Creative Touch Permanent Makeup and Aesthetics

The client narrative around RONS is almost entirely wrong. “Free radicals damage skin; antioxidants prevent that damage” is a framing so compressed it becomes inaccurate – and the inaccuracy matters clinically, because the same reactive species that accelerate photoageing at high concentrations are the signalling molecules that initiate the collagen remodelling cascade after a microneedling session, the antimicrobial agents that sterilise a wound, the vasodilatory NO molecules that support healing angiogenesis, and the entire mechanism of action of cold atmospheric plasma. RONS are not skin’s enemies. They are the redox language through which skin cells communicate about damage, repair, threat, and renewal. The concentration, the species, the location, and the duration of exposure are everything.

The NRF2 antioxidant defence system – the cell’s primary sensor and responder to RONS – is itself activated by low-level ROS. The defence is triggered by the very thing it defends against, at concentrations below the damage threshold. This is the hormetic principle, and it is what makes therapeutic procedures that deliberately generate controlled RONS mechanistically coherent rather than paradoxical. Every practitioner working with CAP, RF microneedling, or laser resurfacing needs a working model of RONS biology – not because the science is needed at the treatment couch, but because clients ask about antioxidants, sun protection, and supplement timing and deserve answers that are accurate, specific, and grounded in something more than “free radicals are bad.”

The dose-response relationship – the organising principle

RONS are not uniformly harmful. They are chemically reactive molecules that act on biological targets – and like every signalling molecule, what they do depends entirely on how much of them is present, where, and for how long. At the low concentrations of normal cellular metabolism, ROS and RNS are second messengers: they activate transcription factors, stabilise hypoxia-inducible proteins, regulate enzyme activity, and coordinate immune responses. At the higher concentrations produced by UV overexposure, chronic inflammation, or dysbiotic microbial activity, the same species outpace the cell’s antioxidant defence capacity, producing cumulative oxidative damage to DNA, membrane lipids, and structural proteins. ^[6]

This dose-response curve is non-linear and biphasic – a pattern called hormesis. The beneficial zone is not zero RONS; it is a controlled low-to-moderate concentration where reactive species activate repair and defence pathways without exceeding the clearance capacity of antioxidant enzymes. Therapeutic procedures that generate controlled RONS – cold atmospheric plasma, RF microneedling, fractional laser resurfacing – are operating deliberately in this hormetic zone. They are not side-stepping the RONS biology; they are using it. Understanding this is the precondition for every other clinical insight this entity provides.

The three transcription factors most directly regulated by RONS concentration form the signalling backbone of this entity:

The relationship between these three is concentration-dependent. Low ROS → primarily NRF2 activation → antioxidant defence, cytoprotection. Moderate-to-high ROS → NF-κB and AP-1 activation → inflammation, MMP production, collagen degradation. Therapeutic RONS targets the NRF2 zone. Chronic sun exposure and uncontrolled inflammation operate in the NF-κB/AP-1 zone.

The NRF2 antioxidant defence system

NRF2 (Nuclear Factor Erythroid 2-Related Factor 2) is the transcription factor that coordinates the cell’s response to oxidative stress – and unlike most stress response systems, it is not dormant until needed. It is constitutively active at a basal level, with its activity continuously modulated by the ambient ROS concentration in the cell. ^[5]

Under resting conditions, NRF2 is held in the cytoplasm by its repressor protein Keap1 (Kelch-like ECH-associated protein 1), which binds NRF2 and targets it for continuous ubiquitin-mediated proteasomal degradation. Keap1 functions as the ROS sensor: it contains reactive cysteine residues – particularly C151, C273, and C288 – whose thiol groups are oxidised or modified by ROS and electrophilic compounds. When RONS concentrations rise, these cysteines are modified, Keap1’s E3 ligase adaptor function is disrupted, NRF2 is no longer ubiquitinated and degraded, and it accumulates in the cytoplasm. It then translocates to the nucleus, heterodimerises with small Maf proteins, and binds the Antioxidant Response Element (ARE) sequence in the promoters of its target genes. ^[5]

NRF2 target genes relevant to skin

The NRF2/Keap1/ARE axis declines with age in skin keratinocytes and fibroblasts – basal NRF2 nuclear occupancy decreases, Keap1 expression increases, and the magnitude of the antioxidant response to a given ROS stimulus progressively diminishes. This age-related NRF2 decline is one mechanistic reason why older skin is more vulnerable to UV-induced oxidative damage, recovers more slowly from procedural stress, and accumulates MMP-driven matrix degradation faster than younger skin at equivalent UV exposure. ^{[2] [5]}

