A Mixture of Nicotinamide Mononucleotide,Decursin,and l -Cysteine Lowered Senescence-Associated Markers In Vitro and Was Effective Against 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis In Vivo: An Application of Network Pharmacology

Abstract

Skin aging, accelerated by oxidative stress from environmental factors, results in the breakdown of collagen and elastin, causing visible signs like wrinkles and inflammation. Natural antioxidants such as nicotinamide mononucleotide (NMN), decursin, and l-cysteine have shown potential in combating oxidative damage and inflammation. This study explored the effects of a formulated mixture of these compounds on skin aging and atopic dermatitis (AD) through a combination of in vitro, in vivo, and in silico methods. Using human keratinocyte cells, we assessed cytotoxicity via a cell viability assay, confirming NMN and l-cysteine were nontoxic up to 100 µM, while decursin exhibited toxicity above 10 µM. The intracellular oxidative stress measurement demonstrated that mixtures A and B, composed of the same concentrations of decursin and l-cysteine but differing in NMN levels (low in mixture A and moderate in mixture B), significantly reduced oxidative stress levels induced by 2,2’-azobis(2-amidinopropane) dihydrochloride, whereas mixture C, which contained the highest NMN concentration, was ineffective. Mixture B further reduced senescence-associated heterochromatin foci formation under oxidative stress, while mixture C caused cell structure disruptions. In the dinitrochlorobenzene-induced AD model in BALB/c mice, both, mixture-L and mixture-H treatments reduced epidermal thickness, scratching behavior, and transepidermal water loss, with mixture-L also lowering dermal thickness and mast cell infiltration. Gene expression analysis confirmed that mixture B decreased proinflammatory cytokines like TNF-α and IL-6, while network pharmacology predicted key antioxidant pathways, validated through restored NOS2 gene expression. Overall, the findings highlight the potential of these compound mixtures to mitigate oxidative stress and inflammation, offering a promising approach for skin aging and dermatitis management, though further validation is needed to optimize efficacy and safety.

INTRODUCTION

The aging process of the skin, known as skin senescence, is influenced by various factors.¹ Oxidative stress accelerates the breakdown of collagen and elastin fibers, leading to wrinkles, and loss of skin elasticity.² In contrast, studies have explored the potential of various natural antioxidants to mitigate damages caused by oxidative stress and to delay aging process.^3,4 Among many natural products, nicotinamide mononucleotide (NMN) participates in the production of nicotinamide adenine dinucleotide (NAD⁺)⁵ that is essential for activating sirtuin and poly (ADP-ribose) polymerase,⁶ all of which maintain genomic stability within cells and prevent aging.⁷ Deficiency of NAD⁺ can be compensated for by supplementing NAD⁺ precursors like NMN that present higher bioavailability than directly consuming the NAD⁺, which is considered impermeable to the plasma membrane, and NAD⁺ administration cannot efficiently increase NAD⁺ levels.⁸

Decursin is a natural coumarin present in Angelica gigas Nakai.⁹ Previous research has shown that decursin has various biological activities, including anti-inflammatory,¹⁰ anticancer,¹¹ and inhibition of melanogenesis.¹² In the context of skin-aging, decursin effectively mitigated ultraviolet B-induced photoaging,¹³ lowered allergic reaction (that mimicked atopic dermatitis [AD]) by reducing histamine secretion,¹⁴ and exhibited anti-inflammatory through suppressing nitric oxide production in raw 264.7 cells.¹⁵ However, it is not fully understood how this naturally occurring coumarin derivative will elicit protection against skin-aging phenotypes when combined with NMN.

Last, l-cysteine acts as a precursor to glutathione, a critical antioxidant that protects skin cells from oxidative damage induced by reactive oxygen species (ROS) generated through environmental stressors. Furthermore, l-cysteine has been shown to support wound healing and skin regeneration by facilitating cellular homeostasis and reducing inflammation.¹⁶ Thus, the inclusion of l-cysteine in skin-aging research may offer a comprehensive approach to delaying photoaging by enhancing the skin’s intrinsic antioxidant defenses and promoting recovery from stressors. Individual studies have explored their respective effects on skin aging or protection,¹⁷ yet the mode of actions of these compounds with distinct effects remain elusive. To this end, we have tested a mixture of compounds to examine whether complementary mechanisms thereof will elicit significant protection against cellular aging markers in vitro and in vivo.

