https://orcid.org/0000-0002-6517-7830
Abstract
Keratinocytes are the primary component of the epidermis, so maintaining the precise balance between proliferation and differentiation is essential for conserving epidermal structure and function. Rosae multiflorae fructus extract (RMFE) has wide application in the cosmetic industry, but the molecular mechanisms underlying beneficial effects on keratinocytes are still not fully understood. In this study, we found that RMFE promoted epidermal differentiation and enhanced the barrier function of normal human epidermal keratinocytes (NHEKs) and three-dimensional epidermis model in culture. In addition, RMFE promoted human umbilical vein endothelial cell (HUVEC) proliferation and angiogenesis, whereas the conditioned medium from RMFE-treated HUVECs further promoted NHEK proliferation and increased wound healing ability. Analysis of constituent bioactivities identified a quercetin derivative as a potential mediator of NHEK and HUVEC responses to RMFE. Taken together, these results suggest that RMFE enhances epidermal functions through both direct effects on keratinocytes and indirect effects mediated by endothelial cells.
Rosae multiflorae fructus extract (RMFE) regulates epidermal differentiation and vessel-mediated epidermal proliferation.
Human skin consists of three main tissue layers, namely epidermis, dermis, and hypodermis. In the epidermis, the main cellular components are keratinocytes organized in 4 distinct layers that act as a barrier against physical, biological, and chemical damage to the body. Keratinocytes proliferate in the deep basal cell layer and migrate to the surface, forming a spinous cell layer, then to the granular cell layer, and finally to a cornified cell layer (Kanitakis 2002). Postmitotic keratinocytes enter a progressive differentiation program during migration, leading to the formation of a superficial protective barrier (Simpson, Patel and Green 2011). Tight junctions are necessary for maintaining barrier functions. They consist of different specific transmembrane proteins, such as claudins (CLDN) and occludins (OCLN), and peripheral membrane proteins, such as tight junction protein 1 (TJP1), also known as ZO-1.
Efficient epidermal wound healing is critical for restoring skin defects and regaining lost integrity and barrier function. Furthermore, interactions of multiple cell types such as keratinocytes, adipocytes, immune cells, and vascular endothelial cells (ECs) are required (Leonardo et al. 2020), and various tight junction genes are regulated during wound healing in response to injury (Guo and Dipietro 2010), although the interaction between the epidermis and capillary vessel network has not been clarified because the epidermis is avascular. The dermis has a rich blood vessel network which provides nutrients and oxygen to the epidermis, keeping the skin layers healthy. A recent study reported that vascular aging causes changes in the microenvironment, which in turn leads to epidermal stem cell aging (Ichijo et al. 2022). Therefore, the interactions between vascular ECs and keratinocytes are important for understanding epidermal function.
Rosa multiflora Thunberg (“Eijitsu” in Japanese and “Yingshi” in Chinese), commonly known as “Multiflora Rose” or “Rambling Rose,” is a flowering plant native to Japan and China (Danezan et al. 2015; Huebner et al. 2014). Rosae multiflorae fructus (RMF) has been used as a traditional medicine in Eastern Asia for inflammatory disorders and chronic pain (Kitahiro et al. 2019; Chrubasik, Roufogalis and Chrubasik 2007). Rose extracts are also widely used in cosmetics owing to their documented antioxidant and moisturizing effects. RMF extracts (RMFEs) are reported to contain essential fatty acids, vitamin E, minerals, and several flavonol glycosides such as quercetin, multinoside A, and multiflorin A (Takagi et al. 1976; Kitahiro et al. 2019; Bui et al. 2019), although it has not been determined which components have beneficial effects on keratinocytes.
In this study, we investigated the direct and indirect (vascular EC-mediated) effects of RMFE on human epidermal cell differentiation and stem cell maintenance. We also examined the specific bioactive agents in RMFE that mediate the observed effects on epidermal and EC functions.
