Fetal bovine serum (FBS; cat. no. SH30919.03) and penicillin/streptomycin (P/S; cat. no. SH40003.01) were purchased from Hyclone (Logan, UT, USA). Trypsin-ethylenediaminetetraacetic acid (cat. no. 15400-054) was purchased from Gibco BRL (Gaithersburg, MD, USA). Phosphate-buffered saline (PBS; cat. no. LB 001-02) and Dulbecco's Modified Eagle Medium (DMEM; high glucose, cat. no. LM 001-05; low glucose, cat. no. LM 001-11) were supplied by Welgene (Daegu, Korea). Anti-β-actin antibody (cat. no. A5441), anti-DNP IgE antibody (cat. no. D8406), p-nitrophenyl-N-acetyl-β-D-glucosaminide (cat. no. N9376), Tween-20 (cat. no. P2287), sucrose (cat. no. S1888), polyvinylidene fluoride (PVDF) membrane (cat. no. IPVH00010), dimethyl sulfoxide (DMSO; cat. no. D2650), LY294002 (cat. no. 440202), SB203580 (cat. no. S8307), and PD98059 (cat. no. P215) were obtained from MilliporeSigma (St. Louis, MO, USA). Recombinant human keratinocyte growth factor (rhKGF; cat. no. 251-KG), recombinant human epidermal growth factor (rhEGF; cat. no. 236-EG) (purity >97%), Recombinant human tumor necrosis factor-α (rhTNF-α; cat. no. 210-TA), and anti-ICAM-1 antibody (cat. no. BAF 796) were acquired from R&D Systems (Minneapolis, MN, USA). The EZ-CyTox kit (cat. no. EZ-3000) and bovine serum albumin (BSA; cat. No. A0100-010) were supplied by DoGenBio (Seoul, Korea) and GenDEPOT (Katy, TX, USA), respectively. Type I collagen (rat tail; cat. no. 354236) was sourced from Corning Life Sciences (Corning, NY, USA). The 5-bromo-2′-deoxyuridine (BrdU) kit (cat. no. 11669915001) and streptavidin-peroxidase (POD) conjugate (cat. no. 11089153001) was obtained from Roche (Indianapolis, IN, USA). DNP-BSA (cat. no. A23018), monoclonal anti-type I and IV collagen antibodies (cat. no. MA1-26771 and cat. no. MA1-22148, respectively) and chemiluminescent substrate (cat. no. 37069) were obtained from Thermo Scientific (Waltham, MA, USA). The antibodies for phospho Syk (cat. no. 2711), ERK1/2 (cat. no. 9102), phospho ERK1/2 (cat. no. 9101), p38 MAPK (cat. no. 9212), phospho p38 MAPK (cat. no. 9211), JNK (cat. no. 9258), phospho JNK (cat. no. 4668), PI3K (cat. no. 4292), phospho PI3K (cat. no. 4228), AKT (cat. no. 9272), phospho AKT (cat. no. 9271), horseradish peroxidase conjugated rabbit immunoglobulin G (cat. no. 7074), and horseradish peroxidase conjugated mouse immunoglobulin G (cat. no. 7076) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). RIPA buffer (cat. no. 9806) was also purchased from the same company. The antibodies for Syk (cat. no. ab3993), VAMP7 (cat. no. ab36195), VAMP8 (cat. no. ab76021), Syntaxin 1a (cat. no. ab170890), Syntaxin 4 (cat. no. ab184545), SNAP 25 (cat. no. ab109105), and polyclonal anti-type I and IV collagens (cat. no. ab6577 and cat. no. ab6581, respectively) were obtained from Abcam (Cambridge, UK). The antibodies for SNAP 23 (cat. no. sc-166244) and filaggrin (cat. no. sc-66192) were supplied by Santa Cruz Biotechnology (Dallas, TX, USA). Anti-HAS-2 (cat. no. NBP2-37446) and anti-HAS-3 (cat. no. NBP1-86328) antibodies were purchased from Novus Biologicals (Littleton, CO, USA). n-Hexane (cat. no. 4081-4110) and ethanol (cat. no. 4204-4410) were obtained from DAEJUNG (Siheung, Korea) and piceatannol was from TCI (Tokyo, Japan).