MATERIALS AND METHODS

Cell culture

The human skin keratinocyte (HaCaT) cell line was obtained from the American Type Culture Collection (PCS-200-011; Manassas, VA). The cells were cultured following a method largely adapted from previously published studies.¹⁸

3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate sample cytotoxicity as described in previous studies with slight modifications.^19,20

Dichlorodihydrofluorescein diacetate assay: Intracellular oxidative stress assessment

To assess intracellular oxidative stress, we used a dichlorodihydrofluorescein diacetate (DCF-DA) cellular ROS assay kit (Abcam; Cambridge, UK) per manufacturer’s instruction.

Wound healing assay

The wound healing potential was evaluated using a scratch assay as described elsewhere²¹ with slight modifications.

Immunohistochemical staining and confocal fluorescence microscopy

HaCaT cells were treated with 10 mM 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) (Sigma-Aldrich; St. Louis, MO) for 24 h, alone or with sample mixtures. Fixed cells were stained with tri-methyl-histone H3 primary (Cell Signaling Technology; Danvers, MA) and Alexa Fluor 488-conjugated secondary (Invitrogen; Carlsbad, CA) antibodies for senescence-associated heterochromatin foci (SAHF) detection. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich), and images were captured using a confocal microscope (Zeiss LSM 900; Oberkochen, Germany), showing SAHF as green fluorescence and nuclei as blue.

Quantitative real-time PCR

Total RNA was extracted using RNeasy Plus Kits (Qiagen; Hilden, Germany) with minor manufacturer protocol adjustments. The mRNA expression levels were determined using a SYBR Green quantitative real-time polymerase chain reaction assay with PowerUp SYBR Green Master Mix (Thermo Scientific), following the manufacturer’s instructions. It was performed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems; Foster City, CA, USA), and data were analyzed with QuantStudio Design and Analysis Software v1.5.1. Primer details are available in Supplementary Table S1.

Dinitrochlorobenzene-induced dermatitis model in vivo

Male BALB/c mice (6–8 weeks, 20–25 g) were used to model AD through dinitrochlorobenzene (DNCB) application. A sample size of n = 10 per group was accepted based on a statistical power calculation (80% power, significance level [α] = 0.05). All animal experiments were conducted in accordance with the Institutional Guidelines for the Use and Care of Laboratory Animals and were approved by the Korea University Institutional Animal Care & Use Committee (Approval Number: KUIACUC-2024-0043). The mice were housed in a controlled facility under standardized environmental conditions, including a temperature of 22 ± 2°C, relative humidity of 50 ± 5%, and a 12-hour light/dark cycle. Animals were provided ad libitum access to food and water and were acclimatized to the facility for 7 days prior to the initiation of the experiments. The mice were divided into five groups: negative control (NEG), positive control (POS), low-dose mixture (mixture-L), high-dose mixture (mixture-H), and prednisolone (Pred). Sensitization was induced by applying 0.5% DNCB to shaved dorsal skin on Days 1, 4, and 7, followed by weekly 0.2% DNCB applications on Days 14 and 21, and daily 0.5% DNCB from Day 21 to Day 35 to exacerbate symptoms. The mixture-L group received 25 mg/kg NMN, 0.15 mg/kg decursin, and 60 mg/kg l-cysteine, while the mixture-H group received 250 mg/kg NMN, 1.5 mg/kg decursin, and 600 mg/kg l-cysteine. The Pred group received 3 mg/kg Pred. NEG received no DNCB or treatment, while POS received DNCB without intervention. Daily treatments were administered orally from Day 7 to Day 35. After euthanasia by CO₂ asphyxiation, tissues were collected for further evaluation.

Assessment of ear and epidermal thickness

Ear thickness was measured to quantify the degree of inflammation in the AD model. A hand-held caliper was used to measure ear thickness, ensuring consistent application pressure. The epidermal thickness measurement method was adapted with slight modifications based on previously published methods.²²

Measurement of scratch behavior

After a 24-h acclimation in observation cages, scratching bouts, defined as continuous hind limb movements toward irritated areas, were recorded for 10 min and analyzed independently by two researchers.

