Unveiling the 28-Year Threshold: Region-Specific Lipid alteration as Early Markers of Skin Aging

Unveiling the 28-Year Threshold: Region-Specific Lipid alteration as Early Markers of Skin Aging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Unveiling the 28-Year Threshold: Region-Specific Lipid alteration as Early Markers of Skin Aging Meiting Yi, Qian Jiao, Jianbiao He, Huiliang Li, Yangyang Fang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7446551/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Traditional Chinese Medicine (TCM) describes a seven-year physiological cycle in women, with age 28 marking the onset of functional decline. However, objective dermatological evidence supporting this theory is limited. Objective To identify early skin aging signatures around age 28 by integrating physiological assessments with skin surface lipid (SSL) profiling. Methods Eighty healthy Chinese women with combination skin were stratified into pre-threshold (22–28 years) and post-threshold (29–35 years) cohorts. Facial elasticity (R2, Q1), hydration (CM), transepidermal water loss (TEWL), pH, and sebum were measured, alongside SSL lipidomics using UPLC-QTOF-MS. Differential lipids were screened by VIP > 1, p 2 or < 0.5. Results Conventional barrier metrics (TEWL, CM, pH) showed no group differences. However, post-threshold skin exhibited region-specific lipid remodeling, characterized by decreased ceramides, triglycerides, diglycerides, and long-chain fatty acids—most pronounced on the cheeks—accompanied by increased cholesteryl esters. These molecular shifts suggest compensatory adaptation to sustain barrier integrity. Conclusion Early alteration of lipid metabolism precedes measurable barrier decline, supporting the TCM concept of an aging threshold at 28. Region-specific lipid markers may inform precision strategies to preserve skin barrier and delay aging. Biological sciences/Biochemistry Health sciences/Biomarkers Health sciences/Diseases Health sciences/Health care Health sciences/Medical research Biological sciences/Physiology skin aging skin surface lipids combination skin Huangdi Neijing lipidomics zonal skincare Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Skin aging manifests as wrinkles, reduced elasticity, epidermal thinning, and barrier dysfunction due to progressive physiological decline 1 – 3 . Longitudinal data from Chinese women indicate that signs of skin aging become more pronounced between the ages of 24 and 30, with changes being more evident in the cheek region than in the forehead 4 . This empirical window aligns with Traditional Chinese Medicine’s “Huangdi Neijing,” which identifies age 28 as a critical physiological turning point in women 5 , 6 . With advancing age, the skin barrier weakens, shown by lower moisture retention and a higher risk of infection. Since skin lipids (like ceramides, cholesterol, and fatty acids) are key for barrier function, their significant decline during aging impairs the skin barrier's structure and function 7 . Specific lipid metabolism genes (e.g., AGPAT1, AGPAT3, ELOVL5, AWAT2, FA2H, and FADS2) are activated to a greater extent in individuals who respond more quickly to nonablative fractional laser (NAFL) treatment, aiding in restoring and maintaining barrier function 8 . Thus, modulating lipid metabolism is vital for enhancing skin barrier function and slowing skin aging. Skin surface lipids (SSLs), comprising sebaceous and intercellular components, are essential for water retention, structural cohesion, and antimicrobial defense 9 . Studying the changes in SSLs during aging helps understand the molecular mechanisms of skin aging. Combination skin, the most common facial skin type, exhibits distinct oil-dry zoning, with the T-zone (forehead, nose, chin) being lipid-rich and the U-zone (cheeks) more susceptible to dryness 10 . The spatial SSL heterogeneity across facial zones and age-related SSL alterations in combination skin populations are still not fully understood. Lipidomics, a mass spectrometry–based technique, enables high-throughput profiling of lipid classes and molecular species to reveal metabolic alterations 11 . It has been widely used to study skin lipid changes in diseases such as atopic dermatitis 12 , acne 13 , and rosacea 14 . In this study, Chinese women aged 22–35 years with combination skin were divided into two groups (22–28 and 29–35 years) based on questionnaire results. Through analyzing skin physiological parameters and skin surface lipid (SSL) profiles, we explored how skin changes with age. Drawing on the Traditional Chinese Medicine theory of the seven-year cycle—which marks age 28 as a key turning point—this work aims to identify region-specific lipid aging markers and provide a more precise foundation for personalized skincare. 2 Materials and methods 2.1 Chemicals and reagents Ammonium formate, acetonitrile, isopropanol, methanol, formic acid, and distilled water are LC-MS grade and were purchased from Thermo Fisher Scientific Technologies. Sebutape® for skin surface lipid adsorption tape was purchased from CuDerm. 2.2 Study subjects Eighty healthy Chinese women with combination skin were recruited using a validated self-administered questionnaire, which has shown good concordance with objective skin type classifications 15 . Participants were stratified into two age groups: 22–28 years (Y group; n = 40, mean age 24.16 ± 1.46) and 29–35 years (O group; n = 40, mean age 32.39 ± 1.92). Exclusion criteria included active dermatological conditions, recent facial medication use, light therapy exposure, smoking, or alcohol consumption. All participants provided informed consent. The study protocol received formal approval from the Beijing Technology and Business University Institutional Research Ethics Committee (Approval No. 2025 − 107). All procedures performed in this study were in accordance with the principles of the Declaration of Helsinki. 2.3 Sample collection and preparation Participants were assigned to Y or O groups based on age. After facial cleansing, subjects acclimated for 30 min under controlled conditions (21 ± 2°C, 50 ± 10% RH). Skin measurements were taken on the left cheek using standardized instruments: hydration (CM825, CK), transepidermal water loss (TEWL; TM300, CK), surface pH (PH905, CK), skin tone parameters (a*, b*, L*, ITA°; CL400, CK), and elasticity (R2, Q1; MPA580, CK). Sebum levels were measured at the cheek (“C”) and forehead (“F”) using SM815. Skin surface lipids (SSLs) were collected using Sebutape® patches and stored at − 80°C. Lipid extraction was performed via the Bligh–Dyer method. Prior to UPLC-QTOF-MS analysis, pooled quality control (QC) samples were prepared to ensure analytical consistency. 2.4 Instrument parameters Chromatographic separation was performed on a Waters UPLC system using a CSH C18 column (1.7 µm, 2.1 × 50 mm). The mobile phases were: A, acetonitrile/water (40:60, v/v) with 0.1% formic acid and 10 mM ammonium formate; B, acetonitrile/isopropanol (90:10, v/v) with identical additives. The flow rate was 0.4 mL/min, injection volume 2 µL, and column temperature 50°C. Mass spectrometry was conducted using a Waters Xevo G2-XS QTOF equipped with an electrospray ionization (ESI) source in positive ion mode. Full-scan spectra were acquired over m/z 50–1200, with leucine enkephalin (m/z 554.2771) as the lock mass. Data were processed using MassLynx 4.1. (Waters Corp.). 2.5 Data acquisition and analysis methods Raw data were processed with Progenesis QI v2.0 and EZinfo 3.0 (Waters Corp., Milford, MA, USA). Statistical analyses were conducted using unpaired t -tests or non-parametric tests, as appropriate. Significance was set at * p < 0.05 (** p < 0.01, *** p < 0.001 for higher confidence levels). 