Materials and methods
Reagent
RMFE was obtained using 50% ethanol extraction and prepared as a freeze-dried powder (Maruzen Pharmaceuticals Co., Ltd.). We redissolved the RMFE powder in an assay buffer or culture medium for use in our experiments. For comparison, we used multiple lot numbers (Lab. No. 180 408, 180 409, and 191 002) but all extracts exerted similar effects (Figure S1a). The chemical compounds comprising RMFE were also purchased, namely, quercetin 3-O-β-D-glucopyranoside (isoquercitrin, IQ; #20311-96, Kanto PPC Technology Co., Ltd.), quercetin 3-O-β-D-glucuronide (miquelianin, Q3GA; #1027S, Cayman Chemical), quercetin 3-O-β-D-galactoside (hyperoside, Q3Gal; #21 289, Extrasynthese), and ellagic acid (EA; #057-08751, FUJIFILM Wako Chemical Corporation). Ellagic acid 4-O-β-D-xylopyranoside (EADX), ellagic acid 4-O-α-L-arabinofuranoside (EALA), and agrimoniin (AM), which were isolated, purified, and their chemical composition identified using nuclear magnetic resonance (NMR) analysis of RMFE by Maruzen Pharmaceuticals, were used in our experiments. Calcium concentration in freeze-dried RMFE powder was measured using a Metallo Assay LS trace metal assay kit for calcium (CPZ III; MG Metallogenics Co., Ltd.) according to the manufacturer's protocol.
Cell culture and ECs-derived conditioned medium (ECCM) preparation
Neonatal normal human epidermal keratinocyte (NHEK), human umbilical vein endothelial cell (HUVEC), and normal human dermal fibroblasts (NHDF) were purchased from Lonza Japan and cultured at 37 °C under a 5% CO2 atmosphere. Keratinocytes were maintained in KGM-Gold with an additive factor kit (#00 192 060, Lonza), HUVECs in EGM-2 medium with an additive factor kit (#CC-3162, Lonza), and NHDFs in FGM-2 medium with an additive factor kit (#CC-3132, Lonza). The medium was replaced every 2 days during the experiment. To avoid interdonor variation, all experiments were performed using NHEK pooled from 3 donors.
To prepare ECs-derived conditioned medium (ECCM), passage 4 (P4) HUVECs were cultured to 50%-60% confluency in a 100-mm cell culture dish in 10 mL of EGM-2 medium with or without RMFE for 24 h. Then, the medium was replaced with 10 mL of KGM-Gold for 24 h, and the ECCM was harvested. After centrifugation at 3000 min-1for 10 min to remove the cell debris, the ECCM was filtered through a 0.22 μm filter (Millipore) and directly used for NHEK culture (P4). The KGM-Gold medium (10 mL) incubated for 24 h in a culture dish without cells was used as the control. Conditioned medium (CM) from RMFE-treated NHDFs (RMFE-DFCM) was also prepared using the same procedure.
Culture of a three-dimensional skin epidermis model
The LabCyte EPI-MODEL24 6D skin culture model (#401124E6), was purchased from Japan Tissue Engineering and maintained at 37 °C under a 5% CO2 atmosphere in culture medium supplied by the manufacturer. For experiments, RMFE was applied to the stratum corneum side of the model, followed by incubation at 37 °C for 2 or 7 days. Medium with or without RMFE was changed every day.
Cell proliferation assay
Cells were seeded in 96-well plates and incubated for 24 h at 37 °C under a 5% CO2 atmosphere prior to experiments. Cells were then treated with RMFE or the indicated compound for 24 h under the same culture conditions. Proliferation rate was measured using Cell Counting Kit-8 (#343-07623, DOJINDO), and the absorbance (optical density) at 450 nm was measured using a microplate reader (Multiskan FC, Thermo Fisher Scientific) according to the manufacturer's protocol. The background control value was subtracted from each measured absorbance value, and the average absorbance from each triplicate set of wells was calculated.
Cell cytotoxicity and dead cell assay
Cell cytotoxicity and dead cells were determined using the WST-1 assay reagent (#ab155902, Roche Diagnostics GmbH) and a Muse Annexin V & Dead Cell Kit (#MCH100105, Luminex Corporation) according to the manufacturer's instructions. Briefly, NHEKs were seeded in 96-well plates at a density of 5.8 × 103. The next day, they were exposed to different concentrations of RMFE for 24 h. WST-1 reagent was added and incubated for 30 min, and the absorbance was measured at 450 nm with a multiplate reader (Infinite F200, Tecan). The cytotoxicity was calculated using the following equation: cytotoxicity (%) = (absorbance in treated group − absorbance in blank)/(absorbance in control group − absorbance in blank) × 100. Additionally, NHEKs were seeded in 12-well plates at a density of 4 × 104. The next day, they were exposed to different concentrations of RMFE for 72 h. Annexin V and Dead Cell reagent were added and incubated for 20 min, and apoptosis and cell death ratio were detected using a Guava Muse Cell Analyzer (Luminex Corporation).