Measurement of transepidermal water loss and erythema index

The transepidermal water loss (TEWL) and erythema index were measured weekly, with dorsal skin shaved prior to each assessment. TEWL was measured using the SKIN-O-MAT (Cosmomed; Ruhr, Germany), while the erythema index was assessed using the Mexameter® MX18 (Courage + Khazaka electronic GmbH; Cologne, Germany). Each site was measured three times, and averages were recorded for accuracy. Dermatitis scores were calculated according to Scoring Atopic Dermatitis Index (SCORAD) criteria, evaluating erythema, scarring, edema, and excoriation on a scale of 0 (none) to 3 (severe), following manufacturer protocols.

Assessment of plasma IgE level

Approximately 500 µL of whole blood per animal was drawn into ethylenediaminetetraacetic acid-containing tubes to prevent coagulation. Samples were placed on ice and centrifuged at 3,000 rpm for 10 min at 4°C to separate plasma. The plasma was carefully collected and used to quantify immunoglobulin E (IgE) levels using a commercial kit (BD Bioscience; Franklin Lakes, NJ).

Mast cell count

The dorsal and ear skin tissues were processed into sections for mast cell analysis. Slides were stained with 0.5% Toluidine Blue O (Sigma-Aldrich), highlighting mast cells as metachromatically purple-blue due to their granules. After slides were washed with distilled water, dehydrated through graded ethanol, cleared with xylene, and mounted using synthetic medium. Mast cells were identified under a light microscope (LEICA DM500; Leica Microsystems, Germany) at 200× magnification, using Leica Application Suite (LAS) version 4.12 for imaging. They appeared as purple-stained cells with granular cytoplasm. Five random fields per section were analyzed, and mast cells were manually counted to ensure regional variability was accounted for.

In silico prediction of target genes: Network pharmacology

The potential targets of the active compounds in the mixture (i.e., NMN, decursin, and l-cysteine) were identified using three well-known databases: PharmMapper (http://lilab-ecust.cn/pharmmapper), SwissTargetPrediction (http://swisstargetprediction.ch), and STITCH (http://stitch.embl.de). For PharmMapper, targets with a normalized fit score >0.7 were selected. In SwissTargetPrediction, a probability threshold of >0.7 was applied. In the STITCH database, compound-target interactions were filtered by setting the species to “Mus musculus” and applying a high confidence score threshold of >0.7. The names of the collected targets were then standardized using the UniProt database (https://www.uniprot.org/).

Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis

Using ClueGO 2.5.8, a Cytoscape plug-in, gene ontology (GO) analysis was conducted to investigate functions across three categories: biological process, molecular function (MF), and cellular component (CC). A bubble chart illustrating the results of the GO and Kyoto Encyclopedia of Genes and Genomes enrichment analyses was generated using an online R package (http://www.ehbio.com/ImageGP/index.php/Home). In the chart, the X-axis represents the percentage of associated genes, while the Y-axis corresponds to the description of the terms. The significance threshold for identifying relevant terms and pathways was set at P value <0.001.

Statistical analysis

Statistical evaluations were conducted using GraphPad Prism version 8.0.2 software (Boston, MA). We assessed whether the data followed a normal distribution using the Shapiro–Wilk test. When the data did not fit a normal distribution, the Mann–Whitney test was applied. Conversely, for data that conformed to a normal distribution, we used a two-tailed unpaired Student’s t-test with Welch’s correction. We presented the data as mean ± standard deviation or standard error of means where appropriate. The results were considered statistically significant if P values were <0.05.

RESULTS AND DISCUSSION

Determination of NMN, decursin, and l-cysteine cytotoxicity

The cytotoxicity of NMN, decursin, and l-cysteine on HaCaT cells was assessed using the MTT assay across concentrations of 0–100 µM Figure 1(A). NMN and l-cysteine showed no significant effects on cell viability across all tested concentrations, whereas decursin significantly reduced cell viability at 10 and 100 µM but had no effect at lower concentrations (0.1 and 1 µM). Based on these results, mixtures of NMN, decursin, and l-cysteine were formulated to minimize cytotoxicity while maintaining potential efficacy (Table 1). The cytotoxicity of these mixtures (A, B, and C) was also evaluated on HaCaT cells, showing no significant reduction in viability compared to the NEG Figure 1(B). All mixtures exhibited cell viabilities of nearly 100%, supporting their use in further studies without cytotoxicity concerns.