3 Result 3.1 Age-related changes in skin physiological parameters Cheek elasticity parameters (R2 and Q1) were significantly lower in the 29–35 age group (O group) compared to the 22–28 group (Y group) ( p < 0.001), indicating an age-related decline in viscoelastic performance (Fig. 1 A). Skin tone analysis showed increased b* and decreased ITA° values in the O group ( p < 0.05), suggesting darker and less radiant skin compared to the younger group (Fig. 1 C). No significant differences were observed between groups in skin hydration (CM), transepidermal water loss (TEWL), or surface pH (Fig. 1 B), indicating overall stability in static barrier parameters. Sebum levels on both the forehead and cheek were significantly higher in the Y group ( p < 0.05), reflecting a measurable reduction in sebum secretion with age (Fig. 1 D). 3.2 Analysis of skin surface lipid metabolism based on nontargeted lipidomics Non-targeted lipidomics of skin surface lipids (SSL) from the forehead and cheek of combination-skin females (aged 22–28 [Y] and 29–35 [O]) was performed using UPLC-QTOF-MS. A total of 1,372 lipids were identified and classified into eight LIPID MAPS categories: SP, GL, GP, PK, PR, ST, FA, and SL (Fig. 2 ). OPLS-DA modeling with orthogonal signal correction revealed clear separation between Y and O groups along the t[1] axis (R²Y, Q² >0.5), indicating age- and region-dependent lipidomic profiles. Differential lipids between the Y and O groups were identified based on VIP > 1, fold change > 2 or < 0.5, and p < 0.05. A total of 59 and 30 significant SSL species were detected in the forehead and cheek, respectively, among Chinese women aged 22–28 (Y) and 29–35 (O) with combination skin (Fig. 3 ). These lipids may serve as potential biomarkers of age-related alterations in SSL. In the forehead, 59 lipids were altered, spanning 7 primary categories (GL, SP, GP, FA, PR, PK, ST) and 9 main classes, including CER, GlcCer, sphingoid bases, PG, FFA, and flavonoids, which were mostly upregulated in the O group. Phytosphingosine and C16 Sphinganine may play key regulatory roles (Fig. 3 A). In the cheek, 30 differential lipids were identified, with a marked reduction in CER in the Y group (Fig. 3 B). CER[EOP] was significantly expressed in both regions of the Y group. Heatmaps revealed increased TG in YF and higher SM in OF (Fig. 4 A). 3.3 Correlation of ceramides with age and skin regional characteristics Ceramides (CER), which are essential components of the stratum corneum, play a key role in maintaining skin barrier integrity by promoting lamellar bilayer formation and reducing transepidermal water loss (TEWL). In this study, CER levels were found to vary significantly according to both facial region and age (Fig. 5 A). Specifically, both the young (Y) and old (O) groups exhibited higher CER levels in the cheek compared to the forehead, a difference that was statistically significant ( p < 0.001). Additionally, within each facial region, the Y group had significantly higher CER levels than the O group, with this age-related difference being significant in both the forehead ( p < 0.05) and the cheek ( p < 0.001). These results indicate that ceramide metabolism is influenced in an age-dependent and region-specific manner, underscoring the potential value of spatially targeted skincare interventions. Further supporting these observations, linear regression analysis confirmed that ceramide concentration declined significantly with age (β = -0.023, p < 0.001) and that levels were consistently lower on the forehead relative to the cheek (β = -0.93, p < 0.001). Moreover, a significant interaction between age and region was detected (Age × Region, β = 0.015, p = 0.029), indicating that the age-dependent reduction in ceramide levels was more pronounced on the cheek than on the forehead (Fig. 5 B). Nineteen ceramide (CER) subclasses were identified in SSL. In the forehead, 14 subclasses (e.g., CER[AP], CER[AS]) showed significantly higher abundance in the YF group than in the OF group ( p 0.05) (Fig. 5 C). CER[ADS] exhibited a non-significant increase in the OF group, possibly due to individual variability, warranting further validation. In the cheek, 16 CER subclasses were significantly elevated in the YC group compared to the OC group ( p < 0.05), showing a more marked age effect than in the forehead (Fig. 5 D). Notably, five O-acyl CERs (CER[EOP], CER[EOH], CER[EOS], CER[EODS], CER[EOSD]) were consistently higher in both YF and YC groups. CER[EOP], with VIP > 1 and p < 0.05, was significantly upregulated in both regions of the Y group, highlighting its potential as a biomarker for age-related changes in facial SSL metabolism. 3.4 Pronounced age-related changes in cheek skin surface lipid composition Fatty acid (FA) chain length and unsaturation degree are key determinants of skin barrier integrity and function. Long-chain (C12–C20) and very-long-chain fatty acids (C > 20) support barrier structure and flexibility, while polyunsaturated fatty acids (PUFAs) such as EPA and DHA modulate lipid metabolism and anti-inflammatory responses 16 . In this study, free fatty acid (FFA) profiles were assessed in forehead and cheek regions (Fig. 6 A). The YC group showed significantly higher levels of unsaturated FAs, including monounsaturated and polyunsaturated species, in the cheek compared to the OC group ( p 0.05). Age-related decreases in LCFA and VLCFA levels were observed in both facial regions. Additionally, long-chain PUFAs (LC-PUFAs) declined with age in the cheek but not in the forehead. These results suggest that the cheek region may be more susceptible to age-related lipid alterations and barrier dysfunction due to a more pronounced decline in structural and functional FAs. Sebum supports skin barrier function, antimicrobial defense, and immune modulation, with alterations implicated in atopic dermatitis (AD) pathogenesis 12 . In this study, age-stratified analysis of sebaceous gland-derived lipids in the forehead and cheek revealed age-related dynamics in TG, diacylglycerol (DG), and CE levels (Fig. 6 B). A significant decline in TG and DG was observed in the O group compared to the Y group in both regions ( p < 0.01), suggesting reduced sebaceous lipid synthesis with aging. Conversely, CE levels were significantly elevated in the O group ( p < 0.001), indicating an opposing trend. These findings suggest differential regulation of sebaceous lipids during aging, with potential implications for skin barrier maintenance and age-associated dermatoses. In the cheek region, several lipid classes that declined significantly with age also showed specific correlations with physiological parameters (Fig. 6 C). Multiple ceramide subclasses (Cer[NS], Cer[NDS], Cer[AH]) were positively correlated with sebum levels and stratum corneum hydration (CM), while polyunsaturated and long-chain fatty acids (PUFA, LCFA, MUFA) displayed strong negative correlations with TEWL. Glycerolipids (TG, DG) also correlated positively with sebum production. These findings suggest that a subset of ceramides, fatty acids, and glycerolipids represent cheek-specific lipid markers linking lipid decline to reduced sebum secretion, impaired barrier function, and diminished hydration. 4 Discussion Guided by the traditional Chinese medicine (TCM) concept of 7-year physiological cycles in women, this study focuses on the transition period around age 28, integrating modern assessments of skin physiology and surface lipidomics to explore early aging patterns in women with combination skin. By comparing females aged 22–28 (Y group) and 29–35 (O group), we identified subtle but significant age-related shifts in skin elasticity, pigmentation, and lipid metabolism. Significant declines in skin elasticity parameters (Q1, R2) were observed in the O group, particularly in the cheek area ( p < 0.001), suggesting dermal matrix degradation due to reduced fibroblast activity and increased matrix metalloproteinase (MMP) expression with age 17 . The marked decrease in sebum levels in both facial regions ( p < 0.05) further reflects age-related sebaceous gland functional alteration 18 , which compromises the lipid reservoir essential for barrier resilience and may indirectly worsen elasticity loss 19 . Increased b* and decreased ITA° values ( p < 0.05) point to early pigment accumulation and skin dullness in the O group, consistent with prior findings that link photoaging, oxidative stress, and hormonal changes to increased melanin production after age 30 20,21 . These results reinforce the importance of targeted photoprotection