Effects of Triacetin on AMPK Activation and Immune Responses in Allergic Contact Dermatitis

Abstract

Background/Objectives: Allergic contact dermatitis (ACD), an inflammatory skin condition, is commonly treated with topical corticosteroids; however, long-term use of these drugs is associated with various risks, such as skin atrophy and steroid resistance. Triacetin (TA), a triglyceride metabolized to acetate, exerts anti-inflammatory affects by activating AMP-activated protein kinase (AMPK) and suppressing mast cell degranulation. Here, we aimed to assess the immediate and long-term effects of TA on ACD suppression, focusing on AMPK activation, using a 2,4-dinitrofluorobenzene-induced rodent model. Methods: Various concentrations of TA were topically applied to rats with 2,4-dinitrofluorobenzene-induced dermatitis. Ear thickness was measured, and histological analysis was performed to assess the inflammation, mast cell infiltration, and degranulation in the established models. AMPK activation was analyzed via Western blotting, and TA degradation was assessed via gas chromatography-mass spectrometry. Dorsomorphin (an AMPK inhibitor) was used to evaluate the effects of AMPK on ACD. Results: TA significantly inhibited inflammation and mast cell degranulation in a dose-dependent manner, with 0.25 mmol/L showing the most potent effects. It also activated AMPK activation. Notably, AMPK inhibition reversed the effects of TA. Conclusions: Overall, TA exerted immediate and long-term anti-inflammatory effects via AMPK activation and inhibition of mast cell degranulation, showing potential as a non-steroidal therapeutic for ACD.

1. Introduction

Allergic contact dermatitis (ACD) is a common inflammatory skin disorder caused by repeated exposure to small molecular allergens, known as haptens, that penetrate the epidermis and trigger a type IV hypersensitivity reaction [1,2]. Clinically, ACD manifests as pruritic erythematous lesions that significantly impair the patient quality of life. Common allergens include metals (e.g., nickel), fragrances, and preservatives commonly found in personal care products [3,4,5,6]. ACD exhibits a high incidence, affecting approximately 15–20% of the general population worldwide [7]. Topical corticosteroids (TCSs) are the first-line treatment for ACD [8]. Most patients diagnosed with ACD are prescribed TCSs for treatment, making them the standard of care for managing ACD symptoms [9]. TCSs mitigate inflammation; however, their long-term use may cause adverse effects, such as skin atrophy, delayed wound healing, and systemic complications, such as adrenal suppression [10]. Furthermore, the emergence of steroid phobia, which is defined as the fear or hesitation to use TCSs due to concerns regarding potential side effects, such as skin thinning, delayed wound healing, and systemic complications, has resulted in inconsistent treatment adherence [11,12]. Therefore, non-steroidal therapeutic alternatives are urgently needed for ACD.

We recently demonstrated that the inclusion of TA in drinking water increases the serum acetate levels, ultimately suppressing ACD symptoms [13]. Therefore, TA possibly exerts anti-inflammatory effects via systemic acetate release, providing a new avenue for ACD treatment. Upon enzymatic breakdown, TA releases acetate, a short-chain fatty acid that plays complex roles in inflammation. Acetate exhibits anti-inflammatory activity [14,15,16]. However, high concentrations of acetate exacerbate skin inflammation in psoriasis [17,18]. Effects of low acetate concentrations on skin inflammation, particularly in conditions such as ACD, remain unclear, warranting further investigation. Acetate activates the AMP-activated protein kinase (AMPK) [19,20], a key metabolic regulator maintaining energy homeostasis by promoting glucose uptake, fatty acid oxidation, and mitochondrial biogenesis [21]. In addition to its metabolic functions, AMPK plays key roles in anti-inflammatory processes by reducing pro-inflammatory cytokine production via nuclear factor-κB signaling inhibition [22,23]. AMPK also contributes to skin homeostasis by stabilizing the skin barrier and enhancing keratinocyte differentiation and lipid synthesis [24]. AMPK activation suppresses mast cell degranulation [25], a critical step in allergic responses [26], thereby mitigating allergic inflammation.

Direct application of acetic acid to the skin poses serious risks, such as skin corrosion and irritating odors. In contrast, TA exerts localized anti-inflammatory effects by gradually releasing acetate in the skin. However, the specific roles of TA in the management of allergic reactions, particularly ACD, remain ambiguous. Therefore, in this study, we aimed to investigate the anti-allergic effects of TA in a 2,4-dinitrofluorobenzene (DNFB)-induced ACD rodent model to assess its potential as a non-steroidal therapeutic alternative for ACD.

