Effect of a hyaluronic acid-based mesotherapeutic injectable on the gene expression of CLOCK and Klotho proteins, and environmentally induced oxidative stress in human skin cells.

OBJECTIVE: Normal circadian rhythms are essential to the repair mechanisms of oxidative stress implicated in skin aging. Given reports that hyaluronic acid (HA) homeostasis exhibits a different profile in chronological skin aging, as compared to environmental or extrinsic aging, an improved understanding of the way HA interacts with its surroundings, and the impact of HA injectables in replacing lost HA and encouraging rejuvenation, is of key benefit to skin aging treatments. The objectives of these current studies were twofold. Firstly, to demonstrate the in vitro effects of two lightweight hyaluronic-based injectables on the expression of CLOCK protein in human skin fibroblasts, and their effects on Klotho protein expression as a marker for circadian rhythms in a combined human keratinocyte and Merkel cell model. Secondly, to ascertain whether these findings could be correlated with in vitro effects on various environmental oxidative stress aging markers (blue light, UVA/UVB, Urban Dust, and IR exposures). METHODS: Oxidative stress studies were aimed to highlight possible protective effects through different challenge conditions in two models, ex vivo human skin explants and in vitro monolayer cultures of normal human dermal fibroblasts (NHDF). The protective effects of the test products were evaluated against an increase of cyclobutene pyrimidine dimers (CPDs) abundance within epidermal section of ex vivo skin explants after UVA/UVB radiation; effects of blue light on gene expression from NHDFs fibroblasts; effects of pollutants (Urban dust, UbD) on gene expression in NHDFs fibroblasts; and an increase of reactive oxygen species (ROS) production by NHDFs fibroblasts after infrared-A radiation. Gene expression was assayed and analyzed utilizing microfluidic TaqMan qPCR arrays. CLOCK expression was measured in young and senescing NHDFs by immunostaining, and Klotho and melatonin expression by immunostaining in Merkel cell-enriched normal adult human epidermal cell cultures. RESULTS: In an aging culture of mixed keratinocyte and Merkel skin cells, activation of Klotho expression was induced by the application of both HA test products. Moreover, the HA products increase Klotho protein expression in both Merkel cells and keratinocytes. The observed positive effect of the tested products on melatonin receptors 1A and 1B expression in aging Merkel cell culture and keratinocytes is also interesting. HA-Y (developed for patients 25+ years old) stimulated melatonin receptors type 1B expression in aging cell cultures more strongly than HA-S (developed for patients 35-65 years old). In age (stressed) cells, a lower expression of Klotho protein and melatonin receptors 1A and 1B is apparent. The addition of HA-Y and HA-S stimulates their expression thus providing a "protective" effect. The blue light irradiation at 40 J/cm2 performed in NHDF fibroblast cultures led to a modification of the expression of several genes, all involved in mechanisms known to be modulated in case of solar radiation stress. CONCLUSIONS: Although these are preliminary findings, they are the first we know of that demonstrate HA facial injectables having a benefit and possibilities beyond the "physical filling" of the skin. As regards the beneficial effects against blue light-induced oxidative stress, and a return to cellular homeostasis, there is a need to conduct further and more precise investigations into HA-S. Furthermore, the benefit of these HA injectables (Novacutan®) in the modulation of oxidative stressed circadian rhythms widens their potential benefit.

Signaling Molecules of Human Skin Cells as the Targets for Injection Cosmetology.

INTRODUCTION: Skin aging is a natural process that cannot be stopped. However, there are many ways to help attenuate premature aging of the skin and reduce the signs that have already appeared. One of them is the subcutaneous administration of preparations containing a combination of hyaluronic acid, active amino acids, and peptides providing an anti-aging clinical effect. The purpose of this research is to study in vitro new signaling molecules with the anti-aging effects and influence of hyaluronic acid fillers on its expression. METHODS: The study was conducted using cell cultures of human facial skin: 1) mixed culture of human facial skin keratinocytes and fibroblasts, and 2) culture of human facial skin keratinocytes enriched with Merkel cells. Immunocytochemistry, confocal microscopy and Western blot were used to identify markers of aging. RESULTS: HA-Y and HA-S activated the expression of Klotho in the case of aging mixed culture of human skin keratinocytes and Merkel cells. The increase in expression of MTH-1 with aging of cultures provides evidence of activating defense mechanisms against reactive oxygen species that are accumulating with aging, under the action of HA-S and HA-Y. There was a statistically valid increase in the area of expression of melatonin receptor 1A and 1B markers when adding both HA-S and HA-Y to cultured cells. CONCLUSION: This investigation showed that the studied fillers have biological effects, testifying the stimulation of reparative processes in the skin under their control.

Cosmeceutical product consisting of biomimetic peptides: antiaging effects in vivo and in vitro.

BACKGROUND: Biomimetic peptides are synthetic compounds that are identical to amino acid sequence synthesized by an organism and can interact with growth factor receptors and provide antiaging clinical effects. PURPOSE: The purpose of this study was to investigate the effects of biomimetic peptides on the repair processes in the dermis using a model of cell cultures and in vivo. PATIENTS AND METHODS: Five female volunteers were subjected to the injection of biomimetic peptides 1 month prior to the abdominoplasty procedure. Cell culture, immunocytochemistry, and confocal microscopy methods were used in this study. RESULTS: Biomimetic peptides regulate the synthesis of proteins Ki-67, type I procollagen, AP-1, and SIRT6 in cell cultures of human fibroblasts. They contribute to the activation of regeneration processes and initiation of mechanisms that prevent aging. Intradermal administration of complex of biomimetic peptides produces a more dense arrangement of collagen fibers in the dermis and increased size of the fibers after 2 weeks. The complex of biomimetic peptides was effective in the in vivo experiments, where an increase in the proliferative and synthetic activities of fibroblasts was observed. CONCLUSION: This investigation showed that the studied peptides have biological effects, testifying the stimulation of reparative processes in the skin under their control.