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Skin is the barrier separating the body from the outer environment, protecting against water loss and external aggressions. Skin’s condition is the most visible indicator of health and general status, and of age…or youth.1 Extrinsic and intrinsic factors affect skin aging.2 Extrinsic factors include exposure to sunlight or pollution, and repetitive muscle movements. Intrinsic aging represents physiological changes over time, occurring at variable, genetically determined rates. The combined effects of aging over the human lifespan lead to a loss of structural integrity and physiological function in the skin. Aged skin is susceptible to dryness, wrinkling, loss of elasticity and hyperpigmentation, among others.3, 4
On a cellular level, intrinsic aging was first described by Hayflick and Moorhead,5 who demonstrated that somatic mammalian cells enter an irreversible growth arrest (cellular senescence) after a finite number of cell divisions. Senescent cells are characterized by their inability to proliferate and their secretion of factors that promote inflammation and tissue deterioration. Watson6 and Olovnikov7 later proposed that the shortening of chromosome terminal sequences, termed telomeres, is the main underlying mechanism of replicative senescence (depicted in Figure 1); and that telomere length is the molecular clock counting down toward a programmed limit on cumulative population doublings. Critically shortened telomeres trigger a persistent activation of DNA damage response pathways, thereby resulting in cellular senescence.8
The mechanistic target of the rapamycin (mTOR) signaling pathway: serves as a central regulator of cellular senescence; coordinates basic cellular responses such as cell
proliferation, apoptosis and inflammation; and is closely related to the DNA damage response.9 DNA damage results in the phosphorylation of mTOR complex 1 (mTORC1), leading to cellular senescence and downstream aging.10, 11
A growing list of evidence suggests that mTOR signaling influences longevity and aging, and the inhibition of mTORC1 with rapamycin is currently the only known pharmacological treatment that increases lifespan in all model organisms studied. As such, the current authors hypothesized that slowing cell proliferation should preserve the telomeres, reduce the activation of mTORC1, and thus preserve “younger,” more optimal cellular functions.
In relation, as previous work describes,12 the Narcissus tazetta daffodil bulb is an underground storage organ for nutrients and energy. It enables the plant to survive adverse environmental conditions by cycling annually between periods of growth and dormancy. When dormant, the plant produces dormancy-inducing factors, i.e., dormins—growth inhibitors that reversibly inhibit cell proliferation. In traditional medicine, the plant bulb is used to treat wounds and inflammation, for antibacterial and antifungal properties, and even as an anti-cancer agent.13
Described here is work to assess how a natural aqueous extract of dormant Narcissus tazetta bulbs captures and transfers the plant dormancy concept to skin fibroblasts. Previously observed to slow cell proliferation in vivo,12 here, testing in vitro in aged human dermal fibroblast cultures confirmed restrained proliferation rates, in addition to preserved telomere length, decreased mTORC1 activation and increased procollagen 1 production, relative to an untreated aged control. In addition, a clinical study showed the extract significantly ameliorated key aging skin parameters including wrinkling, hyperpigmentation, barrier function and elasticity.
In vitro Materials and Methods
The studies described throughout utilize the specified aqueous extracta of dormant Narcissus tazetta bulbs.
Gene expression: Cysteine-rich C-terminal 1 (CRCT1) is encoded by the epidermal differentiation complex (EDC). The EDC comprises numerous genes that are crucial for the maturation of the human epidermis. In fact, it has been shown that CRCT1, regulated by microRNA-520 g, inhibits proliferation.
In order to assess whether Narcissus extracta could enhance CRCT1 gene expression, Normal Human Epidermal Keratinocytes (NHEK) were incubated for 24 hr at 37°C, 5% CO2 either in the presence of 0.15% Narcissus tazetta bulb extract or left untreated (negative control). RNA was then extracted and treated with a 33P label. Labeled cDNA targets were hybridized to specific cDNA probes covalently fixed to a minichip. Finally, target-probe hyprodization was revealed by phosphor imaging; the significance threshold was set at a twofold change vs. the control.
Cellular proliferation, senescence and function in fibroblasts: In order measure the effects of the Narcissus extracta on cell proliferation and cellular senescence, Normal Human Dermal Fibroblasts (NHDF) were grown over 28-30 cell doublings either in the presence of 0.01% or 0.03% of the extract, or left untreated (negative control). Fresh samples were introduced into the culture every three days. After incubation, cells were collected, gDNA was extracted and telomere length was quantified by RT-qPCR.
Protein also was extracted and levels of phosphorylated mTOR and procollagen-1 were quantified by ELISA. As a matter of explanation, Type 1 collagen is a fundamental connective protein, abundant in the dermis and one of the main products of fibroblasts. Reduced levels may contribute to wrinkles through weakening of the bond between the dermis and epidermis. This, therefore, represents a good indicator of fibroblast cellular function. The number of cell doublings was calculated through cell counting.