Reduction of senescence-associated secretory phenotype and exosome-shuttled miRNAs by Haritaki fruit extract in senescent dermal fibroblasts.

OBJECTIVE: Skin ageing is linked to the accumulation of senescent cells and a "senescence-associated secretory phenotype" (SASP). SASP factors include chemokines, cytokines, and small extracellular vesicles (EVs) containing miRNAs. We characterized SASP profile markers in normal human dermal fibroblasts (HDFs) and evaluated the effect of Haritaki fruit extract on these senescence markers. METHODS: Senescence was induced in HDFs by ionizing radiation (X ray), followed by 14 days of culture. Parallel incubations included fibroblasts treated for 12 days with 10 or 100 µg/mL Haritaki (a standardized extract of Terminalia chebula fruit). Senescence was assessed on Day 14 according to cell morphology, ß-galactosidase activity, RT-qPCR measurement of SASP genes, as well as semi-quantitative (RT-qPCR) expression of miRNAs contained in EVs isolated from the medium. The size and distribution of EVs were measured by Nanoparticle Tracking Analysis. RESULTS: Human dermal fibroblasts exhibited a senescent phenotype 14 days after ionizing-radiation, demonstrated by a flattened and irregular shape, increased ß-galactosidase activity and over-expression of SASP genes. CSF3, CXCL1, IL1ß, IL6 and IL8 genes were increased by 1492%, 1041%, 343%, 478%, 2960% and 293%, respectively. The cell cycle inhibitor, CDKN1A, was increased by 357%, while COL1A1, was decreased by 56% and MMP1 was increased by 293%. NTA analysis of the EVs size distribution indicated a mix of exosomes (45-100 nm) and microvesicles (100-405 nm). miRNA expression in EVs was increased in senescent fibroblasts. miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p and miR 186-5p were increased in senescent HDF by 4.17-, 2.43-, 1.17-, 2.01, 12.5-fold, respectively. Incubation of senescent fibroblasts with Haritaki extract strongly decreased SASP mRNA levels and miRNA expression in EVs. CONCLUSION: Haritaki strongly reduced SASP expression and EV-shuttled miRNAs in senescent fibroblasts. These results indicate that Haritaki has strong senomorphic properties and may be a promising ingredient for the development of new anti-ageing dermo-cosmetic products by inhibiting deleterious effects of senescent cells.

OBJECTIF: Le vieillissement cutané est lié à l'accumulation de cellules sénescentes et à un « phénotype sécrétoire associé à la sénescence ¼ (SASP). Le SASP est constitué de chimiokines, cytokines et de petites vésicules extracellulaires (VE) contenant des miARN. Nous avons caractérisé les marqueurs du SASP dans des fibroblastes dermiques humains normaux (HDF) et évalué l'effet d'un extrait de fruit d'Haritaki sur ces marqueurs de la sénescence. MÉTHODES: La sénescence a été induite dans les HDF par des rayonnements ionisants (rayons X), suivis de 14 jours de culture. Parallèlement, des HDF ont été traités pendant 12 jours avec 10 ou 100 µg/mL d'Haritaki (un extrait standardisé de fruit de Terminalia chebula). La sénescence a été évaluée au jour 14 en fonction de la morphologie cellulaire, de l'activité ß-galactosidase, de la mesure des gènes du SASP par RT-PCR, ainsi que de l'expression semi-quantitative (RT-qPCR) des miARN contenus dans les VE isolées du milieu. La taille et la distribution des VE ont été mesurées par Nanoparticle Tracking Analysis (NTA). RÉSULTATS: Les HDF ont présenté un phénotype sénescent 14 jours après le rayonnement ionisant, en effet, ils avaient une forme aplatie et irrégulière, une activité ß-galactosidase accrue et une surexpression des gènes du SASP. Les ARNm de CSF3, CXCL1, IL1ß, IL6 et IL8 ont été augmentés de 1492%, 1041%, 343%, 478%, 2960% et 293%, respectivement. L'inhibiteur du cycle cellulaire, CDKN1A, a été augmenté de 357%, tandis que le COL1A1 a diminué de 56% et la MMP1 a augmenté de 293%. L'analyse NTA de la distribution de taille des VE a montré un mélange d'exosomes (45-100 nm) et de microvésicules (100-405 nm). L'expression des miARN dans les VE a augmenté dans les fibroblastes sénescents. Les miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p et miR 186-5p ont été augmentés dans le HDF sénescent de, respectivement, 4,17-, 2,43-, 1,17-, 2,01 et 12,5- fois. L'incubation de fibroblastes sénescents avec l'extrait de Haritaki a fortement diminué les niveaux d'ARNm du SASP et l'expression de miARN dans les VE. CONCLUSION: L'extrait d'Haritaki a fortement réduit l'expression du SASP et de miARN contenus dans les VE des fibroblastes sénescents. Ces résultats indiquent que Haritaki possède de fortes propriétés sénomorphiques et pourrait être un ingrédient prometteur pour le développement de nouveaux produits dermo-cosmétiques anti-âge en inhibant les effets délétères des cellules sénescentes.

