Basic Research on the Effects and Molecular Mechanisms of FORSCI. Cold Atmospheric Plasma Beauty Device for Skin Anti-Aging

Abstract

With the rapid progression of population aging and the increasing demand for healthy and youthful skin, the development of safe and effective strategies to delay skin aging has become an important research focus in the fields of life sciences, aesthetic medicine, and dermatology. Skin aging mainly consists of intrinsic aging and extrinsic aging, among which photoaging caused by long-term exposure to ultraviolet (UV) radiation is one of the major factors affecting skin structure and function. UV irradiation induces excessive production of reactive oxygen species (ROS) in skin tissues, resulting in oxidative stress imbalance, chronic inflammatory responses, cellular senescence, and degradation of the extracellular matrix (ECM). These pathological changes ultimately lead to reduced dermal collagen content, decreased skin elasticity, wrinkle formation, and impaired tissue repair capacity. Therefore, the development of novel anti-aging technologies capable of regulating cellular functions and improving the tissue microenvironment is of great scientific and clinical significance.

Low-temperature atmospheric plasma, also known as cold atmospheric plasma (CAP), is an emerging physical technology for biological regulation. CAP generates a variety of reactive oxygen and nitrogen species (RONS), charged particles, and electromagnetic effects under near-room-temperature conditions, enabling interactions with biological tissues without causing significant thermal damage. In recent years, CAP has been widely applied in wound healing, antimicrobial therapy, tissue regeneration, and the treatment of skin diseases. Previous studies have demonstrated that appropriate CAP stimulation can modulate the cellular redox state, promote cell proliferation and migration, facilitate tissue repair, and enhance collagen synthesis, thereby improving skin function. However, despite these promising findings, the efficacy of CAP in skin rejuvenation and its underlying molecular mechanisms in alleviating skin photoaging have not yet been systematically investigated.

This study focused on the scientific question of the effects and molecular mechanisms of FORSCI. cold atmospheric plasma beauty device in improving skin photoaging. An in vitro fibroblast senescence model and an in vivo ultraviolet (UV)-induced mouse model of skin photoaging were established to systematically evaluate the anti-photoaging effects of CAP at the cellular, tissue, and molecular levels.

In the in vitro experiments, mouse fibroblast cell lines L929 and NIH-3T3 were used to establish oxidative stress-induced cellular senescence models through hydrogen peroxide (H₂O₂) treatment, followed by CAP intervention. Cellular senescence was assessed using senescence-associated β-galactosidase (SA-β-gal) staining. Cell proliferation was evaluated by the Cell Counting Kit-8 (CCK-8) assay, while cell migration was analyzed using the wound-healing assay. DNA replication activity was determined by 5-ethynyl-2′-deoxyuridine (EdU) staining, and intracellular ROS levels were measured using the DCFH-DA fluorescent probe. Furthermore, quantitative real-time PCR (qRT-PCR) was performed to determine the expression levels of senescence- and collagen metabolism-related genes, including COL1A1, p16, and p21, thereby evaluating the regulatory effects of CAP on senescent fibroblasts at both the phenotypic and molecular levels.

For the in vivo study, a UV-induced skin photoaging model was established in C57BL/6 mice, followed by treatment with the CAP beauty device. The therapeutic effects of CAP were comprehensively evaluated through gross observation, skin appearance assessment, dermoscopy, hematoxylin and eosin (HE) staining, Masson's trichrome staining, immunohistochemical staining, and qRT-PCR analysis. In addition, transcriptome sequencing of skin tissues was performed to identify differentially expressed genes and enriched biological pathways following CAP treatment, providing further insight into the underlying molecular mechanisms.

The results demonstrated that H₂O₂ treatment successfully induced senescence in both L929 and NIH-3T3 fibroblasts, as evidenced by enlarged and flattened cell morphology and a significant increase in the proportion of SA-β-gal-positive cells. CAP treatment markedly reduced SA-β-gal positivity, restored normal cellular morphology, and significantly improved both proliferative and migratory capacities of senescent fibroblasts. Further analyses showed that CAP increased the proportion of EdU-positive cells, enhanced DNA replication activity, reduced excessive intracellular ROS accumulation induced by oxidative stress, and restored intracellular redox homeostasis. At the molecular level, H₂O₂ stimulation significantly decreased COL1A1 expression while markedly increasing the expression of the senescence-associated genes p16 and p21. CAP treatment effectively restored COL1A1 expression and suppressed the abnormal upregulation of p16 and p21, suggesting that CAP alleviates cell-cycle arrest and promotes the functional recovery of fibroblasts.

The in vivo findings further confirmed that CAP effectively ameliorated UV-induced skin photoaging in mice. Compared with the UV model group, mice receiving CAP treatment exhibited fewer wrinkles, smoother skin surfaces, and reduced tissue damage. Histological analyses revealed that CAP alleviated UV-induced epidermal hyperplasia and dermal structural disorganization while promoting collagen fiber regeneration and increasing dermal collagen content. Immunohistochemical and gene expression analyses further demonstrated that CAP reduced the expression of inflammation- and senescence-related markers, including IL-6, p16, and p21, while significantly enhancing COL1A1 expression. Transcriptomic analysis revealed that the differentially expressed genes following CAP treatment were predominantly enriched in pathways associated with collagen biosynthesis, extracellular matrix organization, collagen fibril formation, and tissue repair, indicating that CAP promotes skin regeneration through the coordinated regulation of multiple biological processes.

In conclusion, this study demonstrated that the FORSCI. cold atmospheric plasma beauty device effectively ameliorates skin photoaging by regulating oxidative stress, restoring fibroblast function, promoting collagen metabolism, and modulating senescence-related signaling networks. These findings provide experimental evidence supporting the application of CAP technology in aesthetic anti-aging therapy and offer a theoretical basis for the development of safe, non-invasive, and highly effective skin rejuvenation strategies.

Keywords: cold atmospheric plasma; skin photoaging; fibroblasts; cellular senescence; collagen; oxidative stress; extracellular matrix (ECM).

1. Background

1.1 Mechanisms of Skin Aging and Photoaging

The skin is the largest organ of the human body and serves as the primary interface between the body and the external environment. It plays essential roles in maintaining the skin barrier, regulating water homeostasis, participating in immune defense, and preserving tissue homeostasis. With advancing age, the skin gradually exhibits characteristic features of aging, including reduced epidermal renewal capacity, impaired barrier function, decreased dermal thickness, loss of elasticity, wrinkle formation, and diminished wound-healing ability. In recent years, advances in the 

biology of aging have revealed that skin aging is not merely a consequence of chronological time but rather a multifactorial process driven by genomic instability, mitochondrial dysfunction, loss of proteostasis, cellular senescence, chronic inflammation, and altered intercellular communication ¹.

The concept of the hallmarks of aging has further advanced our understanding of the biological mechanisms underlying aging. Aging is now recognized as a complex process involving multiple interconnected events, including genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and chronic inflammation. As the body's outermost barrier organ, the skin is continuously exposed to both intrinsic metabolic changes and extrinsic environmental insults, making it an ideal model for aging research. Recent studies have further identified several key characteristics of skin aging, including the accumulation of oxidative damage, increased cellular senescence, and alterations in the tissue microenvironment, all of which contribute to progressive deterioration of skin structure and function ².

According to the underlying causes, skin aging can generally be classified into intrinsic aging, which occurs as a consequence of chronological aging, and extrinsic aging, which is induced by environmental factors. Among the latter, photoaging resulting from chronic exposure to ultraviolet (UV) radiation is considered one of the major determinants of skin appearance and functional decline. Compared with intrinsic aging, photoaging is characterized by more pronounced histological and molecular alterations, including wrinkle formation, skin laxity, hyperpigmentation, collagen depletion, and abnormalities in elastic fibers ³.

Upon UV exposure, keratinocytes, dermal fibroblasts, and immune cells perceive ultraviolet radiation and subsequently generate excessive amounts of reactive oxygen species (ROS). When ROS production exceeds the endogenous antioxidant capacity of the skin, redox homeostasis is disrupted, leading to persistent oxidative stress ⁴. Oxidative stress not only directly induces DNA damage, lipid peroxidation, and protein dysfunction but also activates multiple inflammation-related signaling pathways, including the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways. Activation of these pathways promotes the secretion of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α), thereby maintaining the skin in a state of chronic low-grade inflammation and accelerating tissue aging ⁵. Furthermore, persistent oxidative stress drives dermal fibroblasts into a senescent state. Through the paracrine effects of the senescence-associated secretory phenotype (SASP), senescent fibroblasts further exacerbate extracellular matrix (ECM) imbalance and chronic local inflammation, ultimately establishing a positive feedback loop that promotes skin aging ⁶.

Fibroblasts are the principal functional cells within the dermis responsible for maintaining skin structural integrity. They synthesize type I collagen (COL1A1), type III collagen, elastin, and other essential components of the extracellular matrix (ECM). Under physiological conditions, fibroblasts maintain a dynamic balance between collagen synthesis and degradation, thereby preserving skin elasticity and mechanical strength. However, chronic UV irradiation impairs fibroblast function, suppresses collagen synthesis, and simultaneously upregulates the expression of matrix metalloproteinases (MMPs), resulting in accelerated collagen degradation. Among these enzymes, MMP-1, the major collagenase responsible for type I collagen degradation, plays a pivotal role in UV-induced photoaging and is considered one of the primary contributors to dermal matrix destruction and wrinkle formation ⁷. Increasing evidence indicates that skin aging is characterized not only by collagen loss and structural deterioration but also by the progressive accumulation of cellular senescence. Cellular senescence is defined as a stable state of irreversible cell-cycle arrest and is characterized by reduced proliferative capacity, increased senescence-associated β-galactosidase (SA-β-gal) activity, activation of the p16 and p21 signaling pathways, and enhanced production of the senescence-associated secretory phenotype (SASP) ^8 9. Senescent cells continuously secrete IL-6, IL-8, MMPs, and numerous other inflammatory mediators, thereby remodeling the local tissue microenvironment, promoting chronic inflammation, and impairing the function of neighboring healthy cells. Consequently, cellular senescence has emerged as one of the most important therapeutic targets in contemporary anti-aging research ¹⁰.