Reactive oxygen species

ROS are oxygen-derived molecules carrying unpaired electrons or capable of generating them. Four species are primary in skin biology:

Superoxide (O₂·⁻)

The primary ROS product of cellular metabolism – generated by the incomplete one-electron reduction of molecular oxygen at mitochondrial Complex I and Complex III during ATP synthesis, and by NADPH oxidase (NOX) enzyme complexes in the plasma membrane. O₂·⁻ itself is relatively membrane-impermeant and short-lived, but it is the precursor of the more biologically significant H₂O₂ (via SOD dismutation) and the highly damaging peroxynitrite (via reaction with NO). NOX enzymes in skin keratinocytes (NOX1, NOX4) are the primary source of extramitochondrial O₂·⁻, activated by growth factor signalling, cytokine stimulation, and mechanical stress – including the mechanical microtrauma of microneedling. ^[11]

Hydrogen peroxide (H₂O₂)

The most stable and most biologically significant ROS signal. Unlike O₂·⁻, H₂O₂ is membrane-permeable – it crosses cell membranes through aquaporin channels (particularly AQP3 and AQP8) and can therefore act as a diffusible second messenger between cells. At nanomolar-to-low-micromolar concentrations, H₂O₂ activates NRF2 (via Keap1 cysteine oxidation), modulates NF-κB, and coordinates wound healing responses through PI3K/Akt and MAPK pathway activation. At higher concentrations it drives the Fenton reaction (see below) and protein oxidation (cysteine and methionine sulfoxidation, carbonylation). H₂O₂ is the primary antimicrobial species in CAP output and the species primarily responsible for CAP-mediated NF-κB modulation in rosacea and AD skin. ^[8]

Hydroxyl radical (·OH)

The most reactive and most damaging ROS – generated by the Fenton reaction: Fe²⁺ + H₂O₂ → Fe³⁺ + ·OH + OH⁻. Because ·OH reacts within nanoseconds of generation, essentially at its point of production, it produces highly localised damage – DNA strand breaks (8-OHdG formation), membrane lipid peroxidation chain reactions, and irreversible protein carbonylation. There is no enzymatic scavenger for ·OH; the antioxidant defence against it operates indirectly by maintaining low H₂O₂ concentrations and limiting free iron availability. Melanin acts as a ·OH scavenger in UV-exposed epidermal keratinocytes – one functional role of melanin that extends beyond simple UV light absorption. ^[12]

Singlet oxygen (¹O₂)

An excited-state oxygen molecule generated principally by UV irradiation in the presence of endogenous photosensitisers (porphyrins, flavins, melanin intermediates) and by certain plasma discharge reactions in CAP. ¹O₂ has a short lifetime in aqueous environments (microseconds) but reacts rapidly with guanine bases in DNA and with polyunsaturated fatty acids in membrane phospholipids. In photoageing, ¹O₂ contributes to the 8-OHdG DNA damage signature and to lipid peroxidation-mediated membrane disruption. ^[12]

Reactive nitrogen species

RNS are nitrogen-derived reactive molecules whose biology in skin is dominated by nitric oxide (NO) and its downstream chemistry. They are not simply a parallel category to ROS – at higher concentrations they intersect directly through peroxynitrite formation, making the ROS/RNS boundary functionally blurred in inflamed tissue.

Nitric oxide (NO). A short-lived, membrane-permeable gas synthesised by three nitric oxide synthase (NOS) isoforms present in skin: ^[14]

NO’s biological effects are concentration-dependent in the same way as ROS: at eNOS-level concentrations (pM–nM range), NO is vasodilatory, cytoprotective, and pro-angiogenic; at iNOS-level concentrations (μM range), it contributes to inflammatory cytotoxicity and can react with O₂·⁻ to form peroxynitrite. ^{[14] [13]}

NO and melanogenesis

NO generated by keratinocyte NOS directly stimulates melanogenesis through a tyrosinase-dependent mechanism, and acts on melanocytes to increase TYRP1 (tyrosinase-related protein 1) expression and melanin output. UV-induced keratinocyte NO production contributes to the post-UV tanning response through this paracrine pathway. In the context of treatments that generate or modulate NO – including CAP and LED photobiomodulation – the melanogenic potential of the NO signal is worth noting in clients with melasma or post-inflammatory hyperpigmentation risk, particularly with higher-energy or higher-frequency treatment schedules. ^[14]

Peroxynitrite (ONOO⁻)

