{"paper_id":"346219fa-dc6d-4755-83fb-6731a3e881eb","body_text":"=== R E V I E W   C O M M O N S   M A N U S C R I P T ===\nIMPORTANT:\nManuscripts subm itted to Review Com m ons are peer reviewed in a journal-agnostic way.\nUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor.\nThe identity of the referees will NOT be com m unicated to the authors unless the reviewers choose to sign their report.\nThe identity of the referee will be confidentially disclosed to any affiliate journals to which the m anuscript is transferred.\nGUIDELINES:\nFor reviewers: https://www.reviewcom m ons.org/reviewers\nFor authors: https://www.reviewcom m ons.org/authors\nCONTACT:\nThe Review Com m ons office can be contacted directly at: office@reviewcom m ons.org\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n1 \n \nTrehalose promotes wound healing in vitro by enhancing the migration of human \nkeratinocytes via the VEGF/JNK/PI3K pathway \n \nKeigo Taneda a, Xiuju Dai a, Kenji Watanabe b, Teruko Tsuda a, Hideki Mori a, Ken \nShiraishi a, Yoichi Mizukami b, Yasuhiro Fujisawa a, Jun Muto a# \n \na  Department of Dermatology, Ehime University Graduate School of Medicine, \nShitsukawa, Toon, Ehime, Japan. \nb  Institute of Gene Research, Yamaguchi University Science Research Center, \nYamaguchi, Japan. \n \n#To whom correspondence should be addressed: Jun Muto, Department of Dermatology, \nEhime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791 -0295, \nJapan. \nE-mail: junmuto@m.ehime-u.ac.jp; Tel: +81 (89) 960-5350; Fax: +81 (89) 960-5352 \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n2 \n \nAbstract \nBackground: Trehalose is a naturally occurring disaccharide found in invertebrates but \ncannot be synthesized by vertebrates. We previously reported that h igh-concentration \ntrehalose induces a transient senescent-like state in fibroblasts, leading to cell cycle arrest \nand growth factor secretion via CDKN1A/p21 , and this process promoted keratinocyte \nproliferation, enhancing capillary formation and wound closure in vivo. \nObjective: This study aimed to investigate the effect of trehalose on human keratinocytes. \nMethods: Previously published RNA -seq data of cytokine-untreated samples from our \ngroup of trehalose-treated human keratinocytes were re-analyzed, and an in vitro scratch \nassay was performed using cells treated with mitomycin C. \nResults: The trehalose-treated group exhibited increased wound closure. A significantly \nincreased secretion of vascular endothelial growth factor (VEGF)  was observed  in \nkeratinocytes treated with high-concentration trehalose, which is one of the most crucial \nmolecules inducing angiogenesis in the skin. Significant upregulation of mRNA level and \nprotein secretion of VEGF was confirmed using qPCR and ELISA, respectively. \nFurthermore, treatment with axitinib, a VEGF receptor inhibitor, significantly suppressed \ntrehalose-induced activation of keratinocyte migration . Additionally, the increase in \ntrehalose-induced migration activity was significantly inhibited by the J un N-terminal \nkinase (JNK) inhibitor SP600125 and the PI3K inhibitor LY294002. \nConclusion: Trehalose promotes wound healing via VEGF secretion from keratinocytes \nand the PI3K and JNK pathways. The findings of this study may lead to the development \nof novel therapeutic agents that can alter the wound healing process. \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n3 \n \nKeywords: Keratinocytes, Trehalose, Vascular Endothelial Growth Factor A , Wound \nhealing \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n4 \n \n1. Introduction \nWound healing is a complex and dynamic process that requires the coordinated efforts of \nvarious cellular and molecular mechanisms to restore tissue integrity after injury. This \nimportant biological process usually involves four stages:  hemostasis, inflammation, \nproliferation, and remodeling[1]. After injury, vascular permeability increases, and blood \ncomponents are exuded, leading to platelets being concentrated at the wound site to seal \nthe wound and repair the vascular damage [2]. The subsequent inflammatory phase \ntriggers an immune response to protect against injury and infection. This phase is \ncharacterized by  blood vessel expan sion, increased blood flow, neutrophil and \nmacrophage recruitment, and cytokine production. The next proliferative phase initiates \ntissue regeneration at the wound site. This phase is characterized by  the progression of \nangiogenesis and the proliferation of fibroblasts around the wound , which produce \ncollagen and extracellular matrix components for wound repair. At the same time, \nkeratinocytes proliferate to form new skin that covers the wound surface [3]. The final \nremodeling phase involves remodeling and strengthening the tissue at the wound site. \nNew blood vessels are no longer needed,  and the blood supply to the wound area  is \nreduced [1]. \nSuccessful wound healing requires a process called \"re -epithelialization.