In addition to oxidative stress and cellular senescence, impairment of the skin barrier is another hallmark of photoaging. Keratinocytes play a central role in maintaining epidermal barrier integrity, whereas chronic UV exposure disrupts keratinocyte differentiation, alters epidermal lipid composition, and increases transepidermal water loss (TEWL), ultimately leading to skin dryness, hypersensitivity, and impaired repair capacity. Recent studies have demonstrated that restoration of skin barrier function not 

only improves the overall appearance of the skin but also attenuates inflammation and oxidative damage, making barrier repair an important objective of modern anti-aging therapies. Moreover, advances in single-cell sequencing, multi-omics technologies, and aging biology have revealed that skin aging is a highly complex process involving multiple cell populations and signaling networks. Aberrant communication among senescent fibroblasts, immune cells, and epidermal cells establishes a persistent inflammatory microenvironment, thereby promoting ECM degradation and progressive tissue dysfunction. Therefore, future anti-aging strategies should move beyond merely improving superficial skin appearance and instead focus on comprehensive interventions that regulate oxidative stress, restore cellular function, improve the tissue microenvironment, and maintain ECM homeostasis ¹¹.

In summary, skin photoaging is a complex biological process driven by UV-induced oxidative stress, chronic inflammation, cellular senescence, and abnormal extracellular matrix remodeling. The development of novel therapeutic strategies capable of simultaneously targeting these interconnected pathological processes is of great significance for improving skin health and delaying functional skin aging. These advances also provide a strong theoretical foundation for the application of emerging physical bioregulatory technologies, such as cold atmospheric plasma (CAP), in the field of skin rejuvenation and anti-aging therapy.

1.2 Current Advances and Limitations of Anti-Aging Technologies

With the acceleration of population aging and the increasing demand for skin rejuvenation, a variety of anti-aging interventions have been developed, including topical bioactive agents, energy-based aesthetic devices, and regenerative medicine technologies. However, skin aging is not simply a phenotypic alteration caused by a single factor but rather a complex biological process involving collagen loss, impaired cellular function, oxidative stress, chronic inflammation, and alterations in the tissue microenvironment. Consequently, the focus of anti-aging research has gradually shifted from merely improving skin appearance to regulating cellular function, promoting tissue repair, and maintaining skin homeostasis ¹².

In conventional skincare, bioactive ingredients such as retinoids, antioxidants, niacinamide, peptides, and hyaluronic acid have been widely incorporated into skin rejuvenation products. Among these compounds, retinol and its derivatives promote epidermal renewal and improve fine wrinkles and skin laxity by regulating collagen 

metabolism. Antioxidants primarily alleviate ROS-mediated oxidative damage, whereas hyaluronic acid exerts its beneficial effects mainly by enhancing skin hydration and strengthening epidermal barrier function ^13 14. In recent years, advances in regenerative medicine have stimulated increasing interest in skincare strategies containing growth factors, cytokines, and cell-derived bioactive components. The rationale underlying these approaches is to facilitate tissue repair and functional recovery through the modulation of intercellular communication. Nevertheless, the effectiveness of conventional topical formulations is limited by the barrier properties of the stratum corneum. Most bioactive compounds, particularly macromolecules such as peptides and growth factors, exhibit poor penetration into the dermis, thereby providing limited improvement in the deeper pathological alterations associated with photoaging, including collagen depletion, fibroblast dysfunction, and extracellular matrix (ECM) degradation. Although emerging transdermal delivery technologies, including nanocarrier systems, liposomes, and microneedle-assisted delivery, have significantly enhanced the transdermal transport of bioactive agents, their clinical application remains constrained by complex manufacturing procedures, insufficient long-term safety evidence, and limited translational potential.

In addition to topical skincare products, medical aesthetic technologies such as laser therapy, radiofrequency (RF), high-intensity focused ultrasound (HIFU), and microneedling have been widely employed for skin rejuvenation. These energy-based modalities primarily induce controlled thermal stimulation or microinjury to activate endogenous tissue repair processes, thereby promoting collagen remodeling and skin tightening. They have demonstrated favorable efficacy in improving wrinkles, skin laxity, and decreased skin elasticity. However, these treatments are frequently associated with adverse reactions, including erythema, edema, pain, and postoperative recovery periods. Furthermore, their therapeutic outcomes are influenced by treatment parameters and interindividual variability, and the long-term efficacy as well as optimal treatment protocols remain to be further optimized.

Microneedling enhances transdermal delivery by creating transient microchannels within the epidermis while simultaneously activating the skin's intrinsic wound-healing response. When combined with bioactive substances such as growth factors, microneedling can further stimulate collagen synthesis and improve skin texture ¹⁵. Nevertheless, microneedling remains an invasive procedure that inevitably disrupts the 

skin barrier and carries potential risks of infection, as well as increased requirements for postoperative care.

Overall, although current anti-aging technologies can improve skin appearance and partially restore tissue architecture, most available approaches primarily target a single aspect of the aging process. For example, topical skincare products mainly improve epidermal function, whereas laser and radiofrequency therapies promote collagen remodeling principally through stimulation of tissue repair. Regenerative medicine approaches, including exosome-based therapies, primarily exert their effects by modulating intercellular communication. However, skin aging results from the combined actions of oxidative stress, chronic inflammation, cellular senescence, and abnormal extracellular matrix remodeling. Therefore, future anti-aging strategies should evolve toward comprehensive therapeutic approaches capable of simultaneously targeting multiple aging-associated biological processes rather than focusing on a single pathogenic mechanism. 

1.3 Cold Atmospheric Plasma Technology: A Novel Approach for Skin Anti-Aging

Cold atmospheric plasma (CAP) is an emerging physical bioregulatory technology that has developed rapidly in recent years. Unlike conventional high-temperature plasma, CAP can be generated under atmospheric pressure at temperatures close to that of the human body. Owing to its relatively low gas temperature, CAP can interact with biological tissues without causing significant thermal damage. CAP is composed of multiple bioactive components, including reactive oxygen and nitrogen species (RONS), electrons, ions, ultraviolet photons, and electromagnetic fields. These active constituents can interact with cell membranes, proteins, and intracellular signaling pathways, thereby regulating cell proliferation, migration, inflammatory responses, and tissue repair processes ^16 17.

With the rapid development of plasma medicine, the biomedical applications of CAP have expanded considerably. At present, CAP has been extensively investigated for the treatment of skin diseases, chronic wound healing, antimicrobial therapy, and tissue regeneration ¹⁸. These studies have demonstrated that CAP possesses excellent biocompatibility and therapeutic potential, making it a promising non-pharmacological strategy for tissue repair and regenerative medicine.

The biological effects of CAP are primarily attributed to the generation of reactive oxygen and nitrogen species (RONS). Traditionally, reactive oxygen species (ROS) 

were regarded mainly as mediators of oxidative damage. However, accumulating evidence indicates that physiological levels of ROS also function as essential intracellular signaling molecules involved in regulating cell proliferation, migration, differentiation, and tissue repair. Moderate oxidative stimulation induced by CAP can activate adaptive cellular responses, enhance endogenous antioxidant defense systems, and improve cellular resistance to oxidative stress. This phenomenon is generally explained by the concept of hormesis, whereby low-intensity stress elicits beneficial biological adaptations that enhance cellular resilience ^19 20 21. Therefore, the therapeutic effects of CAP are not simply achieved by reducing oxidative stress but rather by restoring redox homeostasis and promoting normal cellular function.

Within the skin, CAP exerts biological effects on multiple cell types, including keratinocytes, dermal fibroblasts, and immune cells. Previous studies have shown that CAP promotes keratinocyte migration and accelerates the restoration of epidermal barrier function while simultaneously stimulating fibroblast activation and enhancing tissue repair. In addition, CAP modulates inflammation-related signaling pathways, thereby improving the local tissue microenvironment and creating favorable conditions for tissue regeneration ²². These biological characteristics make CAP particularly attractive for the treatment of skin photoaging. Photoaged skin is characterized by excessive ROS accumulation, persistent chronic inflammation, impaired fibroblast function, and increased collagen degradation. Because CAP simultaneously regulates oxidative stress, inflammatory responses, and cellular activity through multiple bioactive components, it offers a distinct advantage over conventional anti-aging approaches that target only a single pathogenic mechanism. Consequently, CAP may represent a more effective strategy for comprehensively modulating the complex biological processes involved in skin aging.

In recent years, plasma-activated liquids (PALs) and plasma-activated media (PAM) have emerged as new directions in plasma medicine research. During direct CAP treatment, many reactive species exhibit relatively short lifetimes and limited diffusion distances. By transferring CAP-generated reactive species into liquid systems, these bioactive molecules can be preserved for extended periods, thereby providing more stable and controllable biological effects. Plasma-activated liquids have been shown to promote cell migration and angiogenesis through RONS-mediated mechanisms while simultaneously reducing microbial burden, demonstrating considerable potential for applications in cutaneous wound healing ²³.

Furthermore, the integration of CAP with tissue engineering biomaterials has become an increasingly active area of investigation. Incorporating CAP-generated reactive species into biomaterial platforms, such as hydrogels, enables localized storage and sustained release of bioactive molecules, thereby improving treatment stability while providing a favorable microenvironment for cell adhesion, proliferation, and tissue regeneration ²⁴. Recent studies suggest that the combination of CAP with biomaterials may represent an important future direction in skin repair and regenerative medicine ²⁵.

From the perspective of aesthetic medicine, one of the greatest advantages of CAP lies in its non-thermal and multi-target regulatory properties. Unlike conventional energy-based technologies such as lasers and radiofrequency devices, which primarily rely on thermal stimulation to induce tissue remodeling, CAP promotes skin rejuvenation by modulating cellular redox balance, enhancing tissue repair, and improving the tissue microenvironment. Consequently, CAP possesses considerable potential as a non-invasive, safe, and effective therapeutic modality for long-term skin rejuvenation and anti-aging management.

2. Research Content and Technical Roadmap

This project was designed to investigate the potential of a cold atmospheric plasma (CAP) beauty device in improving skin photoaging. A comprehensive research strategy combining in vitro cellular models and in vivo animal models was employed to systematically evaluate the anti-aging effects of CAP from three perspectives: cellular functional regulation, tissue structural restoration, and molecular mechanism elucidation.

First, in the in vitro study, mouse fibroblast cell lines L929 and NIH-3T3 were selected as experimental models. An oxidative stress-induced cellular senescence model was established through hydrogen peroxide (H₂O₂) stimulation to mimic fibroblast dysfunction caused by excessive ROS accumulation during the skin photoaging process. Subsequently, CAP treatment was applied, and multiple biological indicators, including cellular proliferation activity, migration capacity, senescence status, and oxidative stress levels, were evaluated to determine whether CAP could reverse the senescent phenotype and restore the functional properties of aging fibroblasts.

