Effect of glabridin combined with bakuchiol on UVB‐induced skin damage and its underlying mechanism: An experimental study

Research has demonstrated the anti‐photoaging properties of glabridin and bakuchiol.


| INTRODUC TI ON
3][4] Ultraviolet A (UVA) and ultraviolet B (UVB) are the primary forms of UV radiation, with wavelengths ranging 315-400 nm and 290-320 nm, respectively, 5 UVB radiation exerts deleterious effects on the structural integrity of proteins and DNA within skin cells, with the most significant impact being evident in epidermal cells. 6The research also revealed that UVB exposure resulted in an excessive production of reactive oxygen species (ROS), disturbances in mitochondrial fusion, and the activation of mitogen-activated protein kinase (MAPK) signaling pathways, along with the activation of transcription factors such as nuclear factor-kappa B (NF-κB) and activated protein-1 (AP-1).][9][10][11] Licorice is a well-known Chinese herbal remedy, typically derived from the dried roots and stems of Glycyrrhiza glabra L, Glycyrrhiza uralensis Fisch, and Glycyrrhiza inflata Bat.3][14][15] Licorice possesses the properties of invigorating the spleen, replenishing qi, purging heat, detoxifying, nourishing the lungs, alleviating cough, relieving pain, and harmonizing other medicinal compounds. 12Glabridin has been recognized as the predominant polyphenolic flavonoid in G. glabra L, according to various studies. 16Glabridin showcases an array of biological properties, including antimicrobial effects, 17 antiinflammatory effects, 18 suppression of hepatocellular carcinoma proliferation, 19 cardiovascular safeguarding effects, 20 and antineoplastic effects against liver cancer, breast cancer, lung cancer, leukemia, and other malignancies. 21Furthermore, glabridin has been observed to exhibit estrogenic effects, 22 hepatoprotective properties, 23 neuroprotective activities, 24 inhibition of muscle atrophy, 25 effects on insulin and diabetes, 26 reduction of hyperpigmentation, 27 anti-obesity effects, 28 and antioxidative effects. 29 1998, Yokota et al. unveiled that dermal administration of 0.5% glabridin ameliorated UVB-induced cutaneous pigmentation and erythema in guinea pigs. 30Subsequently, Wang 31 and Du 27 et al. demonstrated that glabridin has the ability to counteract UV-induced photoaging, encompassing oxidative impairment and phototoxicity, while diminishing hyperpigmentation.In 1923, it was initially determined that the seeds of Psoralea corylifolia Linn contain an unsaponifiable oil. 27Mehta 32 identified a unique monoterpene phenol in the oil and named it bakuchiol in Sanskrit.Bakuchiol has emerged as a substitute for topical retinoids, offering a plethora of advantageous effects on the skin.
Research has unveiled its anti-photoaging properties, 33 as well as its ability to combat skin aging. 34It showcases antioxidant, 35 anti-inflammatory, 36 anti-proliferative, 37 and hepatoprotective qualities. 38Additionally, bakuchiol demonstrates antimicrobial, 39 anti-inflammatory, 40 antiviral, 41 anti-osteoporosis 42 and antiacne activity. 43Several studies have indicated that bakuchiol's capacity to ameliorate photoaging is akin to that of retinol, yet it surpasses retinol in terms of photostability and hydrolytic stability.Unlike retinol, bakuchiol does not induce burning, irritation, or redness, rendering it an alluring option for shielding the skin from UV damage.Its photostability allows for daytime use as well. 33Bakuchiol has been demonstrated to offer effective shielding against UV damage and is suitable for daytime use due to its photostability.Consequently, there is a compelling case for considering bakuchiol as a promising alternative to retinol, with fewer associated side effects. 33th glabridin and bakuchiol are acknowledged for their antiaging properties.Nevertheless, the synergy of glabridin and bakuchiol in the context of photoaging remains unexplored.Hence, this study endeavors to delve into the mechanism governing the impact of the combination of glabridin and bakuchiol on UVB-induced photoaging in mice.We utilized UVB-irradiated dorsal skin of mice to examine the effects of glabridin, glabridin + bakuchiol, and bakuchiol on the levels of interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) in fibroblasts, the visible effects on the skin tissue of mice, the involvement of fibronectin (FN), interferonγ (IFNγ), interleukin 22 (IL-22), and transforming growth factorβ (TGFβ) in the tissue, and the impact on the levels of enzymes in the skin.This study will delve into the mechanism of the anti-photoaging effects of glabridin and bakuchiol on the skin.