FIG. 1.

Effect of nicotinamide mononucleotide, decursin, and l-cysteine mixtures on cell viability and intracellular reactive oxygen species (ROS) levels in human skin keratinocyte (HaCaT) cell lines. HaCaT cells were seeded into 96-well plates and cultured to measure cell viability. (A) The effects of each compound on HaCaT were measured using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay; (B) the cell viability following the treatment with the mixtures (see Table 1 for the respective composition) in HaCaT cells was measured using the MTT assay; (C) intracellular ROS levels were measured in cells treated with 24-h mixtures, followed by 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH, 3 mM) treatment for 1 hour, using the dichlorodihydrofluorescein diacetate assay. Results are expressed as means ± standard deviations (n = 5–8). Normality was assessed using the Shapiro–Wilk test. Differences between groups were evaluated using Welch’s t-test for independent samples. ** and **** denote P < 0.01 and P < 0.00001, respectively, compared to nontreated groups (i.e., 0 μM groups) in the panel (A); **** denotes P < 0.00001 compared to the negative control (NEG) while ^# denotes P < 0.05 compared to the positive control (POS) in the panel (C); n.s. denotes no significance in the figure.

Table 1.

The Composition of Nicotinamide Mononucleotide (NMN)-Based Natural Product Mixture

Composition	A (μM)	B (μM)	C (μM)
NMN	1	10	100
Decursin	1	1	1
l-cysteine	100	100	100

Protective effects of the mixtures against AAPH-induced intracellular ROS

The antioxidant effects of NMN, decursin, and l-cysteine mixtures (A, B, and C) were tested by measuring intracellular ROS levels in HaCaT cells using the DCF-DA assay. AAPH treatment (POS group) significantly increased ROS levels compared to NEG, confirming oxidative stress induction (P < 0.00001; Figure 1(C)). Mixtures A and B significantly reduced ROS levels compared to the POS group (P < 0.05), whereas mixture C showed no protective effect. These findings indicate that mixtures A and B effectively mitigate ROS production.

ROS play a critical role in skin aging by damaging CCs and accelerating senescence, leading to visible aging signs (e.g., wrinkles and reduced elasticity).²³ While previous studies highlighted the antioxidative roles of NMN, decursin, and l-cysteine individually,^24,25 our data present the effects of different formulations of the compounds which were not reported previously. Future studies should evaluate the dose-dependent effects and potential synergistic or additive interactions of single compounds, as these aspects could provide deeper insights into their combined efficacy.

Wound healing efficacy of the mixtures in the HaCaT cells

Following the creation of a uniform scratch to simulate a wound, the HaCaT cells were treated with each mixture and incubated for 11 h to observe wound closure (Fig. 2). Quantitative analysis of the wound healing area revealed differences in wound closure among the groups. In this, the mixtures B and C showed enhancement in wound closure, with a statistically significant increase in wound healing area compared to the NEG (P < 0.05 for both; Fig. 2). No difference was noted with mixture A.

FIG. 2.

Effects of mixtures of nicotinamide mononucleotide, decursin, and l-cysteine on wound healing in human skin keratinocyte (HaCaT) cell lines. HaCaT cells were seeded into 6-well plates and cultured to confluency. A uniform scratch was created using a 200 μL pipette tip to simulate a wound. Cells were treated immediately with mixtures A, B, and C then incubated for 11 h to allow recovery (see the main text for the respective composition of the mixtures). Representative images of the wound areas were taken, while quantitative analysis was performed by measuring the wound healing area. Results are expressed as means ± standard deviations (n = 3). Due to the small sample size, normality was not assumed; hence, a nonparametric approach was used for statistical analysis. Differences between groups were evaluated using the Mann–Whitney U test for independent samples. * denotes P < 0.05 compared to the negative control (NEG).