For the in vivo experiments, a UV-induced skin photoaging model was established in C57BL/6 mice. Combined UVA and UVB irradiation was applied to simulate chronic ultraviolet exposure-induced skin damage. Following successful establishment of the photoaging model, mice received intervention using the CAP beauty device. The effects of CAP on structural and functional recovery of photoaged skin were comprehensively evaluated through gross skin observation, dermoscopic examination, histological staining, and molecular analyses.

For the mechanistic investigation, quantitative real-time PCR (qRT-PCR) analysis and transcriptome sequencing were integrated to systematically characterize gene expression alterations in skin tissues following CAP treatment. Particular attention was focused on biological processes associated with oxidative stress regulation, inflammatory response modulation, cellular senescence control, and collagen metabolism remodeling, thereby elucidating the potential molecular mechanisms underlying the protective effects of CAP against skin photoaging.

3. Materials and Methods

3.1 Experimental Materials

Table 1. Major Experimental Reagents and Materials

3.2 Experimental Instruments

Table 2. Major Experimental Instruments

 

3.3 Experimental Methods

3.3.1 Establishment of an In Vitro Fibroblast Senescence Model and CAP Intervention

To simulate fibroblast dysfunction caused by oxidative stress during the process of skin photoaging, an oxidative stress-induced cellular senescence model was established using hydrogen peroxide (H₂O₂) stimulation. L929 and NIH-3T3 fibroblasts were cultured under appropriate conditions until reaching a stable growth state. Subsequently, cells were treated with 200 μmol/L H₂O₂ for 8 h, after which the culture medium was replaced with fresh complete medium, allowing cells to gradually develop into a stable senescent state.

The effectiveness of model establishment was evaluated through morphological observation and senescence-associated β-galactosidase (SA-β-gal) staining. Four experimental groups were established: the normal control group, CAP-only treatment group, H₂O₂-induced senescence group, and H₂O₂ combined with CAP treatment group. Comparisons among these groups were performed to investigate the regulatory effects of CAP on oxidative stress-induced cellular senescence.

To determine appropriate CAP treatment parameters, normal fibroblasts were first exposed to CAP for different durations. Cell proliferation and viability were evaluated using the CCK-8 assay to determine the effects of different CAP exposure times on cellular activity. Based on preliminary experiments, CAP treatment conditions that demonstrated beneficial effects without inducing significant cellular stress were selected for subsequent in vitro experiments.

3.3.2 Detection of Cellular Function and Senescence Status

To comprehensively evaluate the effects of CAP on the functional recovery of senescent fibroblasts, multiple parameters were assessed, including cell viability, migration capacity, proliferative activity, oxidative stress levels, and expression of senescence-associated molecules.

The CCK-8 assay was performed to evaluate changes in cellular proliferation activity under different treatment conditions. Cellular metabolic activity was measured to determine the effects of CAP treatment on the growth capacity of both normal and senescent fibroblasts. A wound-healing assay was conducted to assess cell migration ability. The closure of the scratched area was monitored at different time points to determine whether CAP could restore the repair capacity of fibroblasts following oxidative damage.

Cellular senescence was evaluated using SA-β-gal staining. SA-β-gal activity is one of the most widely used biomarkers for detecting cellular senescence, and increased SA-β-gal positivity generally indicates the establishment of a stable senescent phenotype. By comparing the proportion of SA-β-gal-positive cells among different experimental groups, the effects of CAP on H₂O₂-induced senescence phenotypes were assessed.

Furthermore, EdU staining was performed to detect DNA replication activity and evaluate changes in cellular proliferative potential following CAP intervention. The proportion of EdU-positive cells was analyzed to determine whether CAP could alleviate oxidative stress-induced cell-cycle arrest and promote the re-entry of senescent cells into the proliferative state.

3.3.3 Detection of Senescence-Associated Gene Expression

To further investigate the regulatory effects of CAP on fibroblast senescence at the molecular level, quantitative real-time PCR (qRT-PCR) was performed to analyze changes in gene expression profiles.

The analysis focused on genes closely associated with cellular senescence and collagen metabolism, including the cell-cycle inhibitors p16 and p21, as well as the type I collagen-encoding gene COL1A1. Among these markers, p16 and p21 are classical regulators involved in senescence-associated cell-cycle arrest, and their abnormal upregulation typically indicates the establishment of a stable senescent state. COL1A1 serves as an important indicator of fibroblast-mediated maintenance of skin structural integrity and extracellular matrix (ECM) synthesis capacity.

By comparing gene expression differences among various treatment groups, this study evaluated whether CAP could suppress activation of senescence-related signaling pathways while restoring collagen synthesis capacity in fibroblasts.

3.3.4 Establishment of UV-Induced Mouse Skin Photoaging Model and CAP Treatment

To further validate the anti-aging effects of CAP within a complex tissue environment, a UV-induced mouse skin photoaging model was established. Six-week-old male C57BL/6 mice were used in this study. Prior to model induction, dorsal hair removal was performed to ensure consistent exposure of the irradiation area. Subsequently, photoaging was induced through combined UVA and UVB irradiation. The UVB dose was set at 120 mJ/cm² and the UVA dose at 20 mJ/cm². Mice received irradiation once daily for 8 consecutive weeks to establish a stable photoaging model. According to the experimental design, animals were randomly divided into four groups: the normal control group (NC), CAP treatment group (CAP), UV-induced photoaging model group (UV), and UV combined with CAP treatment group (UV+CAP).

After successful establishment of the photoaging model, the dorsal skin of mice in the CAP and UV+CAP groups received treatment using the FORSCI. cold atmospheric plasma beauty device twice daily, with each treatment lasting 20 s, for 7 consecutive days. The effects of CAP on UV-induced skin photoaging were evaluated by comparing differences in skin appearance, tissue structure, and molecular expression profiles among experimental groups.

3.3.5 Histological and Molecular Analysis of Mouse Skin Tissue

To evaluate the effects of CAP on tissue architecture and collagen metabolism in photoaged skin, multiple histological and molecular approaches were applied for comprehensive analysis.

During the experiment, gross observation and dermoscopic examination were first performed to record skin appearance changes in each group, including wrinkle formation, roughness, desquamation, and pigmentation, which are typical manifestations associated with photoaging.

Histological alterations were evaluated using hematoxylin and eosin (HE) staining, including epidermal thickness, dermal structural integrity, and inflammatory cell infiltration. Masson's trichrome staining was performed to assess dermal collagen fiber distribution and content. Collagen-positive staining areas were quantified to determine whether CAP could promote restoration of collagen architecture following UV-induced damage.

At the molecular level, immunohistochemical staining was conducted to detect changes in markers associated with senescence, inflammation, and collagen metabolism, with particular emphasis on P16, P21, IL-6, and COL1A1 expression. In addition, qRT-PCR analysis was performed to further evaluate transcriptional changes of related genes and verify the regulatory effects of CAP on skin aging-associated molecular pathways at the tissue level.

3.3.6 Transcriptome Sequencing and Bioinformatics Analysis

To further elucidate the potential molecular mechanisms underlying CAP-mediated improvement of skin photoaging, transcriptome sequencing was performed using skin tissues collected from the UV model group and the UV+CAP treatment group. By comparing gene expression profiles between the two groups, differentially expressed genes (DEGs) induced by CAP intervention were identified. Subsequently, functional enrichment analyses were conducted to investigate the biological processes and signaling pathways associated with these DEGs. The analysis focused particularly on biological processes closely related to skin aging regulation, including oxidative stress responses, inflammatory regulation, collagen synthesis, extracellular matrix organization, and tissue repair pathways. In addition, protein–protein interaction (PPI) network analysis was performed to identify potential regulatory nodes involved in the anti-aging effects of CAP, providing further evidence for elucidating the molecular mechanisms by which CAP improves skin photoaging.

3.3.7 Safety Evaluation of CAP Treatment

To evaluate the safety of the FORSCI. cold atmospheric plasma beauty device during skin treatment, assessments were performed from two perspectives: local thermal effects and systemic physiological responses. Infrared thermal imaging was used to continuously monitor changes in skin surface temperature during CAP treatment to determine whether CAP exposure resulted in significant thermal accumulation. During the experimental period, changes in body weight were continuously recorded. In addition, blood biochemical parameters and hematoxylin and eosin (H&E) staining of major organs were analyzed to evaluate whether CAP treatment caused systemic toxicity or tissue damage. These safety evaluations provide experimental evidence supporting the further application and clinical translation of CAP-based beauty devices in the field of skin anti-aging.

3.4 Ethics Statement

This study was approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (Approval No. HUST-IACUC-2025-0076).

4. Experimental Results and Analysis

4.1 Results of Cellular Experiments

4.1.1 Establishment and Validation of the Fibroblast Senescence Model

To evaluate the regulatory effects of CAP on senescent fibroblasts, an oxidative stress-induced cellular senescence model was first established in vitro (Figure 1). SA-β-gal activity staining was performed to evaluate the establishment of the senescence model. Following treatment with 200 μmol/L H₂O₂ for 8 h and subsequent culture in fresh complete medium, both L929 and NIH-3T3 mouse fibroblasts exhibited typical senescent morphological characteristics, including enlarged cell bodies, flattened and expanded cellular morphology, and extensive accumulation of dark blue SA-β-gal-positive granules in the cytoplasm. Quantitative image analysis further demonstrated that the proportion of SA-β-gal-positive cells in the untreated normal control (NC) group remained below 10%, whereas H₂O₂ stimulation significantly increased SA-β-gal positivity to over 55% (p < 0.0001). This alteration showed highly consistent patterns between the L929 and NIH-3T3 fibroblast cell lines. These findings confirmed the successful establishment of a stable, highly positive, and reproducible in vitro fibroblast senescence model, providing a reliable experimental platform for subsequent CAP intervention studies.

Figure 1. SA-β-gal staining results of fibroblasts after H₂O₂ treatment.

(A) SA-β-gal staining of L929 cells after H₂O₂ treatment (scale bar: 200 μm); (B) Quantitative analysis of SA-β-gal-positive L929 cells; (C) SA-β-gal staining of NIH-3T3 cells after H₂O₂ treatment (scale bar: 200 μm); (D) Quantitative analysis of SA-β-gal-positive NIH-3T3 cells. (n = 9, mean ± SD; ns indicates no significance; ****p < 0.0001, one-way ANOVA).

