研究背景

机体衰老过程呈现复杂多维度特性，在面部皮肤呈现的典型老化表现包含表皮层变薄、胶原蛋白损耗、皮肤松弛以及皱纹加深，导致视觉年龄变化。其中相关变化不仅引起皮肤形态学改变，还与表皮屏障损害、慢性亚急性炎症等相关临床变化。具体分子机制涉及基因表达异常、抗氧化防御系统和免疫稳态失调构成的体系，但其具体原理仍有待研究。

既往针对皮肤衰老研究多聚焦于单纯年龄改变、单一细胞类型的功能衰退或细胞外基质的静态结构改变，而对真皮组织内的多细胞组分，包括内皮细胞、免疫细胞、成纤维细胞的协同作用、免疫-代谢网络动态调控机制及关键枢纽基因的系统解析仍存在空白。此外，成纤维细胞作为真皮核心功能细胞，其与微环境动态互作在免疫代谢信号传递、氧化应激响应等的具体分子机制尚未阐明。基于此，本研究整合单细胞多组学技术与功能验证实验，解析真皮组织的细胞异质性、免疫代谢网络及关键基因的增龄性相关动态变化，旨在为皮肤衰老的多维度干预提供新理论依据。

研究目的

1.基于GSE249225数据集，通过视觉年龄差异显著的皮肤组织转录组数据差异分析，解析皮肤衰老相关的关键差异基因及功能调控网络。

2.通过收集14例不同年龄组样本及先天性皮肤松弛症面部来源真皮组织，进行单细胞测序解析增龄性真皮组织的细胞异质性，结合配体-受体互作分析与随机森林算法，检测成纤维细胞功能衰退及免疫微环境失调的动态变化，揭示关键基因在皮肤老化中的调控机制。

3.基于前期分析筛选以PAX6作为关键基因，通过体外培养成纤维细胞并建立衰老关键PAX6基因过表达及敲低体外细胞模型，结合衰老标志检测（β-半乳糖苷酶染色、p53/p21通路分析），研究PAX6对细胞增殖、迁移及衰老表型的影响，探究其通过调节p53依赖通路及氧化应激反应抑制皮肤衰老的分子机理。

研究方法

1.视觉老化转录组差异数据的相关基因网络解析：利用GSE249225中的25例女性表皮RNA-seq资料，利用R环境调整p值<0.05和|log₂FC|>2为阈值鉴定差异表达基因，采用DAVID与Cytoscape联用策略，开展GO功能和KEGG通路的富集研究，采用STRING数据库解析蛋白质相互作用（PPI）网络，运用Cytohubba插件的Degree算法，识别出前15位核心Hub基因。

2.单细胞组学下解析增龄性真皮异质性：采集14份0-18岁、18-35岁、35-50岁及50岁以上各年龄段，以及1例先天性皮肤松弛症患者的耳前皮肤真皮组织标本，经胰蛋白酶与胶原酶共同消化后制成单细胞悬液，整合单细胞测序技术，借助Seurat工具实施数据优化（过滤异常细胞）和UMAP降维聚类分析，明确10个细胞亚群，采用CellChat技术分析细胞互作关系，采用随机森林分类器挖掘标志基因，利用SCENIC阐明基因调控网络，系统性揭示成纤维细胞功能衰退与免疫代谢失衡的动态协同机制。

3.PAX6基因功能验证与分子机制研究：采集不同年龄梯度皮肤组织，采用HE和Masson染色法检测表皮厚度及胶原纤维排列，采用IHC技术检测PAX6的表达水平，成纤维细胞经体外分离后实施培养，通过构建PAX6过表达及敲低成纤维细胞模型，结合衰老表型评估及p16、p53、p21通路检测，阐明PAX6调控皮肤衰老的多维度分子机制。