Inhibitory effects of mixtures on the formation of senescence-associated heterochromatin foci induced by AAPH in HaCaT

The effects of mixtures of NMN, decursin, and l-cysteine (mixtures A and B) on the senescence-associated heterochromatin foci (SAHF) in HaCaT cells were assessed following exposure to oxidative stress induced by AAPH (Fig. 3). SAHF-positive cells were characterized by the presence of condensed heterochromatin foci, as indicated by the red arrow in Figure 3, whereas such structures were relatively less prominent in SAHF-negative cells. Quantification of SAHF-positive cells was performed based on these morphological features, using randomly selected fields and independent of the representative images shown. During the experiments, treatment with mixture C resulted in noticeable alterations in cell shape and cytoskeletal structure (Supplementary Fig. S1). These morphological changes, which were not present in cells treated with mixtures A or B, suggested potential cytotoxic effects on cellular architecture. Due to these observed changes, SAHF quantification was not performed for cells treated with mixture C. The POS group, treated with AAPH alone, displayed a significant increase in SAHF-positive cells compared to the NEG, indicating successful induction of cellular senescence by AAPH (P < 0.05). Mixture A did not decrease the percentage of SAHF-positive cells compared to the POS, whereas the mixture B showed a substantial inhibitory effect on SAHF formation, with a statistically significant reduction in SAHF-positive cells relative to the POS (P < 0.05; Fig. 3). These findings suggest that mixture B may be effective in reducing SAHF formation in HaCaT cells.

FIG. 3.

Inhibitory effects of mixtures of nicotinamide mononucleotide, decursin, and l-cysteine on the formation of senescence-associated heterochromatin foci (SAHF) induced by 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) in human skin keratinocyte (HaCaT) cell lines. Confocal microscopy images (scale bar: 20 μm, magnification: 40×) were obtained using HaCaT cell lines after 24 h of treatment with AAPH (10 mM) or AAPH (10 mM) combined with mixtures A and B (see the main text for the respective composition of the mixtures). Green fluorescence represents tri-methyl-histone H3 (Lys9, K9HM3), an epigenetic marker associated with heterochromatin formation, while blue fluorescence represents cell nuclei stained with TO-PRO-3, a fluorescent dye that binds to DNA. Red arrows indicate intact SAHF-positive cells that remain structurally preserved, highlighting the presence of condensed heterochromatin. Quantification of SAHF-positive cells (% of total cell count) was also provided in which results were presented as means ± standard deviations (n = 4). Due to the small sample size, normality was not assumed hence a nonparametric approach was used for statistical analysis. Differences between groups were evaluated using the Mann–Whitney U test for independent samples. * denotes P < 0.05 compared to the negative control (NEG) while ^# denotes P < 0.05 compared to the AAPH-treated control (POS). POS, positive control.

Effects of mixture B on the expressions of inflammatory genes in HaCaT

The protective effects of mixture B against AAPH-induced SAHF formation and ROS were confirmed by analyzing the expression of key inflammation and oxidative stress-related genes: interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nuclear factor erythroid 2-related factor 2 (NRF2). We found a significant increase in IL-6 and TNF-α in the POS group compared to the NEG, indicating successful ROS induction and activation of inflammatory pathways (P < 0.01 for both genes; Fig. 4). Treatment with mixture B, however, significantly reduced the genes’ expression compared to the POS group (P < 0.0001 for IL-6 and P < 0.01 for TNF-α), suggesting an anti-inflammatory effect under oxidative stress conditions. Furthermore, NRF2 gene expression, a key regulator of antioxidant responses, was notably elevated in the POS group relative to the NEG while no difference was noted in the mixture B treatment group (Fig. 4). Excessive ROS levels in the skin contribute to cellular senescence and the degradation of structural proteins, leading to visible aging signs. Elevated IL-6 and TNF-α levels are typical markers of inflammation in skin cells subjected to oxidative stress, promoting collagen degradation and impairing skin barrier function, both of which accelerate the aging process.²⁶ In this study, on the other hand, mixture B significantly downregulated IL-6 and TNF-α mRNA levels in AAPH-treated HaCaT cells, suggesting its potential to reduce oxidative stress-induced inflammation, which is crucial for slowing skin aging.

