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. 2023 Jan 10;29(1):e13275. doi: 10.1111/srt.13275

Functional validation of co‐culture model of human keratinocytes and neuronal cell line for sensitive skin by using transient receptor potential channel vanilloid subfamily member 1 antagonist

Sun Mee Shin 1,*, Eun Joo Baek 2,*, Dong Yeol Oh 2, Kwang Ho Kim 2, Kwang Joong Kim 2, Eun Joo Park 2,✉
PMCID: PMC9838752  PMID: 36704884

Abstract

Background

Sensitive skin is a subjective cutaneous hyper‐reactivity that occurs in response to various innocuous stimuli. Keratinocytes have recently been shown to participate in sensory transduction by releasing many neuroactive molecules that bind to intra‐epidermal free nerve endings and modulate nociception. In the literature, the characterization of these interactions has been based on the co‐culture of keratinocyte and mammalian‐origin neuronal cell lines. In this study, we established an in vitro model based on a co‐culture of primary human keratinocytes and differentiated SH‐SY5Y cells, a human neuronal cell line.

Methods

Human epidermal keratinocytes and SH‐SY5Y cells were monocultured and co‐cultured. Changes in calcium influx, substance P, inflammatory cytokines, and neuropeptides between the monoculture and co‐culture groups treated with capsaicin only and capsaicin with transient receptor potential channel vanilloid subfamily member 1 (TRPV1) antagonist, trans‐4‐tert‐butylcyclohexanol (TTBC), together. In addition, the difference in stinging sensation was evaluated by applying it to the volunteers.

Results

When SH‐SY5Y cells were co‐cultured with keratinocytes, they had no significant effect on axonal development. Substance P was also released after capsaicin treatment and reduced by TTBC under co‐culture conditions. Moreover, the expression of inflammatory cytokines and neuropeptides was significantly increased in co‐cultured keratinocytes compared to that under monoculture conditions. In addition, the stinging sensation was significantly induced after the application of capsaicin in vivo and was relieved after the application of the TRPV1 antagonist.

Conclusion

We demonstrated that the novel co‐culture model is functionally valid through capsaicin and TRPV1 antagonist. We also confirmed that TTBC could be used for the treatment of sensitive skin through a co‐culture model and in vivo tests. This co‐culture model of keratinocytes and SH‐SY5Y cells may be useful in vitro alternatives for studying the close communication between keratinocytes and neuronal cells and for screening therapeutic drugs for sensitive skin.

Keywords: cell culture, claim substantiation, keratinocyte, neuronal cell, sensitive skin, skin physiology

1. INTRODUCTION

The skin is a sensory organ densely innervated by primary sensory nerve endings while protecting the body from external stimulation. 1 , 2 , 3 Sensitive skin is a subjective cutaneous hyper‐reactivity to environmental factors that are typically not accompanied by distinct physical signs of skin irritation. 4 , 5 Although the pathophysiology of sensitive skin is not fully understood, neurosensory dysfunctions, such as altered nerve endings, increased neurotransmitter release, unique central information processing, chronic nerve‐ending trauma, and slower neurotransmitter removal, are among the most important causes. 4 , 5

Various neuromediators and specific receptors expressed on both neuronal and epidermal cells mediate communication between sensory and epidermal cells. In particular, substance P (SP) and calcitonin gene‐related peptide (CGRP) play an important role in the communication between epidermal cells and sensory neurons in the skin. Activated sensory neurons induce the secretion of these neuropeptides, and SP and CGRP receptors are expressed in keratinocytes. 3 , 6 , 7

Activation of the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) induces cellular calcium influx, producing an action potential in sensory fibers leading to stinging and/or itching sensations. 8 , 9 TRPV1 is also expressed in many cells such as keratinocytes, fibroblasts, mast cells, and endothelial cells. 12 Capsaicin activates TRPV1 in keratinocytes and induces keratinocytes to release PGE2 and interleukin (IL)−1, which induces the activation of sensory nerve endings. 10 , 11 The over‐activation of TRPV1 in sensitive skin caused by an increased expression level or potentiation of signal intensity can be relieved by trans‐4‐tert‐butylcyclohexanol (TTBC). 13