研究结果

1.生物信息学分析提示PAX6为皮肤视觉衰老差异的核心枢纽基因：基于GSE249225数据集，首次通过多组学整合策略筛选出312个视觉年龄相关差异基因（DEGs），其中PAX6在蛋白互作网络（PPI）中位居核心枢纽基因首位（Degree值最高）。在黑色素合成、氧化应激防御系统及表观遗传修饰中起关键作用。色素代谢障碍、蛋白质平衡破坏以及异常糖基化过程与视觉老化显著相关。

2.单细胞测序真皮组织细胞异质性结果：对14例年龄梯度样本（G1-G4组）及1例先天性皮肤松弛症（CCL组）真皮组织进行单细胞测序，获得113,782个高质量细胞，UMAP聚类鉴定10类细胞群体，其中成纤维细胞与内皮细胞为信号交互核心节点。以二者为核心细胞共筛选得到24个差异表达基因中，利用随机森林算法筛选出5个关键基因（AKAP12、FABP5、CDKN1A、NHERF2、SLC25A6），在核糖体合成与IL-17/Toll样受体通路上呈现明显富集。

3.单细胞测序成纤维细胞聚类及PAX6动态表达规律：发现关键基因KRF和PAX6分别通过TNFA/NF-κB及p53信号通路调控免疫代谢。PAX6在成纤维细胞亚群中呈现显著年龄依赖性下降（G1至G4组表达量降低，CCL组完全缺失），且与关键基因中FABP5、CDKN1A成相关调控。成纤维细胞功能下降、慢性炎症累积叠加代谢通路紊乱，共同驱动皮肤的多维衰老进程。

4.拟时序分析显示先天性皮肤松弛症细胞分化程度最低，但发育潜能较强，主要分布于分化轨迹末期。免疫浸润显示，老龄样本显示成纤维细胞功能急剧下降，免疫微环境失调还表现为抗原呈递细胞协同抑制合并I型干扰素反应增强。

5.PAX6与组织学增龄性变化关联性验证：组织学分结果显示，随着年龄增长，皮肤组织出现表皮厚度降低、真皮胶原含量下降和弹性纤维排列异常。PAX6免疫组化染色显示PAX6表达随年龄增长而下降，主要定位于细胞核。

6.PAX6通过p53/氧化应激轴抑制成纤维细胞衰老的分子机制：Pax6 成功在HFSs内过表达及敲降成功，qPCR显示：Pax6/HFSs组内Pax6 mRNA表达增高，shPax6/HFSs组内Pax6 mRNA表达降低，差异有显著性意义(P < 0.05)；Western blot显示：Pax6/HFSs组内Pax6 蛋白表达增高，shPax6/HFSs组内Pax6 蛋白表达降低，差异有显著性意义(P < 0.05)。β-糖苷酶染色显示：Pax6/HFSs组内阳性细胞较少，shPax6/HFSs组内阳性细胞增高，差异有显著性意义(P < 0.05)；CCK8实验显示：Pax6/HFSs组内增殖能力增强，shPax6/HFSs组内增殖能力减弱，差异有显著性意义(P < 0.05)；流式细胞术结果显示：Pax6/HFSs组内细胞凋亡减少，shPax6/HFSs组内细胞凋亡增加，差异有显著性意义(P < 0.05)；qPCR显示：Pax6/HFSs组内p16、p53、p21 mRNA表达增高，col-1mRNA表达降低，shPax6/HFSs组内p16、p53、p21 mRNA表达降低，col-1mRNA表达增高，差异有显著性意义(P < 0.05)；Western blot显示：Pax6/HFSs组内p16、p53、p21 蛋白表达增高，col-1蛋白表达降低，shPax6/HFSs组内p16、p53、p21 蛋白表达降低，col-1mRNA表达增高，差异有显著性意义(P < 0.05)。