2. Materials and Methods

2.1. Rats, Experimental Diets, and Sucrose Solution

Five-week-old male Sprague–Dawley rats and Institute of Cancer Research mice were obtained from Japan SLC Inc., Shizuoka, Japan, and housed under controlled conditions under a 12/12 h light/dark cycle at 22.5 ± 0.5 °C temperature and 55 ± 5% humidity. The animals were provided standard laboratory chow (CE-2; Japan CLEA Inc., Tokyo, Japan) and water ad libitum. All experimental procedures were approved by the Animal Experimentation Committee of the Kobe Gakuin University (approval nos. 21-17, 22-09, 23-05) and conducted in accordance with the institutional guidelines.

2.2. Induction of Allergic Dermatitis

Allergic dermatitis was induced using a well-established DNFB model. Animals were first sensitized by applying 50 μL of 0.5% DNFB (Nacalai Tesque, Kyoto, Japan) solution (prepared in a 3:1 mixture of acetone [Nacalai Tesque] and olive oil [Nacalai Tesque]) to the shaved backs for two consecutive days. After one week, the rats were challenged by applying 20 μL of 0.2% DNFB solution to the right ear. The left ear served as a control and was treated only with the vehicle solution. Various concentrations of TA (Nacalai Tesque) were applied topically to both ears in a solution of 2-propanol (Nacalai Tesque) and propylene glycol (Nacalai Tesque) in a 7:3 ratio at 20 μL for two days: one day before and one day after the DNFB challenge. Ear thickness was measured using an electronic caliper before and after the challenge, and change in ear thickness was used as an indicator of inflammation. To investigate the role of AMPK activation, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 15 μg/20 μL; Abcam, Cambridge, UK) or dorsomorphin (Dor; 45 μg/20 μL; Abcam) solution was subcutaneously injected into the occipital region 1 h prior to the application of DNFB and induction of allergic response.

2.3. Histological Analysis

After the DNFB challenge, the animals were euthanized, and both ears were harvested for histological analysis. Briefly, ear tissues were fixed with 4% paraformaldehyde phosphate-buffered saline (Nacalai Tesque) overnight at 4 °C, resin-embedded using Technovit 7100 (KULZER GmbH, Hanau, Germany), and sectioned. The sections were stained with hematoxylin and eosin to visualize inflammatory cells or toluidine blue to observe the mast cells. Inflammatory cells were identified in HE-stained sections as cells with bright red-stained cytoplasm, whereas mast cells were identified via intense purple staining. The numbers of inflammatory cells and mast cells in each section were counted under a microscope at 40× magnification, but their specific identification as eosinophils could not be confirmed using HE staining alone. Mast cell degranulation was assessed by categorizing the mast cells as intact or degranulated based on the presence of cytoplasmic granules. Then, the degranulation rate was calculated as the percentage of degranulated mast cells relative to the total number of mast cells.

2.4. Immunofluorescence Assay

Fixed tissues were immersed in 10 and 30% sucrose solutions, embedded in the Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA), frozen, and sectioned at a thickness of 10 μm. Air-dried frozen sections were re-fixed with 4% paraformaldehyde phosphate-buffered saline and washed with 0.1% Triton X-100, 50 mmol/L NaF in Tris-buffered saline (TBSTx-NaF; Nacalai Tesque). Then, 0.18 mg/mL proteinase K (Nacalai) solution was applied, and the tissue was washed with TBSTx-NaF. After blocking with 3% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO, USA)/TBSTx-NaF for 1 h at room temperature, the tissue sections were incubated with a solution containing 3% BSA/TBSTx-NaF and primary antibodies for approximately 16 h at 4 °C. After washing thrice with TBSTx-NaF, the tissue sections were incubated with a solution of 3% BSA/TBSTx-NaF containing the corresponding secondary antibodies and Hoechst 33258 at room temperature. After washing thrice, the tissues were washed with TBSTx for 5 min. Then, Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) was applied dropwise, and the tissues were covered with a cover glass (Matsunami Glass, Kyoto, Japan) and sealed. Anti-phospho-AMPKα (Thr172; 40H9) rabbit mAb (#2535) primary antibodies were purchased from Cell Signaling, and the tryptase alpha/beta 1 (NBP2-26444) secondary antibodies were purchased from Novus Biologicals (Centennial, CO, USA). Anti-rabbit IgG Alexa Fluor 568 (ab175471) was procured from Abcam, and Goat anti-mouse IgG Alexa Fluor 448 (A28175) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Hoechst 33258 was purchased from Dojindo Laboratories (Kumamoto, Japan).

