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Article

Retinol and Hydroxyasiaticoside Synergistically Relieve Histamine-Induced Atopic Dermatitis Activity by Repressing TRPV1, L1R1, and CD130 Targets

by
Qin Liu
1,
Peng Shu
1,2,3,4,5,
Qingle Song
1,
Zhenhai Huang
6,
Jiyu Weng
6,
Lanyue Zhang
6,
Yuan Wang
1,* and
Qingqing Liu
7,*
1
HBN Research Institute and Biological Laboratory, Shenzhen Hujia Technology Co., Ltd., Shenzhen 518000, China
2
State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Urumqi 830000, China
3
CAS Key Laboratory of Chemistry of Plant Resources in Arid Regions, Urumqi 830000, China
4
Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830000, China
5
University of Chinese Academy of Sciences, No. 19 (A) Yuquan Road, Shijingshan District, Beijing 100049, China
6
School of Biomedical and Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of Plant Resources Biorefinery, Guangdong University of Technology, Guangzhou 510006, China
7
School of Public Health, Southwest Medical University, Luzhou 646000, China
*
Authors to whom correspondence should be addressed.
Cosmetics 2024, 11(6), 203; https://doi.org/10.3390/cosmetics11060203
Submission received: 15 October 2024 / Revised: 13 November 2024 / Accepted: 21 November 2024 / Published: 25 November 2024
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Abstract

:
Background: Retinol, an important bioactive substance with multiple physiological functions such as promoting collagen synthesis, inhibiting matrix metalloproteinase activity, alleviating oxidative stress, regulating gene expression, and promoting epidermal cell proliferation, has a significant effect on skin damage recovery. Hydroxyasiaticoside, a triterpenoid saponin derived from Centella asiatica (L.) Urb., is closely related to the secretion of collagen types I and III, and possesses multiple biological activities, including moisturizing, antioxidants, anti-apoptosis, neuroprotection, anti-inflammation, and the promotion of wound healing. It plays a particularly prominent role in reducing oxidative stress in wounds and inducing vasodilatation. Objective: The aim of this study was to investigate the therapeutic efficacy of retinol combined with hydroxyasiaticoside in histamine-induced atopic dermatitis. Materials and Methods: The experiment was carried out using three different concentrations of a retinol and hydroxyasiaticoside mixed solution: low, medium, and high concentrations. After inducing atopic dermatitis in mice through histamine administration, these solutions were applied to the skin surface of the mice, and a comparative analysis was conducted with both the control group and the model group. The effect of combination therapy on atopic dermatitis was evaluated through histopathology, immunohistochemistry, and transcriptomic analysis. Results: The combination of retinol and hydroxyasiaticoside significantly attenuated histamine-induced scratching behaviors, alleviated the phenomenon of epidermal hyperplasia, and effectively reduced the proliferation, infiltration, and degranulation of mast cells. In addition, the combination inhibited the expression of relevant pro-inflammatory cytokines. Quantitative RNA-seq analysis revealed that the gene expression patterns were similar in different concentration groups. However, the medium dose group may be able to regulate skin inflammation by regulating upstream genes to inhibit autophagy-related pathways. Further GO analysis revealed that the low-dose group mainly affected metabolism-related genes, the medium-dose group affected more genes related to body systems, and the high-dose group was dominated by genes related to human diseases.