The flowers of Paulownia tomentosa were harvested in Daepyeong-ri, Gwangdeok-myeon, Cheonan, Korea (36.678200°N, 127.161120°E). Plant collection was conducted in accordance with the guidelines of the Korea Forest Service (Daejeon, Korea). Paulownia tomentosa is not classified as a protected or endangered species. Its conservation status is listed as ‘Least Concern’ in the IUCN Red List (26). It is classified not included in any protected or threatened category in national databases, including the Korean Red Data Book of Vascular Plants (27). The plant material was taxonomically authenticated by Dr. Hyun-Jun Kim at the Forest Medicinal Resources Research Center (Seoul, Korea). The voucher specimen (No. PCU-0001) has been deposited in publicly accessible herbaria at the Korea Essential Oil Resource Research Institute, Hoseo University (Asan, Korea), and in the Korea Forest Plants Essential Oil Bank of the National Institute of Forest Science (Seoul, Korea). The flower essential oil of Paulownia tomentosa was obtained as an absolute by organic solvent extraction. Briefly, 4 kg of Paulownia tomentosa flowers were subjected to extraction with n-hexane to obtain a concrete, which was then dissolved in ethanol. The solvent was evaporated, leaving 5 g of the absolute type of essential oil. The resulting absolute type of essential oil was a yellow, viscous liquid, with a final yield of 0.125% (w/w). The extracted absolute type of essential oil was stored at −80°C until further use. For subsequent cell-based experiments, the PTFAb sample was dissolved in DMSO, to prepare a stock solution before treatment. The final concentration of DMSO was adjusted to be identical in all experimental groups, including the PTFAb-treated and control groups. For RBL-2H3 cell experiments, the final DMSO concentration was maintained at 0.1%, whereas for HaCaT cell experiments, it was maintained at 0.5%. The corresponding DMSO concentrations were used in the control groups as vehicle controls.

PTFAb analysis was conducted by NICEM (the National Instrumentation Center for Environmental Management, Seoul National University, Seoul, Korea). Its components were identified by GC/MS analysis using a TRACE 1310 GC unit coupled to an ISQ LT single-quadrupole mass spectrometer (Thermo Scientific, Waltham, MA, USA), as reported elsewhere (28). Briefly, the derivatized samples were subjected to separation on a DB-5MS column (60 m × 0.25 mm, 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) at a constant flow rate of 1 ml/min using the following program: 50°C for 5 min, 50 to 65°C at 10°C/min, 65 to 210°C at 5°C/min, 210 to 310°C at 20°C/min, and 310°C for 10 min. The mass spectra were acquired in the range of m/z 35 to m/z 550 at 0.2 scans/sec. The transfer line and ion source temperatures were 300°C and 270°C, respectively. The detected compounds were identified by comparing the mass spectra and retention indices (RIs) with reference standards in the NIST/NIH/EPA mass spectral library (NIST 11, version 2.0 g) and by matching the retention times and spectra with those of commercially available standards. A solution of C₇-C₃₀ n-alkanes was used as the standard to calculate the RIs.

RBL-2H3 cells (rat basophilic leukemia; cat. no. 22256), which were commonly used as a mast cell model (29,30), were obtained from the Korea Cell Line Bank (Seoul, Korea) and the HaCaT human keratinocyte cell line (Cellosaurus CVCL_0038) was obtained from the National Institute for Korean Medicine Development (Gyeongsan, Korea). According to the institute, the cells were originally sourced from Keimyung University (Daegu, Korea), which had purchased them from CLS Cell Lines Service (Cytion; cat. no. 300493, Eppelheim, Germany), the official DKFZ-approved supplier (31,32). Cells were maintained under standard culture conditions. Their identity and integrity were routinely confirmed by morphological assessment and monitoring of growth characteristics. All cultures were regularly tested for mycoplasma contamination, and cells between passages 28 and 35 were used in experiments. The cells were cultured in DMEM (Welgene Inc., Daegu, Korea) supplemented with 10% FBS (HyClone, Logan, UT, USA) and 1% P/S (HyClone) at 37°C in a humidified incubator containing 5% CO₂.

The RBL-2H3 and HaCaT cells viability was assessed using a water-soluble tetrazolium salt (WST) assay with the EZ-CyTox kit (cat. no. EZ-3000; DoGenBio, Seoul, Korea). The cells were seeded at 5×10³ cells per well in 96-well microtiter plates and incubated overnight in a humidified atmosphere containing 5% CO₂ and 95% air at 37°C. Cells were treated with various concentrations of PTFAb for 24 h, while vehicle control cells received the corresponding concentration of DMSO. Finally, 10 µl of EZ-Cytox reagent (cat. no. EZ-3000; DoGenBio, Seoul, Korea) was added, and the plate was incubated at 37°C for 30 min. The absorbance at 450 nm was determined using a multi-well plate reader (Synergy 2; BioTek Instruments, Winooski, VT, USA).