| Principal reagent
The ELISA kit for IFNγ, FN, IL-

| Grouping of laboratory animals
The SPF KM mice used in this experiment were all 5-week old, female and weighing 34-38 g.After an adaptive feeding period, the mice were kept at an ambient temperature of 23°C, with approximately 60% humidity, a 12-h light-dark cycle, and acclimatized for a minimum of 7 days. 44Subsequently, the mice were then randomized (n = 5): control group, model group (UVB), GL + BA group (UVB + 0.5% glabridin and 0.5% bakuchiol mixed solution), GL group (UVB + 0.5% glabridin solution) and BA group (UVB + 0.5% bakuchiol solution).

| Treatment of laboratory animals
Prepare a solution of 0.5% glabridin and 0.5% bakuchiol in DMSO at a dosage of 150 mg/kg.The day before the experiment, hair on the dorsal skin of the mice was removed using depilatory cream.The UVB (313 nm) photoaging model was established, and drug administration was initiated and continued for 21 days.Daily administration was given to all groups except the blank and model groups.The administration modes were as follows: • GL + BA group: The mice in this group were smeared with 0.5% glabridin and 0.5% bakuchiol mixed solution (200 μL/mice) on the back of the depilated area.
• GL group: The mice in this group were smeared with 0.5% glabridin solution (200 μL/mice) on the back of the depilated area.
• BA group: The mice in this group were smeared with 0.5% bakuchiol solution (200 μL/mice) on the back of the depilated area.
Control group and model group: The mice in this group were smeared with physiological saline (200 μL/mice) on the back of the depilated area.
After 30 min of drug application, the GL + BA, GL, BA, and model groups were irradiated with 100 mJ/cm 2 of UVB for 2 h, while the control group does not receive irradiation, lasting for 8 weeks.UVB irradiation was performed using a UVP CL-1000 ultraviolet crosslinker.Following completion of UVB irradiation, photographs of the irradiated skin of the mice were taken.The mice were then euthanized by cervical dislocation.The dorsal skin tissue was fixed in paraformaldehyde and frozen for further analysis. 45

| Histological and immunohistochemical analyses
The dorsal skin tissues of mice were embedded in paraffin wax and subjected to analysis using hematoxylin and eosin (H&E) 45 and toluidine blue (TB). 46After staining, the sections were observed using a standard light microscope to describe the histological characteristics of the cells in the dorsal skin of mice.Immunocombination analysis was performed following the protocol described by Huang, 47 using specific antibodies to detect specific proteins or markers in the tissue samples.
The specific steps for immunohistochemical analysis are as follows: The tissue sections were treated with sodium citrate buffer (pH 6.0), and the antigens were extracted through microwave heating.The microwave was set to medium for 8 min until boiling, then turned off for 8 min to cool.Subsequently, it was set to mediumlow heat for 7 min to maintain gentle heating and prevent solution evaporation.The slides were allowed to cool naturally and then placed on a PBS (pH 7.4) decolorizing shaker.They were washed three times for 5 min each.Next, the sections were incubated with 3% H 2 O 2 for 25 min in the dark to block endogenous peroxidase activity.Decolorization was repeated with PBS (pH 7.4) three times.
Serum blocking was performed using 3% BSA for 30 min.Then, the tissue sections were incubated overnight in diluted primary antibody (PBS dilution) at 4°C.Following this, The tissue sections were washed with PBS and then incubate with the secondary antibody for 50 min.Finally, DAB was employed for chromogen detection, and hematoxylin counterstaining was utilized for nuclear detection.
The images were acquired using the Axio Scan.Z1 automated digital glass scanner (Carl Zeiss AG, Analytik Jena, Germany), employing a high-fidelity 3-chip CCD camera for bright field scanning (Hitachi HV F202SCL) with a resolution of 1600 × 1200 pixels.Specific parameters included the use of a Plan Apochrome 20 × objective lens (NA = 0.8) and a TL exposure time of 200 μs.