1. Introduction

Atopic dermatitis (AD), also known as atopic eczema, is a common inflammatory skin disease. Clinically, it is characterized by intense itching, recurrent scratching leading to eczematous skin lesions, and exacerbation of skin inflammation [1]. AD affects approximately 20% of children and around 3% of adults. Recent studies indicate that its prevalence is on the rise, particularly in developing countries [2]. The pathogenesis of AD is complex, and influenced by various factors such as genetics, immunity, and the environment. Hanifin and Rajka suggest that AD is not just a series of skin symptoms but also a response pattern to allergens, with different stimuli leading to diverse clinical manifestations [3]. Research has shown that inflammatory factors such as TNF-α, IFN-γ, IL-4, IL-13, IL-31, IL-33, IL-23, IL-22, IL-17, and mast cells are involved in the pathogenesis of AD [4,5]. Furthermore, mutations or deficiencies in epidermal barrier proteins like filaggrin can impair barrier function, promoting inflammation and T-cell infiltration [3]. During the progression of AD, various immune reactions are triggered, ultimately leading to the production of immunoglobulin E (IgE), which in turn triggers IgE-mediated allergic reactions, exacerbating AD symptoms [6].
Histamine (2-(1H-imidazol-5-yl)ethanamine) is a significant chemical mediator, with research indicating its ability to promote inflammatory responses and induce the infiltration of inflammatory cells such as mast cells and eosinophils. Furthermore, histamine can elevate Th2 cell cytokines and serum IgE levels, while also inducing scratching behavior. These actions suggest that histamine may be associated with the development of chronic inflammatory skin diseases, such as atopic dermatitis (AD) [7]. Currently, the pharmacotherapy for AD mainly includes topical corticosteroids, calcineurin inhibitors, oral steroids, and immunosuppressants. These medications can alleviate acute inflammatory symptoms, but prolonged use may lead to side effects such as skin atrophy, pathogenic infections, and immunosuppression [8,9]. Therefore, there is a growing demand for safe and effective alternatives to steroids and antihistamines [10]. In response to this demand, researchers are striving to discover new therapeutic drugs, particularly natural substance extracts. These natural substances may provide greater safety and enhance therapeutic efficacy and, thus, may better meet the needs of patients seeking treatment for AD.
Histamine-induced AD is a disease characterized by the production of proinflammatory cytokines such as TRPV1 (Transient Receptor Potential Cation Channel Subfamily V Member 1), IL1R1, and CD130 in both the epidermis and dermis. TRPV1, a protein-coding gene, transmits information about noxious stimuli to the central nervous system, inducing a burning pain sensation, and has been detected in non-neuronal cells like skin keratinocytes and mast cells [11,12]. IL1R1 belongs to the interleukin 1 receptor family of cytokine receptors and is a receptor for IL-1α, IL-1β, and IL-1RA, which are crucial mediators of cytokine-induced immune and inflammatory responses [13]. CD130, mainly involved in IL-6 family cytokines signaling, forms a functionally complete IL-6 receptor complex with the receptor α subunit upon binding, activating downstream signaling pathways such as the JAK/STAT, MAPK, and PI3K/Akt pathways, with JAK/STAT being the most typical and important [14,15].
Vitamin A (VA) and its derivatives play a crucial role in the maturation and function of the immune system [16]. Vitamin A is a collective term referring to a group of unsaturated organic compounds, including retinol, retinal, and retinoic acid [17]. Vitamin A plays a pivotal role in the human body, intricately linked to physiological functions such as vision, the immune system, cell growth, gene transcription, and protein formation [16,18]. Among these, retinol is a form of vitamin A that has been proven to possess a multitude of physiological functions. Retinol can stimulate the synthesis of collagen, inhibit the activity of matrix metalloproteinases (MMPs), reduce oxidative stress, and regulate gene expression [19]. The application of retinol on the skin can increase the content of mature collagen in the skin, promote the development of dermal blood vessels, decrease the levels of matrix metalloproteinases, and stimulate the growth of fibroblasts [20]. Some researchers have found that in studying the anti-aging effects of retinol, it can enhance the expression of the CRABP2 and HBEGF genes, thereby promoting the proliferation of epidermal cells, restoring the skin’s ability to respond to environmental damage, reducing pigmentation, and ultimately ameliorating signs of skin aging [21].
