Functional properties and skin care effects of sodium trehalose sulfate

Abstract

Background

It is known that heparinoid, a mucopolysaccharide polysulfate, is effective in improving rough skin and promoting blood circulation as medicines for diseased areas. However, heparinoid has a molecular weight of more than 5000 and cannot penetrate healthy stratum corneum.

Objective

We tested the efficacy of sulfated oligosaccharides with a molecular weight of less than 2000 on the human skin barrier function and moisturizing function.

Methods

We measured the transepidermal water loss (TEWL) of a three‐dimensional human epidermis model cultured for 3 days after topical application of sulfated oligosaccharides, then observed the effects on TEWL suppression. The mRNA levels of proteins involved in intercellular lipid transport and storage in the stratum corneum, and moisture retention were measured using RT‐qPCR.

Results

An increase in the mRNA levels of the ATP‐binding cassette subfamily A member 12 (ABCA12), which transports lipids into stratum granulosum, was confirmed. Increases were also observed in the mRNA levels of filaggrin (FLG), which is involved in the generation of natural moisturizing factors, and of caspase‐14, calpain‐1 and bleomycin hydrolase, which are involved in the degradation of FLG. Antibody staining confirmed that the application of sodium trehalose sulfate to 3D model skin resulted in more ABCA12, ceramide, transglutaminase1, and FLG than those in controls. In a randomized, placebo‐controlled, double‐blind study, participants with low stratum corneum water content applied a lotion and emulsion containing sodium trehalose sulfate to their faces for 4 weeks. Sodium trehalose sulfate decreased the TEWL and increased the stratum corneum water content.

Conclusion

These results suggest that cosmetics containing sodium trehalose sulfate act on the epidermis by increasing barrier factors and moisturizing factors, thereby ameliorating dry skin.

1. INTRODUCTION

The skin has an important barrier function that prevents stimulation from the external environment and evaporation of moisture from inside the body. The main factors maintaining this barrier function are the cornified cell envelope (CE) of stratum corneum cells, natural moisturizing factors (NMFs) produced by the breakdown of proteins such as filaggrin (FLG) and the intercellular lipids of the stratum corneum (Figure 1). The main component of the CE of stratum corneum cells is a protein called involucrin (IVL). In the process of keratinization in the stratum corneum, transglutaminase forms cross‐links (isopeptide bonds) between involucrin and other proteins, thus forming insoluble CE. NMFs are primarily composed of amino acids, and their derivatives are essential for skin moisturization. ATP‐binding cassette subfamily A member 12 (ABCA12) is a critical transmembrane lipid transfer protein in keratinocytes which plays an important role in lamellar granule lipid transport. ¹ Spontaneous ABCA12 mutation has been shown to interfere with lipid transport into lamellar granules, thus causing ichthyosis. ¹ ABCA12 is expressed between the stratum spinosum and stratum granulosum, and this transporter helps to localize and store synthesized intracellular lipids, such as ceramides and fatty acids, within lamellar granules and eventually secretes the lipids stored in the cells into the intercellular space of the stratum corneum. ² Spontaneous mutations in, or other damage to, the ABCA12 gene interfere with intercellular lipid transport and proper lipid storage within the lamellar granules. ¹ Eventually, intercellular lipid stores are diminished, thereby impairing barrier function.

FIGURE 1.

Degradation pathway of FLG to NMFs in the stratum granulosum and stratum corneum. FLG, filaggrin.

NMFs are derived from FLG and are closely associated with skin hydration. FLG is a protein involved in differentiation that aggregates keratin fibers and the structural proteins of the stratum corneum and strengthens its internal structure. ³ FLG consisting of 10−12 FLG units is stored in the stratum granulosum and, after keratinization, is excised by kallikrein‐5 (KLK5) and other proteins, thus yielding individual FLGs. ⁴ When FLG is dephosphorylated, it polymerizes with keratin and forms the internal structure of stratum corneum cells, called the keratin pattern. FLG is degraded by proteolytic enzymes, such as calpain‐1 (CAPN1), caspase‐14 (CASP14) and bleomycin hydrolase (BLMH), thus forming NMFs ⁴ (Figure 1). Recently, abnormalities in the FLG gene have been found to be a major factor in the pathogenesis of ichthyosis vulgaris and atopic dermatitis. ⁵ , ⁶