To study the communication dynamics of epidermal cells, it is essential to develop in vitro co‐culture models of keratinocytes and sensory neurons. Many studies have used primary dorsal root ganglion cultures of mammalian origin (rats, mice, or pigs) because of ethical considerations and difficulties in isolation. 14 , 15 , 16 However, such cultures are heterogeneous and unsuitable for many biochemical and molecular studies. To overcome these limitations, the classical PC12 cell line or immortalized clonal cell lines derived from dorsal root ganglion cells, such as 50B11, ND7/23 cell line, or F‐11 neurons, have been used; however, these cell lines are also of mammalian origin. 17 , 18 , 19 , 20 The human neuroblastoma cell line SH‐SY5Y, which is commonly used as a model of adrenergic or dopaminergic neurons, exhibits traits of peripheral sensory neurons such as the functional expression of the sensory neuron‐specific sodium channel. 21 , 22 , 23 Here, we report the establishment of a human co‐culture model of primary human epidermal keratinocytes (HEKs) and the human neuronal cell line SH‐SY5Y to enable the screening of large libraries of compounds in the future. We first studied the morphological and functional properties of SH‐SY5Y cells in a co‐culture by analyzing neuronal markers. Subsequently, we evaluated the effects of human keratinocytes on SH‐SY5Y cell viability. Keratinocytes co‐cultured with differentiated SH‐SY5Y cells were compared with monocultured keratinocytes in terms of SP release and the expression of pro‐inflammatory cytokines and neuropeptides. In addition, we also evaluated the stinging sensation induced by the application of capsaicin to activate TRPV1 in vivo and the change in stinging sensation when a TRPV1 antagonist was applied to determine whether the co‐culture model corresponded to the in vivo sensations.

2. MATERIALS AND METHODS

2.1. Cell culture

HEKs were isolated from foreskins obtained from circumcision. This study was approved by the institutional review board (IRB) of Hallym University Sacred Hospital, Korea (IRB number: HALLYM 2019‐07‐029). The epidermal layer was separated from the dermis by enzymatic digestion. The tissue was incubated with dispase (25 U/ml in Hank's balanced salt solution) for 20 h at 4°C. The following day, the epidermis was removed, and keratinocytes were dissociated from the epidermal layer by digestion (15 min) using a 0.05% trypsin‐EDTA solution. The cells were cultured in a 154CF medium (Gibco/Fisher Scientific, Waltham, MA, USA) (0.07 mM Ca2+) containing a human keratinocyte growth supplement (HKGS). For the experiments, HEKs at passages 2–8 were used, with enzymatic dissociation using trypsin‐EDTA for each passage.

The human neuroblastoma cell line SH‐SY5Y was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and grown in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan UT, USA) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin, Gibco).

For the co‐culture of HEKs and neuronal differentiated SH‐SY5Y cells, SH‐SY5Y cells were centrifuged and plated at 20 000 cells/cm2 in an appropriate proliferation medium. After 24 h, the supernatant was removed and replaced with MEM containing 1% FBS and 10 µM retinoic acid for 4 days. Thereafter, the supernatants were removed and the HEKs (20 000 cells/cm2) were placed in 154CF medium containing HKGS and SH‐SY5Y cells for varying lengths of time. All the cell types were incubated in a humidified 5% CO2 incubator at 37°C. Cells were visualized using a phase‐contrast microscope (TS100; Nikon, Tokyo, Japan). Cell images were captured to measure neurite length.

2.2. Immunocytochemistry

After co‐culture on culture slides for 24, 48, and 72 h, the cells were rinsed three times with 1 × phosphate‐buffered saline (PBS) for 10 min each and then fixed using 4% paraformaldehyde for 10 min. The fixed cells were rinsed with 1 × PBS three times for 10 min each and then permeabilized using 1 × PBS containing 1% Triton‐X100 for 10 min at room temperature (RT). Before antibody binding, non‐specific sites were blocked by treating the cells with PBS containing 1% bovine serum albumin for 1 h at RT after rinsing with 1 × PBS three times for 10 min each. The cells were incubated with a primary antibody against MAP2 in 0.1% Triton X‐100 overnight at 4°C and incubated with Alexa Fluor 568 Goat Anti‐Rabbit IgG in PBS for 1 h at RT in the dark. After rinsing with PBS, the cells were mounted in 4,6‐diamidino‐2‐phenylindole‐containing Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) to label the nuclei and visualized using a confocal laser scanning microscope (LSM 700; Carl Zeiss, Oberkochen, Germany).

