Living, quiescent Lactobacillus plantarum Lp90 probiotic, delivered topically to full thickness tissues in vitro via a just-add-water cream delivery system, stimulates the expression of elastin protein.

INTRODUCTION: Delivering living probiotics to the skin can be challenging as most water-containing cosmetic products require preservatives to maintain product stability. A recently introduced powdered technology [Stratabiosys™, Vantage Personal Care] allows for quiescent probiotic powders to be stored for extended periods of time. The powders can then be reconstituted to creams at the point of use by adding water and mixing and were examined in vitro on reconstructed human full thickness tissues to see if the probiotic had any influence of several important biomolecules expressed in the skin. MATERIALS AND METHODS: A probiotic powder containing 200 M CFU/gram of living quiescent Lactobacillus plantarum Lp90 was reconstituted to a cream by adding ultrapure water and gently mixing the components at room temperature to quickly produce a cream. The resulting cream was tested topically on Epiderm® Full Thickness Tissues by treating the tissues for 24 h, removing the cream with a PBS rinse and then repeating the treatment for another 24 h. The resulting tissues were examined for four strategically important skin biomolecules including Type 1A collagen, elastin, filaggrin and hyaluronic acid. The probiotic-containing powder was tested against untreated tissues and powders not containing probiotics and powders containing measured amounts of one of two cryoprotectants known to be used to maintain the integrity of the quiescent probiotics during drying of the quiescent probiotic powders. RESULTS: It was found that topical treatment on Epiderm® tissues with creams containing 2 M (1%), 4 M (2%) and 6 M (3%) CFU/gram prepared from a base powder containing 200 M CFU/gram of Lactobacillus plantarum Lp90 stimulated elastin expression in a dose dependent fashion. There was no effect on the other biomolecules examined in the studies. In addition, it was found that creams made from powders containing only the known cryoprotectants, not bacteria, had no influence on elastin expression. CONCLUSION: The results of this study demonstrate that topical delivery of probiotics is possible from powders containing quiescent probiotic powders converted to creams just prior to application to the tissues. In the case of a powder containing Lactobacillus plantarum Lp90, topical application significantly increased expression of elastin in the skin replicants after 48 h of exposure to the cream made with the probiotic. The elastin-stimulating effects are not coming from the oligosaccharide cryoprotectants used to maintain the probiotic powders in their quiescent, dried state. The results indicate that it is the living Lactobacillus plantarum probiotic that is stimulating the elastin expression in the skin tissues.

Pyrroloquinoline Quinone Disodium (PQQ2Na) Has an NLRP Inflammasome-Induced Caspase-1 Release Influence in UVB-Irradiated but Not ATP-Treated Human Keratinocytes but Has No Influence in Increasing Skin Cell Mitochondrial Biogenesis in Either Human Keratinocytes or Fibroblasts.

INTRODUCTION: Pyrroloquinoline quinone is a bacterial-derived redox factor that has been shown to have numerous benefits in humans. Recently, a model for examining the ability of normal human epidermal keratinocytes (NHEKs) to demonstrate anti-inflammatory benefits via nod-like receptor protein (NLRP)-activated caspase-1 release was reported. The question of whether PQQ2Na might have anti-inflammatory benefits that function through NLRP-activated release of active caspase-1 has not been explored. In addition, it has been reported that PQQ2Na will induce mitochondrial biogenesis in humans when taken orally. Whether or not this effect occurs in skin cells is presently unknown. METHODS: The inflammation studies followed previously published methods that demonstrated both UVB and ATP were able to upregulate the NLRP-activated release of caspase-1 in NHEKs. In addition, NHEK and normal dermal human fibroblasts (NHDF) were treated with PQQ2Na to see if the molecule might stimulate mitochondrial biogenesis measured by increased expression of cyclooxygenase-1 (COX-1) and succinate dehydrogenase complex, subunit A (SDHA). RESULTS: At non-cytotoxic concentrations between 5 µg/mL and 100 µg/mL in NHEKs and between 0.1 µg/mL and 5 µg/mL in fibroblasts, the PQQ2Na had no influence on cellular mitochondrial biogenesis. In ATP-activated NHEKs at concentrations of PQQ2Na between 0.05 µg/mL and 50 µg/mL, there was no influence of PQQ2Na on release of active caspase-1. In NHEKs irradiated with 60mJ/cm2 of UVB radiation as previously described and treated with 0.05 µg/mL to 50 µg/mL of PQQ2Na, the molecule showed a dose-dependent benefit at reducing the expression of active caspase-1 in the irradiated cells. DISCUSSION: Benefits of PQQ2Na on various skin cell types which had not been investigated previously were addressed. Surprisingly, the PPQ2Na had no apparent influence on skin cell mitochondrial biogenesis. However, the molecule has a strong suppressing influence on UVB-induced active caspase-1 release in UVB-irradiated NHEKs.