The integrated optical density (IOD) values of IL-1β and TNFα in
the murine skin following immunohistochemical staining were quantified using Image-Pro Plus.The resultant data were compiled and visualized using GraphPad Prism.

| ELISA assay
ELISA reagent kits were utilized to evaluate the protein levels of IFNγ, FN, IL-22, and TGFβ in the skin tissue. 48The collected skin tissue was homogenated, and the contents of each index were tested using their respective kits.The detection wavelength employed was 450 nm.To determine the concentration of the samples, the optical density (OD) values obtained from the ELISA assay were compared to a standard curve.This standard curve is established by measuring known concentrations of the target protein and plotting the optical density at various concentrations.Subsequently, the concentration of the sample can be calculated by comparing its OD value to the standard curve.

| Analysis of skin tissue SOD, HYP and MDA
The protein levels of SOD, HYP, and MDA in the skin tissue were assessed using SOD, HYP, and MDA assay kits, respectively. 49Initially, the collected skin tissue underwent homogenized to produce a homogenate.Subsequently, the levels of each index were analyzed using the corresponding kits.The determination wavelength for SOD was set at 420 nm, while for HYP, it was 560 nm.In the case of MDA, dual determination wavelengths were required.The absorbance of the MDA reaction product was measured at both 532 nm and 600 nm.By meticulously following the instructions provided with each kit and employing the appropriate determination wavelengths, precise assessment of the SOD, HYP, and MDA levels in the skin tissue was achieved.

| Principal component analysis
Principal component analysis (PCA) is a widely utilized method for analyzing data.It enables unsupervised pattern recognition in multidimensional datasets, uncovering sample repeatability and distinctions among samples within a group.Following dimension reduction analysis, the coordinate points represent the corresponding principal components.The distance between samples is indicative of the distance of each sample point.Closer distances between samples signify increased similarity and better biological replication between samples.The volcano plot illustrates the total number of genes detected in the differential group, highlighting significant numbers of upregulated and downregulated genes.Additionally, the cluster heatmap displays differential genes and clustering among samples.These visual representations are instrumental in analyzing differentially expressed genes and gene expression levels.

| Network pharmacology
The Gene Ontology (GO) 50 is an internationally recognized classification system for gene function.Developed by the GO Gene Ontology Consortium, it serves as a comprehensive database that aims to establish a standardized vocabulary applicable to various species.The primary objective of GO is to define and describe the functions of genes and proteins, while also allowing for updates as research progresses.GO mainly includes three categories: cellular components, biological processes and molecular functions.A smaller P adjust value corresponds to a greater-log10 (P adjust ) value, signifying a more pronounced enrichment of the KEGG path.

| Statistical analysis
The experimental data was analyzed using GraphPad Prism 8.3, and the outcomes were presented as mean ± SD.ANOVA was utilized to assess the statistical variances between groups.A significance threshold of p < 0.05 was deemed indicative of a statistically noteworthy distinction between the groups.