CASP14, CAPN1 and BLMH are FLG‐degrading enzymes. Low levels of CASP14 are often identified in patients with atopic dermatitis and psoriasis. ⁷ CAPN1, an µ‐calpain, is activated by calcium ions. BLMH is universally present in all tissue, and its highest concentrations are found in the skin. Patients with atopic dermatitis have been reported to have diminished levels of skin BLMH. ⁸

Heparinoid, a mucosulfated oligosaccharide, has a molecular weight of more than 5000 and cannot penetrate healthy stratum corneum. ⁹ It is therefore used in medicine to moisturize and promote blood circulation in the treatment of inflammatory diseases, progressive palmar keratoderma, sebum deficiency (senile xeroderma), chilblains and hypertrophic scars and keloids. ¹⁰ Disaccharide polysulfate, with a molecular weight of less than 1000, can penetrate healthy stratum corneum. ¹¹

The objective of this study was to investigate whether topical application of sulfated oligosaccharides with an average molecular weight of less than 2000, such as sodium trehalose sulfate, sucrose octasulfate K, and nano‐type chondroitin sulfate oligosaccharide, to three‐dimensional reconstructions of the human epidermis (3D epidermis models) might promote the synthesis of FLG. In addition, the contribution of FLG‐degrading enzymes under increasing NMF doses was assessed by measuring the mRNA levels of these enzymes and associated factors. We also examined the effectiveness of the brightening function of disaccharide polysulfate on the skin.

2. MATERIALS AND METHODS

2.1. Chemical substances used in the test

The suppliers of the sulfated oligosaccharides used in the experiment are as follows: sodium trehalose sulfate (Japanese Standards of Quasi‐drug Grade; Fujimoto Chemicals Co., Ltd., Osaka, Japan); sucrose octasulfate K (Japanese Standards of Pharmacopoeia Grade; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan); and nano‐type chondroitin sulfate oligosaccharide (Reagents Grade; Funakoshi Co., Ltd., Tokyo, Japan). Trehalose without sulfonic groups was used for reference (Japanese Standards of Quasi‐drug Grade; Hayashibara Co., Ltd., Okayama, Japan). The chemical structures are shown in Figure 2.

FIGURE 2.

Basic structure of low‐molecular‐weight sulfated polysaccharides.

2.2. Measurement of transepidermal water loss (TEWL) and quantification of mRNA in 3D human epidermis models

2.2.1. 3D epidermis models

Human 3D epidermis models (LabCyte EPI‐MODEL) were purchased from Japan Tissue Engineering Co. Ltd. (Aichi, Japan). Ethical approval and consent for the use of the human 3D epidermis models are not required in accordance with local/national guidelines. The aluminum‐wrapped 3D epidermis models in cups fixed to agar plates were transported at room temperature, transferred to Falcon 24‐well tissue culture plates containing 0.5 mL of LabCyte EPI‐MODEL exclusive medium and used for experiments starting the following day. An agarose plate (prepared at the time of shipment of the 3D epidermis models) was placed in an incubator at 32°C in a room with a temperature of 22°C and humidity of 40%. The LabCyte EPI‐MODEL is a human 3D cultured epidermis model in which normal human epidermal cells are cultured in layers and it can be used to evaluate the toxicity and irritation associated with test substances. Because the cells retain metabolic activity, the model can be used for dermatological research, such as measuring the production of inflammatory factors and cell growth factors, and for pharmacological research on various drugs. ¹² , ¹³ , ¹⁴ , ¹⁵

2.2.2. Application of sulfated oligosaccharides to the 3D epidermis models

Ten milligrams of sulfated oligosaccharides were weighed and dissolved in 2 mL of ultrapure water to prepare an aqueous solution of 5 mg/mL sulfated oligosaccharides. Ultrapure water was used alone as a control. The aqueous solution and control water were sterilized using a sterile filter. Twenty microliters of the prepared aqueous solutions were topically applied to the 3D epidermis models (n  = 3), then incubated at 37°C for 3 days.

2.2.3. Measurement of TEWL

The amount of TEWL was measured using a Tewameter (Courage and Khazaka Electronic GmbH, Köln, Germany) at 25°C and 28%, as previously described. ¹² , ¹⁴

2.2.4. RNA extraction and mRNA quantification using real‐time PCR with the 3D epidermis models

RNeasy Protect Mini Kit (Qiagen N.V., Venlo, Netherlands) was used for RNA extraction and analysis was carried out using a One‐Step SYBR PrimeScript RT‐PCR kit II (Takara Bio Inc. Ltd., Shiga, Japan), Quant Studio 5 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the ΔΔCt method. Primers for IVL, TGM1, ABCA12, FLG, CASP14, CAPN1, BLMH and the housekeeping gene ACTB (Qiagen N.V.) were used. Experiments were performed three times.