研究结论

1.PAX6作为皮肤多维衰老的调控枢纽：从生物信息学分析发现到单细胞组学验证，基于GSE249225数据集的多组学整合分析，首次筛选出PAX6为视觉年龄差异相关基因网络的核心枢纽。单细胞测序进一步证实，PAX6在成纤维细胞中呈现增龄性衰减（G1至G4组表达量降低，CCL组先天性皮肤松弛症患者则完全缺失），并成为连接成纤维细胞功能衰退与免疫微环境失衡的动态生物标志物。

2.单细胞测序研究提示衰老的免疫代谢协同调控：提示老龄样本显示成纤维细胞功能急剧下降，免疫微环境失调还表现为抗原呈递细胞协同抑制合并I型干扰素反应增强。筛选得到5个关键基因，在核糖体合成与IL-17/Toll样受体通路上呈现明显富集，其中成纤维细胞二次聚类发现关键基因KRF和PAX6调控免疫代谢功能，并发现PAX6在组间随增龄而表达降低。成纤维细胞功能下降、慢性炎症累积叠加代谢通路紊乱，共同驱动皮肤的多维衰老进程。

3.PAX6靶向干预：分子机制功能验证PAX6过表达逆转成纤维细胞衰老表型（β-半乳糖苷酶活性降低、增殖能力提升），其分子机制依赖于p53/p21通路抑制。组织学证据显示，PAX6表达与皮肤组织学老化改变表现呈正相关，证实其潜在抗衰作用，为靶向抗衰治疗奠定了基础。

Background

The ageing process is complex and multidimensional, and typical manifestations of facial skin include epidermal thinning, collagen depletion, skin laxity and deepening of wrinkles, leading to changes in visual age. These changes not only lead to morphological alterations, but are also associated with epidermal barrier damage and chronic low-grade inflammation. The underlying molecular mechanisms involve systems comprising dysregulated gene expression, disturbed antioxidant defences and dysregulated immune homeostasis, but the exact principles remain to be elucidated.

Previous investigations into skin aging have predominantly focused on chronological aging, functional decline in single cell types, or static extracellular matrix alterations. Significant knowledge gaps persist regarding the synergistic interactions among multicellular components in dermal tissue (particularly endothelial cells, immune cells, and fibroblasts), the dynamic regulatory mechanisms of immune-metabolic networks, and systematic identification of hub genes governing these processes. Furthermore, while fibroblasts serve as central functional units in the dermis, the molecular mechanisms underlying their dynamic crosstalk with microenvironmental elements in mediating immunometabolic signaling and oxidative stress responses remain poorly characterized. To address these critical gaps, this study employs an integrated approach combining single-cell multi-omics technologies with functional validation experiments to systematically investigate cellular heterogeneity, immune-metabolic network dynamics, and age-associated variations of key regulatory genes in dermal tissue. These findings aim to establish novel theoretical frameworks for multidimensional interventions against skin aging.

Objectives

1. Utilizing the GSE249225 dataset, differential transcriptomic analysis was performed on skin tissues exhibiting marked visual age disparities to identify critical senescence-associated differentially expressed genes (DEGs) and delineate their functional regulatory networks in skin aging.

2. Single-cell sequencing was conducted on 14 age-stratified clinical specimens and congenital cutis laxa-derived facial dermal tissues to resolve cellular heterogeneity in age-progressive dermal tissue. Integrative ligand-receptor interaction analysis and random forest algorithm were employed to characterize fibroblast functional decline and immune microenvironment dysregulation, thereby elucidating regulatory mechanisms of hub genes in cutaneous aging.

3.Building on prior analytical screening identifying PAX6 as a critical regulator, we established in vitro fibroblast models with PAX6 overexpression and knockdown. Combined with senescence marker assessments (β-galactosidase staining, p53/p21 pathway profiling), we systematically evaluated PAX6’s impact on cellular proliferation, migration, and senescence phenotypes. Mechanistic investigations revealed that PAX6 mitigates skin aging by modulating p53-dependent signaling cascades and oxidative stress responses, thereby providing a molecular foundation for its therapeutic potential in senescence intervention.