| Glabridin and bakuchiol suppresses epidermal hyperplasia in the murine
In order to investigate the pathological changes in murine skin following UVB irradiation and assess the impact of glabridin and bakuchiol on photoaged skin, we conducted a histological examination using H&E staining on the dorsal skin tissues of the mice. 45Figure 1 illustrates the results of the H&E staining analysis on the skin tissue obtained from the dorsal region of the mice.Figure 1A  Upon application, the dorsal skin displayed improved pathological alterations, and the potential damage caused by UVB radiation was alleviated.Following UVB irradiation, there was a noticeable increase in epidermal thickness compared to the control group, attributed to a stimulatory response (p < 0.01).However, both the GL + BA and BA groups showed a significant reduction in skin epidermal layer thickness compared to the model group (p < 0.01).Histological scores were recorded based on the dorsal skin tissue in mice, indicating that all treatment groups had lower scores than the model group.This highlights the superior anti-allergic and protective effects of the combination of glabridin and bakuchiol on skin inflammation.As shown in Figure 2B, the model group displayed a higher number of mast cells compared to the control group, indicating that UVB exposure resulted in damage to the dorsal skin of mice.The treatment groups demonstrated protection against UVB-induced skin photoaging in mice.Among them, the GL, GL + BA, and BA groups exhibited significantly lower mast cell infiltration compared to the model group.Particularly noteworthy was the GL + BA group, which demonstrated the lowest mast cell infiltration among all the groups.

| Glabridin and bakuchiol inhibited the production of inflammatory cytokiness
During the process of inflammation, macrophages and lymphocytes in the inflamed intestinal mucosa stimulate the production        As can be seen from Figure 8, the genes significantly expressed in the control group were not significantly expressed in the treated group and were most evident in the 0.5% glabridin + 0.5% bakuchiol group.Figure 10 shows that we performed metabolomic analysis on all samples, starting with PCA analysis (Figure 11A).Then, the Venny 2.1 method was used to screen out the common differential metabolites between the treatment group and the model group, and between the model group and the control group, and the cluster heat map was drawn (Figure 11B).The samples were enriched with differential metabolites, including four pathways, namely (−)menthy lacetate, emetine, protoporphyrinogen IX and saccharonoic acid.

| DISCUSS ION
Certainly, ongoing research is dedicated to identifying compounds capable of mitigating skin photoaging, particularly emphasizing natural substances and their derivatives renowned for their antioxidant and anti-inflammatory attributes.These include resveratrol-enriched rice, 7 probiotics, 56 flavonoids, carotenoids, 57 ginsenoside, 58 glycyrrhizic acid, 59 and trigonalline, 60 among others.
Research has revealed the anti-inflammatory properties of glabridin and bakuchiol, with the potential to ameliorate hyperpigmentation and cutaneous photodamage. 27,33In this investigation, UVB irradiation was utilized to induce photoaging in the dorsal skin of mice, and the anti-inflammatory effects of glabridin and bakuchiol were evaluated by assessing the levels of pro-inflammatory cytokines such as TNFα, IL-1β, and IL-22 in the three test samples.
Histological examination results demonstrated that all three samples effectively mitigated skin allergic reactions and cortical hyperplasia induced by UVB exposure.Studies have indicated that mast cells play a significant role in inflammatory skin conditions, and an increase in mast cell numbers in skin lesions is one of the underlying mechanisms. 61Our experimental findings also revealed that the The Kyoto Encyclopedia of Genes and Genomes (KEGG) is an exhaustive repository that systematically scrutinizes gene functionality and genomic data.It amalgamates a diverse array of information encompassing genomes, biological pathways, diseases, drugs, chemicals, and related data.KEGG encompasses genomic insights and sophisticated functional data, seamlessly integrating them to facilitate a systematic analysis of extensive datasets derived from highthroughput experimental technologies like genome sequencing.The KEGG pathway is depicted on the vertical axis, while the number of genes/transcripts in the comparison pathway is portrayed on the upper horizontal axis, with distinct points on the line corresponding to them.The level of enrichment significance is indicated by the horizontal axis below, and the column's height corresponds to the level.
displays the H&E staining image of the paraffin section from the dorsal skin tissue of the mice, while Figure 1B presents the histogram illustrating the histopathological scoring of the dorsal skin in mice.The H&E staining image in Figure 1A reveals that the model group demonstrated a heightened level of epidermal hyperplasia compared to the GL + BA and BA groups, indicating that the local application of glabridin in combination with bakuchiol, or their individual application, effectively suppressed the epidermal hyperplasia induced by allergic dermatitis.Notably, the combined application of glabridin and bakuchiol exhibited a more pronounced inhibitory effect.In Figure 1B, the model group exhibited a disrupted surface structure of the dorsal skin and evident epidermal hyperplasia, suggesting that UVB irradiation can damage epidermal cells, subsequently inducing skin photoaging.