Real-time quantitative PCR (RT-qPCR)
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from 250 ng of RNA using the QuantiTect Retrotranscriptase reaction kit (Qiagen). Quantitative PCR (qPCR) was performed using SYBR green labeling (SYBR Premix Ex TaqII, Takara) and a TP850 Real-Time PCR System (Takara), with glyceraldehyde-3-phosphate dehydrogenase expression serving as the internal control. All individual sample reactions were run in triplicate. The relative fold change in target gene expression was then calculated based on the ∆∆Ct method. The qPCR primer pairs listed in Table S1. qPCR was used to analyze the characteristics of epidermal cultures. For instance, P4 NHEKs were seeded in 6-well plates at a density of 1 × 105 cells per well and cultured in KGM-Gold with an additive factor kit with or without 1.2 m m calcium under 5% CO2 atmosphere at 37 °C for 24 h prior to experiments. Cells were then treated with RMFE or the indicated compound for 24 h under the same culture conditions to analyze the epidermal differentiation marker genes and barrier function-related genes by qPCR. On the other hand, we confirmed that serially passaged P6 NHEKs had lower expression of stem cell markers than P4 NHEKs (data not shown), exhibiting profound stem cells aging. Therefore, P4 NHEKs were cultured in the proliferative mode using low-calcium media with or without RMFE, serially passaged until P6, and used to evaluate whether RMFE treatment maintains epidermal stem cell markers such as melanoma chondroitin sulphate proteoglycan (Legg et al. 2003), keratin 15 (K15) (Webb, Li and Kaur 2004), and delta-like 1 (DLL1) (Lowell et al. 2000), through qPCR.
Immunofluorescence staining and imaging analysis
Immunostaining procedures were performed as previously described (Suzuki-Komabayashi et al. 2019). Briefly, the three-dimensional cultured epidermis model or cultured NHEKs on chamber slides were fixed for 15 min in 4% paraformaldehyde, treated with blocking buffer (10% donkey serum and 0.1% Triton X-100, pH 7.4) for 30 min at room temperature, and followed by incubation overnight with anti-TJP1 primary antibody (1:1000, Invitrogen), anti-Loricrin (LOR) primary antibody (1:1000, GeneTex), anti-OCLN primary antibody (1:1000, Invitrogen), and anti-CLDN1 primary antibody (1:1000, Santa Cruz Biotechnology). After washing again in PBS with 0.1% Triton X-100, cells were incubated for 1 h with Alexa Fluor 488-conjugated donkey rabbit anti-IgG secondary antibody (1:2500, Molecular Probes) and Alexa Fluor 594-conjugated donkey mouse anti-IgG secondary antibody (1:2500, Molecular Probes), rewashed extensively with PBS, and counterstained with DAPI.
Cell proliferation was also evaluated by incorporating BrdU (Sigma-Aldrich) into the cell culture system and immunofluorescent staining. Briefly, cells were incubated with 50 μg/mL BrdU for 2 h before fixation. For immunofluorescent staining, the cells were incubated with PBS containing 0.1% Triton X-100 and incubated with mouse monoclonal anti-BrdU (Roche). Cells were then washed in PBS, incubated with Alexa Fluor 488-conjugated donkey mouse anti-IgG secondary antibody (1:2500, Molecular Probes), rewashed extensively with PBS, and counterstained with DAPI. Images were acquired using a confocal microscope (FV3000, Olympus) and processed using Adobe Photoshop.
Transepithelial electrical resistance (TEER)
To examine the barrier function after RMFE treatment, samples of the three-dimensional skin epidermis model in a 24-well plate were cultured with or without RMFE for 2 days. Subsequently, transepithelial electrical resistance (TEER) was measured using an EVOM2 Epithelial Voltohmmeter (World Precision Instruments) with an STX2 electrode. Readings from a blank insert were subtracted from each measurement, and unit area resistance (Ωcm2) was calculated by multiplying resistance readings with the effective surface area. On the other hand, NHEKs were placed on the upper side of cell culture insert membranes with a pore size of 0.4 μm in the 24-well Transwell inserts. Following incubation with or without RMFE for 2 days, the electrical resistance of culture models was measured using a TEER measuring system (Kanto Chemicals).