2.3. Cytotoxicity Assay

Mono‐ and co‐cultured keratinocytes and differentiated SH‐SY5Y cells were seeded (5 × 103 cells/well) in 96‐well culture plates (SPL Life Sciences, Republic of Korea). After incubation (24 h), the cells were treated with capsaicin at different concentrations for an additional 24 h. Cell viability was quantified using 2‐(2‐methoxy‐4‐nitrophenyl)‐3‐(4‐nitrophenyl)‐5‐(2, 4‐disulfophenyl)‐2H‐tetrazolium monosodium salt (WST‐8) according to the manufacturer's protocol. The WST‐8 assay, a modified 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium assay, was performed using a commercially available WST‐8 cell viability kit (Quanti‐Max, BioMax, Republic of Korea). After incubation with the drugs for 24 h, Quanti‐Max reagent was added, and the absorbance of WST‐8 formazan was measured at 450 nm.

2.4. Measurement of calcium influx

After 24 h, the cell culture medium was replaced with a serum‐free medium containing 1 µM calcium fluorescent probe Fluo‐3 and 0.04% Pluronic F127 (Thermo Fisher, Waltham, MA, USA) and incubated for 30 min. Capsaicin (1 µM) was added to 10 µM of BAPTA‐AM (1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid tetrakis(acetoxymethyl ester)) as a negative control. To confirm the role of TRPV1, 1 mM TTBC with 1 µM capsaicin was added; 20 min later, the cells were observed under a fluorescence microscope. The ImageJ software was used to analyze the fluorescence intensity of each image. The experiment was repeated at least four times for each culture condition.

2.5. Enzyme‐linked immunosorbent assay of SP

Cells (3 × 105 cells/well) were seeded in 6‐well culture plates (SPL Life Science, Republic of Korea) in the growth medium. After starving the cells for 5 h, capsaicin (1 µM) was added to TTBC (1 mM) for 24 h. Thereafter, the culture supernatant was collected for enzyme‐linked immunosorbent assay (ELISA). The cells were homogenized and stored overnight at −80°C. After two freeze‐thaw cycles to lyse the cell membranes, the homogenates were centrifuged for 5 min at 5000xg at 4°C. The supernatant was assayed using a commercially available SP ELISA kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) in accordance with the manufacturer's instructions. Measurements were obtained in triplicate, at minimum.

2.6. Quantitative real‐time polymerase chain reaction analysis

Inflammatory cytokines and neuropeptides known to cause itching in sensitive skin were examined by quantitative real‐time polymerase chain reaction (qRT‐PCR). Total RNA was isolated using Tri reagent (Molecular Research Center, Cincinnati, OH, USA) in accordance with the manufacturer's protocol. Complementary DNA (cDNA) was synthesized using 1 µg of RNA per 20 µl reaction volume with Maxime RT PreMix (iNtRON, Republic of Korea). Quantitative PCR was performed using SYBR Green dye and the KAPA SYBR FAST qPCR Master Mix (2X) Kit. For the relative quantitation of gene expression, the comparative Ct method (ΔΔCt) was used and β‐actin was used as an endogenous reference control for all transcripts. The primers were designed according to published cDNA or genomic sequences (see Table S1 for primer details).

2.7. In vivo analysis of capsaicin stinging test, and TRPV1 antagonist

This study was approved by the IRB of Hallym University Sacred Hospital, Korea (IRB number: HALLYM 2020‐03‐013). Volunteers who visited the dermatology clinic at Hallym University Sacred Hospital between May 2020 and December 2021, and were >19 years of age, were included. Those with facial dermatitis were excluded. A 7‐point questionnaire described by Buhé et al. 24 was administered to diagnose sensitive skin among the enrolled patients. For volunteers with a score ≥5, to induce the stinging sensation, we applied 1 cotton swab unit of capsaicin 50 parts per million (ppm) cream (0.075% capsaicin cream diluted with petroleum jelly) to both nasolabial folds for 5 min. 25 At 0.5, 2.5, and 5 min after application, the stinging sensation was evaluated by scoring from 0 to 5 (0 = no stinging sensation, 5 = very severe stinging sensation). After inducing a stinging sensation, volunteers applied 100 µl of TTBC 1% (diluted with a base of moisturizing cream) on the right side and a placebo (base of moisturizing cream [Candle House, Korea]) on the left side. Changes in the stinging sensation score were measured at 1, 2, 5, 10, and 15 min. The experiment was performed in a single‐blinded manner.