2.5. Western Blotting

To assess AMPK activation, ear tissues were homogenized in the radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. Protein concentrations of the lysates were determined using the BCA assay (Nacalai Tesque). Equal amounts of protein (20 μg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibodies against AMPK (Cell Signaling, Danvers, MA, USA), pAMPK Thr172 (Cell Signaling), and lamin (Santa Cruz Biotechnology) overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature). After washing thrice with TBST, chemiluminescence assay was performed for 5 min with ImmunoStar LD (Fujifilm Wako, Osaka, Japan), according to the manufacturer’s recommendations, and chemiluminescence signals were visualized and quantified using LumiCube Plus (Liponics, Osaka, Japan) and ImageJ software. Relative expression levels of pAMPK were normalized to those of total AMPK.

2.6. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

Persistence or degradation of TA in the skin was analyzed via GC-MS. Skin samples were collected from the backs of 7-week-old male Sprague–Dawley rats. The skin was divided into six pieces (approximately 1 cm × 1 cm) and kept on ice until the experiment. Skin pieces were coated with 20 μL of 10 mmol/L TA solution (2-propanol/propylene glycol = 7:3 mixture). TA-coated tissue pieces were placed in 2-mL tubes, incubated at 30 °C (Dry Thermo Unit DTU-18; TAITEC corp., Saitama, Japan) for specific time points (0, 0.5, 1, 2, and 4 h), flash frozen using liquid nitrogen, and stored at −80 °C until lipid extraction. Subsequently. lipids were extracted from the skin samples using a mixture of diethyl ether and hydrochloric acid with 1 mmol/L 4-methylvaleric acid as an internal standard (Nacalai Tesque), followed by centrifugation to separate the organic phase. The organic layer was dried over anhydrous sodium sulfate (Hayashi Pure Chemical, Osaka, Japan) and analyzed using a GC-MS instrument (GCMS-QP2010; Shimadzu, Kyoto, Japan) equipped with an HP-5 column (Agilent Technologies, Santa Clara, CA, USA) at an injector temperature of 275 °C, detector temperature of 250 °C, MS ion source temperature of 250 °C, MS interface temperature of 250 °C, and temperature program at 70 °C with 2 min hold, followed by 70–90 °C for 10 min, 90–180 °C for 32 min, 180–250 °C for 36 min. The amount of TA in each sample was quantified using an internal standard, and the percentage of TA remaining was calculated relative to the initial amount.

2.7. Statistical Analyses

Data are represented as the mean ± standard error of the mean. Statistical analyses were conducted using one-way analysis of variance, followed by Tukey’s post hoc test for multiple comparisons. Differences were considered statistically significant at p < 0.05. All statistical analyses were conducted using the R and RStudio/2023.12.1+402 software (R Foundation for Statistical Computing).

3. Results

3.1. TA Suppresses Ear Swelling in DNFB-Induced Allergic Dermatitis

TA (20 μL) was applied topically at various concentrations (0.1, 0.25, 0.5, and 1.0 mmol/L) to assess its efficacy in reducing DNFB-induced inflammation (Figure 1a). The rats treated with DNFB to induce allergic responses (Figure 1d,e) exhibited significantly redder and more swollen auricles than those treated with the vehicle (Figure 1b,c), confirming the successful induction of allergic inflammation in auricular tissues. In contrast, rats treated with 5 nmol TA one day prior to DNFB application exhibited no swelling, similar to the negative control (Figure 1f,g). Ear thickness measurements demonstrated a statistically significant reduction in swelling in the TA-treated groups compared to that in the DNFB-only group (Figure 1h). Application of TA at concentrations of 5–20 nmol (0.25–1.0 mmol/L) effectively suppressed DNFB-induced allergic inflammation. However, higher concentrations (e.g., 10–100 mmol/L) reduced this effect, suggesting the possibility of saturation or substrate inhibition due to lipase activity (Figure A1). Lower concentrations, such as 0.025–0.1 mmol/L, did not exhibit a significant anti-inflammatory effect, indicating that TA’s efficacy follows a specific concentration-dependent profile. These results suggest that the effect of TA on ear swelling is concentration-dependent, with 0.25–1.0 mmol/L identified as the optimal concentration range to suppress DNFB-induced inflammation, while higher concentrations likely result in reduced efficacy due to substrate inhibition.