Centella asiatica (L.) Urb., a perennial herb of the Apiaceae (Umbelliferae) family, is predominantly found in tropical and subtropical regions such as China, South Africa, Australia, Malaysia, and the United States. Its medicinal significance has been well-documented within the Apiaceae (Umbelliferae) family [22]. Centella asiatica (L.) Urb. is rich in a diverse array of phytochemicals, including glycosides, triterpene saponins, alkaloids, monoterpenes, diterpenes, sesquiterpenes, tetraterpenes, polyacetylenes, and phenolic compounds [23]. Triterpenoid saponins, recognized for their pharmacological potency, stand out as the most efficacious constituents [23]. Triterpenoid saponins in Centella asiatica (L.) Urb. mainly include asiaticoside, hydroxyasiaticoside and cysticercoside [24]. Notably, hydroxyasiaticoside is intricately linked to the synthesis of type I and Type III collagen [24]. This compound exhibits a range of beneficial properties, including moisturization, antioxidant effects, anti-apoptotic activity, neuroprotection, anti-inflammatory properties, promotion of wound healing, reduction of oxidative stress in wounds, and induction of vasodilation [25,26]. Hydroxyasiaticoside has been scientifically demonstrated to expedite the healing of burn wounds [25], address acne concerns [27], manage conditions like atopic dermatitis [28] and vitiligo [29], as well as treat various other ailments.
In recent years, remarkable progress has been made in research on the application of hydroxyasiaticoside in dermatology. Scientific studies have demonstrated that hydroxyasiaticoside can shorten wound healing time without causing side effects [30,31], providing new ideas and methods for stimulating wound healing using natural products. Furthermore, hydroxyasiaticoside has also shown significant effects in addressing acne problems, with its anti-inflammatory and antibacterial properties making it a potential drug for acne treatment [32,33]. In the treatment of atopic dermatitis, the moisturizing and anti-inflammatory effects of hydroxyasiaticoside can significantly alleviate patients’ symptoms and improve their quality of life [34]. It is also noteworthy that hydroxyasiaticoside has been found to have a therapeutic effect on vitiligo, aiding in the restoration of normal skin pigmentation by promoting the proliferation and differentiation of melanocytes [29]. Aside from these conditions, hydroxyasiaticoside has been extensively studied for the treatment of various other skin diseases, such as eczema and psoriasis, demonstrating its broad application prospects in dermatology. Thus, as a natural compound with multiple pharmacological activities, hydroxyasiaticoside continues to be deeply researched in dermatology, offering new options and hope for the treatment of skin diseases.
In our study, we observed elevated expression of TRPV1, IL1R1, and CD130 in the skin of mice with histamine-induced AD. Notably, the combination of retinol and hydroxyasiaticoside significantly inhibited the expression of these cytokines, with the inhibitory effect increasing with concentration. Given that both retinol and hydroxyasiaticoside possess antioxidant, collagen-promoting, and anti-inflammatory physiological functions, and considering that studies have shown hydroxyasiaticoside’s inhibitory effect in treating AD induced by both phthalic anhydride and 2,4-dinitrochlorobenzene [28,34], we further investigated their potential beneficial impact on histamine-induced AD using a mice model. Our research aimed not only to explore the underlying mechanisms by which these compounds may influence signaling pathways such as JAK/STAT and PI3K/Akt, but also to investigate any potential synergistic effects between retinol and hydroxyasiaticoside.

2. Materials and Methods

2.1. Principal Reagents

Retinol and hydroxyasiaticoside were purchased from Sigma-Aldrich (Shanghai, China). Hematoxylin and Eosin staining (H&E), Masson Trichrome staining, and Toluidine Blue staining (TB) kits were provided by Servicebio (Wuhan, China). Anti-FLG rabbit polyclonal antibody (pAb), anti-nerve growth factor (NGF) rabbit pAb, anti-IL-1β rabbit pAb, horseradish peroxidase-conjugated goat anti-rabbit IgG, and 3,3′-diaminobenzidine were from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China), and TRIzol reagent was purchased from Thermo Fisher Scientific (Shanghai, China)

2.2. Laboratory Animals

For the purposes of this experiment, female KM mice, aged four weeks, with an average body weight exceeding 15 g, were selected. These mice were procured from the Guangdong Experimental Animal Center, where they were assigned the Experimental Animal License number SCXK/2013-0002. Prior to the commencement of the experimental procedures, the mice underwent an adaptive feeding period in a controlled environment that maintained an indoor temperature of 25 ± 2 °C and a relative humidity ranging from 55% to 65%. Furthermore, the animals were subjected to a 12-h light-dark cycle to mimic natural diurnal patterns. During this period, the mice can eat and drink autonomously to ensure their wellbeing and physiological stability.

2.2.1. Experimental Animal Grouping

After a seven-day period of adaptive feeding, the mice were randomly and evenly divided into five groups, each containing four animals (n = 5). During the grouping process, we referenced relevant literature to establish the concentrations of retinol and asiaticoside [35,36]. The specific grouping details are as follows: a blank control group, a histamine group (HA), a retinol-hydroxyasiaticoside group with concentrations of 0.025% and 0.1% (R&H-L group), a retinol-hydroxyasiaticoside group with concentrations of 0.05% and 0.25% (R&H-M group), and a retinol-hydroxyasiaticoside group with concentrations of 0.1% and 0.5% (R&H-H group). The randomization process was conducted to ensure that the groups were comparable in terms of baseline characteristics, thus, minimizing any potential biases in the experimental outcomes.