2.8. Test products

  1. 0.075%capsaicin cream (Diaxen cream, Dalim BioTech, Korea) Sorbitol solutation, aqua, benzyl alcohol,cetyl alcohol, PEG‐40 stearate, white petrolatum, glyceryl monostearate,isopropyl myrisatate, and capsaicin.

  2. Whitepetrolatum (White Vaseline, GUMI, Korea) White petrolatum.

  3. Base of moisturizing cream (Candle House, Korea) Aqua, butylene glycol,vegetable glycerin, niacinamide, hydroxyethyl acrylate/sodiumacryloyldimethyltaurate copolymer, polydimethylsiloxane, 1,2‐Hexanediol,betaine, cetyl stearyl alcohol.

2.9. Statistical analysis

All experiments were performed at least three times and data are expressed as mean ± standard error of the mean (SEM). Statistical significance between groups was determined using two‐tailed Student's t‐test. In the in vivo study, data were analyzed using the Wilcoxon two‐tailed test; differences with p < 0.05 were considered to be statistically significant.

3. RESULTS

3.1. Morphological characteristics of differentiated SH‐SY5Y cells in HEK co‐culture

SH‐SY5Y neuronal cells were typically grown in MEM supplemented with 10% FBS and were differentiated in a medium containing 1% FBS and 10 µM retinoic acid for 4 days. Thereafter, the supernatants were removed and keratinocytes were placed in a keratinocyte growth medium with differentiated SH‐SY5Y cells for 3 days. Neurite shapes were apparent, and axonal outgrowth was progressive in both monocultured and co‐cultured conditions (Figure 1A). After 3 days of co‐culture, microscopic analysis revealed a connection between keratinocytes and a dense neural network expressing MAP2 (Figure 1B). Axonal lengths reached in monoculture using keratinocyte growth medium were similar to those in the coculture group. The addition of HEKs had no effect on axonal outgrowth (Figure 1C). These results suggest that keratinocytes and keratinocyte growth medium introduced into the culture had no trophic effect on pre‐differentiated SH‐SY5Y neuronal cells after three days of co‐culture.

FIGURE 1.

FIGURE 1

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Co‐culture of differentiated SH‐SY5Y and primary epidermal keratinocytes. Three days after co‐culture (A), the cells were fixed and the neuronal cells were labeled with antibodies against MAP2 (red). Cell nuclei were labeled with 4,6‐diamidino‐2‐phenylindole (DAPI) (blue). The images in B show the spread of the neurite network (original magnification: X40). On days 1, 2, and 3 of culture, the average neurite length (µm) was calculated (C). Data are presented as mean ± SEM (n = 3). The average axon length was not significantly different between monocultured SH‐SH5Ys and co‐cultured cells

3.2. Capsaicin at non‐toxic concentration

In functional studies of co‐culture systems, capsaicin was used to induce an itching sensation. The cytotoxic effects of capsaicin were demonstrated in mono‐ and co‐cultured keratinocytes (Figure S1). Mono‐cultured SH‐SY5Y cells survived well in a medium supplemented with 50 µM capsaicin; however, for keratinocytes, cytotoxicity was observed at a capsaicin concentration of 5uM. Next, we determined the optimal concentration of capsaicin between the cytotoxicity and the intensity of the response of the cells: 1 µM capsaicin for mono‐and co‐cultured keratinocytes corresponded to calcium influx and SP release, with no toxicity.