The convergence point of ROS and RNS chemistry – formed by the near-diffusion-limited reaction of superoxide (O₂·⁻) with nitric oxide (NO): O₂·⁻ + NO → ONOO⁻. Peroxynitrite is more reactive than either precursor. It nitrates protein tyrosine residues (3-nitrotyrosine formation – a marker of nitrosative stress detectable in tissue biopsies), oxidises methionine and cysteine residues, and causes DNA single-strand breaks. Critically, ONOO⁻ formation is preferential at high ROS/RNS concentrations – it becomes the dominant product when both O₂·⁻ and NO are being generated simultaneously at elevated levels. In chronically inflamed skin where NOX-derived O₂·⁻ and iNOS-derived NO are both elevated, ONOO⁻ production becomes a genuine pathological species contributing to matrix damage and sustained inflammatory signalling, distinct from either oxidative or nitrosative stress alone. ^[10]

NO₂ and HNO₂ (nitrous acid)

Secondary RNS formed from NO oxidation in aqueous environments. Less physiologically prominent than NO and ONOO⁻ in skin biology, but relevant in CAP plasma chemistry where NO₂ and HNO₂ are measurable components of the reactive species output and contribute to the acidified aqueous environment that has antimicrobial properties at the treatment surface. ^[8]

RONS in skin ageing

The progressive accumulation of oxidative damage over a lifetime is one of the primary drivers of intrinsic skin ageing – and the UV-driven amplification of that accumulation is the primary driver of photoageing. The mechanism is not simply that ROS “damage” the skin; it is that specific ROS-activated signalling pathways produce specific, predictable structural changes that are visible, palpable, and progressive. ^[9]

The UV/MAPK/AP-1/MMP axis

UV irradiation (UVB particularly) generates ROS in keratinocytes and fibroblasts within seconds of exposure, activating three MAPK kinase cascades simultaneously – ERK, JNK, and p38. All three converge on AP-1 transcription factor assembly. AP-1 (c-Jun/c- Fos) drives expression of MMP-1 (interstitial collagenase – cleaves native fibrillar collagen I and III), MMP-3 (stromelysin – degrades fibronectin, laminin, proteoglycans), and MMP-9 (gelatinase B – degrades denatured collagen and elastin). A single minimal erythemal UV dose produces measurable collagenase activity in sun-exposed skin within hours. Repeated UV exposures do not allow full MMP clearance and collagen resynthesis between them – the deficit accumulates, the collagen and elastin matrix progressively degrades, and the structural thinning, laxity, and rhytid formation of photoageing results. ^{[7] [7]}

The mitochondrial ROS perpetuation cycle

Mitochondria are both the primary endogenous source of ROS and the primary target of ROS damage in ageing skin. Mitochondrial Complex I and Complex III generate O₂·⁻ as a normal byproduct of electron transport. With age, accumulating mitochondrial DNA damage (from decades of ROS exposure, with mitochondrial DNA particularly vulnerable due to its proximity to the electron transport chain and relative lack of protective histones) impairs electron transport chain efficiency – producing more electron leak, more O₂·⁻ generation per unit ATP synthesised, and further mitochondrial DNA damage. This self-amplifying cycle progressively increases the mitochondrial ROS baseline in dermal fibroblasts over time, elevating the AP-1/NF-κB signalling environment without any external UV trigger. Age-related intrinsic skin changes – fibroblast functional decline, reduced procollagen synthesis, increased baseline MMP expression – are partly explained by this progressive mitochondrial ROS escalation operating independently of sun exposure. ^{[1] [9]}

Fibroblast senescence and SASP

Chronic ROS exposure – from the mitochondrial perpetuation cycle, from UV accumulation, and from sustained inflammatory cytokine environments – drives dermal fibroblasts into a senescent state: irreversible cell cycle arrest accompanied by the Senescence-Associated Secretory Phenotype (SASP). Senescent fibroblasts secrete IL-6, IL-8, TNF-α, MMP-1, MMP-3, and further ROS into their tissue microenvironment – a pro-inflammatory, pro-degradative output that compromises neighbouring healthy fibroblasts and accelerates matrix breakdown around the senescent cell. SASP-driven ROS then promote further fibroblast senescence in adjacent cells, propagating senescence as a spreading tissue state rather than a contained cellular event. This is the mechanism linking accumulated oxidative stress to the “spreading slowness” of aged skin’s repair response – not individual fibroblasts failing, but a growing population of senescent cells actively degrading the ECM around them. ^{[7] [3]}

RONS in wound healing

The wound healing role of RONS is the clearest demonstration of the dose-response principle in action. The acute ROS burst at a fresh wound site is not an unfortunate side effect of tissue damage – it is a coordinated signalling event that initiates the healing programme. ^[11]