2.2.2. Animal Handling

On the day before the commencement of the experiment, the hair on the dorsal skin of the mice was removed using a depilatory cream. The entire experiment comprised a seven-day drug administration phase followed by a seven-day acute sensitization phase. For the various R&H groups, retinol and hydroxyasiaticoside were dissolved in propylene glycol to prepare solutions with concentrations of 0.025 + 0.11%, 0.05 + 0.25%, and 0.1 + 0.5%. These solutions, in volumes of 200 µL each, were applied to the shaved areas on the backs of the mice. For the control and HA groups, 200 µL of propylene glycol solution was administered to the shaved areas. All treatments were administered once daily for seven consecutive days.
Subsequently, an AD mice model was established through histamine induction. On the seventh day of drug administration, the R&H and HA groups received an intraperitoneal injection of 100 uL of histamine saline solution (1 mg/mL in normal saline) 30 min after drug administration, while the control group received 100 uL of normal saline. The mice were video-recorded for 30 min to observe the frequency of scratching events. Following this, the mice were euthanized using cervical dislocation, and the dorsal skin tissues were fixed in paraformaldehyde for subsequent analysis.

2.2.3. Scratching Behavior Analysis

Itching is a hallmark symptom of AD [3]. After establishing the AD mice model through histamine induction, mice were video-recorded for 30 min to quantify the number of scratching events. Typically, deliberate scratching of the treated skin using the forepaws or hind paws was considered a single scratching event [37].

2.3. Hematoxylin and Eosin Staining

Mice skin tissue sections were prepared from paraffin blocks and thoroughly immersed in hematoxylin solution for staining. Following precise differentiation procedures, the sections were further stained with eosin. Under a microscope, the cellular morphology and tissue structure were carefully observed to record and analyze various phenomena and data, providing insights into the relevant characteristics and changes in the mice’s skin tissue.

2.4. Masson Staining

After pre-treatment, mice skin tissue sections were immersed in a series of reagents, including hematoxylin for staining cell nuclei. After differentiation, bluing, and subsequent staining with ponceau fuchsin acid fuchsin, the sections were treated with phosphomolybdic acid solution and counterstained with aniline blue. Microscopic examination allowed for a detailed observation of the morphology, distribution, and staining characteristics of collagen fibers and other structures in the tissue. The observed details were accurately recorded and analyzed to assess the status and changes of relevant components in the mice’s skin tissue.

2.5. Toluidine Blue Staining

The mice’s skin tissue sections were first subjected to deparaffinization and hydration steps. The skin tissue sections were rinsed with water, then soaked in toluidine blue solution for 2 to 5 min, differentiated with 0.1% glacial acetic acid, dried, and transparently affixed. Under a microscope, the stained tissue structure was carefully observed, focusing on cellular morphology, cytoplasm, and nuclear staining characteristics. The observations were recorded in detail for subsequent in-depth analysis and research.

2.6. Immunohistochemical Analysis

After dewaxing and hydrating paraffin-embedded skin tissue sections with gradient ethanol, we successfully carried out antigen extraction by microwave heating in sodium citrate buffer (pH 6.0). After cooling the sections, we dripped them with normal goat serum blocking solution and incubated them at room temperature for 20 min. After removing the blocking solution, we dropwise added rabbit polyclonal antibodies against NGF and IL-1β, which we had properly diluted, and incubated the sections overnight at 4 °C or for 2 h at room temperature. Subsequently, we washed the sections with PBS and incubated them with HRP-labeled goat anti-rabbit IgG for 30 min at room temperature. Finally, we stained the sections with 3,3′-diaminobenzidine (DAB) and hematoxylin [38].
The sealed sections were placed under a microscope to observe the morphology, structure, and staining intensity and distribution of the cells. The Integral Optical Density (IOD) values of TRPV1, IL1R1, and CD130 were measured using Image-Pro Plus 6.0 software. Finally, GraphPad Prism was used to plot the collected data.

2.7. Transcriptome Analysis

Total RNA was extracted from samples using TRIzol reagent and verified for integrity and concentration through 1.0% agarose gel electrophoresis and Bioanalyzer analysis. Subsequently, poly(A)+ mRNA was isolated using a magnetic bead enrichment method, followed by reverse transcription to synthesize cDNA. The cDNA was then fragmented, adapters were added, and a sequencing library was constructed through PCR amplification. After the library was qualified by Qubit quantification and Bioanalyzer fragment analysis, sequencing was performed using the Illumina platform to obtain raw data. FastQC was employed for quality control to remove low-quality and adapter-contaminated sequences, while STAR v2.7.6a Software was used for precise alignment of high-quality reads to the reference genome. Finally, tools such as FeatureCounts were utilized to quantify gene expression, and differential expression analysis, functional annotation, and pathway enrichment analysis were conducted to comprehensively characterize the transcriptome features.