2.3. Immunohistochemical staining of the 3D epidermis models

2.3.1. Culture of the 3D epidermis models

Twenty microliters each of the 5 mg/mL aqueous solution of filter‐sterilized sodium trehalose sulfate and ultrapure water (control) was topically applied to the 3D epidermis models (n  = 3) and incubated at 37°C for 3 days.

2.3.2. Culture of the 3D epidermis models

Embedding agent for frozen tissue section production (Tissue‐Tek O.C.T. Compound; Sakura Finetek Japan Co., Ltd., Tokyo, Japan) was added to an embedding dish. After being completely frozen, the samples were thinly sliced to 10 µm using a cryostat (Leica Biosystems Nussloch GmbH, Wetzlar, Germany) and collected on glass slides.

2.3.3. Immunofluorescence of TGM1, ABCA12, ceramide, and FLG

Human anti‐TGM1 rabbit polyclonal antibody (Proteintech Group, Inc., Rosemont, IL, USA), anti‐ABCA12 polyclonal antibody, Alexa Fluor 488 conjugated (Bioss Inc., Boston, MA, USA), anti‐ceramide antibody (Glycobiotech GmbH, Hamburg, Germany), and human anti‐FLG rabbit polyclonal antibody (Atlas Antibodies AB, Bromma, Sweden) in PBS solution were added to the tissues placed on glass slides and incubated for 1 h. After the samples were washed with PBS, the secondary antibody (Alexa Fluor 488 goat anti‐rabbit IgG (H+L); Thermo Fisher Scientific Inc.) in PBS solution was added and allowed to stand for 1 h. After a washing step, nuclear staining was performed using DAPI (1000‐fold dilution; Dojindo Laboratories, Kumamoto, Japan) for 5 min. Each sample was washed with pure water and drained. The samples were observed and photographed under an inverted fluorescence microscope (IX70, Olympus Corporation, Tokyo, Japan). The experiment was performed on three tissue sections.

2.4. Effects of topical application of disaccharide polysulfate solution on the amounts of free amino acids in the 3D epidermis models

2.4.1. Topical application of disaccharide polysulfate solution on the 3D epidermis models

The 3D epidermis models (24‐well, Japan Tissue Engineering Co., Ltd.) were cultured in dedicated medium for 1 day in an incubator. The medium was changed, 50 µL of aqueous solutions of disaccharide polysulfate (0.05%, 0.1% and 0.2%) were applied, and the cells were cultured for 3 days.

2.4.2. Measurement of free amino acids and total amino acids

The 3D epidermis models were washed twice with PBS, and then 500 µL of 10 mM HCl (550‐fold dilution of 20% HCl) was added to the epidermis model for 24 h at room temperature to extract free amino acids. Subsequently, 20 µL of the extract or amino acid standard diluted with 10 mM HCl was placed in a 96‐well plate, 80 µL of fluoraldehyde reagent solution (Thermo Scientific) was added, and the amount of free amino acids was measured at 360 nm excitation and 460 nm fluorescence. From the calibration curve, the amount of amino acids in the sample was determined. To correct for variations due to differences in the number of cells analyzed, the amount of free amino acids was normalized to the total amino acid amount. The quantity of total amino acids was determined by adding 1 mL of 6 M KOH and hydrolyzing at 95°C for 24 h.

2.5. Effects of topical application of sodium trehalose sulfate solution on the water content of human stratum corneum and transepidermal water loss (TEWL) in the 3D epidermis models

2.5.1. Effects of topical application of sodium trehalose sulfate solution on the water content of human stratum corneum

Stratum corneum water content was measured using a SKICON‐200EX (Yayoi Co. Ltd., Tokyo, Japan) instrument at high‐frequency conduction at 24°C and 40%. After measurement of the stratum corneum water content, 10 µL of test solution (water, sodium trehalose sulfate 0.05%, 0.1% or 0.2%) or commercially available heparinoid 0.3% lotion (Shinshin Pharmaceutical Co.,Ltd., Toyama, Japan) was topically applied to five locations (1.5 cm × 1.5 cm) on the flexed sides of the forearms of five healthy adults and spread with the side of a pipette. The change in the moisture content of the stratum corneum was measured using a SKICON‐200EX instrument after the product was allowed to naturally penetrate the skin for 15 min, and this was repeated after 30 min.