Figure
Figure 2A depicts the image of the toluidine blue staining on the paraffin section of the dorsal skin tissue, while Figure 2B presents the histogram illustrating the histopathological scoring of the dorsal skin in mice.The toluidine blue staining was employed to evaluate the infiltration of mast cells.The protective effects of glabridin and

F I G U R E 1
This is a figure schemes follow the same formatting.The results of hematoxylin and eosin (H&E) staining on the dorsal skin of mice.(A) Histological section of the back skin of mice stained with H&E (magnification ×200).(B) Histological scores of dorsal skin slices of mice.Compared with UVB group, compared with the model group, **p < 0.01.F I G U R E 2 Results of Toluidine blue (TB) staining on the dorsal skin of mice.(A) Histological section stained with TB skin tissue of mice dorsal (magnification × 200).(B) Histological scores of dorsal skin tissue in mice.Compared with UVB group, compared with the model group ****p < 0.0001.ofproinflammatory cytokines, such as TNFα and IL-1β.51These cytokines play a crucial role in perpetuating the inflammatory response.52Specifically, IL-1β can worsen the inflammatory response by initiating a cascade of cellular activation.53Conversely, TNFα can trigger the activation and aggregation of inflammatory cells, and it can also work in conjunction with IL-1β to further amplify the inflammatory response.54

Figure
Figure 3A,B depicts a significant increase in the expression of the pro-inflammatory cytokine IL-1β (p < 0.01) in the UVB-exposed group compared to the control group.Treatment with glabridin and bakuchiol resulted in a substantial reduction in IL-1β expression in the skin tissue compared to the model group (p < 0.0001).Both the GL + BA and BA groups effectively suppressed the expression of IL-1β, with tartary bakuchiol alone exhibiting the most pronounced inhibitory effect.Figure 3C,D shows that the levels of TNFα in the control group did not differ significantly from the model group.However, treatment with glabridin and bakuchiol led to notably lower expression of TNFα in the GL + BA group compared to the model group (p < 0.01), indicating the effective reduction of TNFα expression by the combination of glabridin and bakuchiol.The results indicate that both glabridin and bakuchiol have the ability to inhibit the expression of TNFα.Of all the groups, the GL group exhibited the most potent suppressive effect on TNFα expression.

3. 4 |Figure 4
Figure 4 depicts that the levels of FN, IFNγ, interleukin-22 (IL-22), and TGFβ in the skin tissue of mice in the model group were elevated in comparison to those in the control group.In contrast to the model group, the levels of FN, IFNγ, and IL-22 in the skin tissues of the GL, GL + BA, and BA groups exhibited a significant decrease (p < 0.01), and the levels of TGFβ in the skin tissues of the GL + BA and BA groups were reduced compared to the model group (p < 0.05).Upon analysis of the results, it was observed that in comparison to the model group, the levels of FN, IFNγ, IL-22, and TGFβ in the skin tissue of mice in the BA group experienced the most significant decrease, indicating that the use of bakuchiol alone can notably reduce the levels of FN, IFNγ, IL-22, and TGFβ.

Figure 5
Figure5illustrates that UVB radiation led to changes in the skin aging tissues of mice, characterized by a significant increase in MDA levels and a notable decrease in SOD and HYP levels.However, the results from the GL, GL + BA, and BA groups suggest that glabridin and bakuchiol were able to reduce MDA levels and enhance SOD and HYP levels.Among these groups, the combination of glabridin and bakuchiol showed a superior effect in rejuvenating photoaged skin.Essentially, glabridin and bakuchiol demonstrate antioxidant properties by boosting the levels of antioxidant enzymes and suppressing oxidative stress markers.