Transepidermal water loss (TEWL)
Transepidermal water loss (TEWL) was measured using a handheld TEWL measuring instrument (VAPO SCAN AS-VT100RS, Asahi Techno Lab). Measurements were conducted 2 days after treating samples of the three-dimensional skin epidermis model in a 24-well plate. Then, the three-dimensional skin epidermis model was placed on a hot plate at 37 °C for 30 min on a clean bench. Each value was determined by the same investigator.
Tube formation assay
Procedures were performed as previously described (Iwai et al. 2021). Briefly, 48-well plates were coated with Matrigel (Corning) and incubated for 30 min at 37 °C under a 5% CO2 atmosphere. HUVECs were plated on the gel and cultured in a medium with RMFE. After 6 h of additional incubation, microscopic images of the tubes were acquired using a phase contrast microscope. Five images were then captured per well and analyzed by ImageJ software. HUVECs from passage number 4 were used for this assay. All experimental conditions were replicated in triplicate.
Scratch assays
NHEKs were seeded on a 24-well culture plate in KGM-Gold medium at a cell density of 5 × 105 cells/well, and the cells formed a monolayer after an overnight culture. The cell monolayer was scratched with a micropipette tip. Next, the medium was replaced with fresh KGM-Gold medium with or without RMFE, control ECCM medium, or RMFE-ECCM medium (as shown above), and maintained for 48 h. Additionally, the effect of inhibiting cell proliferation was evaluated by adding 10 μg/mL Mitomycin C (MC; #20898-21, Nacalai Tesque) to RMFE-ECCM medium. Time-lapse imaging was taken by All-in-one fluorescence microscope (BZ-X800, KEYENCE). The ratio of wound closure of NHEKs was calculated by the following equation: relative wound closure = [(initial wound area − final wound area)/initial wound area].
Liquid chromatography–mass spectrometry (LC–MS) analysis
LC–TOF/MS was performed using a XEVOTM G2 TOF MS System (Waters Japan) equipped with an ACQUITY UPLC® System (Waters Japan). The mass conditions were as follows: The ESI capillary voltage was set at 2.6 kV in the positive ion mode. The source and desolvation temperatures were set at 120 °C and 450 °C, respectively. The desolvation and cone gas flows were 800 L/h and 50 L/h, respectively. The sample and extraction cone voltages were set at 40 V and 4.0 V, respectively.
Quantification and statistical analysis
All findings are presented as the mean ± standard error of the mean (SEM) for datasets containing exactly 2 groups, an unpaired 2-sided Student's t test was used to determine significant differences. In figures, asterisks denote statistical significance at the following levels: ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. All statistical analyses and plotting of quantitative data were performed using Microsoft Excel.
Results
RMFE promotes NHEK differentiation and barrier function
First, we assessed the cytotoxicity of RMFE in NHEK cultures (Figure S1b and c) and evaluated its effects on epidermal differentiation in NHEK at concentrations of 0.25-25 μg/mL. RMFE-treated NHEK culture showed morphological changes (Figure S1d); however, the morphological characteristics were diverse and difficult to distinguish. Therefore the following expression profiling and immunostaining were performed to analyze the effect of RMFE at the molecular level. RMFE-treated NHEK after 24 h culture increased the expression of the early differentiation markers (spinous layer markers) keratin 10 (K10) and small proline rich protein 1B (SPRR1B) (Fujimoto et al. 1993; Poumay and Pittelkow 1995), as well as the late differentiation markers (granular layer markers) involucrin and LOR (Lee, Yuspa and Dlugosz 1998; Wong et al. 2022) (Figure 1a). Moreover, the expression level of K10 was dose-dependently increased by RMFE (Figure 1b). Treatment with RMFE also upregulated expression of the barrier function-related genes (Brandner 2016) OCLN, CLDN1, and TJP1 compared to control NHEK (Figure 1c). Furthermore, immunostaining revealed that LOR, CLDN1, and TJP1 protein expression increased after 24 or 72 h of culture with RMFE (Figure 1d and Figure S1e) compared with that in the control NHEKs, confirming that RMFE not only affects differentiation marker mRNA expression levels but also protein expression levels. Because calcium ions are important for inducing keratinocyte differentiation (Pillai et al. 1988; Jeriha et al. 2020), we measured the calcium concentrations of RMFE and detected 0.068 μg/mL calcium in 25 μg/mL RMFE. This is equivalent to 1.7 μm, which is much lower than the concentration of 1.2 m m calcium ions