and antioxidant interventions in early adulthood. Despite functional and compositional differences, no significant group differences were observed in TEWL, CM, or pH. This underscores the limitations of conventional biophysical measures: TEWL primarily detects acute disruptions 22 , CM is confounded by environmental variability, and pH may remain stable due to compensatory lipid or metabolic adjustments. Indeed, aging studies have documented declines in major stratum corneum lipids, with selective increases in certain ceramide subtypes—suggestive of adaptive lipid remodeling 23 . Moreover, applying lipid mixtures with optimized cholesterol-dominant molar ratios accelerates barrier recovery in aged skin, implying skin’s capacity for functional compensation through lipid adjustment 24 . At the cellular level, autophagy has been shown to influence lamellar body formation and glycerophospholipid profiles, further supporting the notion of dynamic lipid remodeling in barrier maintenance 25 . Therefore, integrating lipidomics—and potentially metabolomics—into barrier studies could uncover these adaptive processes that static measurements fail to detect. The aged skin barrier is more vulnerable to damage and demonstrates decreased repair efficiency compared to younger skin, which correlates with diminished lamellar body secretion and changes in lipid synthesis and composition. The findings indicate that targeted lipid supplementation could be an essential strategy for anti-aging interventions 7 , 8 , 26 . The decline in barrier integrity frequently correlates with notable alterations in skin surface lipids, such as sebum and ceramides 27 . Ceramides, essential constituents of the epidermal barrier, exhibit a variety of molecular types, with more than 20 subtypes recognized in human tissues 28 , 29 . These subtypes create lamellar lipid bilayers in defined molar ratios, affecting barrier permeability and mechanical strength. Ceramide metabolism is associated with several skin conditions 30 – 32 . Alterations in ceramide subclasses and chain lengths influence barrier function, while the ratio of CER[NS] to CER[NP] affects barrier properties by modifying lipid bilayer phase transition temperatures 33 . Ceramides contribute to the structural barrier of the skin and also regulate functions through their metabolites, including sphingosine-1-phosphate (S1P). They engage in immune responses through the activation of Toll-like receptor (TLR) pathways and the modulation of nuclear factor-κB (NF-κB) signaling to regulate inflammation 34 . Ceramides serve as dynamic regulators of epidermal homeostasis, with roles that extend beyond mere static barrier components 35 . Free fatty acids (FFAs) constitute another critical lipid class essential for maintaining normal skin barrier integrity, serving as a vital component of barrier homeostasis. In the stratum corneum (SC), ceramides, cholesterol, and FFAs constitute the lipid matrix's primary components, a crucial structural factor in epidermal barrier function 36 . Variations in FFA chain length and composition significantly influence barrier performance. Experimental lipid models replicating atopic dermatitis-affected skin indicate that increased levels of short-chain fatty acids (SCFAs) considerably compromise barrier integrity. A diminished chain length is associated with a lower lipid packing density, heightened conformational disorder, and weakened interactions between free fatty acids and other structural lipids such as ceramides 29 . In contrast, long-chain fatty acids (LCFAs) improve barrier function by decreasing TEWL and preventing pathogenic infiltration 37 . Polyunsaturated fatty acids (PUFAs) regulate epidermal homeostasis, inflammation, and barrier maintenance through genomic modulation and lipid mediator synthesis 27 . This mechanistic understanding has led to clinical trials of PUFA-based barrier-related dermatoses 38 . Skin biosynthesis of very long-chain saturated (VLC-SFA) and polyunsaturated (VLC-PUFA) fatty acids is tightly regulated by the elongation enzyme ELOVL4. ELOVL4 deficiency impairs VLCFA production, O-acyl ceramide synthesis, and skin barrier architecture and function 39 , 40 . Photodamage studies show significant reductions in FFAs and TGs in UV-exposed epidermis, suggesting their role in photoaging 41 . These findings demonstrate the structural and functional importance of fatty acid composition in skin barrier regulation. Sebum is a complex lipid mixture composed primarily of triglycerides (TG), diacylglycerols (DG), wax esters, squalene, cholesteryl esters (CEs), and cholesterol 42 . Sebum is proposed to facilitate cutaneous antioxidant protection through α-tocopherol (vitamin E). The antioxidant system is proposed to neutralize reactive oxygen species (ROS) at the skin surface, thus reducing barrier degradation and photoaging 43 . With advancing age, there is a reduction in sebaceous gland activity, primarily attributed to decreased activity of HMG-CoA reductase, an enzyme critical for lipid synthesis. This decline compromises the skin's barrier function, accelerating the aging process 42 . Lipidomics revealed marked age- and region-dependent declines in ceramides (CERs), particularly in the cheek. Among 19 identified subclasses, ω-O-acyl ceramides (EO-CERs), including CER[EOP], were significantly reduced in the older group. EO-CERs are vital for forming multilamellar lipid lamellae and preventing TEWL, and their depletion has been recognized as a molecular signature of early barrier impairment 44 , 45 . Simultaneous accumulation of sphingoid intermediates (e.g., phytosphingosine, C16 sphinganine; VIP > 1) suggests impaired ceramide biosynthesis, possibly due to reduced CerS or ELOVL4/CYP4F22 activity, which are critical for producing long-chain fatty acids and ω-hydroxy components of acylceramides 46 , 47 . Importantly, correlation analysis further supported these molecular shifts. Ceramide subclasses (e.g., Cer[NS], Cer[NDS], Cer[AH]) showed positive associations with sebum and stratum corneum hydration, while polyunsaturated and long-chain fatty acids (PUFA, LCFA, MUFA) were negatively correlated with TEWL, highlighting their protective contribution against water loss. In contrast, glycerolipids (TG, DG) displayed positive correlations with sebum, underscoring the sebaceous contribution to structural and barrier lipids. These results indicate that the decline of ceramides, fatty acids, and glycerolipids in the cheek is functionally linked to reduced sebaceous activity and barrier weakening, despite stable physiological readouts such as TEWL and CM. Region-specific analysis revealed that the cheek underwent more pronounced lipid depletion than the forehead, particularly in unsaturated and long-chain fatty acids, TGs, DGs, and EO-CERs. This asymmetry may be explained by higher sebaceous gland density in the forehead, where continuous sebum secretion buffers against ceramide loss via alternative lipid components 19 , 48 , 49 . Such findings establish the cheek as a sensitive site for early molecular monitoring of skin aging. In parallel, cholesteryl esters (CEs) increased significantly with age in both regions. Given their structural role in barrier lipids, this rise may reflect compensatory remodeling aimed at sustaining barrier homeostasis, as suggested in previous experimental models 50 . While speculative, such compensation could partly mask early lipid deficiencies and delay functional decline. Longitudinal and mechanistic studies, including in vitro 3D epidermal models, will be required to validate this hypothesis and clarify its biological relevance. 