\" This \nprocess requires the directional migration of keratinocytes toward the wound center [4]. \nChronic wounds or excessive scarring  can occur when  the wound  healing process is \nimpaired, posing significant clinical challenges. The c-Jun N -terminal kinase (JNK) \npathway is a key signaling pathway involved in wound healing. JNK is a member of the \nmitogen-activated protein kinase (MAPK) family  and plays a critical role in regulating \ncellular responses to stress, inflammation, and apoptosis [5]. The JNK pathway is \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n5 \n \ninvolved in several key processes in wound healing, including regulating inflammatory \nresponses, promoting keratinocyte migration, and re -epithelialization. JNK activation \nenhances keratinocyte migration, which is essential for covering the wound surface, and \npromotes fibroblast activity, which is crucial for extracellular matrix production and \ntissue remodeling [6]. \nSimilarly, the phosphoinositide 3 -kinase (PI3K) pathway  plays a critical role in \nwound healing . PI3K is a family of lipid kinases that plays an important role in the \nregulation of various cellular functions, including cell growth, proliferation, survival, and \nmigration. Upon PI3K activation by growth factors, cytokines, or other extracellular \nsignals, it generates phosphatidylinositol (3,4,5)-trisphosphate, which serves as a docking \nsite for proteins with pleckstrin homology domains, such as Akt (also known as protein \nkinase B) [7, 8] . Akt activation leads to the modulation of downstream targets that \npromote cell survival and growth . Therefore, the PI3K/Akt pathway plays a key role in \ntissue regeneration and repair. Recent studies have highlighted the importance of the PI3K \npathway in wound healing and have shown that the downregulation of PTEN, a negative \nregulator of PI3K/Akt, promotes Akt activation and enhances wound healing [9]. The \nproper regulation of these pathways is essential for efficient wound healing and can be a \npotential therapeutic target, especially in treating chronic wounds. \nTrehalose is a naturally occurring disaccharide consisting of two glucose molecules \nlinked by an α,α-1,1-glycosidic bond. Trehalose is widely found in plants, fungi, bacteria, \nand some invertebrates  and serves as a source of energy and a protective agent under \nstress conditions [10]. Trehalose is unique due to its remarkable stability and ability to \nprotect cellular structures and proteins from damage caused by dehydration [11], freezing \n[12], and oxidative stress [13]. Furthermore, trehalose acts as a bioprotectant , which is \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n6 \n \none of its most remarkable properties  [14]. During extreme environmental conditions \nsuch as drought and freezing, organisms that accumulate trehalose can stabilize their \ncellular membranes and proteins, allowing them to survive and recover once  the \nconditions improve. This protective effect has led to the application of trehalose in various \nfields, including food preservation, pharmaceuticals, and cosmetics, where it enhances \nproduct stability and shelf life [15]. \nThe beneficial effects of trehalose on skin health have been investigated. Trehalose \nis used in skincare products due to its moisturizing and antioxidant properties, which help \nprotect and repair the skin barrier. Trehalose may have potential applications in wound \nhealing and tissue regeneration  due to its ability to stabilize proteins and cellular \nstructures under stress conditions. \nGenerally, trehalose is a versatile molecule with wide-ranging applications from the \nfood and cosmetic industries to potential therapeutic medical uses [16]. Given its unique \nproperties, trehalose has become an important focus of ongoing