Methods

1. Transcriptome analysis and functional analysis:RNA-seq data from 25 female epidermal samples (GSE249225) were used to identify differentially expressed genes (DEGs) using the R package, with thresholds of adjusted p-value <0.05 and |log₂FC|>2. Functional enrichment analyses of the GO terms and KEGG pathways were performed using DAVID, and visualised by Cytoscape. Protein-protein interaction (PPI) networks were constructed using the STRING database and the top 15 centred genes were identified using the Degree algorithm in Cytohubba.

2. Single-Cell Omics Profiling of Age-Related Dermal Heterogeneity:
Dermal tissues from 14 age-stratified donors (0–18, 18–35, 35–50, and >50 years) and one congenital cutis laxa patient were enzymatically dissociated (trypsin/collagenase digestion) into single-cell suspensions. Single-cell sequencing data were processed using Seurat for quality control (aberrant cell filtration) and UMAP-based clustering, resolving 10 distinct cellular subpopulations. CellChat analysis mapped intercellular communication networks, while a random forest classifier identified cell-type-specific marker genes. SCENIC (Single-Cell Regulatory Network Inference and Clustering) further delineated gene regulatory networks, systematically uncovering dynamic interplay between fibroblast functional decline and immunometabolic dysregulation during aging.

3. Functional Validation and Mechanistic Exploration of PAX6:
Skin tissues across age groups were histologically assessed via H&E and Masson's trichrome staining to quantify epidermal thickness and collagen organization. PAX6 expression was localized using immunohistochemistry (IHC). Primary fibroblasts were isolated and cultured to establish PAX6-overexpressing and PAX6-knockdown models. Senescence phenotypes were evaluated through β-galactosidase staining and p16/p53/p21 pathway analyses. This multidimensional approach elucidated PAX6’s regulatory role in skin aging, demonstrating its capacity to mitigate senescence via p53-dependent signaling and oxidative stress modulation.

Results

1. PAX6 Emerges as a Central Hub Gene in Visual Skin Aging via Bioinformatics Analysis: Integrated multi-omics analysis of the GSE249225 dataset first identified 312 visual aging-associated differentially expressed genes (DEGs), with PAX6 ranking as the top central hub gene (highest Degree score) in the protein-protein interaction (PPI) network. Functional annotation revealed critical role of PAX6 in melanogenesis, oxidative stress defense, and epigenetic regulation. Dysregulated pigment metabolism, proteostasis imbalance, and aberrant glycosylation processes were strongly linked to visual aging phenotypes.

2. Using Single-Cell Profiling Uncovers Dermal Cellular Heterogeneity:Single-cell sequencing of dermal tissues from 14 age-stratified samples (Groups G1-G4) and one congenital cutis laxa case (Group CCL) yielded 113,782 high-quality cells. UMAP clustering resolved 10 distinct cell populations, with fibroblasts and endothelial cells serving as central signaling hubs. Among 24 DEGs identified in these core populations, random forest analysis prioritized five key genes (AKAP12, FABP5, CDKN1A, NHERF2, SLC25A6), which were significantly enriched in ribosome biogenesis and IL-17/Toll-like receptor signaling pathways.

3. Fibroblast Subclustering and PAX6 Dynamics in Aging: Subpopulation analysis revealed that KRTAP5-AS1 (KRF) and PAX6 regulate immunometabolic processes via TNF-α/NF-κB and p53 signaling, respectively. PAX6 expression exhibited age-dependent decline across fibroblast subpopulations (reduced in G1-G4, absent in CCL) and showed positive correlation with FABP5 and CDKN1A. The convergence of fibroblast functional decline, chronic inflammatory accumulation, and metabolic pathway dysregulation collectively drives multidimensional skin aging.