5 Conclusion Our findings identify age 28 as a critical threshold for early skin aging, characterized by region-specific alterations in lipid metabolism that precede measurable barrier dysfunction. These results suggest that lipidomic shifts, particularly in the cheek, represent sensitive molecular markers of early aging and may provide novel targets for preventive intervention. Declarations Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement Meiting Yi : Conceptualization,Methodology , Formal analysis, Writing – Original Draft, Visualization, Writing – Review & Editing. Qian Jiao: Conceptualization, Writing – Original Draft, Methodology, Data Curation, Writing – Review & Editing. Jianbiao He: Conceptualization, Methodology, Investigation, Data Curation, Writing – Review & Editing. Huiliang Li: Resources, Writing-review & editing, Project administration, Methodology, Funding acquisition. Yangyang Fang : Resources, Writing-review & editing, Project administration, Methodology, Funding acquisition. Youjie He: resources, Writing-review & editing, Project administration, Methodology, Funding acquisition.Huaming He : Supervision, Resources, Writing-review & editing, Formal analysis, Investigation, Methodology, Funding acquisition. Yan Jia : Supervision, Resources, Writing-review & editing, Formal analysis, Investigation, Methodology, Funding acquisition. 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7446551","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":517688238,"identity":"61f437d1-1c8f-48f1-ad6b-22410ac6f1b2","order_by":0,"name":"Meiting Yi","email":"","orcid":"","institution":"Beijing Technology and Business University","correspondingAuthor":false,"prefix":"","firstName":"Meiting","middleName":"","lastName":"Yi","suffix":""},{"id":517688239,"identity":"9e5bb27c-a7e2-45af-aa9d-ff035fc9f32c","order_by":1,"name":"Qian Jiao","email":"","orcid":"","institution":"Beijing Technology and Business University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Jiao","suffix":""},{"id":517688240,"identity":"67339488-0ba3-4547-aa6b-d0900d7759d6","order_by":2,"name":"Jianbiao He","email":"","orcid":"","institution":"Beijing Technology and Business University","correspondingAuthor":false,"prefix":"","firstName":"Jianbiao","middleName":"","lastName":"He","suffix":""},{"id":517688241,"identity":"a171b32f-a5f2-4cec-a8cd-9367012ce00a","order_by":3,"name":"Huiliang Li","email":"","orcid":"","institution":"Hangzhou Huaningxiang Biotechnology Co., Ltd, Hangzhou, Zhejiang, 310000, China","correspondingAuthor":false,"prefix":"","firstName":"Huiliang","middleName":"","lastName":"Li","suffix":""},{"id":517688244,"identity":"47dce955-0c5f-4c3c-a198-0445c90b1010","order_by":4,"name":"Yangyang Fang","email":"","orcid":"","institution":"Hangzhou Huaningxiang Biotechnology Co., Ltd, Hangzhou, Zhejiang, 310000, 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11:28:32","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1165235,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/274dc369d887e30847552b94.png"},{"id":92079177,"identity":"2ce6c4bd-5ffe-4775-a43f-dcd684fac7b3","added_by":"auto","created_at":"2025-09-24 11:28:33","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108004,"visible":true,"origin":"","legend":"","description":"","filename":"4c9e341ab0b74d5196351e9eb5df45911structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/919589fe98498db59660743c.xml"},{"id":92079170,"identity":"43d04978-0b5f-4760-ada0-e26d34a7d742","added_by":"auto","created_at":"2025-09-24 11:28:32","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123120,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/87304855f3b4fa5105a559b5.html"},{"id":92079173,"identity":"e525ac9a-b59e-4851-8ce0-9899ab9487a3","added_by":"auto","created_at":"2025-09-24 11:28:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32415395,"visible":true,"origin":"","legend":"\u003cp\u003eAge-related differences in skin physiological parameters. (A) Cheek elasticity: Q1 (recovery efficiency) and R2 (elastic deformation recovery). (B) Barrier function : stratum corneum hydration (CM), transepidermal water loss (TEWL), and skin surface pH. (C) Skin tone : a* (redness), b* (yellowness), L* (brightness), and ITA° (skin tone index). (D) Sebum secretion on the forehead (Sebum[F]) and cheek (Sebum[C]). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/7f2c21ac4441db397fa6c9e9.png"},{"id":92079160,"identity":"8b3652b3-1b01-44b2-b654-3b1ef52df0d9","added_by":"auto","created_at":"2025-09-24 11:28:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9603449,"visible":true,"origin":"","legend":"\u003cp\u003eOPLS-DA score plots of skin surface lipid (SSL) profiles in women aged 22–28 (Y) and 29–35 (O). (A) Forehead: R²Y = 0.85, Q² = 0.74; (B) Cheek: R²Y = 0.84, Q² = 0.70. Groups are clearly separated. Y: young group; O: older group; F: forehead; C: cheek.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/3dc6a08c6f2c397231bf3df1.png"},{"id":92079121,"identity":"f2b60318-9856-40eb-bfa7-6905bd9ec43b","added_by":"auto","created_at":"2025-09-24 11:28:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7055054,"visible":true,"origin":"","legend":"\u003cp\u003eAge-related differential lipids in the forehead and cheek. (A) Forehead; (B) Cheek. Based on VIP \u0026gt; 1, p \u0026lt; 0.05, and fold change \u0026gt; 2 or \u0026lt; 0.5, 59 and 30 differential lipids were identified in the forehead and cheek, respectively. The right side of the figure shows lipid class distribution (Class II on x-axis). Lipids above the dashed line are upregulated in the Y group; those below are downregulated.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/9212d38c06c40c97f8c2da80.png"},{"id":92079120,"identity":"3d505cd4-8af2-44f6-9cc1-01e3184b60e5","added_by":"auto","created_at":"2025-09-24 11:28:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31628077,"visible":true,"origin":"","legend":"\u003cp\u003eAge-related differential lipids in the forehead and cheek. VIP \u0026gt; 1, p \u0026lt; 0.05, and fold change \u0026gt; 2 or \u0026lt; 0.5, 59 and 30 differential lipids were identified in the forehead and cheek, respectively. (A) Heatmap of forehead lipids; (B) Heatmap of cheek lipids. The color scale represents relative abundance (blue: low, red: high).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/6d8b46332cee71044ef2d1e2.png"},{"id":92079109,"identity":"e8b336af-ac5a-4696-a378-74c3b214ade4","added_by":"auto","created_at":"2025-09-24 11:28:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1573305,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Ceramide distribution varies significantly by facial region and age in this non-invasive study. (B) Linear regression of ceramide levels across age and facial region reveals a significant decline with age (β = -0.023, p \u0026lt; 0.001), region-specific differences (forehead vs. cheek: β = -0.93, p \u0026lt; 0.001), and a significant interaction indicating a steeper age-related decline in the cheek (β = 0.015, p = 0.029). Age-dependent alterations in ceramide subclasses across the forehead (C) and cheek (D). A total of 19 CER subclasses were identified in SSL, including CER[NH], CER[NP], CER[NS], CER[EOS], CER[EOP], and others. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/1523b07a598529651895b6eb.png"},{"id":92079112,"identity":"b0a83ffc-0a3b-4237-9069-910669d94948","added_by":"auto","created_at":"2025-09-24 11:28:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3665830,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Age- and region-dependent variations in fatty acid chain length and degree of unsaturation. UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acids; \u0026nbsp;LCFA, C12-C20, long-chain fatty acids; VLCFA, C \u0026gt; 20, very-long-chain fatty acids; LC-PUFA, long chain polyunsaturated fatty acids; (B) Age-dependent dynamic characteristics of sebaceous gland-derived lipids. Triacylglycerol (TG); diacylglycerol (DG); cholesteryl ester (CE); (C) Heatmap of Pearson’s correlation coefficients between significantly decreased lipids in the cheek region and physiological parameters (sebum, TEWL, CM, pH). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/4525f245adedf9e19d8bbbc6.png"},{"id":95221143,"identity":"daf9e3a6-e353-4cf6-8e69-01e3d73b705b","added_by":"auto","created_at":"2025-11-05 16:18:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":65928167,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7446551/v1/fcce17ce-9b83-4431-917d-474e6548956b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":" Unveiling the 28-Year Threshold: Region-Specific Lipid alteration as Early Markers of Skin Aging","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eSkin aging manifests as wrinkles, reduced elasticity, epidermal thinning, and barrier dysfunction due to progressive physiological decline \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Longitudinal data from Chinese women indicate that signs of skin aging become more pronounced between the ages of 24 and 30, with changes being more evident in the cheek region than in the forehead \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This empirical window aligns with Traditional Chinese Medicine\u0026rsquo;s \u0026ldquo;Huangdi Neijing,\u0026rdquo; which identifies age 28 as a critical physiological turning point in women \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWith advancing age, the skin barrier weakens, shown by lower moisture retention and a higher risk of infection. Since skin lipids (like ceramides, cholesterol, and fatty acids) are key for barrier function, their significant decline during aging impairs the skin barrier's structure and function \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Specific lipid metabolism genes (e.g., AGPAT1, AGPAT3, ELOVL5, AWAT2, FA2H, and FADS2) are activated to a greater extent in individuals who respond more quickly to nonablative fractional laser (NAFL) treatment, aiding in restoring and maintaining barrier function \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Thus, modulating lipid metabolism is vital for enhancing skin barrier function and slowing skin aging. Skin surface lipids (SSLs), comprising sebaceous and intercellular components, are essential for water retention, structural cohesion, and antimicrobial defense \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Studying the changes in SSLs during aging helps understand the molecular mechanisms of skin aging.\u003c/p\u003e\u003cp\u003eCombination skin, the most common facial skin type, exhibits distinct oil-dry zoning, with the T-zone (forehead, nose, chin) being lipid-rich and the U-zone (cheeks) more susceptible to dryness \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The spatial SSL heterogeneity across facial zones and age-related SSL alterations in combination skin populations are still not fully understood.\u003c/p\u003e\u003cp\u003eLipidomics, a mass spectrometry\u0026ndash;based technique, enables high-throughput profiling of lipid classes and molecular species to reveal metabolic alterations \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It has been widely used to study skin lipid changes in diseases such as atopic dermatitis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, acne \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and rosacea \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In this study, Chinese women aged 22\u0026ndash;35 years with combination skin were divided into two groups (22\u0026ndash;28 and 29\u0026ndash;35 years) based on questionnaire results. Through analyzing skin physiological parameters and skin surface lipid (SSL) profiles, we explored how skin changes with age. Drawing on the Traditional Chinese Medicine theory of the seven-year cycle\u0026mdash;which marks age 28 as a key turning point\u0026mdash;this work aims to identify region-specific lipid aging markers and provide a more precise foundation for personalized skincare.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e\u003cp\u003eAmmonium formate, acetonitrile, isopropanol, methanol, formic acid, and distilled water are LC-MS grade and were purchased from Thermo Fisher Scientific Technologies. Sebutape\u0026reg; for skin surface lipid adsorption tape was purchased from CuDerm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Study subjects\u003c/h2\u003e\u003cp\u003eEighty healthy Chinese women with combination skin were recruited using a validated self-administered questionnaire, which has shown good concordance with objective skin type classifications \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Participants were stratified into two age groups: 22\u0026ndash;28 years (Y group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40, mean age 24.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46) and 29\u0026ndash;35 years (O group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40, mean age 32.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92). Exclusion criteria included active dermatological conditions, recent facial medication use, light therapy exposure, smoking, or alcohol consumption. All participants provided informed consent. The study protocol received formal approval from the Beijing Technology and Business University Institutional Research Ethics Committee (Approval No. 2025\u0026thinsp;\u0026minus;\u0026thinsp;107). All procedures performed in this study were in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Sample collection and preparation\u003c/h2\u003e\u003cp\u003eParticipants were assigned to Y or O groups based on age. After facial cleansing, subjects acclimated for 30 min under controlled conditions (21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% RH). Skin measurements were taken on the left cheek using standardized instruments: hydration (CM825, CK), transepidermal water loss (TEWL; TM300, CK), surface pH (PH905, CK), skin tone parameters (a*, b*, L*, ITA\u0026deg;; CL400, CK), and elasticity (R2, Q1; MPA580, CK). Sebum levels were measured at the cheek (\u0026ldquo;C\u0026rdquo;) and forehead (\u0026ldquo;F\u0026rdquo;) using SM815. Skin surface lipids (SSLs) were collected using Sebutape\u0026reg; patches and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\u003cp\u003eLipid extraction was performed via the Bligh\u0026ndash;Dyer method. Prior to UPLC-QTOF-MS analysis, pooled quality control (QC) samples were prepared to ensure analytical consistency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Instrument parameters\u003c/h2\u003e\u003cp\u003eChromatographic separation was performed on a Waters UPLC system using a CSH C18 column (1.7 \u0026micro;m, 2.1 \u0026times; 50 mm). The mobile phases were: A, acetonitrile/water (40:60, v/v) with 0.1% formic acid and 10 mM ammonium formate; B, acetonitrile/isopropanol (90:10, v/v) with identical additives. The flow rate was 0.4 mL/min, injection volume 2 \u0026micro;L, and column temperature 50\u0026deg;C.\u003c/p\u003e\u003cp\u003eMass spectrometry was conducted using a Waters Xevo G2-XS QTOF equipped with an electrospray ionization (ESI) source in positive ion mode. Full-scan spectra were acquired over m/z 50\u0026ndash;1200, with leucine enkephalin (m/z 554.2771) as the lock mass. Data were processed using MassLynx 4.1. (Waters Corp.).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Data acquisition and analysis methods\u003c/h2\u003e\u003cp\u003eRaw data were processed with Progenesis QI v2.0 and EZinfo 3.0 (Waters Corp., Milford, MA, USA). Statistical analyses were conducted using unpaired \u003cem\u003et\u003c/em\u003e-tests or non-parametric tests, as appropriate. Significance was set at *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (**\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for higher confidence levels).