FIG. 4.

Quantification of target gene mRNA expressions following reactive oxygen species induction in human skin keratinocyte (HaCaT) cell lines. HaCaT cells were seeded in 60 × 15 mm plates and treated with the mixture B (see the main text for the composition) for 24 h, followed by stimulation with 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH, 3 mM) or not for 1 h. The cells were harvested, and total RNA was extracted for quantitative PCR analysis of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nuclear factor erythroid 2-related factor 2 (NRF2) mRNA levels, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as an internal reference. Results are expressed as means ± standard deviations (n = 4–6). Normality was assessed using the Shapiro–Wilk test. Differences between groups were evaluated using Welch’s t-test or Mann–Whitney U test for independent samples. ** denotes P < 0.001 compared to the negative control (NEG) while ^## and ^#### denote P < 0.01 and P < 0.0001, respectively, compared to the positive control (POS).

Effects of oral administration of mixture B on DNCB-induced AD: Primary phenotypes

The DNCB-induced AD model visualized in Figure 5(A) showed that the POS group displayed typical AD symptoms, including erythema, dryness, and crust formation²⁷ as well as significantly increased spleen weight, ear thickness, and scratching behavior compared to the NEG group Figure 5(B), (C), (D). Spleen analysis, indicative of immune involvement in AD,²⁸ showed significantly increased spleen weights in the POS group compared to the NEG, while the reference control (i.e., Pred group) exhibited significant reductions and the mixture groups (low and high doses) showed non-significant reductions Figure 5(C). Ear thickness was significantly greater in the POS group compared to the NEG, with no significant improvement noted in the mixture or Pred groups Figure 5(C). However, mixture and Pred groups reduced scratching frequency compared group to the POS Figure 5(D).

FIG. 5.

Effects of mixtures of nicotinamide mononucleotide, decursin, and l-cysteine in a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis model in skins of BALB/c mice. (A) The BALB/c mice were randomly divided into five groups (n = 10 per group): Negative control group (NEG), positive control group (POS), a low-dose mixture treatment group (mixture-L), a high-dose mixture treatment group (mixture-H), and a prednisolone treatment group (PRED). To induce sensitization, 0.5% DNCB was applied to the shaved dorsal skin of the mice in the POS, mixture-L, mixture-H, and PRED on D1, D4, and D7. After, the mice received weekly applications of 0.2% DNCB to the same dorsal skin area to maintain dermatitis phenotypes (i.e., D4, and D21). Starting D21, to simulate exacerbated dermatitis symptoms, the concentration of DNCB was increased to 0.5%, with daily applications continuing until the end of the experiment. Concurrently, the treatment groups received their respective oral administrations (see the method for the respective composition of each groups) each day for the duration of the 4-week period (namely D7 through D35); (B) representative images of skin lesions in the ears and dorsal skin and spleen in DNCB-induced dermatitis mice; (C) spleen weight and ear thickness of animals; and (D) number of scratches of each animal was video recorded and analyzed for 10 min by two independent researchers. Results are expressed as means ± standard deviations. Normality was assessed using the Shapiro–Wilk test. Differences between groups were evaluated using Welch’s t-test. * and **** denotes P < 0.05 and P < 0.0001 compared to the NEG, respectively. ^{#, ##, ###} and ^#### denote P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 compared to the POS, respectively.

Histological analysis showed substantial increases in epidermal and dermal thickness and elevated mast cell counts in the POS versus NEG groups Figure 6(A) and (B). The mixture groups significantly reduced epidermal thickness, while the mixture-L decreased dermal thickness and mast cell infiltration; the reference group (i.e., Pred group) effectively improved all markers. Further, the mixture groups significantly improved skin barrier function, as evidenced by reduced TEWL, though benefits in SCORAD scores and erythema were less pronounced Figure 6(C). Repeated DNCB application caused systemic immune activation and chronic inflammation, as evidenced by spleen enlargement and various skin manifestations.²⁸ Mixture groups, particularly mixture-L, demonstrated decreases in scratching behavior, epidermal thickness, TEWL, dermal thickness, and mast cell counts, with similar patterns observed in ear tissues Figure 6(D, E). These results highlight the potential efficacy of NMN, decursin, and l-cysteine mixtures in mitigating inflammation and enhancing skin barrier integrity in AD.