4.1.2 Optimization of CAP Treatment Parameters and Regulation of Fibroblast Proliferation and Migration

After successful establishment of the cellular senescence model, preliminary experiments were performed to determine the optimal CAP treatment parameters and evaluate its effects on fibroblast proliferation and migration recovery (Figure 2).

The CCK-8 proliferation assay demonstrated that immediately after cell seeding, the optical density values (OD450) among all groups were comparable, indicating consistent initial cell densities. Following 24 h and 48 h of culture, normal fibroblasts treated with CAP for 1 s, 5 s, or 10 s exhibited enhanced proliferative activity compared with the untreated control group.

Among different treatment durations, 5 s CAP exposure consistently increased the OD450 values of both L929 and NIH-3T3 fibroblasts (p < 0.01). However, cells receiving 10 s CAP treatment showed a slight reduction in proliferation rate at 48 h, suggesting that prolonged CAP exposure may induce mild cellular stress. Therefore, a 5 s CAP treatment duration was selected as the standard parameter for subsequent in vitro intervention experiments.

In the proliferation assay of senescent fibroblasts, H₂O₂ stimulation markedly suppressed cellular proliferation at both 24 h and 48 h. Following 5 s CAP treatment, the H₂O₂+CAP group exhibited a significant increase in OD450 values compared with the H₂O₂-only group. At 48 h, proliferative activity recovered to approximately 80% of that observed in normal fibroblasts, demonstrating a clear time-dependent recovery trend.

Figure 2. Evaluation of CAP-induced effects on fibroblast proliferation.

(A) CCK-8 assay of L929 cells treated with CAP at 0 h, 24 h, and 48 h; (B) CCK-8 assay of NIH-3T3 cells treated with CAP at 0 h, 24 h, and 48 h; (C) CCK-8 assay of H₂O₂-induced L929 senescent cells treated with CAP at 0 h, 24 h, and 48 h; (D) CCK-8 assay of H₂O₂-induced NIH-3T3 senescent cells treated with CAP at 0 h, 24 h, and 48 h. (n = 9, mean ± SD; ns indicates no significance; ***p < 0.001, ****p < 0.0001, one-way ANOVA).

Furthermore, the wound-healing assay further confirmed the restorative effects of CAP on fibroblast migration capacity (Figure 3). H₂O₂ treatment markedly impaired cellular migration, resulting in delayed wound closure, with the wound-healing rate remaining below 20% after 48 h of culture. In contrast, CAP treatment enhanced intrinsic migratory capacity in normal fibroblasts and significantly promoted wound closure in H₂O₂-induced senescent fibroblasts. The wound-healing rate of the H₂O₂+CAP group increased to over 60% at 48 h, indicating that CAP effectively alleviated oxidative stress-induced impairment of fibroblast migration and tissue repair capacity.

Figure 3. Evaluation of fibroblast migration capacity.

(A) Representative wound-healing images of H₂O₂-induced L929 cells after CAP treatment (scale bar: 500 μm); (B) Quantitative analysis of wound closure in L929 cells; (C) Representative wound-healing images of H₂O₂-induced NIH-3T3 cells after CAP treatment (scale bar: 500 μm); (D) Quantitative analysis of wound closure in NIH-3T3 cells.

4.1.3 Alterations in Cellular Phenotype and Intracellular Microenvironment Following CAP Treatment

To further investigate the effects of CAP on cellular physiological status and intracellular microenvironment remodeling, changes in senescence phenotype, DNA replication activity, and oxidative stress levels were comprehensively analyzed among different experimental groups. SA-β-gal staining demonstrated that CAP intervention significantly improved H₂O₂-induced fibroblast senescence phenotypes (Figure 4). In the normal control group, only a small proportion of cells exhibited SA-β-gal positivity, with limited blue cytoplasmic staining, indicating a low basal level of cellular senescence. Following H₂O₂ stimulation, fibroblasts displayed prominent senescent characteristics, including a substantial increase in SA-β-gal-positive cells and extensive accumulation of blue-stained granules within the cytoplasm, confirming successful induction of oxidative stress-mediated cellular senescence. In comparison, CAP treatment markedly reduced the number of SA-β-gal-positive granules in the H₂O₂+CAP group compared with the H₂O₂ group. The proportion of SA-β-gal-positive cells decreased to below 30%, accompanied by restoration of a more normal cellular morphology. These results indicated that CAP effectively suppressed oxidative damage-induced cellular senescence and reduced the activation of senescence-associated β-galactosidase activity.

Figure 4. SA-β-gal staining analysis of H₂O₂-induced senescent fibroblasts following CAP treatment.

(A) SA-β-gal staining of H₂O₂-induced L929 cells treated with CAP (scale bar: 200 μm); (B) SA-β-gal staining of H₂O₂-induced L929 cells treated with CAP (scale bar: 200 μm); (C) Quantification of SA-β-gal-positive NIH-3T3 cells; (D) Quantification of SA-β-gal-positive NIH-3T3 cells. (n = 9, mean ± SD; ns indicates no significance; ***p < 0.001, ****p < 0.0001, one-way ANOVA).

The EdU incorporation assay was further performed to evaluate changes in DNA replication activity and proliferative capacity of fibroblasts following CAP treatment (Figure 5). The results showed that the proportion of EdU-positive cells in the normal control group remained above 20%, indicating that most cells maintained strong DNA synthesis activity and remained in an active proliferative state. Following H₂O₂ stimulation, the percentage of EdU-positive cells was significantly reduced, suggesting that oxidative stress induced a stable cell-cycle arrest and markedly suppressed DNA replication activity. After CAP intervention, the proportion of EdU-positive cells in the H₂O₂+CAP group was significantly restored and showed a marked increase compared with the H₂O₂-only group. These results indicated that CAP promoted the re-entry of senescent fibroblasts into the cell cycle and restored their proliferative potential. Together with the SA-β-gal staining results, these findings further demonstrated that CAP not only reduced the expression of senescence-associated markers but also alleviated H₂O₂-induced cell-cycle arrest, thereby effectively reversing the senescent phenotype of fibroblasts.

Figure 5. EdU staining results of H₂O₂-induced fibroblasts following CAP treatment.

(A) EdU staining of H₂O₂-induced L929 cells after CAP treatment (scale bar: 200 μm); (B) Quantitative analysis of EdU staining in H₂O₂-induced L929 cells; (C) EdU staining of H₂O₂-induced NIH-3T3 cells after CAP treatment (scale bar: 200 μm); (D) Quantitative analysis of EdU staining in H₂O₂-induced NIH-3T3 cells. (n = 9, mean ± SD; ns indicates no significance; ***p < 0.001, ****p < 0.0001, one-way ANOVA).

4.1.4 Quantitative Analysis of Transcriptional Levels of Senescence- and Collagen Synthesis-Related Genes

At the transcriptional level, quantitative real-time PCR (qRT-PCR) was performed to further investigate the molecular mechanisms underlying CAP-mediated rejuvenation-like effects in fibroblasts (Figure 6). Quantitative analysis demonstrated that compared with the NC group, H₂O₂ stimulation resulted in a marked decrease in the mRNA expression level of COL1A1, a key gene encoding type I collagen. This finding indicated that oxidative stress severely impaired the major extracellular matrix (ECM) synthesis function of fibroblasts. Meanwhile, the mRNA expression levels of p21 and p16, which are classical markers involved in cell-cycle arrest and cellular senescence pathways, were significantly increased by approximately 3–5-fold following H₂O₂ stimulation. After CAP intervention, the expression level of COL1A1 mRNA in the H₂O₂+CAP group was significantly restored compared with the H₂O₂-induced senescence group, approaching the level observed in the NC group. Meanwhile, the abnormally elevated expression levels of p21 and p16 were markedly reduced. These transcriptional data demonstrated that CAP treatment effectively alleviated p16- and p21-mediated cell-cycle arrest, restored collagen synthesis capacity in fibroblasts, and promoted the reversal of senescence-associated transcriptional alterations toward a more youthful cellular state.

Figure 6. mRNA expression levels of COL1A1, p21, and p16 in senescent fibroblasts following CAP treatment.

(A) Relative mRNA expression levels of COL1A1, p21, and p16 in CAP-treated L929 cells; (B) Relative mRNA expression levels of COL1A1, p21, and p16 in CAP-treated NIH-3T3 cells. (n = 3, mean ± SD; ns indicates no significance; ****p < 0.0001, one-way ANOVA).

4.2 Results of Animal Experiments

4.2.1 CAP Treatment Does Not Induce Abnormal Systemic Physiological Changes in Animals

To evaluate the thermal effects generated during CAP treatment and its influence on overall physiological status, infrared thermal imaging was first applied to monitor temperature changes in the treated skin area before, during, and after CAP exposure. In addition, changes in mouse body weight were continuously recorded throughout the experimental period. During CAP treatment, the temperature of the treated skin area remained stable (Figure 7). Before treatment (Pre-CAP), the average temperature of the monitored region was 36.04℃. After initiation of CAP treatment, temperatures at 1 s, 5 s, 10 s, and 20 s were 35.92℃, 36.38℃, 36.22℃, and 36.24℃, respectively. Only minor temperature fluctuations were observed throughout the treatment process, with no continuous increase in temperature detected. These results indicated that CAP exerted its biological effects primarily through non-thermal mechanisms and did not induce significant local temperature elevation. Further analysis of temperature recovery after CAP treatment showed that skin temperatures at 5 s, 30 s, 60 s, and 300 s post-treatment were 36.20℃, 36.22℃, 36.00℃, and 35.96℃, respectively. All values remained within the normal physiological skin temperature range, suggesting that CAP treatment did not cause thermal accumulation or delayed heat-induced tissue damage.

Figure 7. Skin temperature changes in mice before, during, and after CAP treatment.

(A) Temperature changes during 20 s CAP exposure; (B) Temperature changes within 300 s after CAP treatment; (C) Statistical analysis of skin temperature changes throughout the entire CAP treatment process. (n = 5, mean ± SD; ns indicates no significant difference between mouse skin temperature and CAP treatment).

4.2.2 CAP Ameliorates UV-Induced Skin Tissue Damage and Promotes Restoration of Collagen Architecture

To further investigate the histological basis underlying the protective effects of CAP against skin aging, H&E staining and Masson's trichrome staining were performed to analyze changes in skin tissue architecture and collagen fiber distribution among different experimental groups.