2.8. Metabolomics Analysis

Based on previous studies [39], we made appropriate modifications to the metabolomics methodology. An appropriate amount of AD mouse skin tissue homogenate was mixed with chromatography-grade methanol, at a ratio of 0.1 g skin tissue homogenate to 1 mL methanol. After mixing, the samples were left at room temperature for 10 min, then moved to 4 °C and centrifuged at 13,000 rpm for 15 min. The resulting supernatant was purified by a 0.22 µm filter, and finally analyzed by ultra-high performance liquid chromatogre-mass spectrometry (UHPLC-MS).
The chromatographic conditions were as follows: Hydrophilic Interaction Liquid Chromatography (HILIC) column was used, the column temperature was set at 25 °C, the flow rate was controlled to 0.5 mL per minute, and the sample size was ensured to be 2 µL per sample. The mobile phase consisted of two components: Mobile phase A, comprising water with the addition of 25 mmol/L ammonium acetate and 25 mmol/L ammonia water; and Mobile phase B, which was acetonitrile. The gradient elution program was meticulously designed, commencing with an initial condition of 95% B for 0 to 0.5 min, followed by a linear decrease in B from 95% to 65% over the next 6.5 min (0.5 to 7 min). Subsequently, B decreased further from 65% to 40% over the next minute (7 to 8 min), maintaining a constant 40% B for the next minute (8 to 9 min). Immediately thereafter, B increased rapidly from 40% to 95% within 0.1 min (9 to 9.1 min), and finally, the elution was concluded with a constant 95% B for the remaining 2.9 min (9.1 to 12 min).
Parameters and Settings of mass spectrometry: The Electrospray Ionization (ESI) source supports both positive and negative ionization modes. The mass charge ratio (m/z) is scanned in the range of 60–1000 for Time-of-Flight (TOF) scans, providing a broad spectrum of molecular weights for detection. For ion scans, the m/z range is extended to 25–1000, enhancing the sensitivity to smaller fragments and ions; In the implementation of secondary mass spectrometry, we use a data-oriented acquisition strategy coupled with a high-sensitivity operation mode. In this mode, the clustering potential of both positive and negative ions is precisely set at ±60 V, while the collision energy is flexibly adjusted in the range of (35 ± 15) eV to ensure the accuracy and efficiency of the analysis.
Utilizing metabo analysis, a non-targeted metabolomics study was conducted on the metabolites in AD mice skin under the influence of different concentrations of retinol and asiaticoside. Finally, volcano maps are used to visualize p-values and folding change (FC) values for screening differential metabolites. The projected importance analysis value (VIP) is commonly used in multivariate statistical methods such as partial least squares regression to evaluate the importance of variables.

2.9. Statistical Analysis

The experimental data were analyzed using GraphPad Prism 8.3 software, and the results were expressed as the mean ± standard deviation (mean  ±  SD). A one-way ANOVA method was employed, combined with the Dunnett-t test, to assess the statistical differences between groups. When the p < 0.05, the difference between groups was considered statistically significant.

3. Results

3.1. Retinol and Hydroxyasiaticoside Suppress Skin Hyperplasia in AD Mice

In Figure 1, we show the skin repair process of mice in various groups post-treatment and assign scores based on their severity. As can be observed in Figure 2, the skin structure of the mice in the control group remains normal, exhibiting a moderate thickness of the epidermis. However, the stained sections of the HA group reveal severe epidermal hyperplasia in the mice’s skin subsequent to histamine treatment, leading to the disruption of the dermisepidermis junction and a disordered arrangement of the dermal tissue. This finding aligns with the skin-thickening characteristics induced by histamine, as reported in the study by Jin et al. [5]. In comparison to the HA group, the R&H groups administered at three different concentrations (L, M, H) demonstrate varying degrees of improvement, characterized by a thinner epidermis and a more organized arrangement of dermal fibers. Notably, the skin pathological features of the mice in the R&H-H high-dose group show marked improvement, with no evidence of inflammatory cell infiltration. These results indicate that the AD mice model has been successfully established in this ongoing experiment. Additionally, they suggest that the combined administration of retinol and hydroxyasiaticoside holds the potential to effectively alleviate atopic dermatitis triggered by histamine.

3.2. Retinol and Hydroxyasiaticoside Enhance Collagen Fiber Production in AD Mice Skin

As can be seen from the masson staining results in Figure 3, the blue collagen fibers in the skin of mice in the control group were tightly arranged in an orderly pattern, with a wavy dermoepidermal junction and moderate thicknesses of the epidermis and dermis. In comparison to the control group, the collagen fibers in the dermal layer of mice in the HA group were significantly reduced, arranged in a disorganized manner, and both the epidermis and dermis were thicker. When compared to the HA group, the dermal layers of the skin tissue in the R&H-L, R&H-M, and R&H-H groups of mice all showed some improvement in the morphology of collagen fibers, with a certain degree of increase in collagen fiber content. The fibers were more uniformly distributed, and the improvement effect showed a positive correlation with concentration.

3.3. Retinol and Hydroxyasiaticoside Reduce Skin Mast Cell Infiltration in AD Mice

In the present study, mast cell infiltration into the dermis was induced in the skin of AD mice using histamine, as shown in Figure 4. The HA group showed an increase in the total number of mast cells and degranulated cells compared with the control group. When AD mice were treated with retinol and hydroxyasiaticoside, it was observed that the infiltration of mast cells in the dermis layer of the skin tissue of the mice was significantly inhibited and the number of mast cells was reduced. The effect was more pronounced as the concentration of the mixture of retinol and hydroxyasiaticoside was increased; even the infiltration of mast cells in the dermis layer in the high-dose R&H group was lower than that in the control group, which had a better anti-inflammation effect.

3.4. Retinol and Hydroxyasiaticoside Regulate TRPV1, IL1R1, and CD130 Levels

Compared with the control group, the expression levels of TRPV1, IL1R1, and CD130 in the HA group were significantly increased, as shown in Figure 5. The expression of TRPV1, IL1R1, and CD130 was significantly suppressed after the administration of a mixed solution of retinol and hydroxyasiaticoside. It indicates that the mixed solution of retinol and hydroxyasiaticoside can achieve the purpose of soothing histamine-induced skin injury in AD mice by inhibiting the expression of pro-inflammatory cytokines. Moreover, the higher the concentration of the administered drug, the more significant the soothing effect is, within the maximum concentration of the administered drug.