\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Result","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Age-related changes in skin physiological parameters\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCheek elasticity parameters (R2 and Q1) were significantly lower in the 29\u0026ndash;35 age group (O group) compared to the 22\u0026ndash;28 group (Y group) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating an age-related decline in viscoelastic performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Skin tone analysis showed increased b* and decreased ITA\u0026deg; values in the O group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting darker and less radiant skin compared to the younger group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). No significant differences were observed between groups in skin hydration (CM), transepidermal water loss (TEWL), or surface pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating overall stability in static barrier parameters. Sebum levels on both the forehead and cheek were significantly higher in the Y group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting a measurable reduction in sebum secretion with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Analysis of skin surface lipid metabolism based on nontargeted lipidomics\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNon-targeted lipidomics of skin surface lipids (SSL) from the forehead and cheek of combination-skin females (aged 22\u0026ndash;28 [Y] and 29\u0026ndash;35 [O]) was performed using UPLC-QTOF-MS. A total of 1,372 lipids were identified and classified into eight LIPID MAPS categories: SP, GL, GP, PK, PR, ST, FA, and SL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). OPLS-DA modeling with orthogonal signal correction revealed clear separation between Y and O groups along the t[1] axis (R\u0026sup2;Y, Q\u0026sup2; \u0026gt;0.5), indicating age- and region-dependent lipidomic profiles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDifferential lipids between the Y and O groups were identified based on VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, fold change\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0.5, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. A total of 59 and 30 significant SSL species were detected in the forehead and cheek, respectively, among Chinese women aged 22\u0026ndash;28 (Y) and 29\u0026ndash;35 (O) with combination skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These lipids may serve as potential biomarkers of age-related alterations in SSL. In the forehead, 59 lipids were altered, spanning 7 primary categories (GL, SP, GP, FA, PR, PK, ST) and 9 main classes, including CER, GlcCer, sphingoid bases, PG, FFA, and flavonoids, which were mostly upregulated in the O group. Phytosphingosine and C16 Sphinganine may play key regulatory roles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the cheek, 30 differential lipids were identified, with a marked reduction in CER in the Y group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). CER[EOP] was significantly expressed in both regions of the Y group. Heatmaps revealed increased TG in YF and higher SM in OF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Correlation of ceramides with age and skin regional characteristics\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCeramides (CER), which are essential components of the stratum corneum, play a key role in maintaining skin barrier integrity by promoting lamellar bilayer formation and reducing transepidermal water loss (TEWL). In this study, CER levels were found to vary significantly according to both facial region and age (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Specifically, both the young (Y) and old (O) groups exhibited higher CER levels in the cheek compared to the forehead, a difference that was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, within each facial region, the Y group had significantly higher CER levels than the O group, with this age-related difference being significant in both the forehead (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the cheek (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results indicate that ceramide metabolism is influenced in an age-dependent and region-specific manner, underscoring the potential value of spatially targeted skincare interventions. Further supporting these observations, linear regression analysis confirmed that ceramide concentration declined significantly with age (β = -0.023, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and that levels were consistently lower on the forehead relative to the cheek (β = -0.93, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Moreover, a significant interaction between age and region was detected (Age \u0026times; Region, β\u0026thinsp;=\u0026thinsp;0.015, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), indicating that the age-dependent reduction in ceramide levels was more pronounced on the cheek than on the forehead (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eNineteen ceramide (CER) subclasses were identified in SSL. In the forehead, 14 subclasses (e.g., CER[AP], CER[AS]) showed significantly higher abundance in the YF group than in the OF group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while five subclasses (CER[NH], CER[NP], CER[NS], CER[AH], CER[ADS]) showed no significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). CER[ADS] exhibited a non-significant increase in the OF group, possibly due to individual variability, warranting further validation. In the cheek, 16 CER subclasses were significantly elevated in the YC group compared to the OC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), showing a more marked age effect than in the forehead (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, five O-acyl CERs (CER[EOP], CER[EOH], CER[EOS], CER[EODS], CER[EOSD]) were consistently higher in both YF and YC groups. CER[EOP], with VIP\u0026thinsp;\u0026gt;\u0026thinsp;1 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, was significantly upregulated in both regions of the Y group, highlighting its potential as a biomarker for age-related changes in facial SSL metabolism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Pronounced age-related changes in cheek skin surface lipid composition\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFatty acid (FA) chain length and unsaturation degree are key determinants of skin barrier integrity and function. Long-chain (C12\u0026ndash;C20) and very-long-chain fatty acids (C\u0026thinsp;\u0026gt;\u0026thinsp;20) support barrier structure and flexibility, while polyunsaturated fatty acids (PUFAs) such as EPA and DHA modulate lipid metabolism and anti-inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In this study, free fatty acid (FFA) profiles were assessed in forehead and cheek regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The YC group showed significantly higher levels of unsaturated FAs, including monounsaturated and polyunsaturated species, in the cheek compared to the OC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with no significant differences in the forehead (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Age-related decreases in LCFA and VLCFA levels were observed in both facial regions. Additionally, long-chain PUFAs (LC-PUFAs) declined with age in the cheek but not in the forehead. These results suggest that the cheek region may be more susceptible to age-related lipid alterations and barrier dysfunction due to a more pronounced decline in structural and functional FAs.\u003c/p\u003e\u003cp\u003eSebum supports skin barrier function, antimicrobial defense, and immune modulation, with alterations implicated in atopic dermatitis (AD) pathogenesis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this study, age-stratified analysis of sebaceous gland-derived lipids in the forehead and cheek revealed age-related dynamics in TG, diacylglycerol (DG), and CE levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). A significant decline in TG and DG was observed in the O group compared to the Y group in both regions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting reduced sebaceous lipid synthesis with aging. Conversely, CE levels were significantly elevated in the O group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating an opposing trend. These findings suggest differential regulation of sebaceous lipids during aging, with potential implications for skin barrier maintenance and age-associated dermatoses.\u003c/p\u003e\u003cp\u003eIn the cheek region, several lipid classes that declined significantly with age also showed specific correlations with physiological parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Multiple ceramide subclasses (Cer[NS], Cer[NDS], Cer[AH]) were positively correlated with sebum levels and stratum corneum hydration (CM), while polyunsaturated and long-chain fatty acids (PUFA, LCFA, MUFA) displayed strong negative correlations with TEWL. Glycerolipids (TG, DG) also correlated positively with sebum production. These findings suggest that a subset of ceramides, fatty acids, and glycerolipids represent cheek-specific lipid markers linking lipid decline to reduced sebum secretion, impaired barrier function, and diminished hydration.