required to induce keratinocyte differentiation (Pillai et al. 1988; Jeriha et al. 2020). We also compared the effects of RMFE in NHEK culture supplemented with 1.2 m m calcium in the presence and absence of RMFE as the keratinocyte differentiation protocol (Figure 1a and c). In this differentiation protocol, it was also confirmed that the supplementation of 1.2 m m calcium ions promoted the differentiation of NHEK and that the coexistence of RMFE further increased the expression of differentiation marker genes (Figure 1a and Figure S1e) and barrier function-related genes (Figure 1c and Figure S1e). These suggested that RMFE alone showed sufficient physiological activity for NHEK differentiation, even when compared with the effect of calcium ions. Furthermore, treatment of the three-dimensional cultured epidermis model with 0.25 μg/mL RMFE for 7 days significantly increased the expression of SPRR1B, K10, LOR, CLDN1, and TJP1 genes (Figure 1e) and enhanced K10, LOR, and CLDN1 protein expression as confirmed by immunostaining (Figure 1f and Figure S2a). Additionally, culture of a three-dimensional skin epidermis model with medium containing RMFE was thicker than the control culture, indicating the effectiveness of RMFE on enhancing epidermal development (Figure 1f and Figure S2b). Then, we assessed whether RMFE treatment improved barrier function in three-dimensional cultured epidermis models. TEER is based on measuring the Ohmic resistance and is a widely accepted measurement of the integrity of cell–cell junctions (Srinivasan et al. 2015). RMFE treatment showed a significant increase in TEER compared with the control three-dimensional cultured epidermis model (Figure 1g). Next, we evaluated the TEER values of RMFE-treated NHEK to confirm whether the increase of TEER in RMFE-treated three-dimensional culture was due to an increase in the expression of tight junctions or epidermal thickness. Consequently, a significant increase in TEER values due to RMFE treatment was confirmed even in two-dimensional NHEK culture (Figure S2c), suggesting that increased tight junctions may be one of the mechanisms for the effect of RMFE on TEER values. Furthermore, after 0.25 μg/mL RMFE treatment for 2 days, the TEWL value, which is a well-established and widely used parameter for characterizing skin barrier function (Gupta et al. 2008), was significantly decreased compared with that of the control (Figure 1h), indicating a moisturizing effect.
Rosae multiflorae fructus extract (RMFE) promotes epidermal cell differentiation and barrier function in normal human epidermal keratinocyte (NHEK) and three-dimensional human epidermis model culture. (a) Expression levels of keratinocyte differentiation markers were upregulated in 25 μg/mL RMFE-treated NHEKs cultured with or without 1.2 m m calcium compared with that in untreated control NHEKs after 24 h, as measured by real-time quantitative (RT-q) PCR. (b) RMFE treatment upregulated mRNA expression of the early differentiation marker gene K10 dose-dependently from 0.25 to 25 μg/mL. (c) Treatment with 25 μg/mL RMFE also upregulated mRNA expression of the multiple barrier function markers in NHEKs cultured with or without 1.2 m m calcium. (d) Treatment with 25 μg/mL RMFE upregulated expression of the cell junction protein TJP1 after 72 h culture as measured by immunofluorescence staining. (e) Similar results were observed in the three-dimensional cultured human epidermis model of the RMFE treatment at 0.25 μg/mL after 7 days. RT-qPCR analysis of keratinocyte differentiation and barrier function marker genes. (f) Immunofluorescence staining of K10 and LOR in the three-dimensional cultured human epidermis model after 7 days of 0.25 μg/mL RMFE treatment. DAPI is also shown. (a-f) Data represent mean ± SEM of all experiments; n = 5 independent experiments. (g) Transepithelial electrical resistance (TEER) of control and 0.25 μg/mL RMFE-treated three-dimensional cultured human epidermis models was measured after 2 days. (h) Transepidermal water loss (TEWL) values of control and 0.25 μg/mL RMFE-treated three-dimensional cultured human epidermis models were measured on day 2. (g, h) Data represent mean ± SEM of experiments; n = 3 experiments. Scale bar, (f) 50 μm. ***P < 0.001, **P < 0.01, and *P < 0.05.
Conversely, RMFE-treated NHEK culture inhibited the proliferation rate (Figure S2d), and slightly decreased the BrdU-positive ratio (Figure S2e) although the changes did not reach statistical significance. These data suggest that RMFE enhances epidermal differentiation and barrier function in NHEK and three-dimensional cultured epidermis model.