4. Pseudotime Trajectory and Immune Dysregulation in Aging: Pseudotemporal analysis demonstrated that congenital cutis laxa cells occupied the terminal differentiation trajectory with minimal differentiation maturity but retained developmental plasticity. Immune infiltration profiling revealed marked fibroblast functional deterioration in aged samples, accompanied by dysregulated immune microenvironments characterized by suppressed antigen-presenting cell collaboration and heightened type I interferon responses.

5. Histopathological Validation of PAX6-Aging Correlation: Histopathological analysis confirmed age-dependent epidermal thinning, reduced dermal collagen density, and disorganized elastic fiber architecture. Immunohistochemical (IHC) staining revealed progressive nuclear PAX6 downregulation with advancing age, corroborating its role as an aging-sensitive regulator.

6. PAX6 Attenuates Fibroblast Senescence via p53/Oxidative Stress Axis: PAX6 overexpression and knockdown models were successfully established in human foreskin fibroblasts (HFSs). qPCR analysis demonstrated significantly elevated PAX6 mRNA levels in PAX6-overexpressing HFSs (PAX6/HFSs) and reduced expression in PAX6-knockdown HFSs (shPAX6/HFSs) (P < 0.05). Western blot confirmed corresponding changes in PAX6 protein expression (P < 0.05). Senescence-associated β-galactosidase staining revealed fewer positive cells in PAX6/HFSs compared to increased positivity in shPAX6/HFSs (P < 0.05). CCK-8 assays showed enhanced proliferative capacity in PAX6/HFSs and reduced proliferation in shPAX6/HFSs (P < 0.05). Flow cytometry analysis indicated decreased apoptosis in PAX6/HFSs versus increased apoptosis in shPAX6/HFSs (P < 0.05). Mechanistically, qPCR and Western blot analyses demonstrated upregulated mRNA and protein levels of p16, p53, and p21 in PAX6/HFSs, whereas shPAX6/HFSs exhibited significant downregulation of these senescence markers (P < 0.05). These findings collectively establish that PAX6 suppresses fibroblast senescence by modulating the p53-dependent oxidative stress axis.

Conclusions

1. PAX6 as a Central Regulatory Hub in Multidimensional Skin Aging: Integrative multi-omics analysis of the GSE249225 dataset first identified PAX6 as the core hub gene in visual age-associated transcriptional networks. Single-cell sequencing further validated its role, revealing age-dependent attenuation of PAX6 expression in fibroblasts (progressive decline from Groups G1 to G4, with complete absence in congenital cutis laxa, Group CCL). PAX6 emerged as a dynamic biomarker linking fibroblast functional deterioration to immune microenvironment imbalance, underscoring its centrality in orchestrating multidimensional aging processes.

2. Immunometabolic Crosstalk in Skin Aging Revealed by Single-Cell Profiling: Aged samples exhibited pronounced fibroblast dysfunction coupled with immune microenvironment dysregulation, characterized by suppressed antigen-presenting cell collaboration and hyperactivated type I interferon responses. Among five key genes (AKAP12, FABP5, CDKN1A, NHERF2, SLC25A6) enriched in ribosome biogenesis and IL-17/Toll-like receptor pathways, subclustering analysis identified KRTAP5-AS1 (KRF) and PAX6 as immunometabolic regulators. Both genes displayed age-correlated downregulation, mechanistically connecting fibroblast functional decline, chronic inflammation, and metabolic pathway disruption to drive multidimensional skin aging.

3. PAX6-Targeted Intervention Reverses Senescence Phenotypes: Functional validation demonstrated that PAX6 overexpression counteracts fibroblast senescence, evidenced by reduced senescence-associated β-galactosidase activity and enhanced proliferative capacity. Mechanistically, PAX6 suppresses the p53/p21 axis, attenuating oxidative stress and apoptosis. Histopathological correlation confirmed inverse relationship of PAX6 with aging features (epidermal thinning, collagen loss), solidifying its role as a potent anti-aging target. These findings provide a molecular rationale for PAX6-centric therapeutic strategies, laying the groundwork for targeted anti-aging therapies.