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eGuided by the traditional Chinese medicine (TCM) concept of 7-year physiological cycles in women, this study focuses on the transition period around age 28, integrating modern assessments of skin physiology and surface lipidomics to explore early aging patterns in women with combination skin. By comparing females aged 22\u0026ndash;28 (Y group) and 29\u0026ndash;35 (O group), we identified subtle but significant age-related shifts in skin elasticity, pigmentation, and lipid metabolism.\u003c/p\u003e\u003cp\u003eSignificant declines in skin elasticity parameters (Q1, R2) were observed in the O group, particularly in the cheek area (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting dermal matrix degradation due to reduced fibroblast activity and increased matrix metalloproteinase (MMP) expression with age \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The marked decrease in sebum levels in both facial regions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) further reflects age-related sebaceous gland functional alteration \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, which compromises the lipid reservoir essential for barrier resilience and may indirectly worsen elasticity loss \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIncreased b* and decreased ITA\u0026deg; values (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) point to early pigment accumulation and skin dullness in the O group, consistent with prior findings that link photoaging, oxidative stress, and hormonal changes to increased melanin production after age 30 \u003csup\u003e20,21\u003c/sup\u003e. These results reinforce the importance of targeted photoprotection and antioxidant interventions in early adulthood.\u003c/p\u003e\u003cp\u003eDespite functional and compositional differences, no significant group differences were observed in TEWL, CM, or pH. This underscores the limitations of conventional biophysical measures: TEWL primarily detects acute disruptions \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, CM is confounded by environmental variability, and pH may remain stable due to compensatory lipid or metabolic adjustments. Indeed, aging studies have documented declines in major stratum corneum lipids, with selective increases in certain ceramide subtypes\u0026mdash;suggestive of adaptive lipid remodeling \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, applying lipid mixtures with optimized cholesterol-dominant molar ratios accelerates barrier recovery in aged skin, implying skin\u0026rsquo;s capacity for functional compensation through lipid adjustment \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. At the cellular level, autophagy has been shown to influence lamellar body formation and glycerophospholipid profiles, further supporting the notion of dynamic lipid remodeling in barrier maintenance \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, integrating lipidomics\u0026mdash;and potentially metabolomics\u0026mdash;into barrier studies could uncover these adaptive processes that static measurements fail to detect.\u003c/p\u003e\u003cp\u003eThe aged skin barrier is more vulnerable to damage and demonstrates decreased repair efficiency compared to younger skin, which correlates with diminished lamellar body secretion and changes in lipid synthesis and composition. The findings indicate that targeted lipid supplementation could be an essential strategy for anti-aging interventions \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The decline in barrier integrity frequently correlates with notable alterations in skin surface lipids, such as sebum and ceramides \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Ceramides, essential constituents of the epidermal barrier, exhibit a variety of molecular types, with more than 20 subtypes recognized in human tissues \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These subtypes create lamellar lipid bilayers in defined molar ratios, affecting barrier permeability and mechanical strength. Ceramide metabolism is associated with several skin conditions \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Alterations in ceramide subclasses and chain lengths influence barrier function, while the ratio of CER[NS] to CER[NP] affects barrier properties by modifying lipid bilayer phase transition temperatures \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Ceramides contribute to the structural barrier of the skin and also regulate functions through their metabolites, including sphingosine-1-phosphate (S1P). They engage in immune responses through the activation of Toll-like receptor (TLR) pathways and the modulation of nuclear factor-κB (NF-κB) signaling to regulate inflammation \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Ceramides serve as dynamic regulators of epidermal homeostasis, with roles that extend beyond mere static barrier components \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFree fatty acids (FFAs) constitute another critical lipid class essential for maintaining normal skin barrier integrity, serving as a vital component of barrier homeostasis. In the stratum corneum (SC), ceramides, cholesterol, and FFAs constitute the lipid matrix's primary components, a crucial structural factor in epidermal barrier function \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Variations in FFA chain length and composition significantly influence barrier performance. Experimental lipid models replicating atopic dermatitis-affected skin indicate that increased levels of short-chain fatty acids (SCFAs) considerably compromise barrier integrity. A diminished chain length is associated with a lower lipid packing density, heightened conformational disorder, and weakened interactions between free fatty acids and other structural lipids such as ceramides \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In contrast, long-chain fatty acids (LCFAs) improve barrier function by decreasing TEWL and preventing pathogenic infiltration \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Polyunsaturated fatty acids (PUFAs) regulate epidermal homeostasis, inflammation, and barrier maintenance through genomic modulation and lipid mediator synthesis \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This mechanistic understanding has led to clinical trials of PUFA-based barrier-related dermatoses \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Skin biosynthesis of very long-chain saturated (VLC-SFA) and polyunsaturated (VLC-PUFA) fatty acids is tightly regulated by the elongation enzyme ELOVL4. ELOVL4 deficiency impairs VLCFA production, O-acyl ceramide synthesis, and skin barrier architecture and function \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Photodamage studies show significant reductions in FFAs and TGs in UV-exposed epidermis, suggesting their role in photoaging \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings demonstrate the structural and functional importance of fatty acid composition in skin barrier regulation.