3.2. TA Inhibits Eosinophil and Mast Cell Infiltration in DNFB-Induced ACD

Histological analysis of ear sections stained with hematoxylin and eosin and toluidine blue revealed a significant reduction in both eosinophil and mast cell infiltration in the TA-treated groups (Figure 2e,f) compared to the DNFB-only controls (Figure 2c,d). Quantification of inflammatory cells in high-power fields indicated a notable decrease in the number of inflammatory cells in the 5 nmol TA/DNFB group (Figure 2g). Similarly, mast cell infiltration was significantly reduced (Figure 2h), indicating that TA effectively limited the recruitment of immune cells to the site of allergic inflammation. Suppression of cellular infiltration correlated with the observed reduction in ear swelling (Figure 1h), further supporting the ability of TA to mitigate inflammatory responses in ACD.

3.3. TA Inhibits Mast Cell Degranulation

Mast cells play a critical role in the immediate allergic response by releasing histamine and other pro-inflammatory mediators through a process known as degranulation [26]. To evaluate the effects of TA on mast cell stability, we quantified non-degranulated (Figure 3a) and degranulated mast cells (Figure 3b) in ear tissue sections. Mast cell degranulation was markedly reduced in the 5 nmol TA-applicated group compared to that in the DNFB-only controls (Figure 3c), suggesting that TA stabilizes mast cells and prevents the release of inflammatory mediators during allergic responses.

3.4. TA Rapidly Suppressies Allergic Responses

In addition to its long-term anti-inflammatory effects, we determined whether TA suppresses immediate allergic responses. We measured ear thickness and mast cell degranulation 30 min after DNFB application (Figure 4a). Photographic images of the auricular tissues did not demonstrate significant variations among the experimental groups (Figure 4b,d,f). However, TA significantly decreased the ear swelling compared to that in the DNFB-only group at the 30 min time point (Figure 4h). Additionally, histological analysis revealed decreased number of degranulated mast cells in the TA-treated groups at the initial stage of the allergic response (Figure 4i). These findings suggest that TA rapidly suppresses early-phase allergic responses and proinflammatory mediator release.

3.5. TA Is Gradually Degraded in the Auricular Tissues

To assess the breakdown of TA into its metabolite acetate, GC-MS was performed at various time points (0, 0.5, 1, 2, and 4 h) post-application. TA underwent gradual hydrolysis on the skin surface, thereby providing a continuous supply of acetic acid. At 2 and 4 h post-application, approximately 20 and 30% of the applied TA were degraded, respectively (Figure 5). This suggests that TA is efficiently broken down by skin enzymes that are involved in lipase activity [27,28]. Acetate production could not be directly measured owing to its volatility, making accurate quantification challenging. However, TA hydrolysis presumably generates acetate. Acetate activates AMPK and modulates inflammatory responses [19,20,22], consistent with the observed therapeutic effects of TA. Therefore, although acetate measurement was not possible, our findings suggest that TA breakdown into acetate contributed to AMPK activation and subsequent alleviation of allergic inflammation.

3.6. TA Activates AMPK

To investigate the molecular mechanisms underlying the anti-inflammatory effects of TA, we examined AMPK activation in ear tissues. Western blotting analysis revealed increased pAMPK levels in the TA-treated groups (Figure 6a,b). AMPK activation inhibited the proinflammatory pathways, including those involved in mast cell degranulation, explaining the ability of TA to suppress allergic responses. These findings suggested that TA exerts its anti-inflammatory effects partly by activating the AMPK pathway. Immunohistochemistry of ear tissues was performed to determine the pAMPK levels in mast cells. In the DNFB-treated group, mast cells (tryptase-positive) showed reduced pAMPK staining compared to that in the control group, indicating suppressed AMPK activation during allergic responses. However, in the group pretreated with TA followed by DNFB treatment, pAMPK staining in the mast cells was restored to levels similar to those observed in the negative control group (Figure 6c). This suggests that TA maintains AMPK activation in mast cells, facilitating their stabilization and inhibiting their degranulation. Overall, both Western blotting and immunohistochemistry findings confirm our hypothesis that the anti-inflammatory effects of TA are closely associated with AMPK activation, particularly in mast cells, which play key roles in allergic responses.