Hyaluronic acid (HA) stimulates the in vitro expression of CD44 proteins but not HAS1 proteins in normal human epidermal keratinocytes (NHEKs) and is HA molecular weight dependent.

BACKGROUND: In the skin, hyaluronic acid is broken down to smaller fragments by hyaluronidase enzymes, particularly when skin is wounded. The impact of various molecular weight fragments of HA on normal human epidermal keratinocytes (NHEK) with regard to expression of important cellular proteins has not been deeply explored. AIMS: Examination of three molecular weight (Mw) fractions of hyaluronic acid: 1) average Mw of the high fraction: 1.5-2 MDa, 2) average Mw of the medium fraction: 200-500 kDa, and 3) average Mw of the low fraction: 5-10 kDa and a unique 1:1:1 composite complex of the three HA fragments (Triluronic® Acid) was done to examine the influence of the HA on two critical skin cell protein targets: hyaluronan synthase-1 (HAS-1) and the HA binding protein cluster of differentiation 44 (CD44). METHODS: NHEKs were treated in vitro with a 1.0% stock solution of each HA Mw fraction at 1.0, 0.5, and 0.1% concentrations of the 1.0% solution and the polysaccharide composite at the same concentrations for 48 Hrs. The cells were than analyzed by ELISA protein assays for HAS-1 and CD44 protein content. RESULTS: Examination of HAS-1 protein expression indicates that none of the HA test materials influenced the expression of HAS-1 at any concentration. Examination of the CD44 protein expression indicated that the low Mw fraction and the commercial complex of the three Mw fractions upregulated CD44 protein expression in NHEKs, but the medium Mw and high Mw HA fractions did not. CONCLUSIONS: In this work, it was demonstrated that HA can influence the expression of CD44 protein, a critical HA transmembrane HA binding protein, and the influence appears to be molecular weight dependent.

Purified Cannabidiol isolate does not inhibit active caspase-1 release in NLRP inflammasome-mediated UVB or ATP-activated keratinocytes or appear to reduce key inflammatory cytokines in UVB-irradiate keratinocytes.

BACKGROUND: Cannabidiol is a plant-derived cannabinoid that has been suggested to have several human health benefits including potential anti-inflammatory effects. It is now common to find various forms of Cannabidiol, most often referred to as CBD, in nutritional supplements and topical treatments. The mechanisms by which CBD can influence inflammatory pathways in the body, and more particularly in the skin, are presently still unclear. It is known that CBD will bind to cannabinoid receptors, CB1 and CB2, in the body and recent work has shown that in keratinocytes, CBD can regulate inflammation through transcriptional regulation involving the NFÆ™ß nuclear pathways. The fact that CBD operates through the NFÆ™ß pathways suggests that, perhaps, the molecule may influence the expression of active caspase-1 through NLRP inflammasome-mediated pathways. METHODS: Recently, work has published demonstrating that Normal Human Epidermal Keratinocytes (NHEKs) can be activated by UVB and ATP to express active caspase-1 via NLRP inflammasome-mediated pathways. There was a strong interest to see whether highly purified Cannabidiol Isolate (>99% purity) might function to control release of active caspase-1 by testing of NHEKs using the previously described models. In addition, NHEKs expression of non-NLRP inflammasome-induced inflammation markers including IL-6, IL-8 and PGE2 was examined in UVB-activated NHEKs. RESULTS: It was found that purified Cannabidiol Isolate did not influence active caspase-1 release in either UVB or ATP-activated NHEKs suggesting the molecule does not influence the NLRP inflammasome pathways. In addition, it was surprisingly found that the Cannabidiol Isolate did not impact the expression of additional UVB-activated non-NLRP inflammatory markers. CONCLUSIONS: Data presented suggest that if Cannabidiol functions as an anti-inflammatory, it does so through pathways not associated with either the NLRP inflammasome-mediated expression of caspase-1 or through the more commonly known expression of interleukin or prostaglandin inflammatory pathways.