3.5. Gene Expression Pattern Analysis and Differential Gene Identification in R&H-Treated Animals Using RNA-Seq

The degree of difference among each sample was analyzed by principal component analysis (PCA) in Figure 6A, where the contribution of the x-axis was 26.89% and the y-axis was 12.98%. The comparison of the degree of sample aggregation was primarily conducted along the x-axis. It can be observed that the samples within each group were relatively clustered. The closer the correlation coefficient is to 1 in the Pearson correlation coefficient plot (Figure 6B), the higher the similarity of the expression patterns among the samples. The similarity of the samples within each group was good when |R| > 0.8. The similarity and reproducibility among the respective samples of the R&H group and the control group were good, and the best similarity was found among the samples in the R&H group. Figure 6C presents an inter-sample Venn analysis for each group, revealing 49 shared genes in the experimental group and 14,314 shared genes between the R&H group and the control group, indicating that each group represented the same disease model. Figure 6D performs Venn analysis on the differential gene set, showing that the total number of genes in R&H vs. HA was 47, accounting for 17.40% of all groups, indicating that the gene variability in this differential gene set was high.
The expression pattern clustering analysis of the genes in the selected gene set from the RNA-seq clustering heat map revealed that red represented higher gene expression in a sample, while blue represented lower expression. The left side depicted the gene clustering dendrogram, the right side listed the gene names, and the top side displayed the dendrogram of the sample clustering. The closer the two gene branches were, the more similar their expression patterns were. As depicted in Figure 6E, most genes within each R&H concentration group exhibited significant differences in expression compared to the control group. Specifically, genes that were highly expressed in the R&H groups displayed lower expression in the control group. Furthermore, the gene expression patterns among samples within different R&H concentration groups were similar, suggesting that genes with similar expression patterns often have related functions. The genes that were significantly enriched in the clustered heat map and displayed differences were Vcam1, Birc3, Angpt2, Lgf2 (likely a typo, perhaps meant to be Lgf2 or a similar gene name), Camk2d, Tlr2, Zap70, Pik3r5, Sfrp4, Reln, Mras, Pik3cd, Fgf7, F2r, Cd40, and Cacna2d2. Many of these genes were associated with angiogenesis and fibroblast repair, among which Cd40 was considered an important protein for alleviating skin inflammation and promoting epidermal cell proliferation, playing a unique role in skin injury repair [40].
The DEGseq2 difference analysis tool was used to identify differential genes (DEGs), and the adjusted p value < 0.05 and |logFC| ≥ 1.2 were used as the cutoff criteria. The genes with p values in the top 10 were labeled (Figure 7). A total of 1970 DEGs were detected in the R&H-L group compared to the model group, among which 1069 genes were up-regulated and 901 genes were down-regulated. The R&H-M group compared to the model group detected a total of 961 DEGs, of which 208 were up-regulated genes and 753 were down-regulated genes. Compared with the model group, a total of 1664 deg were detected in the R&H-H group, including 589 up-regulated genes and 1075 down-regulated genes. Compared with the model group, the number of differentially expressed genes in the R&H-L group was the highest, indicating that the genetic status of this sample was complex. The three groups had more significantly up-regulated genes of kinin-releasing enzyme (KLK13) and calcium-binding protein A (S100A). Kinin-releasing enzymes are essential epidermal messengers that regulate the skin’s micro-environment during homeostasis, repair, and disease in vivo. Studies have shown the elevated expression of several KLKs in the stratum corneum of AD mice [41], where over-expression of KLK7 leads to symptoms such as skin thickening, hyperkeratosis, inflammatory skin reactions, and itching, which are similar to the clinical manifestations of chronic AD [42]. It has been reported that certain S100 proteins may ensure continuous epidermal renewal and support wound healing, while other S100 proteins are involved in epidermal differentiation or have a protective role [43].
In Table 1, all genes associated with AD skin with high significance and their regulation are listed (Reference site: Database of genes related to the diseases Keratosis, Seborrhea, and Hyperhidrosis, accessible at https://ctdbase.org (accessed on 27 September 2024)). (p < 0.05).

3.6. Retinol and Hydroxyasiaticoside Affect Levels of Differentially Expressed Metabolites (DEMs)

To identify differentially expressed metabolites (DEMs) across different comparison groups, we conducted pairwise comparisons. Compared to the control group, changes in metabolite expression were observed in the HA group, with 127 metabolites up-regulated and 225 down-regulated. When compared to the HA group, the R&H-L group showed an increase to 231 up-regulated metabolites and 387 down-regulated metabolites. Significant alterations in metabolite expression were noted when the R&H-M group was compared to the HA group, with 553 metabolites up-regulated and 258 down-regulated. Similarly, in comparison to the HA group, the R&H-H group exhibited 246 metabolites up-regulated and 506 down-regulated. As shown in Figure 8, this indicated that the medium dose of R&H affected more metabolites to regulate skin inflammation. Then, we collected the DEMs from the R&H-L, R&H-M, and R&H-H dose groups compared to the HA group to remove duplicated items, and obtained a total of 419 DEMs. These were analyzed by Venn analysis along with 358 DEMs from the control and HA groups, resulting in 99 shared DEMs, as shown in Figure 9. Subsequently, 33 endogenous metabolites were screened using metabo analysis, as depicted in Figure 10, which was presented in the form of a clustered heat map. From this heat map, the expression of the shared DEMs can be clearly observed. The red color indicates that the metabolite had a higher expression in that sample, while the blue color indicates that the metabolite had a lower expression in that sample.

3.7. Metabolic Pathway Analysis of the Skin-Soothing Efficacy of Retinol and Hydroxyasiaticoside on AD Mice

We uploaded 33 DEMs to MetaboAnalyst and analyzed them to identify 12 metabolic pathways involved in the alleviation of AD skin by retinol and hydroxyasiaticoside, as shown in Figure 11. Among these, the top five metabolic pathways were purine metabolism, tricarboxylic acid cycle, pyruvate metabolism, glycolysis, and alanine, aspartate, and glutamate metabolism.
Finally, based on PCA, the VIP values, p values, and FC values were visualized using volcano plots for differential metabolite screening (Figure 12). In the volcano plot, each point represents a metabolite, and the size of the point is proportional to the VIP value. The horizontal coordinate represents the FC (taking the logarithm of base 2), while the vertical coordinate represents the p value (taking the negative of the logarithm of base 10). The volcano plot identifies the differential metabolite screening results using dot color, with the condition that VIP > 1 and p < 0.05. Significantly up-regulated metabolites are represented by red dots, significantly down-regulated metabolites by blue dots, and non-significantly different metabolites by gray dots.

3.8. Application of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes in Gene Function Analysis

In order to clarify the effect of pretreatment on the genotype of each group, we performed gene ontology (GO) analysis and made functional annotations based on the differential expression gene (DEGs) screening results. These functions can be subdivided into three major parts: biological processes (BP), molecular functions (MF), and cell composition (CC). In addition, we take advantage of the Kyoto Encyclopedia of Genes and Genomes (KEGG), a comprehensive database that integrates genomic, chemical, and biochemical system function information, providing a valuable resource for in-depth understanding of the functions, interactions, and regulatory mechanisms of genes and metabolic pathways.
In this study, the top five GO terms with a significant level of p < 0.05 were enriched (Figure 13A,D,G). Specifically, the genes in the low concentration group were mainly related to the regulation of cellular biological processes and transport. The genes in the medium concentration group were involved in the positive regulation of cellular energy and cellular processes, as well as the negative regulation of RNA biosynthetic processes. Genes in the high concentration group were linked to positive regulation of signal transduction and nucleic acid transcription, response to redox states, animal organ development, and regulation of transmembrane transporter protein activity.
KEGG pathways with a significant difference (p < 0.05) and a high number of genes (n ≥ 6) were enriched in the experimental vs. control groups (Figure 13B,E,H). The pathways that were significant in the low concentration group were circadian rhythm, S. aureus infection, and the IL-17 signaling pathway. Pathways that were significant in the medium concentration group were circadian rhythm, the interaction of viral proteins with cytokines and cytokine receptors, and the IL-17 signaling pathway. The pathway with significance in the high concentration group was circadian rhythm. Figure 13C,F,I demonstrate the number of up- and down-regulated genes annotated to a pathway and their classification. The highest number of genes related to organismal systems were found in the low and high concentration groups; the highest number of genes related to human diseases were found in the medium concentration group.

