1. Introduction

As the largest organ of the human body, skin health and appearance are of great significance to an individual’s overall health and quality of life. With aging, environmental influences, and diseases, the skin may develop problems such as aging and damage. Both Cold Atmospheric Plasma (CAP) and Vitamin C have shown certain potential in the field of skin treatment, and research on their combined application has gradually become a focus.

2. Characteristics of CAP and Its Mechanism of Action on Skin

(1) Generation and Characteristics of CAP

CAP is a state of matter produced by ionizing neutral gas molecules through energy forms such as electromagnetic fields or thermal energy. It consists of ions, electrons, reactive oxygen species (e.g., OH, H₂O₂), reactive nitrogen species (e.g., NO), electric fields, and heat [1][2]. CAP has important potential in the medical field, especially in dermatology, due to its relatively low temperature, which can avoid severe thermal damage to the skin.

(2) Mechanism of Action of CAP on Skin

① Thermal Effect: When CAP comes into contact with the skin, the skin temperature rises through gas heat transfer. A moderate temperature increase (37-38.5°C) can stimulate the proliferation of skin keratinocytes and promote tissue regeneration. When the temperature reaches a certain level (e.g., 45°C for 8-10 seconds), it can induce fibroblasts to produce a heat shock response, upregulate the expression of type I and type III procollagens, thereby promoting collagen synthesis and enhancing skin elasticity and firmness.

② Action of Reactive Substances: Reactive substances generated by plasma, especially NO, play multiple important roles in the skin. As a signaling molecule, NO participates in regulating the proliferation and differentiation of keratinocytes and fibroblasts, maintaining the normal structure and function of the epidermis [2][3][4][5]. It also has a vasodilatory effect, which can improve skin microcirculation, promote the supply of nutrients and oxygen, and facilitate the metabolism and repair of skin cells [6][7]. In addition, NO plays a role in immune regulation, which can enhance the skin’s immune defense capability and resist the invasion of external pathogens [5].

3. Characteristics of Vitamin C and Its Mechanism of Action on Skin

(1) Properties and Sources of Vitamin C

Vitamin C is a water-soluble vitamin that cannot be synthesized by the human body and must be obtained from the diet. It has a variety of important physiological functions in the body and is also crucial for skin health.

(2) Mechanism of Action of Vitamin C on Skin

① Antioxidant Effect: Vitamin C is a powerful antioxidant that can scavenge free radicals in the body, including endogenous free radicals and exogenous free radicals (generated by ultraviolet radiation, environmental pollution, etc.). Free radicals cause oxidative damage to skin cells, leading to lipid peroxidation of cell membranes, protein oxidation, and DNA damage, which in turn accelerates skin aging, resulting in wrinkles, sagging, pigmentation, and other problems [8][9][10][11][12][13]. By donating electrons to neutralize free radicals and converting them into harmless products, Vitamin C reduces the damage of oxidative stress to the skin and protects the integrity of skin cells [14][15][16].

② Promotion of Collagen Synthesis: As a cofactor for proline and lysine hydroxylase, Vitamin C participates in the collagen synthesis process. These enzymes hydroxylate proline and lysine residues in collagen molecules, stabilize the tertiary structure of collagen molecules, and promote the formation of correct collagen fibers [17][18][19][20][21][22][23][24][25]. Vitamin C can promote the expression of collagen genes and enhance the ability of fibroblasts to synthesize collagen, thereby maintaining skin strength and elasticity [21][26]. It should be noted that the regulation of collagen metabolism by Vitamin C takes a relatively long time to gradually show its effects. A lack of Vitamin C can lead to collagen synthesis disorders, resulting in fragile and easily damaged skin [17][21][26][27][28][29].

4. Synergistic Effects of "CAP + Vitamin C" on Skin

(1) Synergistic Promotion of Collagen Synthesis

① The thermal effect and reactive substances of plasma can stimulate fibroblast proliferation and collagen synthesis, while Vitamin C provides necessary chemical modification and stabilization for collagen synthesis. When the two are used in combination, Vitamin C can enhance the stimulating effect of plasma on fibroblasts, making collagen synthesis more efficient and stable [17][25].

② Studies have found that the collagen level after combined treatment increases significantly in a short period of time, and the structure of collagen fibers is more orderly. This indicates that the synergistic effect of the two helps improve the structural support of the skin, reduce wrinkle formation, and enhance skin firmness and elasticity [30].