The HaCaT cell proliferation level was evaluated using a DNA synthesis-based BrdU incorporation assay with a BrdU kit (cat. no. 11669915001; Roche, Indianapolis, IN, USA). Briefly, 96-well black flat-bottom plates were coated with type I collagen (0.1 mg/ml), sourced from Corning Life Sciences (Corning, NY, USA), for 30 min, and HaCaT cells were seeded at 2×10³ cells/well and incubated for 12 h. The cells were treated with in PTFAb at the same concentrations described above, with rhEGF (50 ng/ml) as a positive control, and incubated for 36 h. Subsequently, BrdU labeling solution (1:200 dilution with serum-free medium) was added, and the cells were incubated for an additional 12 h at 37°C. After incubation, the cells were fixed at room temperature for 30 min and incubated with peroxidase-conjugated anti-BrdU antibody (1:100 dilution with diluent included in the BrdU kit) for 90 min at room temperature. After the substrate reaction, the luminescence was measured using a luminometer (Synergy 2; BioTek Instruments, USA).

The HaCaT cell migration was evaluated using a 48-well microchemotaxis chamber (cat. no. 866-417-0014; Neuro Probe, Gaithersburg, MD, USA). A polycarbonate membrane with an 8 µm pore size (cat. no. 1220686; GVS Life Science, Bologna, Italy) and pre-coated with type I collagen (0.1 mg/ml) was used. The lower chambers were filled with DMEM containing 0.1% BSA, supplemented with rhEGF (1 ng/ml) as a positive control or PTFAb at various concentrations. After assembling the collagen-coated membrane and the upper chamber, the HaCaT cells were seeded into the upper chamber at a density of 5×104 cells/well in 50 µl of medium. The chamber was incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 3 h and 30 min. After incubation, the membrane was removed from the chamber, and the migrated cells were fixed and stained using a Diff-Quick solution (cat. no. 38721; Sysmex Corporation, Kobe, Japan). The migrated cells were visualized under an optical microscope at ×200 magnification, and their number was quantified by manual counting.

HaCaT cells were seeded at a density of 5×10⁵ cells per 100-mm dish and incubated for 12 h at 37°C in a humidified atmosphere containing 5% CO₂. After incubation, the culture medium was removed, and the cells were washed with PBS. The cells were then treated with PTFAb at the indicated concentrations and further incubated for 48 h under the same conditions. The conditioned medium (CM; supernatants) was collected and centrifuged at 500, 800, and 1,000 × g for 10 min each to obtain the supernatant. In parallel, the cells were washed with PBS, lysed using a lysis buffer, and the total protein concentration was determined using a DC protein assay kit (cat. no. 500-0116; Bio-Rad Laboratories, Hercules, CA, USA). For collagen quantification, 96-well black flat-bottom plates (cat. no. 655090; Greiner Bio-One, Kremsmünster, Austria) were coated with monoclonal antibodies against type I and type IV collagen (cat. no. MA1-26771 and cat. no. MA1-22148, respectively; Thermo Scientific, Waltham, MA, USA) at 2 µg/well in 100 µl and incubated at 4°C for 12 h. After washing with PBS, the plates were blocked with PBS containing 1% BSA and 5% sucrose for 1 h at room temperature. The collected CM was then added to each well and incubated for 90 min at room temperature. After washing with PBS, biotinylated polyclonal antibodies against type I and type IV collagen (cat. no. ab6577 and cat. no. ab6581, respectively; Abcam, Cambridge, UK), diluted 1:2,000, were added and incubated for 90 min at room temperature. The wells were then washed and incubated with POD conjugate (Roche, Indianapolis, IN, USA) diluted 1:5,000 for 1 h. After a final wash with PBS, chemiluminescent substrate solution (Thermo Scientific) was added, and the luminescence was measured using a luminometer (Synergy 2; Bio-Tek Instruments).