HE staining revealed that the NC and CAP groups maintained intact skin structures, normal epidermal thickness, and relatively organized dermal collagen arrangements (Figure 8). Following UV irradiation, mice exhibited obvious histological alterations, including epidermal thickening, abnormal keratinization, and disorganization of dermal structures, indicating successful induction of UV-mediated skin aging (Figure 8A). Compared with the UV group, the UV+CAP group showed reduced epidermal thickness and improved tissue architecture, which more closely resembled the normal condition. Quantitative analysis demonstrated that UV exposure significantly increased epidermal thickness, whereas CAP intervention markedly alleviated UV-induced epidermal hyperplasia (Figure 8C). These findings suggested that CAP effectively reduced abnormal epidermal proliferation induced by UV irradiation.

Masson's trichrome staining was further performed to evaluate alterations in dermal collagen content (Figure 8B). In the NC group, abundant blue-stained collagen fibers with dense and continuous distribution were observed in the dermis. After UV irradiation, collagen fibers were markedly reduced and displayed loose arrangement and structural disruption, indicating enhanced collagen degradation caused by UV exposure, which is a major contributor to skin laxity and wrinkle formation. In contrast, the UV+CAP group exhibited increased collagen fiber abundance and improved fiber organization compared with the UV group. Quantitative analysis of Masson's staining demonstrated that collagen content was significantly decreased after UV exposure but was substantially restored following CAP treatment (Figure 8D). These results indicated that CAP effectively attenuated UV-induced dermal collagen loss and contributed to the maintenance of skin structural integrity.

Immunohistochemical analysis was further performed to evaluate expression of skin aging-associated proteins (Figure 9). UV irradiation significantly increased the expression of the inflammatory cytokine IL-6 and senescence-associated markers P16 and P21, while reducing expression of the collagen synthesis-related protein COL1A1. CAP intervention markedly decreased IL-6, P16, and P21 expression levels and increased COL1A1 expression in the UV+CAP group.

These findings demonstrated that CAP not only improved UV-induced structural alterations in skin tissue but also ameliorated photoaging status through regulation of inflammatory responses and senescence-associated molecular pathways.

Figure 8. Histological analysis of mouse skin tissues by H&E staining and Masson's trichrome staining.

(A) H&E staining of mouse skin tissues from different experimental groups (black arrows indicate the epidermis; n = 5; scale bar: 100 μm; magnification: 400×). (B) Masson's trichrome staining of mouse skin tissues from different groups (n = 5; scale bar: 400 μm; magnification: 100×). Enlarged images of Masson's trichrome staining from different groups (n = 5; scale bar: 100 μm; magnification: 400×). (C) Quantification of epidermal thickness based on H&E staining. (D) Comparison of collagen content among different groups based on Masson's trichrome staining. (Data are presented as mean ± SD; n = 5; ****p < 0.0001; ns indicates no significance; one-way ANOVA).

Figure 9. Immunohistochemical analysis of COL1A1, P16, P21, and IL-6 expression in mouse skin tissues from different experimental groups.

Representative immunohistochemical staining images and quantification of positive staining areas for COL1A1, P16, P21, and IL-6 (n = 5; scale bar: 100 μm; magnification: 400×). (Data are presented as mean ± SD; n = 5; *p < 0.05, ***p < 0.001, ****p < 0.0001; ns indicates no significance; one-way ANOVA).

4.2.3 CAP Improves Skin Photoaging by Regulating the Expression of Senescence-Associated Genes

To further investigate the molecular mechanisms underlying the protective effects of the FORSCI. cold atmospheric plasma beauty device against skin aging, qRT-PCR was performed to analyze the mRNA expression levels of collagen synthesis-related genes and cellular senescence-associated genes in mouse skin tissues from different experimental groups.

Compared with the NC group, UV irradiation significantly reduced COL1A1 mRNA expression in mouse skin tissues, indicating that chronic ultraviolet exposure suppressed collagen synthesis capacity in dermal fibroblasts. This reduction in collagen production represents an important molecular basis for decreased skin elasticity and wrinkle formation during photoaging (Figure 10).

In contrast, the CAP-only treatment group exhibited increased COL1A1 expression compared with the NC group, suggesting that CAP itself may promote the expression of genes associated with collagen synthesis.

In the UV-induced photoaging model, the UV+CAP group showed a significant recovery of COL1A1 expression compared with the UV group, approaching the levels observed in normal skin. These results indicated that CAP intervention partially reversed the UV-induced suppression of collagen synthesis.

Analysis of cellular senescence-related markers revealed that UV irradiation significantly increased the expression levels of P16 and P21. Both P16 and P21 are classical markers associated with cellular senescence and cell-cycle arrest, and their upregulation generally indicates the transition of cells into a senescent state. The increased expression of P16 and P21 following UV exposure suggested that ultraviolet irradiation induced senescence responses within skin tissues.

Compared with the UV group, the UV+CAP group exhibited significantly decreased expression levels of P16 and P21, indicating that CAP effectively reduced UV-induced cellular senescence and delayed the progression of skin aging. Meanwhile, expression levels of P16 and P21 in the CAP-only group did not show significant elevation compared with the NC group, further suggesting that CAP treatment itself did not induce cellular senescence in skin tissues.

Consistent with the immunohistochemical findings described above, CAP treatment not only enhanced COL1A1 protein expression but also reduced senescence-associated markers including P16 and P21. These results suggest that the biological effects of CAP may involve both restoration of collagen metabolism and suppression of senescence-associated signaling activation.

Collectively, the qRT-PCR results demonstrated that the FORSCI. cold atmospheric plasma beauty device regulated the expression of genes associated with collagen synthesis and cellular senescence during skin photoaging, restoring the molecular characteristics of damaged skin tissues toward a normal state. These findings provide molecular evidence supporting the role of CAP in improving photoaging-associated skin phenotypes.

Figure 10. Relative mRNA expression levels of COL1A1, p21, and p16 in mouse skin tissues following CAP treatment. (n = 3, mean ± SD; ns indicates no significance; ****p < 0.0001, one-way ANOVA).

4.2.4 Biosafety Evaluation of CAP Treatment

While evaluating the anti-aging efficacy of CAP, further animal safety assessments were performed. The systemic safety of long-term CAP treatment was evaluated through complete blood count analysis and histological examination of major organs.

First, peripheral blood samples from mice in each experimental group were collected for five-part differential blood analysis (Table 3). The results showed that immune-related hematological parameters, including white blood cell count (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mon), eosinophils (Eos), and basophils (Bas), remained within normal physiological reference ranges in all groups. The WBC level was 6.92 ± 1.43 × 10⁹/L in the CAP group and 4.06 ± 0.59 × 10⁹/L in the UV+CAP group, with neither exceeding the normal range. Red blood cell-related parameters, including red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT), showed no abnormal alterations among different groups, indicating that CAP treatment did not affect normal hematopoietic function. Platelet (PLT) counts also remained within the normal range, with no significant fluctuations observed among groups. Further analysis of immune cell proportions showed that although certain variations in leukocyte distribution occurred following UV irradiation and CAP intervention, all values remained within normal physiological ranges. No persistent inflammatory response or immunosuppressive effect was observed. These results indicated that CAP treatment did not induce detectable hematological toxicity.

Table 3. Differential blood count analysis of mice in the NC, CAP, UVB, and UV+CAP groups after completion of 7-day CAP treatment. (Orbital venous blood samples were collected for five-part differential blood analysis; n = 5, mean ± SD)

  Histological analysis of major organs was further performed using H&E staining to evaluate potential tissue alterations following CAP treatment (Figure 11). Five major organs, including the heart, liver, spleen, lung, and kidney, were selected for pathological evaluation. The results showed that mice in the NC group exhibited normal tissue architecture and intact cellular organization in all examined organs. In the CAP group, no obvious pathological alterations were observed in myocardial structure, hepatic cell arrangement, splenic architecture, alveolar structure, or renal tissue morphology. Due to prolonged UV exposure, slight stress-associated alterations were occasionally observed in some tissues of the UV group. However, compared with the UV group, the UV+CAP group showed no additional tissue damage, and the overall morphology of all examined organs remained comparable to that of the NC group. No obvious pathological changes, including inflammatory cell infiltration, tissue necrosis, fibrosis, or structural disruption, were detected in any group based on HE staining analysis.

Figure 11. H&E staining analysis of major organs (heart, liver, spleen, lung, and kidney) in mice following different treatments (Scale bar: 100 μm).

Collectively, the hematological and histological findings demonstrated that under the experimental duration and treatment parameters used in this study, the FORSCI. cold atmospheric plasma beauty device did not induce significant systemic toxicity or detectable damage to major organs. These results indicate that CAP exhibits favorable biocompatibility and application safety for skin anti-aging treatment.

4.3 Transcriptomic Analysis

4.3.1 Analysis of Differentially Expressed Genes in Skin Tissues Following CAP Intervention

Based on high-quality transcriptome sequencing data, gene expression profiles between the UV group and the UV+CAP group were compared to identify key regulatory genes significantly altered following CAP intervention.

Differential expression analysis revealed substantial changes in the gene expression landscape of mouse skin tissues after CAP treatment compared with the UV-induced photoaging model group (Figure 12A). Volcano plot analysis identified statistically significant differentially expressed genes (DEGs), including both upregulated and downregulated genes. These findings suggested that CAP stimulation induced extensive transcriptional regulation within skin tissues and promoted recovery of functional states associated with skin cellular activity.

Furthermore, hierarchical clustering analysis of DEGs demonstrated clear separation between the UV and UV+CAP groups. Samples within each treatment group exhibited high intra-group consistency, indicating that CAP intervention effectively altered the gene expression patterns of UV-damaged skin tissues (Figure 12B).

Figure 12. Differentially expressed gene analysis based on mouse skin transcriptome sequencing.

(A) Volcano plot of differentially expressed genes. (B) Hierarchical clustering heatmap of differentially expressed genes.

4.3.2 Functional Enrichment Analysis of Differentially Expressed Genes Reveals Activation of Anti-Aging-Related Pathways by CAP

To further elucidate the potential mechanisms underlying the protective effects of CAP on skin aging, Reactome pathway enrichment analysis was performed using the identified DEGs (Figure 13).

The results demonstrated that compared with the UV group, DEGs in the UV+CAP group were primarily enriched in pathways associated with: collagen biosynthesis and modifying enzymes, collagen formation, extracellular matrix organization, regulation of collagen matrix degradation, and integrin-mediated cell surface interactions.

