Introduction

Atopic dermatitis (AD), also known as eczema, is a chronic inflammatory skin disease characterized by pruritic and eczematous lesions (Guttman-Yassky et al. 2013; Huang et al. 2022). The activation of T-helper 2 (Th2) cells, mast cells, and eosinophils further amplifies the immune response, which leads to the pathological characteristic features of AD, such as erythema, pruritus, and lichenification (Garcovich et al. 2021). Numerous studies have identified the intricate nature of AD, seeking to unravel its underlying mechanisms. At molecular level, elevated levels of pro-inflammatory cytokines, including interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-ɑ), contribute to the chronic inflammatory milieu (Huang et al. 2022). TNF-ɑ is a pro-inflammatory cytokine elevated in AD. It attracts immune cells to the site of inflammation to amplifying the immune response in AD lesions, which cause skin barrier dysfunction the expression of barrier proteins and promoting epidermal hyperplasia (Kanwal et al. 2021). In particular, IL-4 and IL-13, Th2-mediated cytokines, stimulate various immune cells including mast cells and eosinophils, resulting in the release of inflammatory mediators. Furthermore, these cytokines significantly downregulate the expression of proteins involved in skin barrier functions such as filaggrin (FLG), loricrin (LOR), and involucrin (IVL) via the Janus Kinase (JAK)-Signaling Transducer and Activator of Transcription (STAT) pathway, which merges as a key mediator in orchestrating the intricated mechanisms between the skin barrier and immune dysregulation (Furue 2020; Guttman-Yassky et al. 2013; Huang et al. 2022).

Accumulating evidence demonstrating the effects of natural products on AD has provide valuable insights into potential therapeutic applications. Natural products for AD application exhibit key biological characteristics such as anti-inflammatory and anti-oxidant properties (Saleh et al. 2021; Wu et al. 2021a, b). Natural products contain ingredients like ceramides or fatty acids and enhance the structural integrity of the skin barrier (Rosso et al. 2016). The efficacy of botanical extracts such as chamomile, calendula, and licorice alleviate AD symptoms by reducing inflammatory and pruritic effects (Bhaskaran et al. 2010; Kodiyan & Amber 2015; Sah et al. 2022; Wu et al. 2021a, b; Yang et al. 2017). Epigallocatechin gallate (EGCG) or omega-3-fatty acids from natural products contribute to skin barrier integrity and possess anti-inflammatory properties (Balić et al. 2020; Kim et al. 2018; Yuan et al. 2020). Some natural products with antimicrobial and antifungal properties can maintain the balance of skin microbiome (Kengne et al. 2023; Redondo-Blanco et al. 2020; Suurbaar et al. 2017). Given the general perception as being gentle and safe is important for patients, further investigation to discover new materials with well-tolerated and less likely to cause adverse reactions is necessary.

Rosa rugosa (R. rugosa), commonly known as the beach rose, is a resilient and adaptable plant. It thrives in coastal environments including sandy shores, dunes, and coastal grasslands. Its ability to withstand salt spray and tolerate sandy soils makes it well-suited for beach ecosystems (Woch et al. 2023). Although R. rugosa is primarily appreciated for its ornamental qualities, it has also been traditionally used in various cultures for medicinal purposes. Rosehip oil derived from the fruit of R. rugosa is known for its skin-nourishing properties. It is used in cosmetic products and traditional remedies for conditions like dry skin, scars, and wrinkles, suggesting that the oil can promote skin regeneration and improve elasticity (Lei et al. 2023) (Liu et al. 2022) (Chae et al. 2021). Since the rose hips are known for their high phenolic content (e.g., quercetin, kaempferol, and ellagic acid) and the extract from the rose hip seeds has strong elastase inhibition activity, R. rugosa may offer its potential for anti-oxidant, anti-aging and anti-inflammatory effects (Chae et al. 2021). Thus R. rugosa extract can be adequately incorporated into skincare products to enhance skin health such as moisturization, anti-aging, and soothing effects. Recent evidence reports that R. rugosa extract has skin lighting effects on mouse melanoma B16F10 cells by inhibiting tyrosinase activity (Liu et al. 2022). Moreover, R. rugosa inhibits skin inflammatory responses in keratinocytes using R. rugosa-based gold nanoparticles (Wang et al. 2022). Despite these biological activities, there have been no scientific evidence to demonstrate the potentials for anti-atopic dermatitis.