3.3. Capsaicin‐induced cellular calcium influx and SP release was inhibited by TRPV1 antagonist

Calcium influx in mono‐and co‐culture of SH‐SY5Y cells and keratinocytes was induced by capsaicin 20 min after the application, as observed under a fluorescence microscope using Fluo‐3 staining. BAPTA‐AM was used as a negative control to eliminate basal intracellular calcium. Cellular calcium influx induced by capsaicin treatment decreased in the presence of the TRPV1 antagonist TTBC (Figure 2A,B). The level of SP released was also significantly decreased by TTBC treatment under both culture conditions (Figure 2C). However, the fold change of co‐cultured keratinocytes was lower than that of monocultured keratinocytes (p < 0.05). By examining the reaction of co‐cultured SH‐SY5Y cells and keratinocytes with capsaicin and TTBC, a TRPV1 antagonist, we confirmed that calcium influx was increased by capsaicin and decreased by TTBC. In particular, we confirmed that the TTBC inhibitory response to SP release was more sensitive under co‐culture conditions.

FIGURE 2.

FIGURE 2

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Effect of capsaicin on cellular calcium influx and substance P secretion. Calcium influx 20 min after stimulation with capsaicin (1 µM) was observed under a fluorescence microscope using calcium fluorescent probe Fluo‐3 (A) and relative fluorescence intensity was quantified by comparing control without capsaicin (B). BAPTA‐AM (10 µM) was used as a negative control and trans‐4‐tert‐butylcyclohexanol (TTBC) was used as a transient receptor potential channel vanilloid subfamily member 1 (TRPV1) antagonist. Substance P release was evaluated 24 h after capsaicin treatment in mono‐and co‐cultured cells using enzyme‐linked immunosorbent assay (ELISA) (C). The results were normalized and expressed as fold‐change ± SEM, compared to the capsaicin‐treated control without TTBC.***p < 0.001, **p < 0.01 (n = 4)

3.4. Synergistic expression appeared in differentiated SH‐SY5Y and keratinocytes co‐culture

To determine the differences between mono‐ and co‐cultured keratinocytes, the expression of several genes known to cause itching in sensitive skin was examined (Figure 3). In the absence of capsaicin stimulation, there was no difference in all conditions; however, when capsaicin was used, the expression of pro‐inflammatory cytokines and neuropeptides in co‐cultured keratinocytes increased compared to that in mono‐cultured SH‐SY5Y cells and keratinocytes. There was a tendency toward increased messenger RNA (mRNA) expression in response to capsaicin in mono‐cultured keratinocytes, but the difference was not statistically significant. Moreover, the capsaicin‐mediated increase of mRNA expression in co‐culture conditions was suppressed by treatment with TTBC with a significant difference between the mono‐cultured and co‐cultured groups. Collectively, our results suggest that keratinocytes co‐cultured with differentiated SH‐SY5Y cells exhibit synergistic effects on capsaicin‐evoked neurosensory responses.

FIGURE 3.

FIGURE 3

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Levels of pro‐inflammatory cytokines and neuropeptides in a co‐culture model. Cells were cultured without serum for 5 h, followed by the addition of capsaicin or transient receptor potential channel vanilloid subfamily member 1 (TRPV1) antagonist to the culture media for 48 h. The expression levels of pro‐inflammatory cytokines (A–D) and neuropeptides (E, F) mRNA in mono‐ and co‐cultured SH‐SY5Y cells and keratinocytes were measured by real‐time polymerase chain reaction (RT‐PCR). The levels (mean ± SEM, n = 6) are shown relative to the pre‐induction levels of mono‐cultured SH‐SY5Y

3.5. Stinging sensation induced by application of capsaicin was improved by TRPV1 antagonist

A total of 15 volunteers with complaints of sensitive skin were enrolled (12 women and three men) with a mean (±SD) age of 29.73 ± 8.01 years (range, 22–49). The volunteers scored a mean of 5.93 ± 0.77 points on the 7‐point questionnaire. Five minutes after capsaicin application, the patients complained of a stinging sensation, scoring 3.93 ± 0.70 points (p = 0.001) (Figure 4A). TTBC was applied to the right nasolabial fold and a placebo was applied to the left nasolabial fold. In the TTBC group, the stinging sensation score decreased by 1.13 points at 1 min, 2.40 points at 5 min, 2.80 points at 10 min, and 3.26 points at 15 min, respectively. In the placebo group, the scores decreased by 0.26 points at 1 min, 0.66 points at 5 min, 1.66 points at 10 min, and 2.06 points at 15 min, respectively. A significant difference was observed in the reduction of the stinging sensation 2 and 5 min after application. At 2 and 5 min, the placebo group showed a 7% and 17% decrease in stinging sensation, and the TTBC group showed a 44% and 61% decrease (p = 0.001 at 2 min, p = 0.000 at 5 min) (Figure 4B).