2.5.2. Effects of topical application of sodium trehalose sulfate solution on TEWL in a 3D epidermis models

The 3D epidermis models (24‐well, Japan Tissue Engineering Co., Ltd.) were cultured in dedicated medium in an incubator for 1 day. Aqueous solutions of sodium trehalose sulfate (0.05%, 0.1% or 0.2%) and commercially available heparinoid lotion (0.3%) were topically applied in 40 µL volumes and incubated for 3 days. TEWL was measured using a Tewameter instrument with a 24‐well culture insert probe as previously described.

2.6. Effects of topical application of lotion and emulsion containing sodium trehalose sulfate on dry skin in humans

The human test was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Kirei Testing Labo Co., Ltd. (Tokyo, Japan; protocol code HENM21083001 and date of approval 2021.10.1). After approval from the ethics committee (approval No. RSIN21100101), we sufficiently explained the aim, details and methods of the study to 26 female participants aged 33–59 years, then obtained their written informed consent to participate in the study. The study was conducted on healthy participants with low stratum corneum water content, high water evaporation and low lipid content. The TEWL, water content in the stratum corneum and lipid content of the skin surface were measured in a room maintained at a constant temperature and humidity, and the participants completed questionnaires regarding the condition of their skin. This was a randomized, placebo‐controlled, double‐blind, parallel comparative study on the topical application of lotions and emulsions, conducted over 4 weeks between November and December. Participants were randomly allocated into one of two groups based on their age, TEWL, water content in the stratum corneum and lipid content of the skin surface measured before topical application, which were used as stratification factors for stratified block randomization. There were 13 participants per group. Both test products (active group: lotion and emulsion containing 0.05% sodium trehalose sulfate; placebo group: lotion and emulsion with no active components) were indistinguishable in their external appearance. Participants applied the lotion and emulsion to their faces in the amounts normally used, twice daily, morning and night, for 4 weeks. No other lotion or emulsion was allowed to be used during the study period. The participants were asked to use their usual cleanser, make‐up remover, lotion and foundation, and were not allowed to switch to other products during the study. After the participants washed their faces in the face‐washing room, they acclimated for 30 min in the waiting room. The temperature in the face‐washing room, waiting room and measurement room was set at 21.8°C ±0.2°C, and the humidity was set at 45.8% ±1.3%.

TEWL was measured on the left cheek using a Tewameter TM300 device (Courage + Khazaka electronic GmbH). The water content in the stratum corneum was measured on the cheek using a SKICON‐200EX instrument. The blood oxygen saturation index (HbSO₂ index (%)) was measured using a CM‐700d spectrophotometer (Konica Minolta Inc., Tokyo, Japan) and CM‐SA skin analysis software. The melanin index was measured using a Mexameter (Courage + Khazaka electronic GmbH) instrument.

2.7. Statistical analysis

For numerical data, t‐tests were performed, and p <0.05 was considered statistically significant.For efficacy evaluation in human trials, the difference between each subject before (W0) and after (W4) topical application of the lotion and emulsion was evaluated via a paired t‐test, and the difference between each measurement in the group with the addition of 0.05% disaccharide polysulfate to the lotion and emulsion (active group) and the group without the addition (placebo group) was evaluated via an unpaired t‐test. Non‐numerical data were subjected to the Wilcoxon signed rank test (W0 vs. W4) or Mann–Whitney's U test (active vs. placebo), with p <0.05 being considered statistically significant.

3. RESULTS

3.1. Inhibitory effect of low‐molecular‐weight sulfated polysaccharides on transepidermal water evaporation (TEWL) in the 3D epidermis models

TEWL was measured 3 days after topical application of low‐molecular‐weight sulfated polysaccharides or solvent solution (control) to human 3D epidermis models. Lower TEWL was observed in the groups treated with sodium trehalose sulfate and sucrose octasulfate K compared with the control group. In contrast, no significant difference in TEWL was observed between the sites treated with nano‐type chondroitin sulfate oligosaccharides and the control sites after 3 days (Figure 3). No effect was observed for non‐sulfated trehalose (Figure 3).