The RBL-2H3 cells were seeded at a density of 3×10⁵ cells per 100-mm culture dish and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 12 h. After incubation, the culture medium was removed, and the cells were washed with PBS. The cells were then treated with various concentrations of PTFAb and incubated at 37°C in an atmosphere containing 5% CO₂ for 48 h. The expression of SNARE proteins, including VAMP7, VAMP8, syntaxin 1a, syntaxin 4, SNAP 23 and SNAP 25, was analyzed by immunoblotting.

The RBL-2H3 cells were seeded in 24-well plates at a density of 1.5×10⁴ cells per well and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 12 h. After incubation, the culture medium was removed, and the cells were washed with PBS. The cells were then treated with the indicated PTFAb and incubated for 48 h at 37°C with 5% CO₂. Anti-DNP IgE (cat. no. D8406; MilliporeSigma, St. Louis, MO, USA) was then diluted in DMEM containing 10% FBS and added to the cells at a final concentration of 200 ng/ml, followed by incubation for 10 h at 37°C in a humidified atmosphere containing 5% CO₂. After sensitization, the cells were washed with Siraganian buffer (cat. no. BS067; Biosolution, Seoul, Korea). Degranulation was induced by stimulation with DNP-BSA, diluted in Siraganian buffer to a final concentration of 20 ng/ml, and incubated in a humidified atmosphere containing 5% CO₂ for 1 h at 37°C. The culture supernatants were collected and centrifuged sequentially at 500, 1,000, and 10,000 × g for 10 min each to obtain CM. In parallel, cells were washed with PBS and lysed with a lysis buffer to extract the intracellular proteins, which were quantified using a DC protein assay.

For the β-hexosaminidase release assay, 50 µl of the collected supernatant was mixed with 100 µl of substrate buffer containing 2 mM 4-p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.05 M sodium citrate buffer (pH 4.5) and incubated at 37°C for 3 h. The reaction was quenched by adding 100 µl of a stop solution (0.2 M glycine-NaOH, pH 10.0), and the absorbance was measured at 405 nm using a multi-well plate reader (Synergy2; Bio-Tek Instruments).

Histamine release was quantified from the same conditioned medium using a histamine enzyme-linked immunoassay (EIA) kit (cat. no. A05890; Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer's instructions.

The RBL-2H3 cells were seeded at a density of 8×10⁵ cells per 60-mm culture dish and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 12 h. The culture medium was removed, and the cells were washed with PBS. The cells were then sensitized with Anti-DNP IgE (cat. no. D8406; MilliporeSigma) diluted in DMEM containing 10% FBS to a final concentration of 100 ng/ml and incubated for 12 h at 37°C with 5% CO₂. After sensitization, the IgE-containing medium was removed, and the cells were washed with PBS. The cells were then treated with various concentrations of PTFAb and incubated for 1 h at 37°C. The medium was then removed, and the cells were washed with Siraganian buffer. Degranulation was induced by a treatment with DNP-BSA diluted in Siraganian buffer to a final concentration of 20 ng/ml for 10 min. After stimulation, the cells were lysed using RIPA buffer (Cell Signaling). The lysates were centrifuged at 17,000 × g for 15 min at 4°C to obtain the cell lysates. The protein concentrations were determined using a DC protein assay, and the levels of Syk, MAPKs, and PI3K/AKT phosphorylation were analyzed by immunoblotting.

HaCaT cells were seeded at a density of 1×10⁶ cells per 100-mm dish and incubated for 12 h at 37°C in a humidified atmosphere containing 5% CO₂. After incubation, the culture medium was removed, and the cells were washed with PBS. Except for the negative control group, the cells were treated with different PTFAb concentrations dissolved in culture medium containing TNF-α (5 ng/ml). The control group was treated with the culture medium without TNF-α. The cells were then incubated for an additional 24 h at 37°C in a 5% CO₂ incubator. Filaggrin protein expression was analyzed by immunoblotting.

HaCaT cells were seeded at 1×10⁶ cells per 100-mm dish and cultured for 12 h at 37°C in a humidified incubator containing 5% CO₂. After incubation, the culture medium was removed, and the cells were washed with PBS, followed by serum starvation in serum-free DMEM for 6 h. The cells were treated with rhKGF (R&D Systems, Minneapolis, MN, USA) at 20 ng/ml as a positive control, or with the indicated concentrations of PTFAb, and incubated for an additional 12 h at 37°C under 5% CO₂. Expression of hyaluronan synthesis-related proteins was analyzed by immunoblotting.