research aimed at \nharnessing its full potential in various fields. \nIn our previous study, we reported the effects of high -concentration trehalose on \ndermal fibroblasts . Trehalose induce d a transient senescent -like state in fibroblasts, \nleading to cell cycle arrest and growth factor secretion via CDKN1A/p21 [17]. This \nprocess promoted keratinocyte proliferation in living skin equivalent in vitro, enhancing \ncapillary formation and wound closure in vivo. Therefore, this study aimed to investigate \nthe effects of trehalose on human keratinocytes , specifically assessing changes in cell \nbehaviors, such as cell proliferation and migration, after trehalose treatment. Additionally, \nthis study aimed to investigate the effects of trehalose at the molecular level by examining \ngrowth factor secretion and key markers of the MAPK, JNK, and PI3K/Akt signaling \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n7 \n \npathways. The findings of this study may help develop new therapeutic agents that can \nalter the wound healing process. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n8 \n \n2. Materials and methods \n2.1. Keratinocyte culture and treatment \nThis study was approved in advance by the Ethics Committee of Ehime University School \nof Medicine (Ehime, Japan)  and conducted in accordance with the principles of the \nDeclaration of Helsinki. Written informed consent was obtained from all participants. \nNormal human skin biopsies were obtained from individuals undergoing plastic surgery. \nThe epidermis was separated from the dermis, and normal human epidermal keratinocytes \n(NHKs) were isolated and cultured in a serum-free MCDB medium  as previously \ndescribed [18]. Cells were maintained in a humidified incubator at 37°C with 5% CO₂ \nand 95% air. Cells were preincubated in MCDB containing LY294002 (20 μM; Sigma-\nAldrich, USA) or SP600125 (20 μM; Sigma-Aldrich) for 1 h before trehalose stimulation \nto inhibit the PI3K/AKT or JNK signaling pathways. \n \n2.2. Preparation of RNA and real-time reverse transcription PCR \nAll probes specific for glyceraldehyde 3-phosphate dehydrogenase, vascular endothelial \ngrowth factor (VEGF), epiregulin (EREG), fibroblast growth factor (FGF2) , and stem \ncell factor (SCF) were obtained from Thermo Fisher Scientific (Yokohama, Japan). Total \nRNA was isolated from NHKs and subjected to real-time reverse transcription PCR. Gene \nexpression levels were analyzed as previously described [19, 20]. \n \n2.3. Scratch wound healing assay \nAfter NHKs reached near confluence, the cell monolayers were scratched using a 200 μL \nmicropipette tip, washed twice using phosphate-buffered saline , and incubated in an \nunsupplemented medium containing heparin-binding epidermal growth factor -like \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n9 \n \ngrowth factor, with or without specific inhibitors. Phase-contrast images were captured at \ndefined time points after scratching, and the percentage of the remaining wound area was \ncalculated using ImageJ software (National Institutes of Health , Bethesda, MD, USA), \nrelative to the initial wound area at 0 h (defined as 100%). Similar results were obtained \nin three independent experiments. In some experiments, cells were pretreated with \nLY294002 or SP600125 before scratching. \n \n2.4. Growth factor quantification by LEGENDplex™ Multiplex Assay \nThe concentrations of multiple growth factors in the culture supernatants were measured \nusing a bead -based multiplex immunoassay  (LEGENDplex™ Mouse Growth Factor \nPanel, BioLegend, San Diego, CA, USA)  according to the manufacturer ’s instructions. \nBriefly, 25 μL of each sample or standard was mixed with 25  μL of premixed capture \nbeads in a V-bottom 96-well plate and incubated for 2 h at room temperature with gentle \nshaking in the dark. After washing, 25  μL of detection antibodies were added and \nincubated for 1 h, followed by the addition of 25 μL of streptavidin-PE and an additional \n30 min of incubation. The beads were then washed and resuspended in  an assay buffer. \nData were acquired using a BD FACSCanto™ II flow cytometer (BD Biosciences, San \nJose, CA, USA) . The results were analyzed using the LEGENDplex ™ Data Analysis \nSoftware Suite (BioLegend). \n \n2.5. Cell death assays \nCell viability was assessed using Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according \nto the manufacturer’s instructions. The optical density was measured at 450 nm and was \nnormalized to the corresponding stimulation control. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n10 \n \n \n2.6. Whole-transcriptome analysis using RNA-seq \nThe RNA-seq data of cytokine-untreated samples from our previous study on trehalose-\ntreated NHKs were re-analyzed [21]. Mapped read counts were normalized to transcripts \nper million, incremented by one across all values, and transformed into log2. Genes with \na P-value <0.05 and a fold change >1.2 or <0.8 were selected and analyzed by Ingenuity \nPathway Analysis (Qiagen). Heatmaps of upregulated and downregulated genes were \ngenerated based on transcripts per million values using Prism software (version 9.0; \nGraphPad Software). \n \n2.7. Statistical analysis \nAt least three independent experiments were performed, all of which produced consistent \nresults. Quantitative data were presented as dot plots using GraphPad Prism version 9.5.0 \n(GraphPad Software, San Diego, CA, USA). Each graph presents results from a single \nrepresentative experiment, with 3–6 samples per condition. Individual dots represent the \nvalues of 3 –6 replicates for each test point. The uncovered wound area was calculated \nusing ImageJ version 1.53t (National Institutes of Health) and normalized to the wound \narea at 0 h, which was defined as 100%. Quantitative data were presented as mean ± \nstandard deviation (SD), with n ≥ 3. Statistical comparisons were performed using \nStudent’s t-test. Statistical significance was set at P < 0.05, P < 0.01, and P < 0.001. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n11 \n \n3. Results \n3.1. High-concentration trehalose promotes scratch wound closure in NHK layers \nthrough their ability to stimulate migration \nA wound healing assay was performed under in vitro  conditions to confirm the \nimprovement of wound closure via enhanced migration by trehalose. An artificial wound \nwas created on the NHK monolayer. Wound closure was observed 24 and 48 h after \ntrehalose treatment with the addition of mitomycin C (Fig . 1A). Trehalose treatment \nincreased wound closure by a +22% area ratio at 48 h compared with the untreated \ncontrols (Fig . 1B). High-concentration trehalose enhanced wound closure without \npromoting NHK proliferation, suggesting that its effect is due to increased cell migration \n(Supplementary Fig. 1 ). Trehalose up to 60 mg/mL did not affect cell viability, while \nconcentrations above 100 mg/mL reduced it.  Also, to assess whether the promotion of \ncell migration is specific to trehalose, a scratch assay with sucrose was conducted  \n(Supplementary Fig. 2). Unlike trehalose, sucrose did not promote NHK migration at any \nconcentration, indicating that the effect is specific to trehalose.  These findings indicate \nthat trehalose promotes NHK re-epithelialization by activating migration. \n \n3.2. Trehalose regulates several genes involved in cell migration \nPreviously published RNA-seq data from our group were re-analyzed to examine gene \nexpression changes in trehalose-treated NHKs (60 mg/ml) to explore the mechanism that \nenhances NHK migration in the presence of trehalose . The dataset is available in GEO \n(accession number: GSE244738). Gene expression was significantly up - and \ndownregulated by trehalose on a heat map, indicating that trehalose affects various gene \nexpressions related to skin formation (Fig . 2A). In the function analysis, the genes \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n12 \n \nregulated by trehalose were associated with cell migration (Fig. 2B). Additionally, among \nthe cell migration-related factors, VEGF was identified as a potential upstream factor (Fig. \n2C). \n \n3.3. Trehalose induces an increase in the VEGF secretion from NHKs \nProtein was quantified to elucidate the mechanism by which trehalose increases the \nmigration activity. The c ulture supernatants of trehalose-treated NHKs were collected \nafter 20 h. Protein concentrations in the supernatants were quantified in pg/mL using a \nbead-based multiplex LEGENDplex ™ assay (LEGENDplex™ Custom Human Assay, \nBiolegend, San Diego, CA, USA ) according to the manufacturer ’s instructions. The \nresults showed that trehalose had no effect on many proteins (Fig. 3A–C). Interestingly, \nNHK stimulation with trehalose significantly increased VEGF secretion (Fig. 3D). These \nfindings indicate that VEGF plays a role in the enhanced migratory capacity induced by \ntrehalose. \nqPCR mRNA expression analysis of the wound healing -related genes was \nperformed to confirm the results of the mRNA levels , which revealed that four genes \n(VEGF, EREG, FGF2, and SCF) significantly increased in NHKs treated with trehalose \n(60 mg/ml) for 24 h compared with vehicle control NHKs (Fig . 