③ Experimental studies have shown that the collagen level decreases after 4 weeks of Vitamin C treatment, and increases slightly at 6 weeks but is still lower than that of the control sample. Due to the incomplete manifestation of the regulatory effect of Vitamin C on collagen metabolism in the early stage, its collagen level is 26% lower than that of the control group; after 4 weeks of CAP treatment alone, the collagen level first decreases and then increases, and is higher than that of the control sample at 6 weeks, increasing by 17% compared with the control group; when CAP and Vitamin C are used simultaneously, the collagen level shows an upward trend without an initial decrease, increasing by 40% compared with the control group [31].

【See Figure 1 for the collagen comparison experiment under the combined treatment of "CAP + Vitamin C" [30]】

Figure 1 Collagen Comparison Experiment Under Combined Treatment of "CAP + Vitamin C"

(2) Synergistic Enhancement of Skin Antioxidant Capacity

① Plasma may generate a certain amount of reactive oxygen species during treatment, but at the same time, reactive substances such as NO generated by it also have a certain antioxidant regulatory effect. As a classic antioxidant, Vitamin C, when combined with CAP, can further enhance the ability to scavenge free radicals and reduce the damage of oxidative stress to the skin [14][15][16].

② Experiments have shown that after combined application, the oxidative products in skin cells are significantly reduced, and the activity of antioxidant enzymes is enhanced, which effectively protects skin cells from oxidative damage, delays the skin aging process, and improves skin color and texture [8][9][10][11][12][13].

(3) Synergistic Promotion of Skin Cell Proliferation and Repair

① CAP activates the proliferation signaling pathways of skin cells through its thermal effect and reactive substances, promoting the proliferation of keratinocytes and fibroblasts. Vitamin C provides nutritional support and a suitable intracellular environment for cell proliferation, which is conducive to the growth and differentiation of new cells [32][6].

② In wound healing experiments, the combined application of CAP and Vitamin C can accelerate the wound healing process, promote epithelial cell regeneration, reduce inflammatory reactions, improve healing quality, and reduce the risk of scar formation [33][34].

(4) Improvement of Skin Barrier Function

① CAP can regulate the balance of the skin surface microbial community and enhance the skin’s immune defense function. Vitamin C helps maintain the intercellular junction structure of skin cells, such as promoting the formation of desmosomes between keratinocytes and enhancing the integrity of the skin barrier [35].

② After combined treatment, the skin’s moisture retention capacity is enhanced, and its resistance to external stimuli is improved, reducing the occurrence of skin allergies, inflammation, and other problems [36].

5. Research Status and Prospects

At present, research on "CAP + Vitamin C" in skin treatment has achieved certain results, but there are still some problems and challenges. Future research directions include: further exploring the molecular mechanism of their synergistic effect, developing more efficient and convenient combined treatment technologies and products, and expanding their applications in other skin diseases and cosmetic fields. With the deepening of research, "CAP + Vitamin C" is expected to provide more innovative and effective solutions for skin health maintenance and disease treatment.

References

[1] Arisi, M. et al. Cold atmospheric plasma (CAP) for the treatment of actinic keratosis and skin field cancerization: clinical and high-frequency ultrasound evaluation. Dermatol. Ther. 11, 855-866 (2021).

[2] Heinlin, J. et al. Plasma applications in medicine with a special focus on dermatology. J. Eur. Acad. Dermatol. Venereol. 25, 1-11 (2011).

[3] Dams, S. The effect of heat shocks in skin rejuvenation. Doctor of Philosophy Thesis, Eindhoven: Technische Universiteit Eindhoven, (2010).

[4] Dams, S., De Liefde - van Beest, M., Nuijs, A., Oomens, C. & Baaijens, F. Pulsed heat shocks enhance procollagen type I and procollagen type III expression in human dermal fibroblasts. Skin Res. Technol. 16, 354-364 (2010).

[5] Cals - Grierson, M. - M. & Ormerod, A. Nitric oxide function in the skin. Nitric Oxide 10, 179-193 (2004).

[6] Suschek, C. V. & Opländer, C. The application of cold atmospheric plasma in medicine: The potential role of nitric oxide in plasma-induced effects. Clin. Plasma Med. 4, 1-8 (2016).

[7] Vasilets, V. N., Shekhter, A. B., Guller, A. E. & Pekshev, A. V. Air plasma - generated nitric oxide in treatment of skin scars and articular musculoskeletal disorders: Preliminary review of observations. Clin. Plasma Med. 3, 32-39 (2015).

[8] Pullar, J. M. et al. The roles of vitamin C in skin health. Nutrients 9, 866 (2017).

[9] Rhie, G. et al. Aging- and photoaging-dependent changes of enzymic and nonenzymic antioxidants in the epidermis and dermis of human skin in vivo. J. Investig. Dermatol. 117, 1212-1217 (2001).

[10] Shindo, Y. et al. Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin. J. Investig. Dermatol. 102, 122-124 (1994).