FIG. 6.

Effects of nicotinamide mononucleotide, decursin, and l-cysteine mixtures on histological markers in a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis model in BALB/c mice. (A) Representative photomicrographs of dorsal skin sections stained with hematoxylin and eosin (H&E) or toluidine blue were provided. The red lines indicate epidermal, and dermal thickness while black arrows show mast cells of dorsal skin; (B) quantitative analysis of epidermal and dermal thickness and number of mast cells in BALB/c mouse dorsal skin. The thickness of the epidermis was measured from the stratum basale to the stratum corneum, excluding the hair follicles; (C) the transepidermal water loss (TEWL), scoring atopic dermatitis index (SCORAD), and erythema index were assessed weekly. Hair was shaved using clippers before measurements for the TEWL and erythema index measurements. (D) Representative photomicrographs of ear tissues stained with hematoxylin and eosin (H&E) and toluidine blue are shown. Red lines indicate the boundaries of the epidermal and dermal layers, while black arrows point to mast cells present in the tissue; (E) quantitative analysis was conducted to assess ear thickness and mast cell count. Ear thickness was measured using a hand-held caliper, applying consistent pressure to prevent influencing edema development. Results are expressed as means ± standard deviations. Normality was assessed using the Shapiro–Wilk test. Differences between groups were evaluated using Welch’s t-test test. *** and **** denotes P < 0.001 and P < 0.0001 compared to the negative group (NEG), respectively. ^{#, ##, ###} and ^#### denotes P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 compared to the positive group (POS), respectively.

Oral administration of mixture B decreased proinflammatory gene expressions in DNCB-induced AD

To explain the mechanisms, a few key anti-inflammatory genes were assessed. In the POS group, as expected, the levels of TNF-α, IL-1β, IL-4, IL-6, and IL-13 were all markedly elevated compared to the NEG group (Fig. 7). Although each sample group showed a trend toward reduced cytokines gene expressions, the results varied depending on the specific cytokine. In particular, TNF-α in the Pred and mixture-H groups demonstrated significant decreases compared to the POS group (P < 0.05 for Pred and P < 0.001 for mixture-H), while mixture-L showed no effect. For IL-1β, mixture-L achieved a statistical decline (P < 0.05), whereas Pred and mixture-H did not show statistical relevance. In the case of IL-4 and IL-13, both mixture groups exhibited substantial reductions while the Pred group failed to recapitulate the effects. Last, the IL-6 decreased in the mixture-L and Pred groups but, unexpectedly, it increased in the mixture-H. These findings indicate that proinflammatory cytokine expression, heightened during DNCB-induced acute and chronic inflammatory responses,²⁹ is effectively modulated. Last, we did not find statistical significance in serum IgE protein levels between the mixture and the POS (data not shown) although our DNCB protocol dramatically increased the serum IgE level in the POS compared to the NEG (P < 0.0001).

FIG. 7.

Quantification of proinflammatory cytokine mRNA expression in 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis BALB/c mouse skin tissues. Effect of nicotinamide mononucleotide, decursin, and l-cysteine mixtures on the expression of diverse inflammatory factors in dorsal skin of BALB/c mice. Total RNA was extracted from the skin tissue for the quantitative real-time polymerase chain reaction (qPCR) analysis of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-13 (IL-13) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. Results are expressed as means ± standard deviations (n = 4). Normality was assessed using the Shapiro–Wilk test. Differences between groups were evaluated using the Mann–Whitney U test. ** and *** denote P < 0.01 and P < 0.001 compared to the negative group (NEG), respectively. ^{#, ##, ###} and ^#### denote P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 compared to the positive group (POS), respectively.