Immunoblotting analysis was performed as described elsewhere (28). The cells were lysed using RIPA buffer (Cell Signaling) and centrifuged at 17,000 × g for 15 min at 4°C. The total protein concentration in the supernatant was determined using a DC protein assay kit. The proteins (30–60 µg per lane) were fractionated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes at 4°C. The membranes were blocked with 3% skim milk or BSA at room temperature for 2 h. Subsequently, the membranes were washed with PBS containing 0.05% Tween-20 and incubated with the primary antibodies against VAMP7 (dilution, 1:2,000), VAMP8 (dilution, 1:2,000), Syntaxin 1a (dilution, 1:1,000), Syntaxin 4 (dilution, 1:2,000), SNAP 23 (dilution, 1:1000), SNAP 25 (dilution, 1:2,000), phospho p38 MAPK (dilution, 1:1,000), p38 MAPK (dilution, 1:1,000), phospho ERK1/2 (dilution, 1:2,000), ERK 1/2 (dilution, 1:1,000), phospho JNK (dilution, 1:1,000), JNK (dilution, 1:1,000), phospho Syk (dilution, 1:1,000), Syk (dilution, 1:500), phospho PI3K (dilution, 1:1,000), PI3K (dilution, 1:1,000), phospho AKT (dilution, 1:1,000), AKT (dilution, 1:1,000), filaggrin (dilution, 1:500), HAS-2 (dilution, 1:2,000), HAS-3 (dilution, 1:1,000), and ICAM-1 (dilution, 1:1,000) at 4°C overnight. β-actin (dilution, 1:5,000) was incubated at room temperature for 30 min. The membranes were then exposed to horseradish peroxidase conjugated rabbit immunoglobulin G (dilution, 1:2,000) or mouse immunoglobulin G (dilution, 1:2,000) at room temperature for 1 h. β-actin was used as a loading control to confirm equal protein loading and for normalization of protein expression levels. Specifically, the levels of phosphorylated proteins were quantified as the ratio to their corresponding total protein levels. Normalization to β-actin was applied only for the quantification of total protein expression. The protein bands were visualized using a chemiluminescence substrate (cat. no. 2332632; ATTO, Tokyo, Japan) and detected with a chemiluminescence imaging system (LuminoGraph, ATTO, Tokyo, Japan).

All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The data are expressed as the mean ± standard error of the mean (SEM). One-way ANOVA followed by Tukey's post hoc test was used for multiple group comparisons. Results in the present study are based on at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

The chemical composition of PTFAb was analyzed by GC/MS, which identified 13 components (Table I and Fig. 1). Among the identified components, methyl undecanoate (45.63%) had the highest content, followed in order by heneicosane (13.62%), tricosane (13.62%), sesamin (10.33%), 1,4-dimethoxybenzene (5.98%), tetracosane (4.79%), methyl anisate (1.79%), eicosanal (1.36%), 1-octen-3-ol (0.73%), methyl benzoate (0.71%), β-ionone (0.68%), nonanal (0.41%), and 3-oxo-α-ionone (0.35%) (Table I).

The PTFAb test concentration for biological analysis was first determined by observing the changes in cell viability in RBL-2H3 mast cells treated with PTFAb. Treatment with PTFAb (1–200 µg/ml) showed a statistically significant decrease in cell viability compared to the control group at 200 µg/ml, but no significant decrease was observed at 1–150 µg/ml (Fig. 2A). Although there was no significant decrease in cell viability at 150 µg/ml, the observed cell morphology appeared less uniformly rounded and slightly more irregular (Fig. S1). Therefore, the PTFAb test concentration for subsequent biological analysis was ≤100 µg/ml, which did not affect the viability of RBL-2H3 cells. SNARE proteins, such as v-SNAREs (VAMP7 and VAMP8) and t-SNAREs (SNAP 23 and syntaxin 4), form trans-SNARE complexes to promote membrane fusion. These proteins help regulate the mast cell degranulation process and the release of inflammatory mediators, such as histamine and cytokines (4). The influence of PTFAb on the expression of SNARE proteins in RBL-2H3 mast cells was investigated by immunoblotting. PTFAb (0.1–100 µg/ml) concentration-dependently inhibited the expression of the v-SNARE proteins VAMP7 (Fig. 2B and C) and VAMP8 in RBL-2H3 cells (Fig. 2B and D). The expression of VAMP7 and VAMP8 in RBL-2H3 cells decreased significantly after the treatment with PTFAb at 50–100 µg/ml. The reduced expression in VAMP7 and VAMP8 showed a maximum at 100 µg/ml of PTFAb 22.02±8.61% (Fig. 2C) and 20.61±7.40% (Fig. 2D) of the untreated control, respectively. On the other hand, the expression of the t-SNARE proteins (syntaxin 1a, syntaxin 4, SNAP 23, and SNAP 25) did not show significant changes after the treatment with PTFAb at 0.1–100 µg/ml (Fig. S2).