In this study, we unveiled the therapeutic potential of R. rugosa extract in addressing the intricate mechanisms of AD. We demonstrated its robust antioxidant activity and capability to strengthen skin barrier functions. By specifically inhibiting Th2-mediated JAK-STAT6 signaling in human keratinocytes, R. rugosa extract can be a promising candidate for modulating the immune response and restoring the damaged skin barrier in AD. These findings not only deepen our understanding of the molecular mechanisms involved but also signify a potential breakthrough in the development of innovative therapeutics for AD.

Materials and methods

Preparation of R. rugosa extract

The 70% ethanol extract of R. rugosa (MABIK NP60230050) was obtained from the National Marine Biodiversity Institute of Korea (MABIK). In May 2021, R. rugosa samples were harvested from Yangyang-gun in Gangwon-do, South Korea with MABIK’s coordination. Thirty grams of freeze-dried whole R. rugosa were subjected to sonication at 40 kHz for one hour using 70% ethanol (400 mL × 3). Afterward, the mixture was evaporated under reduced pressure following reflux heating. The resulting dry extract of 70% ethanol R. rugosa (800 mg dry weight) was stored at -80 °C until its biological activity could be evaluated. For in vitro experiments, the extract was reconstituted in dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA).

Cell culture

Normal Human Epidermal Keratinocyte (NHEK) cells were purchased from Lonza Bioscience and utilized in this study. The cells were cultured in a medium composed of Keratinocyte Basal Medium (KBM, Lonza, 2.2Switzerland) supplemented with KGM SingleQuot™ supplement (Lonza). Cultivation was carried out in a 5% CO2, 37 °C incubator (ARA P150, Hanil Science, Korea). For this study, cells from passages 4 to 7 were used, with the medium being replaced every 2 days. Subculturing and experimentation were performed when the cells reached 70–80% confluency.

Cytotoxicity assay

The cytotoxicity of the samples was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. NHEK cells were seeded in a 96-well plate at a density of 1 × 104 cells/well and cultured for 24 h. After exchanging with fresh medium, the cells were treated with various concentrations (1, 2, 5, 10 μg/mL) of R. rugosa extract and cultured for an additional 48 h. Subsequently, the medium was replaced with a solution containing CCK-8 reagent at a 1:10 ratio, and the mixture was allowed to react for 3 h in a CO2 incubator. After the 3 h reaction, absorbance was measured at 540 nm. Cell viability at each concentration was calculated using the following Eq. (2).

$$Cell~Viability~\left( \% \right) = ~\frac{{A_{{sample}} }}{{A_{{control}} }}~ \times 100$$

RNA isolation and qRT-PCR

NHEK cells were seeded in a 12-well plate at a density of 7 × 104 cells/well and cultured for 24 h. Subsequently, they were treated with R. rugosa extract to a concentration of 20 μg/mL and cultured for an additional 72 h. After 72 h, RNA extraction was performed using the Quick-RNA Micro Prep Kit (ZYMO Research, USA). The concentration of isolated RNA was measured using the Genova Nano spectrophotometer (JENWAY, UK). RNA (up to 2 μg) was reverse transcribed into cDNA using the High-capacity cDNA Kit (ThermoFisher Scientific, USA). The synthesized cDNA was subjected to real-time quantitative polymerase chain reaction (qRT-PCR) using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, USA) and Taqman Probes (ThermoFisher Scientific, USA) following the manufacturer's instructions. Genes used for PCR included KRT1 (Hs00196158_m1), KRT10 (Hs00166289_m1), LOR (Hs01894962_s1), IVL (Hs00846307_s1), FLG (Hs00856927_g1), CLDN1 (Hs00221623_m1), and the housekeeping gene ribosomal protein large P0 (RPLP0, Hs00420895) was used for normalization. To confirm the expression patterns, real-time PCR conditions included an initial step at 50 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 95 °C for 3 s, and extension at 60 °C for 20 s. Quantification of amplified genes was performed using QuantStudio™ Design & Analysis Software with the ddCt method.