CONTEXTE: Le cannabidiol est un cannabinoïde d'origine végétale considéré comme bénéfique pour la santé humaine et présentant notamment des effets anti-inflammatoires potentiels. Il est désormais courant de trouver diverses formes de cannabidiol dans les suppléments alimentaires et les traitements topiques. Les mécanismes par lesquels le cannabidiol peut influencer les voies inflammatoires dans l'organisme, et plus particulièrement dans la peau sont actuellement encore flous. On sait que le cannabidiol se lie aux récepteurs cannabinoïdes, CB1 et CB2 dans l'organisme et des travaux récents ont montré que dans les kératinocytes, le cannabidiol peut réguler l'inflammation par régulation transcriptionnelle impliquant les voies nucléaires NF-ƙß. Le fait que le cannabidiol fonctionne par le biais des voies NF-Æ™ß laisse à penser que la molécule peut influencer l'expression de la Caspase-1 active à travers les voies médiées par l'inflammasome NLRP. MÉTHODES: Récemment, des travaux ont été publiés démontrant que les kératinocytes épidermiques humains normaux (Normal Human Epidermal Keratinocytes, NHEK) peuvent être activés par les UVB et l'ATP pour exprimer la Caspase-1 active à travers les voies médiées par l'inflammasome NLRP. On cherchait surtout à savoir si l'isolat de cannabidiol hautement purifié (pureté > 99 %) pouvait fonctionner pour contrôler la libération de Caspase-1 active en analysant les NHEK à l'aide des modèles décrits précédemment. En outre, l'expression des NHEK des marqueurs de l'inflammation induits par l'inflammasome non-NLRP, notamment : IL-6, IL-8 et la PGE2 ont été examinées dans des NHEK activées par les UVB. RÉSULTATS: Il a été constaté que l'isolat de cannabidiol purifié n'influençait pas la libération active de Caspase-1 dans les NHEK activées par les UVB ou l'ATP, ce qui suggère que la molécule n'influence pas les voies de l'inflammasome NLRP. En outre, il a été surprenant de constater que l'isolat de cannabidiol n'avait pas d'impact sur l'expression des marqueurs inflammatoires non-NLRP activés par les UVB supplémentaires. CONCLUSIONS: Les données présentées suggèrent que si le cannabidiol fonctionne comme un anti-inflammatoire, il le fait par des voies non associées à l'expression de la Caspase-1 médiée par l'inflammasome NLRP ou par l'expression plus connue des voies inflammatoires de l'interleukine ou de la prostaglandine.

In vitro expression of NLRP inflammasome-induced active Caspase-1 expression in normal human epidermal keratinocytes (NHEK) by various exogenous threats and subsequent inhibition by naturally derived ingredient blends.

BACKGROUND: The discovery of the nod-like receptor protein (NLRP) inflammasomes in 2002 has led to the rapid identification of these unique cellular proteins as key targets for studies on innate inflammation pathways. The NLRP inflammasomes have been shown to be expressed in normal human epidermal keratinocytes (NHEK) and human dermal fibroblasts (HDF). NLRP inflammasomes in keratinocytes are interesting as these skin cells are the first living cells in the skin to contact external exogenous threats such as UV energy, chemicals, physical trauma, and bacteria and viruses. Activation of the NLRP Inflammasomes by exogenous threats results in the release of active Caspase-1 (ACasp-1), a key protease enzyme, which targets inactive forms of IL-1ß, IL-18 as well as IL-1α and IL-33. PURPOSE: This article discusses efforts to examine the release of active Caspase-1 from NHEKs activated by various exogenous threats including UVB energy, ATP, Nigericin and Urban Dust. The work further examines if, after inflammasome activation and Caspase-1 release, certain naturally derived botanical ingredients known to have anti-inflammatory effects can function to inhibit upregulation of active Caspase-1. METHODS: NHEK were treated with various doses of UVB, ATP and Nigericin and with a single dose of Urban Dust. ACasp-1 expression was measured after 3 and 20 hours using the Promega Caspase Glo-1 bioluminescent assay. After confirmation that 60 mJ/cm2 of UVB and 5mM of ATP were effective to activate NHEK ACasp-1 release after 20 hrs, these conditions were employed to examine the influence of three botanical blends of ingredients on their ability to inhibit ACasp-1 expression. RESULTS: Initial results demonstrate that NHEKs can be activated to release active Caspase-1 by ATP and UVB, but not by Nigericin or Urban Dust. In addition, it was unexpectedly found that, while ATP and UVB activated NHEKs, the release of ACasp-1-did not happen within the first 3 hours after exposure but did become significant after 20 hours. Additional results indicate that a blend of polysaccharides and two blends of antioxidants, one oil-soluble and the other water-soluble, known for their anti-inflammatory effects, can reduce expression of active Caspase-1 in activated NHEKs when applied extracellularly. CONCLUSION: Expression of NLRP activated release of ACasp-1 was found to be influenced by UVB and ATP but not by Nigericin or Urban Dust. The effects were also time dependent. Several botanical extract blends were found to reduce ACasp-1 expression in previously activated NHEKs. Links between these inflammatory effects and processes of cellular inflammaging are discussed.