4. Discussion

AD is a recurrent inflammatory skin disease that affects up to 25% of children and 2% to 3% of adults [44]. Some studies have shown that AD is strongly associated with race, genetics, environmental, and immunologic factors [45]. The treatment of AD has been recognized as a protracted battle, and conventional treatment usually involves the scheduled, intermittent application of topical corticosteroids (TCS) or topical calcineurin inhibitors (TCIs) to both old and new-onset skin lesions, as well as the use of moisturizers in all affected areas [46]. However, there is always a concern about side effects associated with the use of these drugs. Prolonged use of TCS can cause effects on the epidermis, including atrophy, hypopigmentation, photosensitivity, loss of the skin barrier, and premature aging. It can also adversely affect skin function, such as ulceration, impaired wound healing, Bettman’s purpura, and dermatoglyphic dilatation. In addition, osteoporosis, cardiovascular disease, acne, hirsutism, and alopecia are also common adverse effects of TCS [47]. A meta-analysis on TCIs found that patients with eczema, atopic dermatitis, or other dermatitis treated with TCIs had a significantly increased risk of developing lymphoma, especially non-Hodgkin’s lymphoma [48].
Therefore, people are trying to find natural extracts with high efficacy and low irritation for relieving and treating AD. Some researchers have tried using Noni’s fermented extract and Lithospermum erythrorhizon (LE) extract in the treatment of AD [49,50]. Studies have shown that retinol has the function of maintaining the health of skin surface epithelial cells and mucous membranes, stimulating the immune system, maintaining the integrity of mucous membranes and epithelium, and increasing the formation of collagen and extracellular matrix, fibroplasia, and the synthesis of collagen, glycoprotein, and proteoglycan [51]. In addition, retinol accelerates wound healing by stimulating regeneration, angiogenesis, and anti-inflammatory responses [52]. Several in vitro and in vivo studies have demonstrated its therapeutic potential in the treatment of atopic dermatitis, burns, acne, ulcers, asthma, lupus, psoriasis, scleroderma, and wounds [53,54]. Especially in the treatment of AD, when the ethanol extract of Centella asiatica (L.) Urb. is applied to DNCB-induced AD skin, it not only significantly reduces mast cell infiltration in the tissue, but also reduces the levels of various pathogenic cytokines, such as TNF-α, IL-4, IL-5, IL-6, IL-10, IL-17, iNOS, COX-2, and CXCL9 [34]. In another study, titrated extract of Centella asiatica (L.) Urb. was shown to inhibit NF-κB signaling and NF-κB activity, as well as the release of TNF-α, IL-1β, IL-6, and IgE in phthalic anhydride-induced AD [28]. Therefore, in this study, the combination of retinol and hydroxyasiaticoside was used to investigate the protective effect on AD, and its development potential for improving other skin diseases was also studied.
One of the characteristics of acute AD lesions is epidermal hyperplasia associated with dermal inflammatory infiltration [55]. The H&E staining results of the present study showed that all three concentration (L, M, and H) groups of R&H exhibited different degrees of improvement in AD. Relative to the model group, the epidermal layer of the mice’s lesion site was thinner, and the fibers of the dermis layer were more neatly arranged. Among them, the dermatopathological features of the mice in the R&H-H high-dose group improved most significantly, and no inflammatory cell infiltration was observed. Masson staining revealed that collagen fibers in the dermis of the mice in the AD group were significantly reduced and disorganized, with thinning of the epidermis and dermis. It was observed that the morphology of collagen fibers in the dermis was improved, the distribution became uniform, and the content increased after R&H treatment, with the best improvement seen in the high-dose group. Some studies have found an increase in the number of mast cells in AD skin lesions, so mast cells are usually considered to be related to the pathogenesis of AD [56]. In the present study, R&H attenuated histamine-induced epidermal and dermal thickening as well as mast cell infiltration in the skin of AD mice, and reduced the number of mast cells in the skin lesions of AD mice.
Our results suggest that retinol and hydroxyasiaticoside may exert their inhibitory effects on the expression of TRPV1, IL1R1, and CD130 through the modulation of signaling pathways such as JAK/STAT and PI3K/Akt. Retinol, known for its regulatory roles in cell proliferation, differentiation, and immune function, may directly interact with JAK protein kinases or STAT proteins to inhibit their activity or phosphorylation process, thereby blocking the JAK/STAT signaling pathway [57] Additionally, retinol may affect key molecules within the PI3K/Akt signaling pathway, such as phosphatidylinositol-3-kinase (PI3K) or Akt protein, to inhibit the activation of this pathway. This regulation may be achieved by influencing the expression or activity of upstream or downstream molecules within these signaling pathways, thereby modulating immune cell functions and inflammatory responses [58]. Hydroxyasiaticoside, a natural compound with anti-inflammatory and antioxidant properties, may indirectly affect the JAK/STAT signaling pathway by inhibiting the release of inflammatory mediators such as cytokines [59]. Furthermore, it may influence the activity of the PI3K/Akt signaling pathway through the modulation of upstream signaling molecules (e.g., growth factor receptors) or downstream effector molecules (e.g., mTOR) within this pathway [60]. However, it is important to note that the specific mechanisms by which retinol and hydroxyasiaticoside influence these signaling pathways are not fully understood at present. Future research needs to delve deeper into the interactions between these compounds and key molecules within the signaling pathways, as well as how they synergistically regulate the functions of immune cells and the pathological processes of atopic dermatitis. Through further investigation, we hope to uncover the exact mechanisms of retinol and hydroxyasiaticoside in the treatment of atopic dermatitis and provide stronger support for their clinical application. This will not only advance our understanding of the disease but also pave the way for more effective therapeutic strategies.
In order to determine the metabolite differences between the different groups, we first performed a PCA analysis, which showed a gradual separation between the experimental group samples and the control group samples as the concentration of retinol and hydroxyasiaticoside increased. Pearson correlation coefficient plots indicated good similarity and reproducibility between samples within the groups, with the highest inter-sample similarity in group R&H-L. The results of the PCA analysis revealed that the experimental group samples became increasingly separated from the control group samples as the concentration of retinol and hydroxyasiaticoside increased. Subsequently, we performed an inter-sample Venn analysis for each group, and the results indicated that all groups had the same disease model. Further Venn analysis of the differential gene set showed that the gene similarity was higher in the differential gene set of the R&H-H and HA groups. In the RNA-seq clustering heatmap, there were significant differences in expression between most of the genes in the experimental and control groups, and the gene expression patterns were similar between samples within each experimental group. This result is consistent with the results shown in the clustering heat map for kinin-releasing enzyme 13 (Klk13), human osteocalcin (Bglap3), and CD5 antigen-like protein (Cd5l). Next, differential metabolite screening was performed based on PCA using volcano plots to visualize VIP values, p values, and FC values. We then identified the differentially expressed metabolites between different groups by comparing pairs of groups. The graph showing the number of differentially expressed metabolites between the different groups indicates that the medium dose of retinol and hydroxyasiaticoside ingredients affects a greater number of metabolites regulating skin inflammation. Then, 33 endogenous metabolites were screened by Metabo Analysis, and the expression of shared DEMs can be clearly seen from the clustered heat map.
According to related studies, CD40, as an important protein related to relieving skin inflammation and promoting epidermal cell proliferation, can increase the autophagy flux and further lead to the accumulation of LC3 and LAMP-1 [61]. MAP1LC3, typically abbreviated as LC3, is a ubiquitous 17 kDa soluble protein in mammalian tissues and cultured cells, serving as a key component of autophagy, specifically marking autophagosome formation by integrating into its membranes during biosynthesis, and only the liposomized form enables LC3 to bind to the autophagosome membrane and contribute to the autophagy process. In addition, after the knockout of the ANGPT2 gene enriched by transcriptome, the expression of p-PI3K, P-Akt and p-mTor in fibroblasts decreased significantly [62]. It is suggested that the inhibition of the ANGPT2 gene can activate autophagy and inhibit the proliferation, migration and extracellular matrix accumulation of scar hyperplasia-related fibroblasts. In addition, in HSFs down-regulated by the ANGPT2 gene, mTOR agonist MHT1485 could partially reverse the effect of ANGPT2 gene knockout on HSFs.
In order to study the types of genes altered by pretreatment in each group, we performed GO analysis and annotated the functions based on the screened DEGs. These can be categorized into three levels of terms: biological processes, molecular functions, and cellular components. In this study, the results show that the low concentration group has the most genes related to metabolism, followed by genes related to human diseases. The medium concentration group has the most genes related to organismal systems. Additionally, the high concentration group has the most genes related to human diseases, followed by genes related to organismal systems.