FIGURE 3.

Effect of low‐molecular‐weight sulfated polysaccharides on transepidermal water loss (TEWL) in the 3D epidermis models. n =3, mean ± standard deviation, ^* p <0.05 versus control.

3.2. mRNA quantification of IVL, TGM1, ABCA12, FLG, CASP14, CAPN1, and BLMH in the 3D epidermis models

In the 3D epidermis models, 0.25 mg/mL sulfated polysaccharides solution was topically applied, and the cells were cultured for 3 days. Then, mRNA was extracted, and real‐time PCR was performed. The primers were specific to the differentiation markers IVL and TGM1; the lipid transporter ABCA12; FLG; the FLG‐degradation enzymes CASP14, CAPN1 and BLMH. FLG is responsible for aggregating keratin proteins, the skeletal proteins of the stratum corneum. FLG is degraded into NMFs, which are essential for moisturization.

The results of the quantitative mRNA analysis are shown in Figure 4. All values were normalized to those of ACTB. The mRNA levels of IVL, TGM1, ABCA12 FLG, CASP14, CAPN1, and BLMH were significantly higher in the sodium trehalose sulfate‐treated 3D epidermis models than in the solvent‐treated 3D epidermis models (control). The mRNA levels of TGM1, ABXA12, CAPN1, and BLMH were significantly higher in the sucrose octasulfate K‐treated 3D epidermis models than in the solvent‐treated 3D epidermis models (control). In contrast, the mRNA levels of these enzymes were not significantly higher in the trehalose‐treated epidermis than in the solvent‐treated 3D epidermis models (control).

FIGURE 4.

Effects of topical application of low‐molecular‐weight sulfated polysaccharides on the mRNA levels of IVL, TGM1, ABCA12, FLG, CASP14, CAPN1, and BLMH in the 3D epidermis models. n =3, mean ± standard deviation, ^* p <0.05 versus, control. ^** p <0.01 versus, control. BLMH, bleomycin hydrolase; FLG, filaggrin; IVL, involucrin.

3.3. Immunohistochemical staining of TGM1, ACBA12, ceramide and FLG, in the 3D epidermis models

To examine whether the immunoreactivity of TGM1, ACBA12, ceramide, and FLG were greater in the presence of sodium trehalose sulfate, we used a fluorescent antibodies against TGM1, ACBA12, ceramide, and FLG. The results of TGM1 staining and nuclear are shown below (Figure 5A). ABCA12 staining and nuclear are shown below (Figure 5B). Ceramide staining and nuclear are shown below (Figure 5C). The results of FLG staining and nuclear are shown below (Figure 5D). The fluorescence intensity of immunoreactive TGM1, ACBA12, ceramide, and FLG, were greater close to the stratum corneum in the 3D epidermis models with topically applied sodium trehalose sulfate than in the model with topically applied solvent (control).

FIGURE 5.

Immunoreactive FLG, TGM1, ABCA12 and ceramide in 3D epidermis models with topically applied sodium trehalose sulfate or solvent (control). FLG, filaggrin.

3.4. Free amino acids in the 3D epidermis models with topical application of sodium trehalose sulfate or heparinoid

After the epidermis models had been incubated with sodium trehalose sulfate solution or heparinoid solution for 3 days, the quantity of free amino acids relative to total amino acids was calculated. The groups treated with sodium trehalose sulfate solution at 0.05%, 0.1% or 0.2% showed significantly higher amounts of free amino acids than the control solution groups (Figure 6). Application of 0.3% heparinoid lotion resulted in significantly higher free amino acid levels compared with the control solution (Figure 6).

FIGURE 6.

Free amino acids relative to total amino acids in the 3D epidermis models with topical application of sodium trehalose sulfate solution, heparinoid lotion or solvent solution (control). n =4, mean ± standard deviation, ^* p <0.05 versus, control. ^** p <0.01 versus, control culture.

3.5. Stratum corneum water content and TEWL after topical application of sodium trehalose sulfate solution

At 15 and 30 min after sodium trehalose sulfate solution at 0.05%, 0.1% or 0.2% or solvent solution (control) was topically applied to human skin, the water content of the stratum corneum was significantly greater at the sites treated with the sodium trehalose sulfate solution than at the control sites. In contrast, the water content of the stratum corneum at sites treated with 0.1% and 0.3% heparinoid lotion was significantly higher than that at the control‐treated sites after 15 min, but no significant difference was observed between the heparinoid lotion‐treated and control‐treated sites after 30 min (Figure 7A).