Figure 5
Figure 5 reveals that UVB irradiation induced alterations in the skin aging tissues of mice, marked by a notable elevation in MDA

Figure
Figure 6A depicts the grouping of samples, suggesting the reproducibility of samples within each group.Correlation analysis of all samples was performed to assess the consistency of biological replicates within groups and to ensure the reliability of differentially expressed genes, as well as to identify any aberrant samples.The biological replicate correlation was assessed using Pearson's correlation coefficient R. As the absolute value of R approaches 1, it indicates a higher degree of correlation between the two replicate samples. 55Figure 6B illustrates a correlation ranging from 0.80 to 0.98 (R > 0.80), indicating strong reproducibility within the group.As can be seen from volcanic plot Figure 7, The orderplot represents the gene expression fold change, the ordinate represents the level of gene significance.Differential genes are represented by green dots, differential genes downregulated by blue dots, and non-differential genes by red dots.With corrected P value less than 0.05 and Fold change greater than or equal to 1.2 as screening criteria, compared with the control group, the 0.5% glabridin + 0.5%

Figure 9
Figure9shows that the GO genes were mainly enriched in binding, biological regulation, cellular process, and cell part in the three treatment groups as compared with the control group.
three test samples were capable of attenuating skin allergic reactions by inhibiting the infiltration and degranulation of mast cells, thereby alleviating their inflammatory response.IFNγ, FN, IL-22, and TGFβ are all implicated in the pathological mechanisms of cutaneous inflammation.IFNγ serves as a pivotal proinflammatory agent, mediating and engaging in both local and F I G U R E 9 Gene Ontology (GO) functions of the (A) GL + BA_versus_UV, (B) GL_versus_UV and (C) BA_versus_UV.F I G U R E 1 0 Kyoto Encyclopedia of Genes and Genomes s enrichments of the (A) GL + BA_versus_UV, (B) GL_versus_UV and (C) BA_ vsersus_UV.systemic inflammatory responses.It has the capacity to recruit and initiate inflammatory reactions, while also regulating the expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and TNFα. 62Th22 cells, a subset of helper T cells initially identified in the skin tissues of individuals with inflammatory skin conditions, promote tissue inflammation by secreting the cytokine IL-22.62These cytokines and factors play critical roles in the inflammatory response and can contribute to the initiation and progression of cutaneous inflammation.A comprehensive understanding of their involvement in skin inflammation can aid in the development of targeted therapies for inflammatory skin disorders.63,64In addition to pro-inflammatory cytokines, there are also anti-inflammatory cytokines that play a pivotal role in modulating inflammation.Research has revealed the presence of FN in the extracellular matrix (ECM) of diverse human tissues.Furthermore, TGFβ is a potent multifunctional cytokine.Both FN and TGFβ serve as significant antiinflammatory agents, capable of facilitating wound healing and suppressing inflammatory responses, encompassing oral, cutaneous, and intestinal inflammation.They are also implicated in diminishing the infiltration of inflammatory cells.65

5 |
photoaging of the dorsal skin in mice and alleviate skin inflammation.Histological staining with H&E and TB revealed a significant reduction in the thickness of photoaged skin and a decrease in mast cell infiltration in the skin tissue following treatment with glabridin and bakuchiol.Immunohistochemistry and ELISA analyses indicated a notable decrease in proinflammatory factors such as IL-1β, IL-22, IFNγ, and TNFα, alongside an increase in anti-inflammatory components such as FN and TGFβ.Furthermore, measurements of SOD, HYP, and MDA content in the dorsal skin of mice suggested that glabridin and bakuchiol not only elevated SOD and HYP content but also reduced MDA content.Thus, this study posits that glabridin and bakuchiol could potentially serve as natural ingredients for future interventions targeting photoaging.Nonetheless, additional research is imperative to delve into their underlying mechanisms, laying the groundwork for the development of related products.