An application of network pharmacology

Network pharmacology (NP) is an emerging approach that integrates bioinformatics tools to explore the complex interactions between biological networks,³⁰ enabling comprehensive prediction and mapping of potential targets for active compounds. By using computational databases (e.g., PharmMapper), we identified 61 target genes of NMN, decursin, and l-cysteine (Supplementary Table S2) and further analyzed them through GO to reveal potential pathways affected by the mixture (Supplementary Table S3). The enriched GO: MF terms highlight MFs (Fig. 8) related to skin health, particularly oxidoreductase activity for managing oxidative stress that may protect skin cells from conditions like AD³¹ and NADP/FMNs binding for redox balance and tissue repair.²⁵ Although the top three GO: MF categories showed higher percentages, they were excluded from validation due to their limited associated gene counts, which restricted the possibility of deriving meaningful conclusions. NOS2, ranked fourth, was selected for validation due to its significant role in cellular senescence and aging processes, as well as its association with inflammation-related pathways. To validate, the nitric-oxide synthase (NOS2) gene expression, associated with inflammation Figure 8(A), was assessed because the presence of NOS2 activity suggests a role in the inflammatory responses in the skin.³² NOS2 mRNA levels in the mouse dorsal skin samples were elevated in the POS group but restored in the mixture groups, demonstrating anti-inflammatory effects, unlike prednisone, which showed no impact Figure 8(B). Reduced NOS2 levels align with cytokine downregulation, supporting the therapeutic potential of the mixtures.

FIG. 8.

Gene ontology (GO) enrichment analysis of molecular functions associated with the predicted gene set. (A) Gene ontology (GO) enrichment analysis was performed on predicted genes associated with the molecular mechanisms of nicotinamide mononucleotide, decursin, and l-cysteine mixtures. The bubble size represents the number of genes associated with each molecular function, while the color indicates statistical significance (-Log₁₀ [P value]), with red denoting higher significance. The X-axis shows the percentage of genes associated with each function. (B) NOS2 mRNA expression was quantified using total RNA extracted from dorsal skin tissues of BALB/c mice. qPCR was performed using GAPDH as the internal reference. Results are expressed as means ± standard deviations (n = 4). Normality was assessed using the Shapiro–Wilk test, and differences between groups were evaluated using the Mann–Whitney U test. * denotes P < 0.05 compared to the negative control group (NEG), and ^# denotes P < 0.05 compared to the positive control group (POS). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEG, negative control group; NMN, nicotinamide mononucleotide; NOS2, nitric oxide synthase 2; qPCR, quantitative real-time polymerase chain reaction.

Together, the study highlights the efficacy of NMN, decursin, and l-cysteine mixtures in reducing oxidative stress, inflammation, and skin damage in AD models. While NP predictions and experimental results provide a mechanistic basis for these effects, further in vivo studies are required to confirm long-term safety and optimize formulations for clinical use.

Footnotes

AUTHORS’ CONTRIBUTIONS

H.R.S.: Writing—original draft (lead); formal analysis (lead); and validation (support). L.Y.C.: Writing—original draft (support); formal analysis (lead); and validation (lead). S.H.B.: Writing—original draft (support); formal analysis (support); and visualization (lead). T.G.K.: Funding acquisition (lead) and project administration (support). H.T.C.: Formal analysis (support) and funding acquisition (support). T.J.C.: Resources (support) and review and editing (support). N.S.O.: Resources (support) and review and editing (support). Y.Y.S.: Methodology (support) and review and editing (support). M.J.T.R.: Methodology (support) and review and editing (support). J.H.P.: Investigation (lead) and review and editing (support). J.-O.S.: Conceptualization (equal). E.-C.S.: Conceptualization (equal). S.-G.K.: Methodology (lead). J.P.: Investigation (support) and resources (support). Y.H.: Investigation (support) and resources (support). K.-C.C.: Review and editing (support). Y.J.K.: Supervision (support) and project administration (support). W.H.: Methodology (lead) and review and editing (support). K.L.: Writing—review and editing (equal); formal analysis (lead); and supervision (lead). J.K.K.: Writing—review and editing (equal) and project administration (lead).

AUTHOR DISCLOSURE STATEMENT

No competing ﬁnancial interests exist.

FUNDING INFORMATION

This work was supported by a Korea University Grant (K2317281) and Regional Innovation Strategy (RIS) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE; 2024C0701006).

SUPPLEMENTARY MATERIAL

Supplementary Figure S1

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

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Supplementary Material

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