FIGURE 7.

Increase in stratum corneum water content (A) and decrease in TEWL (B) after topical application of sodium trehalose sulfate solution. n =5, mean ± standard deviation, ^* p <0.05 versus, control. ^** p <0.01 versus, control. TEWL, transepidermal water loss.

TEWL was measured 3 days after topical application of 0.05%, 0.1% or 0.2% trehalose sulfate solution, 0.3% heparinoid lotion, or solvent solution (control) to the 3D epidermis models. TEWL was lower in the treatment groups than in the control group. In contrast, after 3 days, no significant difference in TEWL was observed between the 0.3% heparinoid lotion‐treated sites and the control‐treated sites (Figure 7B).

3.6. Stratum corneum water content and TEWL after topical application of sodium trehalose sulfate solution

Two placebo group members dropped out during the study period and were excluded from the analysis because of a lack of data from the 4 week measurements. Thus, 13 participants in the active group and 11 participants in the placebo group were included in the statistical analysis. The average ages in the active and placebo groups were 44.7 ±9.8 years (mean ± S.D.) and 45.4 ±9.2 years (mean ± S.D.), respectively. No significant differences were observed between the active and placebo groups in terms of age, TEWL, stratum corneum water content, blood oxygenation index or melanin index prior to topical application.

Comparison of the changes in TEWL before and after application (ΔTEWL (W4‐W0)) indicated a significant improvement in TEWL in the active group compared with the placebo group (Figure 8A). The amount of water in the stratum corneum increased significantly in the active group after 4 weeks of daily application, but no significant difference was observed before and after daily use in the placebo group. A significant difference (Δ stratum corneum water content (W4‐W0)) between the active and placebo groups was observed (Figure 8B). The blood oxygenation index increased significantly in the active group after 4 weeks of daily use, but no significant difference was observed before and after daily use in the placebo group. A significant difference in HbSO₂ (Δ HbSO₂ (W4‐W0)) between the active and placebo groups was observed (Figure 8C). The melanin index de‐creased significantly in the active group after 4 weeks of daily use, but no significant difference was observed before and after daily use in the placebo group. A significant difference in the melanin index (Δ melanin index (W4‐W0)) between the active and placebo groups was observed (Figure 8D).

FIGURE 8.

Effects of topical application of lotion and emulsion containing 0.05% sodium trehalose sulfate on dry skin in humans. TEWL (A), water content in the stratum corneum (B), blood oxygenation index (C) and melanin index (D) were analyzed for the groups treated with lotion and emulsion containing 0.05% disaccharide polysulfate (active group) and with the placebo lotion and emulsion (placebo group); n = 13 (active) and n = 11 (placebo). M represents the mean value. TEWL, transepidermal water loss.

4. DISCUSSION

Among the low‐molecular‐weight sulfated oligosaccharides examined in this study, sodium trehalose sulfate and sucrose octasulfate K were found to suppress TEWL and increase TGM1, ABCA12, CASP14, CAPN1, and BLMH mRNA levels in a 3D human epidermis model. In particular, sodium trehalose sulfate was also found to increase mRNA levels of IVL and FLG. On the other hand, nano‐type chondroitin sulfate oligosaccharide and unsulfated trehalose did not show these effects. These results suggest that sulfated oligosaccharides with molecular weights below 1300 were effective in promoting the stratum corneum barrier function and that sodium trehalose sulfate, which has a smaller molecular weight, was more effective. In the experiments in 3D epidermis models, IVL and TGM1 mRNA levels increased after the topical application of sodium trehalose sulfate. The cells of the stratum spinosum differentiate into the stratum granulosum and eventually into the stratum corneum. In the stratum corneum, proteins such as IVL and loricrin are cross‐linked by TGM1 to form a robust CE, which plays a role in the skin barrier function. The stratum granulosum expresses IVL and TGM1. The expression of loricrin is confined to the stratum granulosum of normal human epidermis. The expression of loricrin in human skin did not change after barrier disruption, but the expression of IVL, which is restricted to the stratum granulosum and upper stratum spinosum in normal human skin, has been reported to extend to the lower stratum spinosum 24 h after acetone treatment. ¹⁶ Thus, the application of sodium trehalose sulfate induces the expression of IVL and TGM1 in the stratum granulosum and upper spinous layers, thereby promoting differentiation. Antibody staining confirmed that the application of sodium trehalose sulfate to 3D model skin resulted in more ABCA12, ceramide, TGM1, and FLG than those in controls.