\u003c/p\u003e\u003cp\u003eSebum is a complex lipid mixture composed primarily of triglycerides (TG), diacylglycerols (DG), wax esters, squalene, cholesteryl esters (CEs), and cholesterol \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Sebum is proposed to facilitate cutaneous antioxidant protection through α-tocopherol (vitamin E). The antioxidant system is proposed to neutralize reactive oxygen species (ROS) at the skin surface, thus reducing barrier degradation and photoaging \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. With advancing age, there is a reduction in sebaceous gland activity, primarily attributed to decreased activity of HMG-CoA reductase, an enzyme critical for lipid synthesis. This decline compromises the skin's barrier function, accelerating the aging process \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLipidomics revealed marked age- and region-dependent declines in ceramides (CERs), particularly in the cheek. Among 19 identified subclasses, ω-O-acyl ceramides (EO-CERs), including CER[EOP], were significantly reduced in the older group. EO-CERs are vital for forming multilamellar lipid lamellae and preventing TEWL, and their depletion has been recognized as a molecular signature of early barrier impairment \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Simultaneous accumulation of sphingoid intermediates (e.g., phytosphingosine, C16 sphinganine; VIP\u0026thinsp;\u0026gt;\u0026thinsp;1) suggests impaired ceramide biosynthesis, possibly due to reduced CerS or ELOVL4/CYP4F22 activity, which are critical for producing long-chain fatty acids and ω-hydroxy components of acylceramides \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImportantly, correlation analysis further supported these molecular shifts. Ceramide subclasses (e.g., Cer[NS], Cer[NDS], Cer[AH]) showed positive associations with sebum and stratum corneum hydration, while polyunsaturated and long-chain fatty acids (PUFA, LCFA, MUFA) were negatively correlated with TEWL, highlighting their protective contribution against water loss. In contrast, glycerolipids (TG, DG) displayed positive correlations with sebum, underscoring the sebaceous contribution to structural and barrier lipids. These results indicate that the decline of ceramides, fatty acids, and glycerolipids in the cheek is functionally linked to reduced sebaceous activity and barrier weakening, despite stable physiological readouts such as TEWL and CM.\u003c/p\u003e\u003cp\u003eRegion-specific analysis revealed that the cheek underwent more pronounced lipid depletion than the forehead, particularly in unsaturated and long-chain fatty acids, TGs, DGs, and EO-CERs. This asymmetry may be explained by higher sebaceous gland density in the forehead, where continuous sebum secretion buffers against ceramide loss via alternative lipid components \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Such findings establish the cheek as a sensitive site for early molecular monitoring of skin aging.\u003c/p\u003e\u003cp\u003eIn parallel, cholesteryl esters (CEs) increased significantly with age in both regions. Given their structural role in barrier lipids, this rise may reflect compensatory remodeling aimed at sustaining barrier homeostasis, as suggested in previous experimental models \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. While speculative, such compensation could partly mask early lipid deficiencies and delay functional decline. Longitudinal and mechanistic studies, including in vitro 3D epidermal models, will be required to validate this hypothesis and clarify its biological relevance.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur findings identify age 28 as a critical threshold for early skin aging, characterized by region-specific alterations in lipid metabolism that precede measurable barrier dysfunction. These results suggest that lipidomic shifts, particularly in the cheek, represent sensitive molecular markers of early aging and may provide novel targets for preventive intervention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e\n\u003cp\u003eMeiting Yi\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eConceptualization,Methodology\u003cstrong\u003e,\u003c/strong\u003e Formal analysis, Writing – Original Draft, Visualization, Writing – Review \u0026amp; Editing. Qian Jiao: Conceptualization, Writing – Original Draft, Methodology, Data Curation, Writing – Review \u0026amp; Editing. Jianbiao He: Conceptualization, Methodology, Investigation, Data Curation, Writing – Review \u0026amp; Editing. Huiliang Li: Resources, Writing-review \u0026amp; editing, Project administration, Methodology, Funding acquisition. Yangyang Fang\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eResources, Writing-review \u0026amp; editing, Project administration, Methodology, Funding acquisition. Youjie He: resources, Writing-review \u0026amp; editing, Project administration, Methodology, Funding acquisition.Huaming He\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eSupervision, Resources, Writing-review \u0026amp; editing, Formal analysis, Investigation, Methodology, Funding acquisition. Yan Jia\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eSupervision, Resources, Writing-review \u0026amp; editing, Formal analysis, Investigation, Methodology, Funding acquisition.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data generated during the current study are available from the corresponding author ([email protected]) on reasonable request.\u003c/p\u003e\n\u003cp\u003eDeclaration of Competing\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFranco, A. C., Aveleira, C. \u0026amp; Cavadas, C. Skin senescence: mechanisms and impact on whole-body aging. \u003cem\u003eTrends Mol. Med.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (2), 97\u0026ndash;109 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJin, S. et al. 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ZFP750 affects the cutaneous barrier through regulating lipid metabolism. \u003cem\u003eScience advances\u003c/em\u003e ;\u003cb\u003e9\u003c/b\u003e(17):eadg5423. (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"skin aging, skin surface lipids, combination skin, Huangdi Neijing, lipidomics, zonal skincare","lastPublishedDoi":"10.21203/rs.3.rs-7446551/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7446551/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTraditional Chinese Medicine (TCM) describes a seven-year physiological cycle in women, with age 28 marking the onset of functional decline. However, objective dermatological evidence supporting this theory is limited.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify early skin aging signatures around age 28 by integrating physiological assessments with skin surface lipid (SSL) profiling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEighty healthy Chinese women with combination skin were stratified into pre-threshold (22–28 years) and post-threshold (29–35 years) cohorts. Facial elasticity (R2, Q1), hydration (CM), transepidermal water loss (TEWL), pH, and sebum were measured, alongside SSL lipidomics using UPLC-QTOF-MS. Differential lipids were screened by VIP \u0026gt; 1, p \u0026lt; 0.05, and fold change \u0026gt; 2 or \u0026lt; 0.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConventional barrier metrics (TEWL, CM, pH) showed no group differences. However, post-threshold skin exhibited region-specific lipid remodeling, characterized by decreased ceramides, triglycerides, diglycerides, and long-chain fatty acids—most pronounced on the cheeks—accompanied by increased cholesteryl esters. These molecular shifts suggest compensatory adaptation to sustain barrier integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEarly alteration of lipid metabolism precedes measurable barrier decline, supporting the TCM concept of an aging threshold at 28. Region-specific lipid markers may inform precision strategies to preserve skin barrier and delay aging.\u003c/p\u003e","manuscriptTitle":" Unveiling the 28-Year Threshold: Region-Specific Lipid alteration as Early Markers of Skin Aging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 11:27:38","doi":"10.21203/rs.3.rs-7446551/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d44fda47-e25a-4ce8-b7dc-58fa4932c627","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55018581,"name":"Biological sciences/Biochemistry"},{"id":55018582,"name":"Health sciences/Biomarkers"},{"id":55018583,"name":"Health sciences/Diseases"},{"id":55018584,"name":"Health sciences/Health care"},{"id":55018585,"name":"Health sciences/Medical research"},{"id":55018586,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-11-03T10:08:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 11:27:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7446551","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7446551","identity":"rs-7446551","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}