Among these pathways, collagen metabolism-associated processes exhibited significant enrichment, suggesting that CAP intervention may promote functional recovery of dermal fibroblasts and enhance collagen synthesis and extracellular matrix (ECM) stability. Moreover, activation of pathways associated with collagen synthesis and modification enzymes indicated that CAP may facilitate maturation and processing of newly synthesized collagen, thereby improving collagen fiber integrity and reversing UV-induced structural deterioration of the skin. In addition, DEGs were also enriched in pathways related to ion channel regulation, cytoskeletal organization, muscle contraction, and cellular signal transduction. These findings suggested that CAP may regulate skin homeostasis through multidimensional modulation of cellular functional states.

Collectively, transcriptomic analysis indicated that the CAP beauty device may exert anti-aging effects through multiple mechanisms, including enhancement of collagen biosynthesis, promotion of ECM remodeling, and improvement of the cellular microenvironment.

Figure 13. Reactome pathway enrichment analysis of differentially expressed genes.

(A) Bar plot of Reactome pathway enrichment analysis. (B) Bubble plot of Reactome pathway enrichment analysis.

4.3.3 Protein–Protein Interaction Network Analysis of Differentially Expressed Genes

To further investigate the interaction relationships among proteins encoded by differentially expressed genes, protein–protein interaction (PPI) network analysis was performed (Figure 14). The results showed that differentially expressed proteins formed multiple highly interconnected functional modules. Several core proteins were located at central positions within the interaction network, suggesting that these molecules may play important regulatory roles in CAP-induced skin repair processes. PPI network analysis further demonstrated that protein networks associated with collagen metabolism, extracellular matrix formation, and tissue repair exhibited high interaction density. These findings suggested that CAP intervention may promote skin structural remodeling and functional recovery through coordinated regulation of multiple interacting molecular targets. Based on these analyses, the identified key differentially expressed genes and their interaction networks may serve as important candidate targets for future investigations into the molecular mechanisms underlying the anti-aging effects of CAP.

Figure 14. Protein–protein interaction network of differentially expressed genes.

5. Discussion

Skin aging is a complex biological process driven by multiple interconnected factors. Among these factors, chronic ultraviolet (UV) exposure-induced skin photoaging is considered one of the major contributors affecting skin appearance and functional integrity. Unlike intrinsic aging caused primarily by chronological progression, photoaging is characterized by more pronounced pathological alterations, including enhanced oxidative stress, chronic inflammatory activation, accumulation of senescent cells, and extracellular matrix (ECM) degradation. Therefore, comprehensive regulation of multiple pathological processes involved in photoaging has become an important research direction in the field of skin anti-aging.

In this study, we systematically investigated the effects and underlying molecular mechanisms of a cold atmospheric plasma (CAP) beauty device in improving skin photoaging. By establishing an in vitro oxidative stress-induced fibroblast senescence model and an in vivo UV-induced mouse skin photoaging model, the anti-aging effects of CAP were evaluated at multiple levels, including cellular function, tissue architecture, and molecular regulation. The results demonstrated that CAP effectively alleviated oxidative stress-induced fibroblast senescence, promoted restoration of skin tissue structure, and exerted comprehensive anti-aging effects through regulation of oxidative stress, inflammatory responses, and ECM metabolism.

5.1 CAP Improves Oxidative Stress-Induced Fibroblast Senescence

Dermal fibroblasts are essential functional cells responsible for maintaining skin structural stability and functional integrity. Their primary functions include synthesis of type I collagen, elastin, and various extracellular matrix components. During the progression of skin aging, fibroblasts are continuously affected by oxidative damage, resulting in reduced proliferation capacity, impaired migration ability, and decreased collagen synthesis. These functional abnormalities contribute significantly to skin laxity, reduced elasticity, and wrinkle formation.

In the present study, an oxidative stress-induced fibroblast senescence model was first established using H₂O₂ stimulation. The results demonstrated that H₂O₂ treatment induced typical senescent phenotypes in both L929 and NIH-3T3 fibroblasts, including enlarged cell morphology, cellular flattening, increased SA-β-gal positivity, and significant reductions in proliferative and migratory capacities. These findings confirmed that oxidative stress effectively induced fibroblast senescence.

Based on this model, the regulatory effects of CAP on senescent fibroblast function were further investigated. The results showed that CAP treatment significantly reduced H₂O₂-induced SA-β-gal positivity, improved abnormal cellular morphology, and restored fibroblast proliferation and migration capacities. Furthermore, EdU incorporation analysis demonstrated that CAP enhanced DNA replication activity, indicating that CAP not only reduced senescence-associated phenotypes but also promoted partial recovery of proliferative potential in senescent cells.

These findings suggest that CAP does not simply improve cellular survival but may regulate cellular functional states and promote the restoration of a rejuvenation-associated phenotype in damaged fibroblasts. This mechanism is particularly significant for skin anti-aging applications, as conventional anti-aging strategies are primarily focused on external supplementation or superficial improvement, whereas CAP may directly regulate key functional skin cells and achieve deeper tissue-level modulation.

5.2 CAP Improves Cellular Microenvironment Through Regulation of Oxidative Stress Homeostasis

Our study demonstrated that CAP intervention decreased the expression levels of senescence-associated genes p16 and p21, while restoring COL1A1 expression. The p16/p21 signaling pathway represents a key regulatory mechanism involved in cellular senescence and cell-cycle arrest. Persistent activation of this pathway leads to stable growth arrest, whereas reduced COL1A1 expression reflects impaired ECM synthesis capacity of fibroblasts. Therefore, the regulatory effects of CAP on these molecular markers further suggest that CAP may improve fibroblast functional status by suppressing senescence-associated signaling activation and restoring collagen synthesis capacity.

5.3 CAP Ameliorates UV-Induced Skin Photoaging-Associated Tissue Damage

To further validate the anti-aging effects of CAP in a complex tissue environment, a UV-induced C57BL/6 mouse skin photoaging model was established. Chronic UV exposure induces typical photoaging characteristics, including rough skin texture, increased wrinkle formation, epidermal hyperplasia, and reduced dermal collagen content.

In this study, UV irradiation resulted in obvious photoaging characteristics in mouse skin, whereas CAP treatment markedly improved skin surface appearance, reduced wrinkle formation, and restored more regular tissue texture. These findings indicated that CAP effectively alleviated UV-induced macroscopic skin damage.

Histological analyses further confirmed the tissue repair effects of CAP. H&E staining showed that UV exposure induced epidermal thickening and tissue structural disruption, while CAP treatment significantly restored epidermal architecture. Masson's trichrome staining demonstrated that UV irradiation caused reduction and disorganization of dermal collagen fibers, whereas CAP treatment promoted collagen fiber recovery and increased dermal collagen content.

Collagen depletion represents one of the most important characteristics of skin aging and is a major contributor to reduced elasticity and wrinkle formation. Therefore, the ability of CAP to restore collagen architecture suggests that CAP not only improves superficial skin appearance but also regulates deeper dermal structural remodeling, thereby exerting more profound anti-aging effects.

5.4 CAP May Improve Skin Photoaging Through Multi-Pathway Coordinated Regulation

Skin photoaging is not caused by a single pathological factor but results from the combined effects of oxidative stress, inflammation, cellular senescence, and ECM dysregulation. Therefore, single-target interventions often fail to achieve comprehensive anti-aging outcomes.

In this study, transcriptomic sequencing was performed to systematically analyze gene expression changes following CAP treatment. The results demonstrated that CAP-regulated differentially expressed genes were mainly enriched in biological processes associated with collagen synthesis, extracellular matrix organization, collagen fiber formation, and tissue repair. Combined with the cellular experimental findings, a potential regulatory model for CAP-mediated improvement of photoaging can be proposed. First, CAP-generated bioactive components interact with skin cells to regulate redox balance and reduce oxidative stress-induced cellular damage. Subsequently, CAP suppresses p16/p21-associated senescence signaling and promotes functional recovery of fibroblasts. Meanwhile, enhanced COL1A1 expression and activation of ECM-related processes contribute to dermal structural remodeling. In addition, CAP may improve the chronic inflammatory microenvironment associated with photoaging by reducing inflammatory mediator expression. Therefore, CAP may not act through a single molecular target but rather through a coordinated regulatory network involving oxidative stress regulation, cellular functional restoration, inflammatory control, and tissue remodeling.

5.5 CAP Beauty Device Exhibits Favorable Biosafety for Skin Applications

Safety represents a critical factor for the clinical translation of aesthetic devices. In this study, infrared thermal imaging was used to monitor skin temperature changes during CAP treatment. The results showed that CAP exposure did not induce significant temperature elevation or continuous thermal accumulation under the applied treatment conditions, indicating that CAP primarily functions through non-thermal mechanisms. Meanwhile, body weight monitoring and systemic evaluations in animal experiments showed no obvious physiological abnormalities following CAP treatment, suggesting favorable biological safety under the experimental conditions. Compared with conventional aesthetic devices that rely mainly on thermal injury-induced tissue repair, CAP possesses advantages including non-thermal action, safety, and mild biological regulation, making it potentially suitable for long-term skin care and anti-aging applications. Therefore, this study not only demonstrated the efficacy of CAP in improving skin photoaging but also provided important safety evidence supporting its further development and application.

6. Experimental Conclusion

In this study, the effects and mechanisms of FORSCI. cold atmospheric plasma beauty device in improving skin photoaging were systematically investigated using complementary in vitro cellular models and in vivo animal models.

At the cellular level, CAP effectively improved H₂O₂-induced fibroblast senescence by reducing SA-β-gal positivity, promoting recovery of proliferation and migration capacities, decreasing excessive ROS accumulation, and restoring oxidative stress homeostasis. At the molecular level, CAP reduced the expression of senescence-associated factors including p16 and p21, while promoting recovery of COL1A1 expression, indicating that CAP regulates cellular senescence progression and improves collagen synthesis capacity.

In animal experiments, CAP significantly ameliorated UV-induced skin photoaging phenotypes, including reduction of wrinkles and tissue damage, improvement of epidermal abnormalities, and restoration of dermal collagen structure. Transcriptomic analysis further demonstrated that CAP modulated gene networks associated with collagen formation, extracellular matrix organization, and tissue repair.

Collectively, this study demonstrated that the FORSCI. cold atmospheric plasma beauty device effectively alleviates skin photoaging through regulation of oxidative stress homeostasis, restoration of fibroblast function, promotion of collagen metabolism, and improvement of the tissue microenvironment. Furthermore, this study established an evaluation system for the anti-aging effects of CAP and provided experimental evidence supporting the potential application of cold atmospheric plasma technology in skin rejuvenation and anti-aging fields.