RMFE regulates EC function, and conditioned medium from RMFE-treated ECs promotes NHEK proliferation
Skin tissue contains different cell types, including keratinocytes, fibroblasts, immune cells, and vascular ECs. A recent study reported that an age-dependent decrease in dermal blood vessels induced epidermal stem cell aging (Ichijo et al. 2022), although the underlying signaling mechanisms have not been fully clarified. Therefore, we first examined the effects of RMFE on EC proliferation and tube formation as an estimate of pro-angiogenic activity. Treatment with RMFE significantly promoted HUVECs proliferation (Figure 2a) and two-dimensional tube formation (Figure 2b and c). Moreover, CM from RMFE-treated HUVECs (RMFE-ECCM) increased the proliferation rate (Figure 2d) and BrdU-positive ratio (Figure 2e) in NHEK cultures. After serial passages, RMFE-ECCM-treated NHEKs also showed increased expression levels of epidermal stem cell markers, such as K15 and DLL1 (Gupta et al. 2008), compared to control NHEK cultures (Figure 2f). We similarly assayed the interaction between dermal fibroblasts (DFs) and keratinocytes; however, there was no significant effect of CM from RMFE-treated NHDFs (RMFE-DFCM) on the NHEK culture (Figure S3a and b), indicating that the effect of RMFE was more efficient in EC-mediated action than in DF-mediated action.
RMFE promotes endothelial cell (EC) proliferation, angiogenesis, and ECs-mediated keratinocytes proliferation and wound healing. (a) HUVECs were treated with RMFE (2.5 μg/mL) for 24 h and subjected to the cell proliferation assay. (b, c) HUVECs on the Matrigel were treated with RMFE (0.25-25 μg/mL) for 6 h and subjected to the tube formation assay. Representative images of the untreated and RMFE treatment (b), as well as the quantified tube length, the number of branch points and junctions (c). Cell proliferation assay (d), BrdU incorporation assay (e), and RT-qPCR analysis of epidermal stem cell markers (f) were performed in NHEKs cultured with the conditioned medium from HUVEC-treated with 25 μg/mL RMFE (RMFE-ECCM). (g) NHEKs were treated with control medium or RMFE-ECCM and subjected to the scratch wound healing assay for 48 h. White dotted lines indicate wound margins. Data represent mean ± SEM values of all experiments; n = 5 independent experiments. Scale bars, (b), (g), 200 μm. **P < 0.01, and *P < 0.05.
As epidermal stem cell proliferation is essential for wound healing, we also evaluated if RMFE-ECCM can enhance the proliferation or migration of keratinocytes by performing scratch assays of NHEK culture. Indeed, RMFE-ECCM significantly increased wound healing ability in the NHEK culture (Figure 2g), whereas NHEKs directly treated with RMFE had no such effect on keratinocytes (Figure S3c). In addition, NHEK culture treated with RMFE-ECCM in the presence of cell proliferation inhibitor suppressed its stimulatory effect on wound healing (Figure S3d). This indicated that the enhanced wound healing effect due to RMFE-ECCM is a result of increased cell proliferation, not migratory ability. These results suggest that the interaction between ECs and keratinocytes is strengthened by RMFE treatment, and this enhanced interaction can accelerate epidermal proliferation and skin repair.
RMFE-derived components promote NHEK differentiation and HUVEC proliferation
Many of the beneficial effects of RMFE have been attributed to flavonol glycosides, vitamin E, carotene, and essential fatty acids (Takagi et al. 1976; Bui et al. 2019; Kitahiro et al. 2019). Thus, we investigated the effects of these RMFE-derived components on NHEKs and HUVECs. A recent study classified RMFEs into two groups based on the presence (type I) or absence (type II) of multiflorin A (Kitahiro et al. 2019). The RMFE used in this study was analyzed by LC–MS and identified as type II RMFE because it does not include multiflorin A (Figure 3a) but includes IQ, Q3GA, Q3Gal (Kitahiro et al. 2019) (Figure 3a and Figure S4). As a continuation of our studies to find the active major components, the unidentified fraction from RMFE was purified by reversed-phase and normal-phase silica gel column chromatography and preparative HPLC to furnish EADX (Tanaka, Jiang and Kouno 1997), EALA (Sudjaroen et al. 2012), AM (Okuda et al. 1984), and EA (Figure 3b and Figure S4). EADX, EALA, and AM were identified by comparison of their 1H- and 13C-NMR data with reported values (Sudjaroen et al. 2012; Kitahiro et al. 2019). EA was identified by comparison of the commercial standards. We confirmed the cytotoxicity of RMFE-derived components in NHEK and HUVEC cultures. No significant cytotoxic effects were observed at concentrations of 2.5 μg/mL (Figure S5a and b).