[11] McArdle, F. et al. UVR-induced oxidative stress in human skin in vivo: Effects of oral vitamin C supplementation. Free Radic. Biol. Med. 33, 1355-1362 (2002).

[12] Shindo, Y. et al. Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J. Investig. Dermatol. 102, 470-475 (1994).

[13] Shindo, Y. et al. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light. J. Investig. Dermatol. 100, 260–265 (1993).

[14] Hirschberg, J. et al. Validation of the suitability of stripped lipid as a skin model in plasma medical investigations. Open J. Appl. Sci. 5, 40–49 (2015).

[15] Jadoon, S. et al. Anti-aging potential of phytoextract loaded-pharmaceutical creams for human skin cell longevity. Oxid. Med. Cell. Longev. 2015, 709628 (2015).

[16] Wang, Y. et al. Cold atmospheric-pressure air plasma treatment of C6 glioma cells: Effects of reactive oxygen species in the medium produced by the plasma on cell death. Plasma Sci. Technol. 19, 025503 (2017).

[17] Hinek, A. et al. Sodium l-ascorbate enhances elastic fibers deposition by fibroblasts from normal and pathologic human skin. J. Dermatol. Sci. 75, 173–182 (2014).

[18] Ivanov, V. et al. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate. Am. J. Cardiovasc. Dis. 6, 26–35 (2016).

[19] Kivirikko, K. I., Myllyla, R. & Pihlajaniemi, T. Protein hydroxylation: Prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 3, 1609–1617 (1989).

[20] May, J. M. & Harrison, F. E. Role of vitamin C in the function of the vascular endothelium. Antioxid. Redox Signal. 19, 2068–2083 (2013).

[21] Kishimoto, Y. et al. Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts. Biochem. Biophys. Res. Commun. 430, 579–584 (2013).

[22] May, J. M. & Qu, Z. C. Transport and intracellular accumulation of vitamin C in endothelial cells: Relevance to collagen synthesis. Arch. Biochem. Biophys. 434, 178–186 (2005).

[23] Miller, R. L., Elsas, L. J. & Priest, R. E. Ascorbate action on normal and mutant human lysyl hydroxylases from cultured dermal fibroblasts. J. Investig. Dermatol. 72, 241–247 (1979).

[24] Parsons, K. K. et al. Ascorbic acid-independent synthesis of collagen in mice. Am. J. Physiol. Endocrinol. Metab. 290, 1131–1139 (2006).

[25] Pihlajaniemi, T., Myllyla, R. & Kivirikko, K. I. Prolyl 4-hydroxylase and its role in collagen synthesis. J. Hepatol. 13, 2–7 (1991).

[26] Takahashi, Y. et al. Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells. J. Biol. Chem. 275, 14139–14146 (2000).

[27] Geesin, J. C. et al. Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human skin fibroblast. J. Investig. Dermatol. 90, 420–424 (1988).

[28] Davidson, J. M. et al. Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms. J. Biol. Chem. 272, 345–352 (1997).

[29] Phillips, C. L., Combs, S. B. & Pinnell, S. R. Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts. J. Investig. Dermatol. 103, 228–232 (1994).

[30] Matsumura, Y. & Ananthaswamy, H. N. Toxic effects of ultraviolet radiation on the skin. Toxicol. Appl. Pharmacol. 195, 298-308 (2004).

[31] Shakouri, Reza, et al. In vivo study of the effects of a portable cold plasma device and vitamin C for skin rejuvenation. Scientific reports 11.1 (2021): 21915.

[32] de Araújo, R., Lôbo, M., Trindade, K., Silva, D. F. & Pereira, N. Fibroblast growth factors: A controlling mechanism of skin aging. Skin Pharmacol. Physiol. 4, 275-282 (2019).

[33] Shekhter, A., Kabisov, R., Pekshev, A., Kozlov, N. & Perov, Y. L. Experimental and clinical validation of plasmadynamic therapy of wounds with nitric oxide. Bull. Exp. Biol. Med. 126, 829-834 (1998).

[34] Shekhter, A. B., Serezhenkov, V. A., Rudenko, T. G., Pekshev, A. V. & Vanin, A. F. Beneficial effect of gaseous nitric oxide on the healing of skin wounds. Nitric Oxide 12, 210-219 (2005).

[35] Marionnet, C. et al. Morphogenesis of dermal-epidermal junction in a model of reconstructed skin: Beneficial effects of vitamin C. Exp. Dermatol. 15, 625–633 (2006).

[36] Duarte, T. L., Cooke, M. S. & Jones, G. D. Gene expression profiling reveals new protective roles for vitamin C in human skin cells. Free Radic. Biol. Med. 46, 78–87 (2009).