporter gene constructs containing -1444 bp of the human Sema3A promoter” is shown in Page 11, but more specified promoter sequence is shown in the text.

6. Page 10 line 143-146: Human skin equivalent model is supported by a little bit of more explanation for the readers.

7. Page 11 line 153: Statistical numbers (n) of the experiments are shown in the figures and legends.

8. Page 17 line 240-250: Did authors quantitate baicalein and other components in S. baicalensis extract that authors tested?

9. Page 20 line 296: It is referred that S. baicalensis contains 10 % baicalin and 1% baicalein. Which is the more active component? Do Authors guess beta-glucuronidase degrades baicalin faster, and leads to bioactive component baicalein? If so, smooth beta-glucuronidase degradation must be shown by an enzymatic experiment of culturing of keratinocyte or incubating of cell lysates.　

10. Page 21 line 311-312: Authors reported that Sema3A mRNA expression in NHEKs is regulated by calcium. How is the possibility of activation due to contaminated calcium in the tested samples?

11. Fig. 5 and the legend: Chemicals have been compared at same molar concentration. Chemicals are shown in the figure at each of molar concentrations. In the legend, each name of chemicals is followed by both units of ug/mL and uM.

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PLoS One. 2021 Apr 27;16(4):e0250663. doi: 10.1371/journal.pone.0250663.r002

Author response to Decision Letter 0

2 Apr 2021

Reviewer 1: I have incorporated all of your suggestions into my revision. They were very helpful. Thank you.

Reviewer 2: I have incorporated all of your suggestions into my revision. They were very helpful. Thank you.

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Decision Letter 1

Yi Cao

12 Apr 2021

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

PONE-D-20-28160R1

Dear Dr. Takamori,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yi Cao

Academic Editor

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Reviewer #3: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #3: I Don't Know

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: (No Response)

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Reviewer #3: No

Acceptance letter

Yi Cao

16 Apr 2021

PONE-D-20-28160R1

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

Dear Dr. Takamori:

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Kind regards,

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on behalf of

Dr. Yi Cao

Academic Editor

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. List of concentration units in μg/mL and μM for each compound tested.

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    Data Availability Statement

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