In vitro examination of an oleosome-based sun protection product on the influence of UVB-induced inflammation markers in human epidermal skin equivalent tissue model.

In vitro human epidermal skin equivalent tissues (MatTek EpiDerm™) were employed to examine the influence of UVB radiation on various established inflammation markers in the presence of topically applied sunscreens. MatTek EpiDerm™ tissues were treated with 2.0mg/cm2 of an Experimental oleosome-based SPF 30 product or a commercial SPF 30 beach product. Tissues were irradiated with 300mJ/cm2 of UVB radiation. Inflammatory cytokines IL-1α, IL-6 and IL-8 as well as arachidonic acid cascade marker PGE2 were examined via ELISA-based antibody detection. Untreated tissues irradiated with 300mJ/cm2 of UVB radiation showed statistically significant upregulation of IL-1α, IL-6 and IL-8 as well as PGE2. Application of both the experimental oleosome-based SPF 30 formulation and the commercial SPF 30 formulation demonstrated an ability to prevent the upregulation of all four markers when applied prior to irradiation. The experimental oleosome-based SPF 30 product contained approximately 80% less sunscreen actives than the commercial formulation. This study demonstrates that in vitro reconstructed human tissues can be used to study the influences of sun screen actives in the presence of UVB radiation. The results support previously reported clinical results that demonstrated that oleosome-based sun care formulations can function with high protection effects with significantly less sun care actives.

Reduction of facial wrinkles by hydrolyzed water-soluble egg membrane associated with reduction of free radical stress and support of matrix production by dermal fibroblasts.

OBJECTIVE: The aim of this study was to evaluate the effects of water-soluble egg membrane (WSEM) on wrinkle reduction in a clinical pilot study and to elucidate specific mechanisms of action using primary human immune and dermal cell-based bioassays. METHODS: To evaluate the effects of topical application of WSEM (8%) on human skin, an open-label 8-week study was performed involving 20 healthy females between the age of 45 years and 65 years. High-resolution photography and digital analysis were used to evaluate the wrinkle depth in the facial skin areas beside the eye (crow's feet). WSEM was tested for total antioxidant capacity and effects on the formation of reactive oxygen species by human polymorphonuclear cells. Human keratinocytes (HaCaT cells) were used for quantitative polymerase chain reaction analysis of the antioxidant response element genes Nqo1, Gclm, Gclc, and Hmox1. Evaluation of effects on human primary dermal fibroblasts in vitro included cellular viability and production of the matrix components collagen and elastin. RESULTS: Topical use of a WSEM-containing facial cream for 8 weeks resulted in a significant reduction of wrinkle depth (P<0.05). WSEM contained antioxidants and reduced the formation of reactive oxygen species by inflammatory cells in vitro. Despite lack of a quantifiable effect on Nrf2, WSEM induced the gene expression of downstream Nqo1, Gclm, Gclc, and Hmox1 in human keratinocytes. Human dermal fibroblasts treated with WSEM produced more collagen and elastin than untreated cells or cells treated with dbcAMP control. The increase in collagen production was statistically significant (P<0.05). CONCLUSION: The topical use of WSEM on facial skin significantly reduced the wrinkle depth. The underlying mechanisms of this effect may be related to protection from free radical damage at the cellular level and induction of several antioxidant response elements, combined with stimulation of human dermal fibroblasts to secrete high levels of matrix components.

In vitro and ex vivo examination of topical Pomiferin treatments.

Pomiferin is a unique, prenylated isoflavonoid that can be isolated and purified from the fruits of Maclura pomifera (Osage Orange). The molecule typically is isolated with a small amount of a molecule called Osajin which is structurally similar to Pomiferin but lacks an aromatic hydroxyl group. As a consequence, Osajin has been shown to be a less effective antioxidant than Pomiferin. In vitro studies on Normal Human Dermal Fibroblasts demonstrate that Pomiferin is a potent extracellular matrix protein stimulant, showing increases in collagen, elastin and fibrillin expression comparable or superior to equivalent concentrations of retinol. Ex vivo hair follicle assays demonstrate comparable effects on expression of collagen and elastin at Pomiferin concentrations in the range of 0.05-5 ppm. Taken together, the results from the two assays conducted on different models indicate that Pomiferin may be a very interesting ingredient for topical skin and scalp treatments where modulation of the expression of extracellular matrix proteins is important.