Western blot analysis

NHEK cells were seeded in a 6-well plate at a concentration of 1 × 105 cells/well and cultured for 24 h. Subsequently, they were treated with 10 μg/mL of R. rugosa extract, 10 ng/mL of human recombinant interleukin-4 (rhIL-4, PeproTech, USA), and human recombinant interleukin-13 (rhIL-13, PeproTech, USA), and cultured for an additional 96 h. At the end of experiment, the cells were harvested using a scraper, and proteins were lysed in RIPA lysis buffer (ThermoFisher Scientific, USA) containing protease inhibitor (Gen-Depot, USA) and phosphatase inhibitor (Gen-Depot, USA). Protein quantification was performed using the BCA assay (ThermoFisher Scientific, USA). Subsequently, 20 μg of protein was subjected to electrophoresis on an 8% SDS-PAGE gel. Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Cytiva, Korea) at 30 V, 300 mA, 40 W for 1 h. After transfer, the membrane was blocked in 5% Non-Fat Dried Milk (NFDM, Cellconic, USA) containing Tris-Buffered Saline with Tween (TBS-T, Gen-Depot, USA) for 1 h. Following blocking, the membrane was incubated with the primary antibody (Filaggrin, Santa Cruz, USA) overnight at 4 ℃. After washing with TBS-T, the membrane was incubated with the secondary antibody (goat anti-mouse IgG (H + L), Invitrogen, USA) for 1 h. Protein bands were visualized using the WestGlow Femto ECL chemiluminescent substrate (Biomax, Korea) and observed with an imaging device (iBright CL 750, Invitrogen, USA).

STAT6 phosphorylation

NHEKs cells were seeded at a density of 1 × 105 cells/well in a 6-well plate and treated with 10 ng/ml of rhIL4 and rhIL13 to induce STAT6 phosphorylation. To confirm the inhibitory effect on STAT6 phosphorylation, R. rugosa extract (10 μg/ml) was co-treated with rhIL4/13 and incubated for 30 min (Park et al. 2023). Subsequently, the process followed the same procedure as the Western blot experiment. The primary antibodies used in the experiment were anti-Total-STAT6 (#5397S, Cell Signaling Technology) and anti-phosphorylated-STAT6-Y641 (#558,241, BD Bioscience).

Statistics

The experimental results are presented as the mean ± standard error of the mean (SEM), with each experiment conducted at least three times. Statistical analysis was performed using Student’s t-test to compare two groups. A p-value of less than 0.05 was considered statistically significant.

Results

Cytotoxicity of R. rugosa extract

To determine appropriate dosage levels and preventing unintended cell toxicity, the cell viability of the extract was assessed through a CCK-8 assay (Fig. 1A). NHEK cells were treated with R. rugosa extract at various concentrations (0.1, 0.5, 1, 5, 10 μg/ml) and cultured for 48 h to assess cell viability, with untreated cells serving as the control group. The results revealed that the extract did not significantly affect cell survival rates at all tested concentrations. Additionally, keratinocytes treated with the extract exhibits a flattened and polygonal morphology with increased cell size, comparing to the undifferentiated keratinocytes with a more cuboidal shape (Fig. 1B). This morphological change may reflect the transition from a proliferative to a differentiated state in response to R. rugosa extract.

Fig. 1
figure 1

Viability and morphology of NHEK cells treated with different concentrations of R. rugosa extract. A. CCK-8 assay results at different concentrations of the extract. Values are expressed as mean ± SEM (N = 4). All concentrations are not statistically significant from the control (p > 0.05). B. Keratinocytes morphology treated with the extract (10 μm). Scale bar: 125 μm. n.s. means not statistically significant. DMSO; Dimethyl sulfoxide as a vehicle control

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R. rugosa induces keratinocyte differentiation

To examine the induction of effect of keratinocyte differentiation by R. rugosa extract, the expression of differentiation markers was assessed using qRT-PCR. Keratinocytes were treated with the extract at 20 ug/ml and incubated for 72 h, with untreated cells serving as the control group (Fig. 2). The results indicated a significant increase in the expression of early differentiation markers, KRT1 and KRT10, by about eightfold and 35-fold, respectively in response to the extract. R. rugosa extract markedly enhanced the expression of key components involved in skin barrier function, FLG, LOR and IVL. Additionally, the extract increased the expression of CLDN1, a gene regulating tight junctions that control epithelial permeability (Fig. 2). These findings suggest that R. rugosa extract possesses activity that promotes keratinocyte differentiation and contributes to the enhancement of skin barrier function.

Fig. 2
figure 2

Relative mRNA expression of NHEK cells treated with R. rugosa extract. Values are expressed as mean ± SEM (N = 4). Statistically significant from the control (*p < 0.05, **p < 0.01). Relative mRNA expression was normalized with RPLP0. DMSO is used as a vehicle control

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Induction of filaggrin expression by R. rugosa extract in an in vitro AD model

Through Western blot analysis, we investigated whether the expression of filaggrin protein could be restored by R. rugosa extract (Fig. 3A). To mimic AD condition, FLG expression in keratinocytes was inhibited by 10 ng/mL IL-4 and IL-13 and added with the extract at a concentration of 10 μg/mL for 96 h. Untreated cells served as the negative control group. The results revealed a substantial increase in KRT1, KRT10 and FLG protein expression in cells treated with R. rugosa extract (10 μg/ml) compared to the negative control group. Cells treated with IL-4 and IL-13 showed a decrease in filaggrin protein expression. Furthermore, in cells treated with both IL-4, IL-13, and R. rugosa extract, filaggrin protein expression was higher compared to cells treated only with IL-4 and IL-13. This confirms that R. rugosa extract restores the reduced expression of KRT1, KRT10 and FLG protein inhibited by IL-4/IL-13.