Degranulation of activated mast cells results in the release of β-hexosaminidase and histamine. These mediators are mast cell degranulation markers and have been implicated in the pathogenesis of AD (1,33). The effects of PTFAb (0.1–100 µg/ml) on DNP-BSA (20 ng/ml)-induced β-hexosaminidase and histamine release in anti-DNP IgE (200 ng/ml)-sensitized RBL-2H3 cells were examined to determine if PTFAb inhibits mast cell degranulation. DNP-BSA stimulation increased the levels of β-hexosaminidase (329.09±4.77%) and histamine (193.02±6.64%) compared to the anti-DNP IgE control (Fig. 3A and B, respectively). By contrast, the PTFAb treatment at 1–100 µg/ml significantly reduced these levels in a concentration-dependent manner. In particular, PTFAb exhibited maximum inhibitory activity at 100 µg/ml, reducing β-hexosaminidase and histamine to 146.06±4.34% (Fig. 3A) and 111.63±8.31% (Fig. 3B) of the anti-DNP IgE control, respectively.

During mast cell degranulation and the release of inflammatory mediators, Syk phosphorylation, which accompanies the activation of the IgE receptor FcεRI (Fc epsilon receptor I), plays a key role in the initial signaling (34). PI3K/AKT and MAPK signaling pathways are also involved in this process (35). Thus, the effects of PTFAb on these key signaling molecules in mast cells were examined by observing the phosphorylation of Syk, PI3K/AKT, and MAPKs (p38, ERK1/2, and JNK) induced by DNP-BSA (20 ng/ml) in anti-DNP IgE antibody (100 ng/ml)-sensitized RBL-2H3 cells using immunoblotting to determine how PTFAb affects these key signaling molecules in mast cells. DNP-BSA significantly increased Syk phosphorylation (197.77±13.46%) compared to the anti-DNP IgE control (Fig. 4A and B). On the other hand, treatment with PTFAb at 75 and 100 µg/ml attenuated this level, and the maximum inhibitory effect was observed at 100 µg/ml, reducing Syk phosphorylation to 91.13±5.05% of the anti-DNP IgE control (Fig. 4B). In addition, the DNP-BSA treatment enhanced the phosphorylation levels of PI3K (143.67±17.84%) and AKT (157.02±5.79%) compared to the anti-DNP IgE control (Fig. 4C-E). The PTFAb treatments at 75 and 100 µg/ml and at 50–100 µg/ml significantly attenuated the increased levels of PI3K and AKT phosphorylation, respectively. The maximum inhibitory effects on the phosphorylation of these proteins were observed at 100 and 75 µg/ml, with the PI3K and AKT phosphorylation levels reduced to 60.80±6.24% (Fig. 4D) and 59.42±9.37% of the anti-DNP IgE control group, respectively (Fig. 4E). The total protein levels of Syk, PI3K, and AKT normalized to β-actin are shown in Fig. S3, indicating that the observed effects were not due to changes in total protein expression. On the other hand, PTFAb (0.1–100 µg/ml) did not affect the phosphorylation of MAPKs (p38, ERK1/2, and JNK) induced by DNP-BSA (20 ng/ml) in anti-DNP IgE antibody (100 ng/ml)-activated RBL-2H3 cells (Fig. S4).