7. Application Value and Development Prospects

7.1 Application Value of the CAP Beauty Device

This study systematically investigated the effects and underlying mechanisms of a cold atmospheric plasma (CAP) beauty device in improving skin photoaging. The anti-aging effects of CAP were comprehensively evaluated at the cellular, tissue, and molecular levels. The results demonstrated that CAP not only improved oxidative stress-induced fibroblast senescence but also promoted restoration of UV-induced photoaged skin architecture and collagen remodeling.

These findings further expand the scientific foundation for the application of cold atmospheric plasma in skin health and provide a novel research direction for utilizing physical regulatory technologies to intervene in the skin aging process.

From the perspective of technological application, this study verified the efficacy and safety of the CAP beauty device in improving skin photoaging. Due to its non-thermal characteristics, CAP can exert biological regulatory effects without causing tissue injury, providing advantages including non-invasiveness, safety, and convenience. Therefore, CAP possesses considerable potential for further development as either a home-use beauty device or a professional dermatological care system.

Unlike conventional skincare products that mainly act on the superficial layers of the skin, CAP can regulate cellular redox balance, improve fibroblast function, and promote extracellular matrix homeostasis, thereby influencing aging-related processes at deeper biological levels. This study confirmed the effectiveness of the CAP beauty device in improving photoaging-associated alterations and further demonstrated its favorable safety profile under experimental conditions.

Because CAP does not rely on significant thermal effects and does not cause heat-induced tissue damage, it has promising application potential in non-invasive skin care and anti-aging fields.

Overall, the in vitro cellular senescence evaluation system, animal photoaging model, and multi-level mechanistic analysis platform established in this study provide an experimental foundation for future optimization of CAP devices and product development. Future studies should incorporate human skin samples and clinical evaluation systems to further validate CAP efficacy, application range, and optimal treatment parameters under different scenarios, thereby facilitating translation of this technology into practical aesthetic applications.

7.2 Development Prospects of the CAP Beauty Device

As an emerging physical technology capable of regulating multiple bioactive components, cold atmospheric plasma has broad application potential in skin health management and disease intervention.

This study demonstrated that CAP effectively alleviates skin photoaging by regulating oxidative stress, improving fibroblast function, and promoting collagen metabolism. These findings provide an experimental basis for expanding CAP applications into broader areas of skin care and dermatological treatment.

In the future, the development of CAP beauty devices will not be limited to anti-aging applications alone but may also involve combination strategies with conventional skincare products and therapeutic approaches. Because CAP can improve the skin microenvironment, promote tissue repair, and potentially enhance skin barrier permeability, combining CAP with bioactive ingredients possessing antioxidant, anti-inflammatory, collagen-promoting, or regenerative properties may establish a comprehensive treatment strategy integrating physical regulation with active ingredient synergy.

For example, combining CAP technology with peptides, hyaluronic acid, growth factors, and antioxidant compounds may further enhance the biological effects of these ingredients on deeper skin tissues and improve their utilization efficiency. In addition, for certain dermatological disorders, CAP combined with pharmacological treatments may improve therapeutic outcomes by modulating local microenvironments, regulating inflammatory responses, and promoting tissue recovery.

Furthermore, CAP efficacy is closely associated with plasma generation parameters, including treatment frequency, exposure duration, output intensity, and treatment cycles. Future studies should optimize CAP parameters according to different skin conditions and application requirements to develop personalized treatment protocols.

For skin anti-aging applications, low-intensity and long-term periodic stimulation may be adopted to maintain skin homeostasis and promote collagen metabolism. For inflammatory skin conditions, tissue repair, or pathological damage, treatment intensity and frequency may be adjusted according to therapeutic objectives to achieve more precise intervention.

Beyond skin rejuvenation, CAP has also demonstrated promising potential in various dermatological applications. Previous studies have shown that CAP possesses antimicrobial, anti-inflammatory, and tissue regenerative properties, suggesting potential applications in acne, chronic wounds, alopecia, and fungal nail infections (onychomycosis).

For example, in hair loss-related disorders, CAP may improve scalp microenvironment, promote local angiogenesis, and regulate inflammatory status, thereby providing a novel approach for supporting hair follicle regeneration. In fungal nail infections, CAP-generated reactive species may exert antimicrobial effects while promoting periungual tissue repair, providing an auxiliary strategy for conventional antifungal therapy.

With advances in material science, microelectronics, and intelligent control technologies, future CAP devices are expected to evolve toward miniaturization, intelligence, and home-based applications. Integration with artificial intelligence algorithms, skin condition detection technologies, and adaptive parameter control systems may enable automatic adjustment of treatment modes according to individual skin conditions, improving safety and user experience.

Meanwhile, multi-omics approaches and accumulation of clinical datasets will further clarify CAP targets, optimize treatment parameters, and accelerate the transition of CAP technology from experimental research toward precision skin health management and clinical applications.

Overall, CAP beauty devices not only possess application value for improving skin photoaging but also demonstrate potential for expansion into dermatological disease support, combination therapies, and personalized skin health management. With continuous advances in mechanistic understanding and device optimization, CAP may become an important technological platform for future non-invasive skin care and disease intervention.

8. Recent Research Progress Worldwide

In recent years, with the rapid development of cold atmospheric plasma (CAP) research in the biomedical field, its applications have expanded from initial antimicrobial and wound-healing studies to broader areas, including tissue regeneration, treatment of dermatological disorders, and aesthetic anti-aging applications. Recent studies have demonstrated that CAP does not exert biological effects merely through simple antimicrobial activity or physical stimulation. Instead, CAP regulates multiple biological processes through modulation of reactive oxygen and nitrogen species (RONS), regulation of immune microenvironments, enhancement of cellular functional recovery, and control of tissue regeneration processes. The following section summarizes recent representative advances regarding the applications of CAP in skin health and related biomedical fields.

8.1 Recent Advances in CAP-Mediated Hair Follicle Regeneration and Hair Loss Treatment

CAP and Hair Follicle Regeneration

Core Advantage: ROS + IL-2 drive immune-regulated regeneration, outperforming minoxidil and finasteride.

Hair Follicle Activation: CAP generates ROS + IL-2, expands Tregs, activates DPCs/Wnt signaling, and drives follicles from telogen to anagen.

Key Results: Increased follicle number, length, and diameter; thicker skin and hair shafts; earlier anagen entry; ~100% hair coverage in 15 days.

In recent years, CAP has demonstrated promising potential in the field of hair regeneration. Conventional approaches for treating hair loss mainly rely on pharmacological regulation of the hair growth cycle. However, these strategies primarily focus on promoting hair growth while paying relatively limited attention to abnormalities in the perifollicular immune microenvironment and impaired tissue repair capacity.

Recent studies have revealed that CAP can promote the transition of hair follicles from the resting phase (telogen) to the growth phase (anagen) by regulating the local immune microenvironment, providing a novel physical regulatory strategy for hair loss management. One study developed a CAP-activated hydrogel system that enabled storage and sustained release of CAP-generated bioactive components. The results demonstrated that ROS generated by CAP promoted increased expression of interleukin-2 (IL-2), which subsequently enhanced expansion of regulatory T cells (Tregs).As an important immune cell population maintaining hair follicle immune homeostasis, Tregs can release immunoregulatory factors such as IL-10 and transforming growth factor-β(TGF-β), thereby improving the inflammatory microenvironment surrounding hair follicles and promoting activation of dermal papilla cells (DPCs). Furthermore, CAP-induced immune regulation was shown to activate Wnt-associated signaling pathways, facilitating entry of hair follicles into the anagen phase. Animal studies demonstrated that CAP treatment significantly increased hair follicle number, hair shaft length, and skin hair coverage.This research further expands the application potential of CAP in aesthetic medicine, suggesting that CAP may not only be used for skin anti-aging but may also provide new strategies for scalp care and supportive treatment of hair loss.

Based on the findings of the present study regarding CAP-mediated improvement of skin photoaging, future research may establish dermal papilla cell models, hair follicle stem cell models, and animal hair-loss models to investigate the effects of CAP on hair follicle cycling, stem cell activation, and scalp microenvironment regulation. In addition, the synergistic effects of CAP combined with minoxidil, growth factors, exosomes, and other hair-regenerative agents should be further explored.

8.2 Advances in CAP for Infectious Wound Repair

CAP and Infected Wound Healing

Core Advantage: CAP enables hydrogel formation, activation, and loading with sustained RONS retention for immune-regulated repair, beyond antibacterial action.

Mechanism: CAP generates ROS, inhibits bacteria, improves the wound environment, sustains IL-2 release to expand Tregs, suppresses excessive inflammation, and promotes tissue repair.

Key Results: Sustained RONS (>24 h) increase Tregs, regulate CD4+/CD8+ immunity, reduce inflammation, and enhance tissue regeneration.

Treatment Method: Inject a precursor solution, then apply localized CAP to form an in situ hydrogel with sustained RONS and immune factor release (≥72 h).

Beyond anti-aging and aesthetic applications, CAP-based treatment of infected wounds has become another major research focus. Conventional wound management typically relies on antibiotics to control infection and dressings to promote tissue repair. However, antibiotic resistance, persistent inflammation, and insufficient tissue regeneration remain major challenges limiting the treatment of chronic wounds.

Recent studies have demonstrated that CAP possesses combined antimicrobial, anti-inflammatory, and tissue regenerative properties, allowing simultaneous regulation of multiple pathological processes involved in infected wound healing. CAP-generated RONS can directly interact with bacterial cell membranes, proteins, and genetic materials, causing oxidative damage, reducing bacterial viability, and disrupting biofilm structures. Unlike conventional antimicrobial strategies that mainly target microbial elimination, CAP can additionally regulate wound immune responses by influencing macrophage polarization, promoting angiogenesis, and enhancing fibroblast migration, thereby accelerating wound repair. Recent studies have further shown that integration of CAP with hydrogel materials can generate CAP-active wound dressings with sustained release capabilities, allowing prolonged retention of RONS and related bioactive factors at wound sites and improving therapeutic stability. These advances also provide important implications for aesthetic medicine and skin repair. Many cosmetic procedures, including laser therapy, microneedling, and injectable treatments, are accompanied by temporary skin barrier disruption and inflammatory responses. Therefore, promoting rapid postoperative recovery represents an important clinical challenge.

Future studies may explore combinations of CAP with regenerative hydrogels, growth factors, and anti-inflammatory components to simultaneously enhance collagen remodeling and skin barrier restoration, providing new strategies for postoperative aesthetic care.