3.7. Effects of AMPK Inhibition on the Anti-Inflammatory Activity of TA

To further elucidate the role of AMPK in mediating the effects of TA, the effects of AICAR, an AMPK activator, on DNFB-induced allergic responses were assessed (Figure 7a). AICAR effectively suppressed DNFB-induced allergic inflammation, as indicated by the significantly decreased ear swelling and mast cell degranulation (Figure 7b,c). These findings confirm that AMPK activation attenuates allergic inflammation, highlighting the crucial role of AMPK in controlling immune responses in ACD [25]. To further verify the involvement of AMPK in the action mechanisms of TA, we used Dor to block AMPK activation. Dor was subcutaneously administered prior to DNFB application, and ear thickness was measured 24 h post-treatment (Figure 7a). Dor significantly inhibited the anti-inflammatory effects of TA, increasing the ear swelling and mast cell degranulation compared to that in the TA-treated groups (Figure 7b,c). These findings suggest that the anti-inflammatory effects of TA are largely dependent on AMPK activation, as indicated by the reversal of its therapeutic effects upon AMPK inhibition by Dor.

4. Discussion

This study highlighted the dual anti-inflammatory effects of TA mediated by AMPK activation and inhibition of mast cell degranulation in a DNFB-induced ACD model. Our results suggest that TA facilitates both immediate and long-term suppression of allergic reactions, thereby combating the key pathological features of ACD, including inflammation, immune cell infiltration, and mast cell degranulation.

Notably, TA exerted dose-dependent effects on ear swelling and inflammation, with concentrations of 0.25–1.0 mmol/L (5–20 nmol) showing similar anti-inflammatory efficacy (Figure 1). However, high concentrations, such as 25 mmol/L, reduced the anti-inflammatory effects, possibly due to substrate inhibition. Figure A1 shows data from a wider range of concentrations (0.025–100 mmol/L), confirming this trend. This phenomenon, commonly observed with lipid-based substrates such as TA, is possible due to the excess substrate interfering with the lipase activity in the skin by occupying the active sites or disrupting the enzyme efficiency [29]. Therefore, although TA has therapeutic potential, optimizing its dosage is important to ensure maximum efficacy and avoid potential saturation effects. Future studies should explore the balance between TA hydrolysis and acetate release to better understand the pharmacokinetics and optimize the therapeutic dose of TA.

Mast cells are key mediators of allergic responses that release various pro-inflammatory mediators, including histamine, during degranulation. Here, 0.25 mmol/L TA significantly inhibited mast cell degranulation. This suppression of degranulation possibly contributed to the inhibition of immediate (Figure 4i) and delayed allergic responses (Figure 3) because mast cells are critical players in the early phase of ACD. By stabilizing mast cells, TA prevented the release of pro-inflammatory mediators, thereby reducing inflammation and mitigating symptoms, such as swelling and pruritus, throughout the allergic response (Figure 1, Figure 2 and Figure 4). Mast cell degranulation is a key driver of allergic symptoms [26]; therefore, the ability of TA to suppress this process makes it a valuable candidate for ACD treatment.

AMPK activation is a critical mechanism through which TA exerts its anti-inflammatory effects. Our results showed that TA significantly increased pAMPK levels in the ear tissues (Figure 6). AMPK is a well-known regulator of cellular energy homeostasis [21] that modulates inflammatory pathways by inhibiting pro-inflammatory cytokine production and reducing mast cell activity [23]. Previous studies, including Hwang et al. (2013) [25], have demonstrated that AMPK activation inhibits FcεRI-mediated mast cell degranulation by reducing intracellular calcium influx, a critical trigger for degranulation. Additionally, AMPK downregulates key signaling pathways, including MAPKs (ERK, JNK), mTOR, and IKK, which are involved in mast cell activation and cytokine production. Activation of AMPK by TA provides a mechanistic explanation for the observed reduction in inflammation and immune cell infiltration. Furthermore, the use of Dor, an AMPK inhibitor, substantiated the pivotal role of AMPK in the action mechanisms of TA (Figure 7). The anti-inflammatory effects of TA were significantly diminished when Dor was administered, as evidenced by the increased ear swelling (Figure 7b) and elevated levels of mast cell degranulation (Figure 7c). These findings further support the conclusion that AMPK activation is essential for the therapeutic effects of TA on ACD. Future research should explore whether other upstream or downstream pathways of AMPK are involved in the effects of TA, and whether similar outcomes are observed in other models of allergic inflammation.