Efficacy of a Dermocosmetic Serum Combining Bakuchiol and Vanilla Tahitensis Extract to Prevent Skin Photoaging in vitro and to Improve Clinical Outcomes for Naturally Aged Skin.

BACKGROUND: Skin aging is characterized by slacking and loss of density, especially under ultraviolet (UV) radiation exposure. OBJECTIVE: To investigate the beneficial effects of a combination containing bakuchiol (BK) and vanilla tahitensis extract (VTE) to prevent skin photoaging in vitro and to improve clinical outcomes for naturally aged skin. MATERIALS AND METHODS: Human dermal fibroblasts were treated with active compounds, exposed to an acute dose of UVA and analyzed by confocal microscopy: actin network for morphology, interleukin-8 (IL-8) for inflammation and p16 for senescence. Human skin was used to evaluate chronic UVA-induced glycosaminoglycan (GAG) loss and to assess the benefit of topical application of a BK+VTE serum (Alcian blue staining). An open-label clinical trial was conducted in women applying the serum twice daily for 56 days (n=43). Skin remodeling was assessed by FaceScan®. Firmness was evaluated through Dynaskin® and clinical scoring. Skin radiance was also rated on standardized full-face photographs. RESULTS: UVA induced a significant increase in IL-8 and p16 expression and marked morphological changes in fibroblasts. Treatment with BK or VTE alone prevented both actin network alteration and IL-8 upregulation. Interestingly, BK+VTE demonstrated synergistic protection against IL-8 and p16 overexpression. Serum application prevented GAG loss at the dermo-epidermal junction and increased dermal GAG in UVA-exposed skin explants. In the clinical trial, face ptosis was reduced by 11% on average for 26 responsive subjects and up to 23%. Depth of skin deformation was also reduced by 24% on average for 30 responsive subjects and up to 30%. This firming effect was confirmed by clinical scoring. Radiance was significantly improved by 29% on average for 33 responsive subjects. The serum demonstrated good tolerance/safety. CONCLUSION: BK+VTE combination demonstrated anti-aging efficacy and might provide a substantial benefit in the daily care of naturally aged skin in women, through their synergistic effect on inflammaging and senescence.

Effects of dextran sulfate, 4-t-butylcyclohexanol, pongamia oil and hesperidin methyl chalcone on inflammatory and vascular responses implicated in rosacea.

BACKGROUND: Rosacea is a chronic facial skin disorder characterized by inflammation and vascular abnormalities. The pathophysiology of rosacea involves increased activation of the capsaicin receptor, TRPV1, the vascular endothelial growth factor (VEGF) pathway, and cathelicidin LL-37, MMP-9, and KLKs. We evaluated the activity of four compounds (dextran sulfate, 4-t-butylcyclohexanol [BCH; TRP-regulin®], pongamia oil, and hesperidin methyl chalcone [HMC]) on inflammatory and vascular responses implicated in rosacea. MATERIALS AND METHODS: The anti-inflammatory activity of dextran sulfate was evaluated on PGE2 production after PMA stimulation of NCTC-2544 keratinocytes, and on normal human epidermal keratinocytes (NHEKs) after proinflammatory stimulation to mimic a rosacea environment. The anti-angiogenic activity of dextran sulfate was measured by analyzing pseudotube formation in co-cultured human microvascular endothelial cells/normal human dermal fibroblasts. HMC modulation of vascular responses and IL-8 cytokine production after SP stimulation was evaluated in human skin explants. We also assessed the effect of BCH on TRPV1 activation, and the effect of combined BCH and pongamia oil on the inflammatory response of NHEKs. RESULTS: Dextran sulfate strongly and significantly inhibited PMA-induced PGE2 production, inhibited KLK5 and MMP-9 mRNA expression, and IL-8, IL-1α and VEGF production, and displayed a highly significant inhibitory effect on VEGF-induced pseudotube formation. In SP-stimulated human skin explants, HMC significantly decreased the proportion of dilated vessels, total vessel area, and IL-8 production. BCH significantly and dose-dependently inhibited TRPV1 activation, and BCH and pongamia oil inhibited CXCL1 and CXCL6 mRNA expression and IL-8 production in NHEKs. Combined BCH/pongamia oil inhibited IL-8 production synergistically. CONCLUSION: These in vitro results showed that dextran sulfate, BCH, pongamia oil and HMC, possess complementary soothing and anti-redness properties, supporting their combination in Avène redness-relief cosmetic products for sensitive skin prone to redness, and for topical adjunctive rosacea treatment.