3E–H). FGF2 has been \nreported to promote NHK migration by activating Rac [22]. VEGF has also been reported \nto promote re-epidermalization and enhance wound closure [23]. In this way, the effect \nof trehalose on NHKs was confirmed even at the mRNA level. \n \n3.4. VEGF promotes scratch wound closure in NHK layers through their ability to \nstimulate migration \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n13 \n \nA wound healing assay was performed under in vitro conditions to confirm that VEGF \nmediates improved wound closure via trehalose-promoted migration . NHKs were \nstimulated with trehalose and VEGF in the presence of mitomycin C for 24 h. After \nreplacing the  medium, an artificial wound was created on the NHK monolayer, and \nwound closure was observed at 48 h (Fig. 4A). VEGF treatment increased wound closure \nby a +22% area ratio at 48 h compared with the untreated controls (Fig. 4C). Furthermore, \na comparison of VEGF and trehalose treatment showed no significant difference in wound \nclosure, with only a −1% change after 48 h. These findings indicate that trehalose \nstimulates VEGF secretion, thereby activating migration and promoting NHK re-\nepithelialization. \nFurthermore, experiments were designed using a specific VEGFR  inhibitor to \ndetermine whether trehalose promotes wound closure via VEGF production. After adding \nthe VEGFR inhibitor axitinib (20 μM), scratch wound healing assays were performed on \nNHKs treated with trehalose for 24 h in the same manner as before (Fig . 4B). Similar to \nprevious results, 60 mg/mL of trehalose dramatically promoted significant NHK \nmigration after 48 h of incubation compared with the dimethyl sulfoxide (DMSO)-treated \ngroup (Fig. 4B and D). However, when combined with axitinib, the effect of trehalose in \npromoting wound closure was dramatically reduced ( −42% area ratio). These findings \nindicate that VEGF plays a crucial role in the trehalose-induced enhancement of NHK \nmigration. \n \n3.5. Inhibition of growth and survival signaling pathways suppresses the migration-\nenhancing effects of trehalose \nA scratch wound healing assay was performed using various inhibitors to elucidate how \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n14 \n \ntrehalose enhances the migration activity of NHKs. First, LY294002, a specific inhibitor \nof PI3K, was applied and experimented using the same protocol as before. Trehalose-\ninduced NHK migration activity was suppressed in the presence of LY294002 (Fig. 5A \nand C). \nAdditionally, SP600125, a specific inhibitor of JNK, was applied, and experiments \nwere performed using a similar protocol. SP600125 inhibited trehalose -induced NHK \nmigration activity (Fig. 5B and D). These findings indicate that the effects of trehalose on \nenhancing cell migration activity are mediated through the PI3K and JNK pathways, \nwhich are known as growth and survival signaling pathways. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n15 \n \n4. Discussion \nSignificant attention has been paid to trehalose due to its unique functions. Trehalose has \nbeen reported to have anti-inflammatory effects in femoral fractures [24], antiaging \nproperties via anti -AGE activity [25], and the potential to improve diabetic symptoms \nthrough autophagy activation [26]. Furthermore, trehalose can promote significantly \nextensive spread of the epidermal layer of the living skin equiavalent [17] and enhance \nthe barrier function of keratinocytes [21], with the former representing a groundbreaking \ndiscovery, demonstrating that high concentrations of trehalose can induce fibroblasts to \nenter a temporarily prohealing senescence-like state. A living skin equivalent exploiting \nthis phenomenon may offer significant therapeutic potential for t reating deep ulcers in \nvivo, which have historically been challenging to manage. The latter finding indicates that \ntrehalose can restore the skin barrier by antagonizing IL -4/IL-13 signaling and \nsuppressing STAT3/STAT6 