FIGURE 4.

FIGURE 4

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Effect of capsaicin and trans‐4‐tert‐butylcyclohexanol (TTBC) on stinging sensation score in vivo. After capsaicin application, the stinging sensation score increased; the score was 1.13 points at 30 s, 2.6 points at 150 s, and 3.93 points at 300 s (A). A significant decrease in the stinging sensation score was observed in the group treated with TTBC on skin irritated by capsaicin compared to the group treated with a placebo (B). ***p < 0.001**p < 0.01, *p < 0.05

4. DISCUSSION

In research investigating sensitive skin, most experiments have been conducted only on keratinocytes; however, more complex intercellular communication in the skin was revealed through keratinocytes co‐cultured with other cells constituting the epidermis. Although many co‐culture models have been developed, the co‐culture of human keratinocytes with human neuronal cell lines has not been well established. Some studies have reported functional cross‐talk between keratinocytes and neurons in human co‐culture and the participation of keratinocytes in peripheral sensation by interacting with and developing sensory units with nerve endings. 26 However, these studies have not yet demonstrated a difference between monocultured and co‐cultured keratinocytes, and practical studies aimed at finding a treatment for sensitive skin using a human co‐culture model have, to our knowledge, not been conducted. SH‐SY5Y is a subclone cell line derived from the SK‐N‐SH human neuroblastoma cell line. It serves as a model of adrenergic or dopaminergic neurons because the cells can exhibit traits of peripheral sensory neurons by the addition of specific compounds. 21 , 22 , 23 Therefore, we used SH‐SY5Y cells to establish a keratinocyte co‐culture model, which is an in vitro alternative for studying the close communication between keratinocytes and neuronal cells for sensitive skin therapy.

Capsaicin is known to excite nociceptive neurons by increasing membrane permeability to cations. TRPV1 is a non‐selective cation channel; when activated by capsaicin, it causes an influx of sodium and calcium ions through TRPV1 into the cell to depolarize nociceptive neurons, leading to the generation of an action potential, resulting in a stinging sensation. 27 TRPV1 is one of the most interesting therapeutic targets in dermatology. The first physiological function described for TRPV1 is the recognition of noxious heat, based on the finding that temperatures > 42°C activate this channel. 28 , 29 Heat‐activated keratinocytes transmit temperature information to sensory neurons via adenosine triphosphate secretion, 30 providing a direct connection between keratinocytes and sensory neurons, as described by Peier et al. 31 Moreover, keratinocytes communicate with neurons by releasing substances such as nerve growth factor (NGF) and IL‐1a. 33 Thus, the activation of one keratinocyte leads to the activation of adjacent cells, ultimately inducing the depolarization of nerve endings. 2

TTBC is a TRPV1 antagonist, which has been shown to counteract the hyper‐responsiveness of nerve fibers and, thus, is used in cosmetic products to alleviate sensory effects in individuals with sensitive skin. 32 However, the exact mechanism for the inhibition of TRPV1 by TTBC remains unclear. When TRPV1‐transfected human embryonic kidney 293 cells were treated with capsaicin, significant calcium influx was observed and TTBC significantly reduced capsaicin‐mediated calcium influx. 13 In this study, we also demonstrated that TTBC inhibited TRPV1, and calcium influx and SP secretion decreased in both monoculture and co‐culture conditions.

We confirmed the stability of co‐cultured human keratinocytes and differentiated SH‐SY5Y cells using morphological analysis, which revealed increased neurite length and MAP2 immunocytochemistry. Moreover, the increase in calcium influx and SP secretion by capsaicin treatment revealed that the co‐culture system of keratinocytes and SH‐SY5Y cells was functionally stable. Although we expected a difference in SP secretion between mono‐ and co‐cultures of keratinocytes, no significant difference was observed. However, TTBC reduced capsaicin‐induced calcium influx and SP secretion in co‐cultured keratinocytes compared to monocultured cells.

Unlike calcium influx and SP secretion, the expression of inflammatory cytokines including IL‐4, IL‐8, IL‐31, and IL‐33, and neuropeptides such as NGF and CGRP, increased in the co‐culture model compared to the monoculture, and we confirmed that the inhibition by TTBC was also significant.