Potential bioactive agents in RMFE identified by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS). (a) The profile of RMFE extracts showing absence of multiflorin A and presence of quercetin 3-O-β-D-glucopyranoside (isoquercitrin, IQ), quercetin 3-O-β-D-glucuronide (miquelianin, Q3GA), quercetin 3-O-β-D-galactoside (hyperoside, Q3Gal), and the unidentified fraction. (b) The profiles of unidentified fraction were further isolated and identified as ellagic acid 4-O-β-D-xylopyranoside (EADX) (b-3), ellagic acid 4-O-α-L-arabinofuranoside (EALA) (b-2), agrimoniin (AM) (b-4), ellagic acid (EA) (b-1), and IQ (b-5). Vertical axis indicates the relative intensity, expressed as a percentage of the maximum peak in the total ion current chromatogram.
Finally, we investigated the effects of IQ, Q3GA, Q3Gal, EADX, EALA, AM, and EA on epidermal differentiation and endothelial proliferation. Both IQ and Q3Gal enhanced expression of the early differentiation marker K10 (Figure 4a), and the barrier function-related gene TJP1 (Figure 4b). The expression level of K10 was dose-dependently increased by IQ or Q3Gal (Figure S5c and d). IQ-treated NHEKs under the supplementation of 1.2 m m calcium ions exhibited notable increases in K10 expression compared with that in control or only calcium-supplemented NHEKs (Figure 4c). Immunostaining revealed that OCLN protein expression increased after 24 h of culture with IQ (Figure S5e) compared with that of the control NHEKs. Additionally, treatment of the three-dimensional cultured epidermis model with IQ for 7 days significantly increased keratinocyte differentiation marker and barrier function-related marker expression levels as confirmed by qPCR (Figure 4d) and immunostaining (Figure 4e and Figure S5f). Moreover, after treatment with IQ for 2 days, the TEWL value was significantly decreased, exhibiting a moisturizing effect (Figure 4f). In addition, IQ significantly increased HUVEC proliferation (Figure S5g) and upregulated the angiogenic genes endomucin (Park-Windhol et al. 2017), delta-like 4 (Hellström et al. 2007), and Apelin (Kasai et al. 2004) (Figure 4g). These data strongly suggest that the demonstrated effects of RMFE on epidermal cell proliferation and differentiation are mediated in part by quercetin derivatives and that these effects are both direct and EC-mediated (Figure 4h).
RMFE-derived components promote NHEK differentiation and HUVEC proliferation. (a, b) NHEKs were treated with 2.5 μg/mL each RMFE-derived component for 24 h. Real-time quantitative (RT-q) PCR analysis of keratinocyte differentiation markers (a) and barrier function markers (b) in the RMFE-derived component-treatment and untreated control NHEKs at 24 h of culture. (c) K10 expression level was upregulated in 2.5 μg/mL IQ-treated NHEKs cultured with or without 1.2 m m calcium compared with that in untreated control NHEKs after 24 h, as measured by RT-qPCR. (d) Similar results were observed in the three-dimensional cultured human epidermis model of the IQ treatment at 2.5 μg/mL after 7 days. RT-qPCR analysis of keratinocyte differentiation and barrier function marker genes. (e) Immunofluorescence staining of LOR in the three-dimensional cultured human epidermis model after 7 days of 2.5 μg/mL IQ treatment. DAPI is also shown. (f) Transepidermal water loss (TEWL) of control and IQ (2.5 μg/mL)-treated three-dimensional cultured human epidermis model were measured on day 2. (g) HUVECs were treated with each RMFE-derived component (2.5 μg/mL) for 24 h. RT-qPCR analysis of angiogenesis markers in the RMFE-derived component treatment and untreated control HUVEC at 24 h of culture. (a-g) Data represent mean ± SEM values of experiments; n = 5 independent experiments. (h) A visual summary of our findings. Scale bar, (e) 50μm. ***P < 0.001, **P < 0.01, and *P < 0.05.