8.3 Expansion of CAP Applications in Various Dermatological Disorders

CAP and Other Skin Disorders

Infected & Chronic Wounds: RONS provide antibacterial, anti-inflammatory, and pro-angiogenic effects, reducing infection and accelerating wound healing.

Skin & Radiation Injury: Regulates growth factors to promote cell migration and proliferation, accelerating healing, reducing fibrosis, and supporting hair follicle regeneration.

Atopic Dermatitis: Suppresses mast cells and Th2 responses, reducing inflammation, itching, and skin lesions.

Psoriasis: Inhibits keratinocyte overgrowth, regulates immunity, reduces plaques, and controls chronic inflammation.

Skin Infection (Drug-Resistant Bacteria/Biofilms): Disrupts bacterial structures and enhances antibiotic sensitivity, improving control of persistent infections.

Allergic Contact Dermatitis: Reduces inflammatory mediators and immune activation, relieving inflammation and skin irritation.

Vitiligo: Modulates melanocytes and the local immune environment, supporting repigmentation and regeneration.

Skin Barrier Disorders / Transdermal Delivery: Enhances skin permeability via lipid oxidation and electroporation, improving transdermal drug delivery.

With increasing understanding of CAP biology, its applications have expanded from wound repair to a broader range of dermatological conditions. Recent studies indicate that CAP possesses multiple biological functions, including antimicrobial activity, immune regulation, tissue repair promotion, and barrier restoration. Therefore, CAP has demonstrated potential applications in infectious skin diseases, inflammatory skin disorders, and pigment-related conditions.

In infectious skin diseases, CAP-generated RONS can disrupt microbial structures, including bacteria and fungi. Because its antimicrobial mechanisms involve multi-target oxidative damage, CAP is less likely to induce resistance commonly associated with conventional antimicrobial agents. For example, in the treatment of onychomycosis (fungal nail infection), conventional antifungal therapies often face challenges such as long treatment cycles and insufficient drug penetration. CAP can directly affect fungal cellular structures while improving the local tissue environment, suggesting its potential as an adjunctive antifungal therapy. In inflammatory skin diseases, CAP can regulate immune cell activation and reduce inflammatory mediator release, indicating potential value for chronic inflammatory disorders such as atopic dermatitis and psoriasis. Moreover, CAP may enhance skin barrier recovery by improving keratinocyte function and regulating local redox balance, thereby increasing endogenous repair capacity.

These findings suggest that the future development of CAP will extend beyond aesthetic anti-aging applications and may evolve into a comprehensive skin health regulation platform integrating skincare, disease management, and tissue regeneration.

8.4 Advances in CAP-Based Skin Cancer Therapy

CAP and Skin Cancer

Selective Cytotoxicity: RONS selectively kill melanoma and SCC cells while sparing normal cells with stronger antioxidant defenses.

Multiple Cell Death Pathways: Induces apoptosis, ferroptosis, and immunogenic cell death, enhancing T-cell immunity.

Inhibits Growth & Metastasis: Suppresses PI3K-AKT-mTOR signaling and reduces CD133/CD44 and MMP-2/9, limiting invasion and metastasis.

Treatment Method: Applied as direct CAP, plasma-activated media (PAM), or combined with microneedles.

Combination Therapy: Enhances efficacy when combined with chemotherapy, phototherapy, or nanomedicines.

Activates Antitumor Immunity: Promotes M1 macrophages and CD8⁺ T-cell infiltration, improving the tumor immune microenvironment.

In addition to benign dermatological disorders, CAP has attracted increasing attention in the field of cancer therapy. Recent studies have demonstrated that CAP-generated RONS can selectively induce oxidative damage in tumor cells, while normal cells with stronger antioxidant defense systems may tolerate CAP stimulation to a certain extent. Therefore, CAP exhibits certain selective cytotoxic characteristics toward malignant cells.

Previous studies have shown that CAP can induce tumor cell death through multiple mechanisms, including promotion of DNA damage, enhancement of lipid peroxidation, induction of apoptosis, and activation of immune-related responses. Furthermore, CAP can modify the tumor microenvironment by promoting immune cell recruitment and enhancing anti-tumor immune responses, thereby improving therapeutic efficacy. Currently, CAP has demonstrated promising potential in studies involving various skin malignancies, including melanoma and squamous cell carcinoma.

In the future, combination strategies involving CAP with chemotherapeutic agents, nanomaterials, and immunotherapy may represent important research directions. CAP may enhance drug sensitivity, improve the tumor microenvironment, and reduce certain adverse effects associated with conventional therapies.

Therefore, CAP may not only serve as an independent therapeutic approach but may also function as a platform for drug sensitization and immune modulation in comprehensive cancer treatment strategies.

8.5 merging Strategies of CAP Combined with Biomaterials and Drug Delivery Systems for Skin Repair

CAP for Oral Diseases & Neural Repair

Oral Applications

Advantages: Non-thermal treatment protects oral tissues while precisely targeting infection sites.

Antimicrobial Action: Eliminates Streptococcus, Candida, and disrupts oral biofilms.

Implant Treatment: Improves titanium hydrophilicity, enhancing osteoblast adhesion and osseointegration.

Periodontal Regeneration: Promotes gingival cell growth, angiogenesis, and wound healing.

Neural Repair

Neural Differentiation: Activates Wnt signaling to promote neural stem cell differentiation and axon growth.

Spinal Cord Repair: Reduces apoptosis and glial scarring, supporting axonal regeneration and functional recovery.

Neuroprotection: Activates antioxidant pathways and protective autophagy, reducing ischemic and oxidative injury.

Treatment Method: Applied as direct CAP, plasma-activated saline (PAS), or via local/intrathecal injection.

CAP and Cancer

RONS-Mediated Cytotoxicity: RONS induce DNA damage, lipid peroxidation, and protein inactivation, triggering apoptosis or necrosis.

Immune Activation: Activates antitumor immune responses.

Selective Cytotoxicity: Suppresses tumor growth while minimizing damage to normal cells. Effective against cervical, brain, breast, and other cancers.

Treatment Methods: Delivered by direct application, injection, oral administration, or with medical devices.

Combination Therapy: Combined with nanoparticles, chemotherapy, or immunotherapy for enhanced efficacy.

In recent years, CAP application strategies have gradually evolved from conventional direct plasma exposure toward integration with biomaterials and drug delivery systems. Because CAP-generated RONS generally exhibit short lifetimes and limited diffusion distances, achieving stable preservation and controlled release of these active components has become a major focus in current research. Consequently, combining CAP with hydrogels, biomaterials, and nanocarriers has emerged as an important strategy for improving therapeutic efficiency.

Studies have demonstrated that CAP-activated hydrogels can preserve CAP-generated reactive species and achieve localized sustained release within tissues, thereby extending the biological effects of CAP. Meanwhile, hydrogel materials themselves possess favorable properties, including hydration maintenance, structural support, and tissue regeneration capacity. When combined with CAP, these materials can form novel therapeutic systems integrating biological regulation and tissue protection.

Furthermore, combination strategies involving CAP and pharmaceutical agents represent another important future direction. CAP can improve skin barrier conditions, enhance drug penetration, and activate cellular repair mechanisms through ROS-mediated signaling pathways, thereby potentially increasing the efficacy of bioactive skincare ingredients and therapeutic drugs. For example, CAP combined with hyaluronic acid, peptides, and growth factors may further enhance anti-aging effects, while CAP combined with anti-inflammatory or antifungal agents may improve therapeutic outcomes for dermatological diseases.

For the present research project, future studies may further investigate combinations of CAP with collagen-promoting agents, hyaluronic acid, growth factors, and exosomes. Exploring synergistic anti-aging strategies integrating CAP-mediated physical regulation with bioactive molecules may further enhance the application value of CAP beauty devices in skin rejuvenation.

8.6 Future Development Directions of CAP Technology

CAP Combined with Hydrogels

Current Advances in CAP Therapy

Direct CAP: Direct irradiation generates RONS for antibacterial, anti-inflammatory, and regenerative effects.

Activated Media (PAW/PAH): CAP-activated water or hydrogels provide RONS storage and sustained release for prolonged therapy.

In Situ Gel: CAP induces in situ gelation for local retention, sustained release, and immune regulation.

Drug Penetration: Lipid oxidation and electroporation enhance skin permeability and drug absorption.

CAP + Drugs: Enhances antibiotics and nanomedicines, improving efficacy and overcoming drug resistance.

Material Engineering: Optimizes material properties to increase drug loading and enable controlled release.

Potential Anti-Aging Strategies Combining CAP with Drugs

HA / Hydrogel Systems: CAP-modified HA/hydrogels improve cell adhesion, enable sustained release, and promote ECM remodeling and skin regeneration.

Growth Factors (EGF/FGF): Enhances skin permeability, improving EGF/FGF delivery to stimulate fibroblasts and collagen production.

Nanomedicines: Combined with gold or magnetic nanoparticles to improve drug delivery and therapeutic efficacy.

Antioxidants / Anti-inflammatory Drugs: Low-dose ROS activate repair signaling, while combined drugs reduce oxidative stress and inflammation.

Based on recent advances, CAP technology is gradually evolving from a conventional physical stimulation approach into a multifunctional platform for skin health management. Future development of CAP devices will focus not only on improving single-treatment efficacy but also on establishing precise and personalized treatment strategies according to different skin conditions. Because the biological effects of CAP are influenced by multiple parameters, including discharge mode, output intensity, exposure duration, and treatment frequency, different application scenarios require optimized parameter combinations. For skin anti-aging applications, low-intensity and long-term periodic stimulation strategies may be adopted to promote collagen metabolism, maintain cellular function, and preserve skin youthfulness. For inflammatory conditions, infections, or tissue injuries, treatment intensity and frequency may be adjusted according to specific therapeutic objectives to enhance antimicrobial and regenerative effects. In addition, integration of CAP technology with artificial intelligence (AI) and skin analysis systems will further promote the development of precision aesthetic medicine. Future devices may automatically analyze skin characteristics, including hydration status, elasticity, inflammatory conditions, and age-related changes, and subsequently adjust CAP treatment parameters to achieve individualized skin management.

Based on the findings of the present study demonstrating the beneficial effects of CAP on skin photoaging, future research directions may include: enhancing delivery efficiency of skincare ingredients through CAP-assisted transdermal approaches;investigating CAP-mediated hair follicle regeneration; exploring CAP applications for improving sensitive skin barrier dysfunction; evaluating CAP-assisted recovery following aesthetic procedures. These developments may facilitate the transformation of CAP technology from a single anti-aging device into a comprehensive platform for personalized skin health management.

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