In addition to TTBC, Asivatrep (C21H22F5N3O3S) which is a potent and selective TRPV1 antagonist 34 was treated to see if other TRPV1 antagonists also affected calcium influx and SP release in a co‐culture model (Figure S2). From the results, we found that different type of TRPV1 antagonist also shows more sensitive inhibitory responses in the co‐culture system.

Lactic acid (LA) is known to activate sensitive skin response through acid activation of various receptors and TRPV1 is one of them which is activated by protons at a pH less than 5.9. 35 We investigated the effect of TRPV1 antagonists treatment in LA‐treated co‐cultured keratinocytes. LA‐induced mRNA expression of inflammatory cytokines and neuropeptides was inhibited by TRPV1 antagonists (Figure S3). Our results suggest that keratinocytes co‐cultured with differentiated SH‐SY5Y cells exhibit more meaningful results close to in vivo test compared to the mono‐culture keratinocytes.

Sensitive skin is considered a neuropathic pruritus that occurs due to increased transduction in peripheral neurons, even with mild stimulation that does not cause pain or itching. 36 To date, studies investigating sensitive skin and cytokines are limited; however, studies investigating pruritus and cytokines are in progress. IL‐31 is a major cytokine associated with itching and it is induced by IL‐33. The IL‐31 receptor is expressed in epithelial and neuronal cells. 37 IL‐31 induces the secretion of neuropeptides to induce neurogenic inflammation and activates nearby nerve branches via an axon‐reflex mechanism. 38 IL‐4 and IL‐8 mediate itching in atopic dermatitis, and IL‐4 also plays a key role in chronic itch, epidermal homeostasis, and skin barrier function. 39 , 40 It has also been reported that sensitive skin is more common in patients with atopic dermatitis than in healthy controls. 41 Additionally, CGRP and NGF are neuropeptides associated with the nociceptive pathway. 42 , 43 NGF also plays an important role in the regulation of inflammatory hyperalgesia and TRPV1 42 , 44 and CGRP is known to correlate with inflammatory pain and itching. 43 , 45 In this study, by confirming the increased levels of various cytokines and neuropeptides, it was possible to confirm the occurrence of pruritus and pain in sensitive skin and the importance of cytokines and neuropeptides in sensitive skin.

In our in vivo study, volunteers developed strong stinging and itching sensations after capsaicin application, which were relieved after TTBC application. We assume that this result is due to changes in SP, ILs, and neuropeptides that occurred after capsaicin and TTBC treatment, as in the co‐culture model.

5. CONCLUSION

We established a co‐culture model of primary human keratinocytes and SH‐SY5Y human neuronal cells and confirmed that this model was functionally stable. We compared the differences between mono‐ and co‐cultures and verified the validity of the co‐culture model. The synergistic increase in IL and neuropeptide levels in co‐cultured keratinocytes revealed that our co‐culture model could mimic hypersensitive skin in vivo. We confirmed that TTBC can be used for the treatment of sensitive skin through in vitro co‐culture models of human keratinocytes and SH‐SY5Y cells, and in vivo tests. We expect that our co‐culture model can be used to identify agents that are suitable for the treatment of sensitive skin among other bioactive substances in vitro.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

ETHICS STATEMENT

This experiment was approved by the Institutional Review Board of Hallym University Sacred Hospital, Korea. All procedures in this study were conducted in accordance with the Institutional Review Board of Hallym University Sacred Hospital‐approved protocols.

Supporting information

Supporting Information

Click here for additional data file. (364KB, docx)

ACKNOWLEDGMENTS

This study was supported by the Hallym University Research Fund and the National Research Foundation of Korea grant funded by the Korean government (2019R1G1A100658813).

Shin SM, Baek EJ, Oh DY, Kim KH, Kim KJ, Park EJ. Functional validation of co‐culture model of human keratinocytes and neuronal cell line for sensitive skin by using transient receptor potential channel vanilloid subfamily member 1 antagonist. Skin Res Technol. 2023;29:e13275. 10.1111/srt.13275

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in the reference number.

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Supplementary Materials

Supporting Information

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Data Availability Statement

The data that support the findings of this study are openly available in the reference number.

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