Discussion
Botanical extracts potentially have cosmetic benefits, such as antiaging and anti-inflammatory functions. RMFE has been used in Japanese traditional practice to improve constipation and chronic inflammation without obvious acute toxicity. Rose hip extracts also have wide applications in the cosmetic industry, although the molecular mechanisms of RMFE on keratinocytes remain partially understood. In this study, we focused on the physiological activity of RMFE related to epidermal functions and found that RMFE regulates epidermal differentiation and vessel-mediated epidermal proliferation.
The basal cell layer above the basal lamina comprises proliferating stem and progenitor cells. Those undifferentiated cells exit the cell cycle, keratinocytes migrate upward to form spinous and granular layers, and they trigger the expression of genes related to barrier function (Kanitakis 2002; Simpson, Patel and Green 2011). However, the balance between proliferation and differentiation is progressively impaired during aging as more keratinocytes enter terminal differentiation and the proliferating pool shrinks. In this study, we found that RMFE effectively promoted epidermal differentiation and barrier function in NHEK cultures but also promoted vascular cell-dependent NHEK stem cell proliferation, thereby maintaining this balance. Keratinocytes receive multiple external cues that regulate cell fate, including extracellular matrix constituents (Watt 2002), extracellular calcium concentration (Fuchs 1990; Lee, Yuspa and Dlugosz 1998; Wong et al. 2022), oxygen tension (Ngo et al. 2007), and various signals from capillary vessels. Our results of the tube formation assay revealed that RMFE enhanced EC functions. We also focused on the interaction between keratinocytes and vascular ECs. We confirmed that RMFE treatment did not disrupt the balance between proliferation and differentiation, as epidermal proliferation and stem cell competence were maintained in serially passaged NHEKs by endothelial-CM from RMFE-treated HUVECs, as evidenced by enhanced expression of the epidermal stem cell markers.
RMFE has many reported constituents, including flavanol glycosides, vitamin E, carotene, and essential fatty acids (Takagi et al. 1976; Bui et al. 2019; Kitahiro et al. 2019), but it is still unclear which compounds regulate epidermal function. In this study, quercetin derivatives such as IQ and Q3Gal were found to be active components, consistent with previous studies reporting that quercetin can mitigate UVA-induced damage to keratinocytes and fibroblasts (Rajnochová et al. 2022) and regulate the tight junction barrier properties of kidney cells (Gamero-Estevez et al. 2019). Quercetin is a known regulator of the PI3K/AKT pathway (Jiang et al. 2016), and IQ regulates its signaling (Zhu et al. 2016). A recent study has shown that topical application of quercetin promotes hair regrowth and angiogenesis by activating hypoxia inducible factor-1α in ECs (Zhao et al. 2023). However, the effect of quercetin derivatives on keratinocyte differentiation and proliferation was not clarified. In addition to quercetin derivatives, other candidate compounds, including EA derivatives, regulate the proliferation of NHEKs and HUVECs. Our assays of isolated compounds were performed at 2.5 μg/mL because we had determined that this concentration was not cytotoxic. However, because our analysis estimated the IQ content in RMFE to be approximately 0.002%, we confirmed its effectiveness at low concentrations (Figure S5c and d). Further studies are necessary to determine the synergistic effects of the combination of each RMFE-derived component on epidermal cell function.
In summary, we demonstrate that RMFE has beneficial effects on keratinocyte proliferation, differentiation, barrier formation, and wound healing (Figure 4h). More refined extracts and bioactive component preparations may have even greater cosmetic and medicinal benefits.
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
Author contribution
Conceptualization: H.A., M.I., and K.M.; Methodology: M.Y., Y.N., H.A., and K.M.; Investigation: S.A., T.I., Y.A., K.A., A.I., M.K., A.I., and K.M.; Writing—Original Draft: K.M.; Writing—Review & Editing: all the authors.; Supervision: T.O., M.I., and K.M.
Funding
None declared.
Disclosure statement
The authors T.I. and M.Y. are regular employees of Rosette Co., Ltd. However, T.I. and M.Y. did not have any additional role in the study design, data analysis, decision to publish, or manuscript preparation. This does not alter our adherence to Bioscience, Biotechnology, and Biochemistry policies on sharing data and materials. All other authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.
Acknowledgments
The authors thank Sakiho Koyama for excellent technical assistance.
References
Author notes
These authors contributed equally.