Fig. 3
figure 3

Western blot analysis for protein expression in NHEK cells treated with R. rugosa extract. A. R. rugosa extract induces KRT1 and KRT10 expression and even restores them in in vitro atopic dermatitis model. FLG expression is remarkably restored in in vitro atopic dermatitis model. (N = 3). B. R. rugosa extract inhibits phosphorylation of STAT6 induced by IL4 and IL13 in NHEK. DMSO is used as a vehicle control

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R. rugosa extract inhibits STAT6 phosphorylation induced by IL4/IL13

To explore the potential regulatory effects of the extract on the signaling pathways, we examined its modulation of STAT6 phosphorylation of STAT6 as a downstream signaling cascade of IL4 and IL13. While co-treatment with IL4 and IL13 induced STAT6 phosphorylation compared to the control without treatment, the extract exhibited a significant inhibition on STAT6 phosphorylation in keratinocytes treated with IL4 and IL13 (Fig. 3B). Thus, these results indicate that R. rugosa extract exerts its functions by regulating IL4/Il13-mediated STAT6 signaling pathway and may hold therapeutic potential for AD.

Discussion

The scientific significance of exploring the biological effects of R. rugosa extends across multiple areas of dermatological research. The plant’s rich phytochemical profile, including polyphenols, flavonoids, and terpenoids, has been associated with potent antioxidant activities. Antioxidants play a pivotal role in neutralizing reactive oxygen species, thereby reducing oxidative stress. Like previous studies, our lab confirmed that the robust free radical scavenging activities demonstrated by R. rugosa extract in both DPPH and ABTS assays underscore its potential as a powerful antioxidant. These findings suggest that the extract may effectively neutralize different types of free radicals, indicating its broad-spectrum antioxidant capacity. As shown in accumulated literatures, quercetin, kaempferol, and ellagic acid could be possible candidate molecules. This attribute is particularly valuable in potential applications in various industries, such as cosmetics and pharmaceuticals, where protection against oxidative stress is crucial for product stability and potential health benefits.

Furthermore, the low cytotoxicity of R. rugosa extract is a promising indicator of its safety for cellular environments. The lack of significant impact on cell viability across the tested concentrations suggests that the extract could be applied at various doses without causing unexpected side effects. This is essential for the development of products for human clinicals, which reinforces the potential of R. rugosa extract as a valuable ingredient in skincare or therapeutic formulations.

The ability of the extract to induce keratinocyte differentiation and enhance skin barrier functions is another noteworthy. AD is often characterized by a disrupted skin barrier, which leads to increased permeability and susceptibility to foreign harmful materials from the outside of body. The positive influence of R. rugosa extract on markers of differentiation and skin barrier function suggests its potential in fortifying the skin’s natural defenses and supporting barrier repair in AD patients. Since FLG deficiency is a common clinical and pathological feature in AD patients, the effective restoration of FLG expression in an in vitro AD model is a promising finding. Moreover, the extract’s inhibition of STAT6 phosphorylation induced by IL4 and IL13 provides mechanistic insights into its anti-inflammatory properties. By interfering with signaling pathways associated with Th2-mediated cytokines, R. rugosa extract may contribute to mitigating inflammatory responses in the skin.

Despite our new findings, further investigations could enhance our understanding of how R. rugosa extract exert its effects at the cellular and molecular levels. First of all, analyzing the major ingredients of R. rugosa extract is a crucial aspect that can contribute significantly to understanding its therapeutic potential and optimizing its application in AD. By employing omics techniques such as proteomics or RNA sequencing, we may comprehensively analyze large scaled changes in protein or gene expression. Since R. rugosa extract inhibits STAT6 phosphorylation, in-depth analysis of signaling pathways including JAK-STAT, MAPK, or NF-kB could provide insights into the extract’s biological activities.

Conclusions

Collectively, our study underscores the promising potential of R. rugosa extract as a natural material for treating AD. The biological properties of R. rugosa with antioxidant, anti-inflammatory, and skin barrier enhancing effects position it as a promising candidate for the development of therapeutics targeting AD. Future studies including clinical trials and mechanistic investigations will provide a pivotal link to the translational potential of this natural extract in dermatological applications.