Acefylline activates filaggrin deimination by peptidylarginine deiminases in the upper epidermis.

BACKGROUND: Peptidylarginine deiminases (PADs) catalyze deimination (or citrullination), a calcium-dependent post-translational modification involved in several physiological processes and human diseases, such as rheumatoid arthritis and cancer. Deimination of filaggrin (FLG) by PAD1 and PAD3 during the last steps of keratinocyte differentiation is a crucial event for the epidermis function and homeostasis. This allows the complete degradation of FLG, leading to the production of free amino acids and their derivatives that are essential for epidermal photoprotection and moisturizing of the stratum corneum. OBJECTIVE: To increase the flux of this catabolic pathway, we searched for activators of PADs. METHODS: A large chemical library was screened first in silico and then by using an automated assay based on an indirect colorimetric measurement of recombinant human PAD activity. Potential activators were then confirmed using a recombinant human FLG as a substrate, and secondly after topical application at the surface of three-dimensional reconstructed human epidermis. RESULTS: The data obtained after the library screening pointed to xanthine derivatives as potential PAD activators. Among seven xanthine derivatives tested at 50-300µM, caffeine, theobromine and acefylline proved to be the most potent enhancers of in vitro deimination of FLG by PAD1 and PAD3. After topical application of a gel formulation containing 3% acefylline at the surface of reconstructed epidermis, immunoblotting analysis showed an increase in the total amount of deiminated proteins, and confocal microscopy showed an enhanced deimination in the stratum corneum. This demonstrated the activation of PADs in living cells. CONCLUSION: As a PAD activator, acefylline will be useful to study the role of deimination and could be proposed to increase or correct the hydration of the cornified layers of the epidermis.

Effects of Hydroxydecine(®) (10-hydroxy-2-decenoic acid) on skin barrier structure and function in vitro and clinical efficacy in the treatment of UV-induced xerosis.

10-Hydroxy-2-decenoic acid, a natural fatty acid only found in royal jelly, may be of value in correcting skin barrier dysfunction. We evaluated the activity of Hydroxydecine(®), its synthetic counterpart, in vitro on the regulation of epidermal differentiation markers, ex vivo on the inflammatory response and restoration of skin barrier function, and in vivo on UV-induced xerosis in healthy human volunteers. In cultured normal human keratinocytes, Hydroxydecine(®) induced involucrin, transglutaminase-1 and filaggrin protein production. In topically Hydroxydecine(®)-treated skin equivalents, immunohistochemical analysis revealed an increase in involucrin, transglutaminase-1 and filaggrin staining. In a model of thymic stromal lymphopoietin (TSLP)-induced inflamed epidermis, a Hydroxydecine(®)-containing emulsion inhibited TSLP release. In a model of inflammation and barrier impairment involving human skin explants maintained alive, Hydroxydecine(®) balm restored stratum corneum cohesion and significantly increased filaggrin expression, as shown by immunohistochemistry. It also decreased pro-inflammatory cytokine secretion (IL-4, IL-5 and IL-13). In healthy volunteers with UV-induced xerosis, the hydration index increased by +28.8% (p<0.01) and +60.4% (p<0.001) after 7 and 21 days of treatment with Hydroxydecine(®) cream, respectively. Hydroxydecine(®) thus proved its efficacy in activating keratinocyte differentiation processes in vitro, restoring skin barrier function and reducing inflammation ex vivo, and hydrating dry skin in vivo.