activati on in vitro . These findings indicate  that topical \napplication of trehalose is a promising therapeutic strategy for repairing skin barrier and \npreventing the onset of atopic dermatitis. Furthermore, this study showed that trehalose \nfacilitates keratinocyte cell migration. This effect was mediated by the upregulation of \nVEGF, a key growth factor, which activates the JNK and PI3K signaling pathways. \nNotably, the inhibition of VEGF receptors  and the JNK or PI3K pathways canceled \ntrehalose-induced cell migration  (Fig. 6). Interestingly, trehalose did not affect cell \nproliferation (Supplementary Fig. 1). Given the critical role of cell migration in wound \nhealing [4], these findings indicate that trehalose positively contributes to skin wound \nrepair. Furthermore, this effect was not observed when an equivalent concentration of \nsucrose (60 mg/mL) was administered (Fig. 1). These findings indicate that the observed \nenhancement of cell migration is not attributable to disaccharide-induced osmotic stress. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n16 \n \nFurthermore, no promotion of cell migration was observed at low or high concentrations \nof sucrose, indicating that this is an effect specific to trehalose (Supplementary Fig. 2). \nIn this study, a series of experiments were conducted to elucidate the mechanism \nunderlying the wound  healing effects of trehalose. The findings indicate that trehalose \nenhances VEGF production. In mammals, the VEGF family comprises five members: \nVEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor [27], with VEGF-A \nbeing the central and most widely studied member, commonly referred to as VEGF. \nPrevious studies have shown that keratinocytes express all five VEGF receptors [28]. \nTherefore, VEGF secreted from keratinocytes upon trehalose treatment likely exerts its \neffects in a paracrine manner, thereby promoting cell migration. The expression of VEGF \nreceptors in keratinocytes is crucial for maintaining skin homeostasis during wound \nhealing. Several studies have shown that VEGF contributes to enhancing skin wound \nhealing [27, 29]. These findings indicate that trehalose facilitates keratinocyte migration \nby promoting VEGF production and activating VEGF receptors. \nFurthermore, various inhibitors  were employed in this study to investigate the \nsignaling pathways involved in trehalose -mediated wound healing. The JNK signaling \npathway has been reported to be  critical for wound healing by promoting keratinocyte \nmigration [30]. Additionally, JNK has been reported to enhance the migration of \nkeratinocytes by activating the PI3K/AKT and JNK pathways [31]. Therefore, we \nhypothesized that the JNK and PI3K/AKT pathways contribute to trehalose -induced \nwound healing. This study examined the effects of specific inhibitors  to test this \nhypothesis. Trehalose-induced enhancement of keratinocyte migration was significantly \ninhibited in the presence of the PI3K inhibitor LY294002 and the JNK inhibitor SP600125. \nThese findings indicate that VEGF production in keratinocytes activates the PI3K and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n17 \n \nJNK signaling pathways, thereby facilitating wound healing. \nA key limitation of this study is the lack of in vivo evaluation; therefore, the wound-\nhealing effects of trehalose under physiological conditions remain uncertain. Further \nstudies using animal models, such as murine systems, are needed to validate our \nhypothesis and confirm the in vivo efficacy of trehalose. \nIn conclusion, this study showed that trehalose enhanced VEGF production and \npromoted wound healing in vitro. This effect was significantly suppressed by the VEGF \nreceptor inhibitor  axitinib and by inhibitors of the PI3K and JNK pathways. These \nfindings indicate that trehalose facilitates wound healing by inducing VEGF release, \nthereby activating the downstream PI3K and JNK signaling pathways. Therefore, \ntrehalose is a potential candidate compound for the development of novel therapeutic \nagents for skin wound healing. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n18 \n \nDeclaration of interest \nJM received research funding from ROHTO Pharmaceutical. The remaining authors state \nno conflict of interest. \n \nFunding \nThis work was supported by JSPS KAKENHI Grant Number JP24K11475 for Grant -in-\nAid for Scientific Research (C). \n \nDeclaration of generative AI and AI-assisted technologies in the writing process \nDuring the preparation of this manuscript, the authors used the ChatGPT -4.0 model to \nassist in improving the clarity and accuracy of the language. All content was subsequently \nreviewed and edited by the authors, who take full responsibility for the final version. It is \nimportant to note that no part of the manuscript was generated directly by AI; the tool was \nused solely to refine the presentation of content originally written by the authors. \n \nData availability statement \nAll data generated or analyzed during this study are included in this published article (and \nits supplementary information). \n \nAcknowledgements \nWe thank Eriko Tan for their technical assistance and thank Enago  (www.enago.jp) for \nthe manuscript review and editing support.  \n \nCRediT authorship contribution statement \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n19 \n \nConceptualization: JM;  \nData Curation: KT, XD, YM, KW, JM;  \nFormal Analysis: KT, XD, YM, KW, JM;  \nFunding Acquisition: JM;  \nInvestigation: KT, XD, YM, KW, TT, JM;  \nMethodology: KT, XD, YM, KW, JM;  \nProject Administration: KT, XD, JM;  \nResources: JM;  \nVisualization: KT, XD, YM, KW, JM;  \nWriting e Original Draft Preparation: KT, XD, YM, JM;  \nWriting e Review and Editing: KT, XD, YM, KW, KS, HM, YF, JM \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n20 \n \nFigures \n \n \n \nFig. 1. Scratch wound healing assay to determine whether trehalose promotes wound \nhealing through cell proliferation or migration. (A) Micrographs from a phase-contrast \nmicroscope (10× magnification) show the results of the scratch wound healing assay with \nmitomycin C, capturing the healing rate of primary human keratinocyte monolayer at 0, \n24, and 48 h after treatment with 60 mg/mL trehalose  or sucrose or nontreatment. (B) \nQuantification of the area occupied by primary human keratinocytes after 48 h. Data are \npresented as mean ± standard deviation (SD) and are representative of three independent \nexperiments. *P  <  0.05, **P  <  0.01, ***P  <  0.001, and ****P  <  0.0001 versus the \nvehicle-treated control group versus the sucrose-treated group by Student’s t-test. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n21 \n \n \n \nFig. 2. Trehalose-regulated gene expression in normal human epidermal \nkeratinocytes (NHKs) analyzed by whole -transcriptome RNA-seq analysis. NHKs \nwere treated with trehalose (60 mg/mL) for 18 h, and the total RNA was extracted and \nsubjected to RNA-seq. \n(A) Heatmap representing the gene expressions significantly up - or downregulated by \ntrehalose treatment based on transcripts per million. (B) Functional analysis revealed that \ntrehalose-regulated genes are associated with cell migration.  (C) Among the migration-\nrelated factors, VEGF was identified as a potential upstream regulator. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n22 \n \n \nFig. 3. Trehalose modulates the expression of VEGF and growth factors of mRNA \nlevels. Supernatants were collected and used for LEGENDplex™ assay to determine the \nlevels of secreted: (A) EGF, (B) FGF2, (C) HGF, and (D) VEGF. \n(E) VEGF, (F) EREG, (G) FGF2, and (H) SCF mRNA expressions were assessed by \nqPCR. Data are presented as relative expression to control (vehicle -treated) primary \nhuman keratinocytes. Data are expressed as mean ± SD of triplicate wells and are \nrepresentative of three independent experiments. *P  <  0.05, **P  <  0.01, ***P  <  0.001, \nand ****P  <  0.0001 versus the vehicle-treated control group versus the sucrose-treated \ngroup by Student’s t-test. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n23 \n \n \nFig. 4. Scratch wound healing assay to determine whether VEGF promotes wound \nhealing. (A) Micrographs from a phase -contrast microscope (10× magnification) show \nthe results of the scratch wound healing assay with mitomycin C, capturing the healing \nrate of the NHK monolayer at 0 and 48 h after treatment with 60 mg/mL of trehalose, 650 \npg/mL, or nontreatment. (B) Micrographs from a phase -contrast microscope (10 × \nmagnification) of the scratch wound healing assay with axitinib and mitomycin C over \nthe course of 48 h. (C) Quantification of the area occupied by NHKs after 48 h. Data are \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n24 \n \npresented as mean ± SD and