5. Conclusions

The combined action of retinol and hydroxyasiaticoside on the skin significantly improved AD lesions and symptoms, in terms of scratching behavior, epidermal thickness, and collagen fiber morphology and number. In addition, the combination of retinol and hydroxyasiaticoside reduced the infiltration of inflammatory cells and decreased the levels of the pro-inflammatory factors TRPV1, IL1R1, and CD130. After administration, the skin barrier function in AD was significantly restored. Taken together, this study suggests that the combination of retinol and hydroxyasiaticoside may be a potential therapeutic agent for the prevention and treatment of AD.

Author Contributions

Q.L. (Qin Liu): Data curation (equal); writing—original draft (lead). P.S.: Data curation (equal); writing—original draft (equal). Q.S.: Data curation (equal). Z.H.: Data curation (equal). J.W.: Data curation (equal). L.Z.: Data curation (equal). Y.W.: project administration (equal). Q.L. (Qingqing Liu): writing—review and editing (equal). All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Guangdong University of Technology (protocol code SCXK20230012, approval date 13 June 2023).

Informed Consent Statement

The experiment does not involve human research, therefore this statement is not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Author Qin Liu, Peng Shu, Qingle Song and Yuan Wang were employed by Shenzhen Hujia Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Mice’s back skin tissue image and analysis. (A) The back skin of mice developed erythema and wrinkles after administration. (B) Results of statistical analysis of PASI scores on mice’s backs. Significant differences from the model were observed when assessed using variance analysis and Duncan’s multiple range test. The mean ± SD of 3 mice was represented by each value. ** p < 0.01, *** p < 0.01 and **** p < 0.0001.
Figure 1. Mice’s back skin tissue image and analysis. (A) The back skin of mice developed erythema and wrinkles after administration. (B) Results of statistical analysis of PASI scores on mice’s backs. Significant differences from the model were observed when assessed using variance analysis and Duncan’s multiple range test. The mean ± SD of 3 mice was represented by each value. ** p < 0.01, *** p < 0.01 and **** p < 0.0001.
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Figure 2. H&E staining results of the back skin of mice. (A) Histological sections of the back skin of mice stained with H&E. (B) Histological scores of dorsal skin sections of mice. Compared with the HA group. ** p < 0.01, **** p < 0.0001.
Figure 2. H&E staining results of the back skin of mice. (A) Histological sections of the back skin of mice stained with H&E. (B) Histological scores of dorsal skin sections of mice. Compared with the HA group. ** p < 0.01, **** p < 0.0001.
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Figure 3. Masson trichrome staining of mouse skin tissue. (A) Histological sections of skin tissue of mice with masson trichrome staining. Blue-green represents collagen fibers and red represents myofibrils and cytoplasm. The nucleus is grayish-black. (B) Semi-quantitative analysis of collagen deposition in mice skin tissue sections. Compared with the HA group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3. Masson trichrome staining of mouse skin tissue. (A) Histological sections of skin tissue of mice with masson trichrome staining. Blue-green represents collagen fibers and red represents myofibrils and cytoplasm. The nucleus is grayish-black. (B) Semi-quantitative analysis of collagen deposition in mice skin tissue sections. Compared with the HA group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Figure 4. TB staining of mouse skin tissue. (A) Histological section of TB-stained mouse skin tissue. Blue represents collagen fibers and red represents myofibrils and cytoplasm. The nucleus is dark brown or black. (B) Semi-quantitative analysis of collagen deposition in mice skin sections. Compared with the HA group, *** p < 0.001, **** p < 0.0001.
Figure 4. TB staining of mouse skin tissue. (A) Histological section of TB-stained mouse skin tissue. Blue represents collagen fibers and red represents myofibrils and cytoplasm. The nucleus is dark brown or black. (B) Semi-quantitative analysis of collagen deposition in mice skin sections. Compared with the HA group, *** p < 0.001, **** p < 0.0001.
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Figure 5. (A,C,E) are the skin tissue immunohistochemical sections of TRPV1, IL1R1, and CD130, respectively. (B,D,F) show the expression levels of TRPV1, IL1R1, and CD130, respectively, compared with the HA group. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5. (A,C,E) are the skin tissue immunohistochemical sections of TRPV1, IL1R1, and CD130, respectively. (B,D,F) show the expression levels of TRPV1, IL1R1, and CD130, respectively, compared with the HA group. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Figure 6. (A) Principal Component Analysis Diagram; (B) Pearson Correlation Coefficient Chart; (C) Inter-Sample Venn Diagram; (D) Differential Gene Set Venn Diagram; (E) Clustering heat map.
Figure 6. (A) Principal Component Analysis Diagram; (B) Pearson Correlation Coefficient Chart; (C) Inter-Sample Venn Diagram; (D) Differential Gene Set Venn Diagram; (E) Clustering heat map.
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Figure 7. Volcano Plot of Expression Level Differences. (A–C) represent comparisons of the number of differentially expressed genes between the R&H-L, R&H-M, and R&H-H treatment groups, respectively, and the model group. In the plot, red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots indicate genes that have been detected but exhibit no significant regulatory changes.
Figure 7. Volcano Plot of Expression Level Differences. (A–C) represent comparisons of the number of differentially expressed genes between the R&H-L, R&H-M, and R&H-H treatment groups, respectively, and the model group. In the plot, red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots indicate genes that have been detected but exhibit no significant regulatory changes.
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Figure 8. The number of differentially expressed metabolites in the skin tissues of mice from different groups.
Figure 8. The number of differentially expressed metabolites in the skin tissues of mice from different groups.
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Figure 9. Venn diagram of shared differentially expressed metabolites.
Figure 9. Venn diagram of shared differentially expressed metabolites.
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Figure 10. Heatmap of clustered shared differentially expressed metabolites.
Figure 10. Heatmap of clustered shared differentially expressed metabolites.
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Figure 11. Metabolic pathway map of mouse skin tissue.
Figure 11. Metabolic pathway map of mouse skin tissue.
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Figure 12. The Volcano Plot of Metabolites in Mouse Skin Tissue. (A) Control vs. HA. (B) R&H-L vs. HA. (C) R&H-M vs. HA. (D) R&H-H vs. HA.
Figure 12. The Volcano Plot of Metabolites in Mouse Skin Tissue. (A) Control vs. HA. (B) R&H-L vs. HA. (C) R&H-M vs. HA. (D) R&H-H vs. HA.
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Figure 13. (A–C) GO-enriched chordal maps of the R&H-L group, (D–F) the R&H-M group, (G–I) the R&H-H group, showing KEGG pathway enrichment and metabolic pathway classification.
Figure 13. (A–C) GO-enriched chordal maps of the R&H-L group, (D–F) the R&H-M group, (G–I) the R&H-H group, showing KEGG pathway enrichment and metabolic pathway classification.
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Table 1. Table of Gene Regulation.
Table 1. Table of Gene Regulation.
Gene IDGeneGene Descriptionp-ValueExpression LevelRegulation Results
R&H-LR&H-MR&H-LR&H-LR&H-MR&H-L
ENSMUSG00000059824DbpD site albumin promoter binding protein9.55 × 10−61.82 × 10−193.82 × 10−911.9910.0110.01down
ENSMUSG00000022871Fetubfetuin beta1.08 × 10−31.14 × 10−22.29 × 10−211.009.819.81up
ENSMUSG00000054046Klk13kallikrein related-peptidase 137.07 × 10−258.08 × 10−51.64 × 10−70.660.350.35up
ENSMUSG00000020889Nr1d1nuclear receptor subfamily 1, group D, member 11.76 × 10−28.77 × 10−92.11 × 10−981.5622.7922.79down
ENSMUSG00000037202Prf1perforin 1 (pore forming protein)1.83 × 10−24.11 × 10−62.64 × 10−385.3924.1824.18down
ENSMUSG00000050092Sprr2bsmall proline-rich protein 2B8.02 × 10−132.64 × 10−21.30 × 10−1211.556.826.82up
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MDPI and ACS Style

Liu, Q.; Shu, P.; Song, Q.; Huang, Z.; Weng, J.; Zhang, L.; Wang, Y.; Liu, Q. Retinol and Hydroxyasiaticoside Synergistically Relieve Histamine-Induced Atopic Dermatitis Activity by Repressing TRPV1, L1R1, and CD130 Targets. Cosmetics 2024, 11, 203. https://doi.org/10.3390/cosmetics11060203

AMA Style

Liu Q, Shu P, Song Q, Huang Z, Weng J, Zhang L, Wang Y, Liu Q. Retinol and Hydroxyasiaticoside Synergistically Relieve Histamine-Induced Atopic Dermatitis Activity by Repressing TRPV1, L1R1, and CD130 Targets. Cosmetics. 2024; 11(6):203. https://doi.org/10.3390/cosmetics11060203

Chicago/Turabian Style

Liu, Qin, Peng Shu, Qingle Song, Zhenhai Huang, Jiyu Weng, Lanyue Zhang, Yuan Wang, and Qingqing Liu. 2024. "Retinol and Hydroxyasiaticoside Synergistically Relieve Histamine-Induced Atopic Dermatitis Activity by Repressing TRPV1, L1R1, and CD130 Targets" Cosmetics 11, no. 6: 203. https://doi.org/10.3390/cosmetics11060203

APA Style

Liu, Q., Shu, P., Song, Q., Huang, Z., Weng, J., Zhang, L., Wang, Y., & Liu, Q. (2024). Retinol and Hydroxyasiaticoside Synergistically Relieve Histamine-Induced Atopic Dermatitis Activity by Repressing TRPV1, L1R1, and CD130 Targets. Cosmetics, 11(6), 203. https://doi.org/10.3390/cosmetics11060203

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