In the human tests, the increase in stratum corneum water content resulting from topical application of sodium trehalose sulfate is thought to be due to the promotion of FLG biosynthesis and FLG degradation in the human epidermis, as suggested by increased mRNA levels of FLG and its degrading enzymes in the 3D epidermis models, as well as an increase in FLG protein confirmed by immunofluorescence. The improvement in TEWL after topical application of sodium trehalose sulfate might be due to enhanced lipid transport into the lamellar granules via ABCA12, given the increase in ABCA12 expression.

Furthermore, immunofluorescence staining indicated that topical application of sodium trehalose sulfate increased the fluorescence intensity and area of FLG protein expression in the 3D epidermis models. As the stratum corneum and stratum granulosum decreased, FLG also decreased, thus diminishing the fluorescence intensity and area of FLG protein expression in the control 3D epidermis models. FLG degradation is initiated via partial proteolysis through one or more unknown proteases deep in the stratum corneum. Furthermore, cleavage via CASP14 efficiently degrades FLG fragments into free amino acids which contribute to the production of NMFs. ¹⁷ The proteolytic activation of CASP14 is associated with stratum corneum formation, thus implicating CASP14 in terminal keratinocyte differentiation and cornification. ¹⁸ , ¹⁹ When sodium trehalose sulfate was applied topically in the 3D epidermis models, the mRNA level of CASP14 increased, and its activity increased in the stratum granulosum and stratum corneum. Increased mRNA levels of KLK7, which is involved in the maturation of CASP14, ²⁰ suggested that sodium trehalose sulfate increased the activity of CASP14 by promoting the final differentiation of the epidermis. CAPN1 is also involved in the degradation process of FLG because topical administration of sodium trehalose sulfate to a 3D epidermal model increased CAPN1 mRNA and CAPN1 activity. Calcium ions are required for CAPN activity, ²¹ and accelerated differentiation increases CAPN1 activity in the stratum granulosum and stratum corneum. The increase in calcium ions might have contributed to the increase in CAPN1 activity. In addition, the mRNA level of BLMH, ²² which is thought to be involved in the degradation of deiminated FLG to amino acids, was significantly increased by using sodium trehalose sulfate treatment. Activity staining indicated that BLMH localized to the stratum corneum.

A comparison of cultured keratinocytes with or without siRNA knockdown of ABCA12 indicated granule staining only in the cells without knockdown; therefore, inhibition of ABCA12 decreases granule staining. ²³ Because ABCA12 is involved in lipid transport, ²³ suppression of the ABCA12 gene inhibits normal lipid transport to the lamellar granules, resulting in a loss of stratum corneum intracellular lipids. Therefore, we infer that, in the 3D epidermis models cultured in sodium trehalose sulfate, ABCA12 increased and, finally, the lipid content in the lamellar granules increased.

Although the degree of keratinization varies depending on the area, ²⁴ this article confirms its effectiveness through an efficacy experiment using the face, which is usually used for skin care. Whether it is effective for senile xeroderma of the shins, which is caused by a lack of sebum, is not known in this experiment.

Heparinoid with a molecular weight of 5000 is effective in improving rough skin and as a medicine to promote blood circulation for diseased areas. However, sodium trehalose sulfate, a low‐molecular‐weight sulfated disaccharide with a molecular weight of less than 1000, was found to improve the barrier function of the stratum corneum and moisturizing indicating that it is very useful as an active ingredient in cosmetic products for dry skin.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

ETHICAL APPROVAL AND CONSENT PROCEDURE

The 3D epidermis models used in this study were purchased from Japan Tissue Engineering Co. Ltd., respectively. Ethical approval and consent for the use of these cells are not required in accordance with local/national guidelines. The human study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Kirei Testing Labo Co., Ltd. (protocol code HENM21083001 and date of approval 2021.10.1). Informed consent was obtained from all participants involved in the study. Written informed consent has been obtained from the participants to publish this paper.

Maeda K, Zhou Z, Guo M, Zhang J, Chen L, Yang F. Functional properties and skin care effects of sodium trehalose sulfate. Skin Res Technol. 2024;30:e13666. 10.1111/srt.13666

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.