are representative of three independent experiments. ns: not \nsignificant. *P < 0.05 versus the vehicle-treated control group versus the trehalose-treated \ngroup versus the VEGF -treated group by Student’s t-test. (D) Quantitative analysis of \npercent closure of the scratch wounded areas of the NHK monolayer treated with \ntrehalose with axitinib. Data are presented as mean ± SD and are representative of three \nindependent experiments. *P  <  0.05, **P  <  0.01, ***P  <  0.001, and ****P  <  0.0001 \nversus the DMSO -treated control group versus the trehalose -treated group versus the \naxitinib-treated group versus the trehalose- and axitinib-treated group by Student’s t-test. \nDMSO was used as the negative control. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n25 \n \n \nFig. 5. Scratch wound healing assay for determining the healing-promoting effects \nof trehalose in the presence of specific inhibitors of growth and survival signaling. \n(A, B) Micrographs from a phase-contrast microscope (10× magnification) of the \nscratch wound healing assay with LY294002 or SP600125 and mitomycin C over the \ncourse of 48 h. (C, D) Quantitative analysis of percent closure of the scratch wounded \nareas of the NHK monolayer treated with trehalose in the presence of LY294002 or \nSP600125. Data are presented as mean ± SD and are representative of three independent \nexperiments. *P  <  0.05, **P  <  0.01, ***P  <  0.001, and ****P  <  0.0001 versus the \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n26 \n \nDMSO-treated control group versus the trehalose-treated group versus the LY294002- \nor SP600125-treated group versus the trehalose- and LY294002- or SP600125-treated \ngroup by Student’s t-test. \nDMSO was used as the negative control. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n27 \n \n \nFig. 6. Putative signaling pathways involved in trehalose -induced wound repair in \nNHKs. Trehalose facilitates wound healing in vitro  by the upregulation of  VEGF \nexpression and activation of the PI3K and JNK signaling pathways.  \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n28 \n \nSupplementary information \nEffects of trehalose on NHK cell viability \nCell proliferation rates were measured using Cell Counting Kit-8 (Dojindo, Tokyo, \nJapan) to determine whether the acceleration of wound closure by high-concentration \ntrehalose was due to increased cell proliferation or migration. The results showed that \n60 mg/mL (and below) trehalose did not significantly affect the cell viability of NHKs \ncultured in a serum-free medium compared with the untreated group. However, \ntrehalose concentrations above 100 mg/mL significantly reduced NHK cell viability \n(Supplementary Fig. 1). Additionally, no significant difference in cell viability was \nobserved in cells treated with sucrose, a disaccharide similar to trehalose. These \nfindings indicate that trehalose can enhance wound closure through its ability to \nstimulate NHK migration. \n \nEffects of sucrose on NHK cell migration \nTo confirm whether the promotion of cell migration by disaccharides is specific to \ntrehalose, a scratch assay using sucrose was performed. An artificial wound was created \non the NHK monolayer. Wound closure was observed 24 and 48 h after scurose \ntreatment with the addition of mitomycin C (Supplementary Fig. 2). The addition of \nsucrose, regardless of concentration, did not enhance cell migration. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n29 \n \n \n \n \nSupplementary Fig. 1. Cell viability test in NHKs with various concentrations of \ntrehalose for 24 h. Cell viability was determined and expressed as a percentage of the \ncontrol (without trehalose treatment). Data are presented as mean ± SD (n = 6). \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.25.672108doi: bioRxiv preprint \n\n30 \n \n \nSupplementary Fig. 2. Effect of sucrose on scratch wound closure in NHKs \nmonolayers. (A) Micrographs from a phase contrast microscope (10× magnification, \nscale bar =100 μm) capturing cell monolayer healing rate of NHKs at 0, 24 and 48 h \nafter treatment with 1, 3, 10, 30 and 60 mg/mL of sucrose or untreated. (B) \nQuantification of the area occupied by NHKs after 48 h. Data were expressed as means \n± SD and are representative of three independent. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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