To further investigate the involvement of Syk/PI3K and MAPK signaling pathways in the anti-degranulation activity of PTFAb, pharmacological inhibitors targeting Syk, PI3K, p38, and ERK1/2 were evaluated. PTFAb (50 µg/ml), piceatannol (a Syk inhibitor; 30 µM), and LY294002 (a PI3K inhibitor; 0.1 µM) significantly reduced anti-DNP IgE/DNP-induced β-hexosaminidase release (Fig. S5A). Co-treatment of PTFAb with either piceatannol or LY294002 further reduced anti-DNP IgE/DNP-induced β-hexosaminidase release compared with PTFAb treatment alone (Fig. S5B). In contrast, treatment with SB203580 increased anti-DNP IgE/DNP-induced β-hexosaminidase release (Fig. S5A), whereas co-treatment of PTFAb with SB203580 partially attenuated the inhibitory effect of PTFAb on anti-DNP IgE/DNP-induced β-hexosaminidase release (Fig. S5B). PD98059 alone or in combination with PTFAb had little or no effect on anti-DNP IgE/DNP-induced β-hexosaminidase release (Fig. S5A and B).

The cytotoxic effects of PTFAb on HaCaT cells were first assessed using a WST assay to determine its regenerative potential. The WST assay results showed that PTFAb significantly enhanced cell viability at 250 and 500 µg/ml, while concentrations at 1 to 100 µg/ml showed only a slight, non-significant increase (Fig. 5A). Therefore, the entire concentration range (1–500 µg/ml) of PTFAb was used for further studies. BrdU assays showed that HaCaT cells proliferation was increased significantly by PTFAb at 250 and 500 µg/ml, with the maximum increase occurred at 500 µg/ml (143.98±12.34% compared to the untreated control) (Fig. 5B). Furthermore, the cell migration assay results showed that a PTFAb treatment at 100–500 µg/ml significantly promoted the migration of HaCaT cells, peaking at 840.00±52.46% at the 500 µg/ml (Fig. 5C and D).

Collagen synthesis strengthens the integrity of the dermal and basement membranes, a key component of the skin barrier, supporting keratinocyte migration and proliferation (36). The effects of PTFAb on collagen synthesis in HaCaT cells were evaluated using a sandwich enzyme-linked immunosorbent assay (ELISA). Treatment with PTFAb (10–500 µg/ml) significantly increased type I collagen synthesis at 100 and 500 µg/ml, and type IV collagen synthesis at 500 µg/ml. Specifically, PTFAb at 500 µg/ml had the most prominent effect, with synthesis levels reaching 171.45±7.27 and 154.42±7.53% for type I (Fig. 6A) and IV (Fig. 6B), respectively.

A reduction in the filaggrin levels and HASs (HAS-1, 2, and 3)-mediated HA synthesis impairs the structural stability and moisture retention of the skin barrier, contributing to skin barrier compromise of AD (37,38). Therefore, this study investigated the regulatory effects of PTFAb on the expression of filaggrin and HAS in keratinocytes using immunoblotting. FLG protein expression in HaCaT cells was downregulated by a TNF-α treatment to 42.27±13.23% of the untreated control. Nevertheless, treatment with PTFAb at 250 and 500 µg/ml significantly restored FLG expression, which had been suppressed by TNF-α. In particular, 500 µg/ml PTFAb had a maximum recovery effect (196.08±20.47% of the untreated control) (Fig. 7A and B). In addition, this study observed the influence of PTFAb on HAS expression in keratinocytes. The effects of PTFAb on HAS-2 and HAS-3 expression were evaluated at concentrations ranging from 1 to 500 µg/ml (Fig. 7C-E). The PTFAb treatment significantly increased the HAS-2 levels by 377.11±55.21% compared to the untreated control at 250 µg/ml (Fig. 7D). An increase (260.91±47.65% of the untreated control) was also observed at 500 µg/ml, but did not have statistical significance. In contrast, HAS-3 expression was significantly upregulated at 250 and 500 µg/ml, with a maximum effect of 443.78±61.56% at 500 µg/ml (Fig. 7E).

Increased expression of ICAM-1 in keratinocytes by inflammatory stimuli promotes the skin infiltration of immune cells and local inflammatory responses, which may be associated with abnormal skin barrier functions (15). Therefore, the changes in ICAM-1 protein expression were observed after a PTFAb treatment of TNF-α-stimulated keratinocytes to determine if PTFAb can alleviate skin inflammatory diseases through modulation of ICAM-1 expression. As shown in Fig. 8A and B, the TNF-α (10 ng/ml) treatment significantly increased ICAM-1 expression by 179.08±9.70% compared to the untreated control. Nevertheless, the PTFAb treatment significantly reduced the expression levels at 250 and 500 µg/ml. Specifically, the expression was maximally suppressed, reaching 99.81±19.46% of the untreated control (Fig. 8B).