In addition to its long-term anti-inflammatory effects (Figure 1), TA demonstrated rapid onset of action, significantly suppressing ear swelling and mast cell degranulation within 30 min of DNFB application (Figure 4). This finding suggests that TA not only reduces chronic inflammation, but also has the potential to treat acute allergic reactions, such as those that occur during flares in ACD. Furthermore, the gradual breakdown of TA into acetate in the skin (Figure 5) may provide sustained anti-inflammatory effects, which could be important for preventing ACD flare-ups by maintaining ongoing mast cell stabilization and reducing histamine release. The ability of TA to quickly stabilize mast cells and slowly release acetate makes it a versatile candidate for managing both the acute and chronic phases of allergic skin diseases, and possibly for preventing future reactions. This sustained action may be especially beneficial for patients who experience frequent and severe allergic reactions, offering immediate relief from symptoms such as swelling, itching, and discomfort, while also preventing new episodes. This preventive capacity distinguishes TA from other non-steroidal treatments, which often lack the ability to effectively address both immediate allergic responses and long-term prevention.

Action mechanism of TA, which targets both immediate and long-term allergic responses, highlights its potential as a therapeutic alternative to TCSs. Corticosteroids are the standard of care for ACD [8]; however, their long-term use is associated with side effects such as skin thinning, delayed wound healing, and systemic absorption [10], which can lead to the suppression of the hypothalamic–pituitary–adrenal axis, a key hormonal system that regulates stress responses, immune function, metabolism, and other complications [30]. The emergence of “steroid phobia” among patients further underscores the need for non-steroidal alternatives, such as TA, which offers a safer profile while maintaining efficacy [31].

However, several factors must be considered before TA is recommended as a treatment option for ACD. First, an optimal dosing regimen must be established, as the results of this study indicated that higher concentrations may reduce efficacy due to substrate inhibition. Additionally, long-term safety studies are necessary to assess whether the repeated application of TA to the skin has adverse effects. Although TA is widely used as an excipient in food and cosmetics, its long-term effects, when applied at therapeutic doses to the skin, have not been thoroughly investigated. Such studies are essential to ensure safety and effectiveness in clinical settings.

A limitation of this study is that cytokine and chemokine levels in the auricular tissue were not measured. While we hypothesize that systemic cytokine levels would remain unchanged due to the localized nature of the inflammation, future studies should include an analysis of tissue-specific cytokine and chemokine profiles to further elucidate the molecular mechanisms underlying triacetin’s anti-inflammatory effects. Another limitation of this study is that it could not determine the exact cell types most affected by TA in the skin. Although TA exerts its anti-inflammatory effects primarily through mast cells, other cell types, such as keratinocytes and dendritic cells, may also play key roles in mediating its effects. Keratinocytes are involved in skin barrier functions [32] and cytokine production [33], both of which influence allergic inflammation. Therefore, whether acetate generated from TA enhances keratinocyte functions via AMPK activation, thereby reducing allergen penetration, warrants further investigation. Future studies using isolated MCs and co-culture models with keratinocytes or other skin cells will be essential elucidate whether TA directly targets mast cells or acts indirectly through other pathways.

In addition to AMPK activation, other roles of acetate in immunomodulatory pathways remain unknown. Acetate regulates various metabolic and inflammatory processes. Therefore, its effects on different immune cells, including T cells and macrophages, should be examined to fully elucidate the therapeutic potential of TA.

Finally, long-term safety studies are essential to assess whether repeated application of TA at therapeutic doses causes adverse effects on the skin. Although TA is widely used as an excipient in food and cosmetics, its effects when used therapeutically require further investigation.

5. Conclusions

This study demonstrated the potential of TA as a non-steroidal therapeutic alternative for ACD via AMPK activation and inhibition of mast cell degranulation. Its ability to suppress both immediate and long-term allergic responses makes it a versatile therapeutic, especially for patients seeking TCS alternative. Future studies should further optimize the dose, evaluate the long-term safety, and explore the broader implications of the immunomodulatory effects of TA in other skin conditions.

6. Patents

This study includes compounds for which a patent application is currently pending (Patent Application No. JP2024-030763).