Within seconds of barrier disruption, NADPH oxidase (NOX1 and NOX2) generates a sustained O₂·⁻ burst at the wound edge – rapidly dismutated to H₂O₂, which diffuses into the wound margin as a chemotactic gradient. Keratinocytes and fibroblasts at the wound edge sense this H₂O₂ gradient through redox-sensitive receptor tyrosine kinases and PI3K/Akt pathway activation, triggering proliferation and migration toward the wound centre. Simultaneously: ^{[11] [4]}

• H₂O₂ stabilises HIF-1α (hypoxia-inducible factor) → VEGF transcription → angiogenic ingrowth
• H₂O₂ directly kills bacteria in the wound environment, providing immediate antimicrobial defence before the adaptive immune response arrives
• iNOS induction by wound-site cytokines (IFN-γ, LPS from contaminating bacteria, IL-1β) produces NO → sustained vasodilation maintaining blood flow, collagen crosslinking via NO-modified prolines, and further VEGF signalling
• eNOS-derived NO in endothelial cells produces the vascular smooth muscle relaxation that sustains the increased perfusion at the healing site

The contrast with chronic wound biology is instructive. In chronic wounds – diabetic ulcers, venous leg ulcers – RONS are present at high concentrations rather than controlled acute-phase concentrations. NADPH oxidase is constitutively activated, the antioxidant defence capacity is depleted, and the persistent high-ROS environment activates NF-κB continuously, maintains elevated MMP-9, degrades the provisional matrix as fast as it forms, impairs keratinocyte migration, and prevents re-epithelialisation. The same H₂O₂ that initiates healing at acute concentrations destroys it at chronic excess concentrations. ^[11]

This acute/chronic distinction is directly relevant to procedure planning. Stimulatory aesthetic procedures – RF microneedling, fractional laser, CAP – produce the acute RONS burst biology in controlled, localised form. Pre-existing chronic low-grade inflammation (uncontrolled rosacea, active AD, poorly managed barrier disruption) shifts the baseline RONS environment at the treatment site toward the chronic wound end of the spectrum before the procedure begins – compromising the clean acute-phase response that produces the remodelling benefit.

CAP as therapeutic RONS delivery

Cold atmospheric plasma occupies a unique position in aesthetic medicine: it is the only treatment modality that delivers precisely calibrated RONS directly to the tissue surface as its primary mechanism of action, rather than generating RONS as a secondary consequence of energy delivery. ^[8]

RONS composition of CAP output

CAP generates a defined mixture of reactive species from the ionisation of ambient air or carrier gas during plasma discharge. The primary therapeutically relevant species are: ^{[8] [8]}

What distinguishes CAP RONS from endogenous pathological ROS

Three specific differences define the therapeutic character of CAP-generated RONS and explain why a treatment that generates reactive species at a wound site is mechanistically different from chronic oxidative stress:

1. Controlled species composition. CAP parameters – gas composition, plasma power, exposure time, device geometry – determine the ratio of ROS to RNS and the concentration of each species in the output. The H₂O₂:NO ratio in CAP output can be adjusted to favour antimicrobial (higher H₂O₂) or vasodilatory/reparative (higher NO) applications. Endogenous ROS in chronic inflammation are not compositionally controlled – they reflect whatever upstream activating pathway is driving NOX and NOS activity.

2. Short-lived species acting locally. ·OH, ¹O₂, and ONOO⁻ have nanosecond-to-microsecond half-lives in aqueous environments. CAP-generated versions of these species act at the tissue surface at the moment of treatment and do not accumulate systemically. Contrast with chronic low-grade oxidative stress, where longer-lived species like H₂O₂ diffuse through multiple cell layers and maintain elevated NF-κB/AP-1 signalling persistently.

3. Dose-titration. CAP exposure can be adjusted in real time to maintain RONS delivery within the NRF2-activation/hormetic zone rather than the NF-κB/AP-1/damage zone. The treating practitioner controls the concentration. Endogenous oxidative stress is not clinically titratable.

NRF2 activation as a CAP mechanism. The upregulation of HMOX1 and NQO1 by CAP-generated H₂O₂ is not merely a protective response to the treatment, but a pro-reparative mechanism in its own right. HMOX1 generates cytoprotective bilirubin and CO; CO at low concentrations is anti-inflammatory (suppresses NF-κB, reduces TNF-α) and vasodilatory. NQO1 stabilises p53, supporting normal apoptosis of cells that have accumulated DNA damage. The NRF2 response to CAP is therefore part of its therapeutic mechanism, not a side effect that limits it. ^{[8] [5]}