Examining the impact of skin lighteners in vitro.

Three cosmetically important skin lightening agents, hydroquinone (HQ), kojic acid (KA), and niacinamide (NA), consume the bulk of successful skin lightening ingredients in cosmetic applications. However, the mechanisms by which these ingredients work are still unclear. In this study, melanocytes and keratinocytes were treated with high, nontoxic doses of HQ, KA, and NA and the cells were examined by human microarrays and protein assays for several important targets including cytotoxicity, melanin expression, tyrosinase gene (TYR) and protein expression, melanocortin-1 receptor (MC1R) gene and protein expression, cytochrome c oxidase-1 (COX1) gene and protein expression, and ferritin (FTH1) gene and protein expression. It was found that all the skin lighteners examined showed marked increases in TYR, COX1, and FTH1 gene and protein expression, but not in MC1R expression in melanocytes. Upregulation of COX1 and FTH1 genes and proteins was common across both cell lines, melanocytes and keratinocytes. The results of the tyrosinase expression were somewhat unexpected. The role of iron in the expression of melanin is somewhat unexplored, but common and strong upregulation of ferritin protein in both types of cells due to the treatments suggests that iron plays a more pivotal role in melanin synthesis than previously anticipated.

Modulation of cellular senescence in fibroblasts and dermal papillae cells in vitro.

A hexapeptide (Hexapeptide-11) of structure Phe-Val-Ala-Pro-Phe-Pro (FVAPFP) originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity has demonstrated an ability to influence the onset of senescence in intrinsically aged fibroblasts, extrinsically aged fibroblasts, and extrinsically aged dermal papillae cells in vitro. The mechanism of senescence control is believed to be related to the peptide's ability to reversibly downregulate ataxia telangiectasia mutated (ATM) and p53 protein expression. The importance of p53 as the gatekeeping protein for monitoring cellular DNA damage is strategic for maintaining cellular health. ATM activates p53 by direct phosphorylation, causing cells to move into senescence which effectively moves them out of reproductive processes. Technologies that can influence ATM and p53 expression may offer unique benefits for controlling cellular senescence and effectively delaying cellular aging processes. The influence on ATM and p53 expression is noted to occur in both cell lines at peptide concentrations between 0.1% and 1.0%. The implications of these effects for aging benefits for skin and hair is important as, to date, no known small peptide has been suggested to demonstrate this effect in such a reversible and dose-dependent fashion.

Examining the genomic influence of skin antioxidants in vitro.

A series of well-known, purified antioxidants including: Resveratrol, Epigallocatechin Gallate (EGCG), Genistein, Rosavin, Puerarin, Chlorogenic Acid, Propolis and two newer unexplored isoflavonoids isolated from Maclura pomifera (Osage Orange) including Pomiferin and Osajin, were applied to Normal Human Dermal Fibroblasts (NHDF) and Normal Human Dermal Keratinocytes (NHEK) for 24 hours. The resulting treated cells were then examined using human gene microarrays supplied by Agilent. These chips typically have somewhere on the order of 30,000 individual genes which are expressed in the human genome. For our study, this large list of genes was reduced to 205 principal genes thought to be important for skin and each individual ingredient was examined for its influence on the culled list of genes. Working on a hypothesis that there may be some common genes which are either upregulated or downregulated by all or most of these ingredients, a short list of genes for each cell line was developed. What appears to emerge from these studies is that several genes in the gene pool that was screened are influenced by most or all of the molecules of interest. Genes that appear to be upregulated in both cell lines by all the ingredients include: ACLY, AQP3, COX1, NOS3, and PLOD3. Genes that appear to be downregulated in both cell lines by all ingredients include only PGR.

Influence of an extract from kudzu symbiosomes containing leghemoglobin on in vitro cutaneous procollagen production.

Cytoglobin is a hexacoordinate globin protein that was recently discovered in mammals. Interestingly, of the four human globin proteins that are now known, hemoglobin, myoglobin, neuroglobin and cytoglobin, the latter appears to have the closest resemblance to strikingly similar proteins expressed in plants. In legumes, these proteins accumulate in symbiosomes (root nodules) of various legumes and are called leghemoglobin. The paper will discuss the ability of an aqueous extract from Pueraria lobata (kudzu) symbiosomes that contains leghemoglobin to stimulate procollagen production in human dermal fibroblasts. This effect may be partly due to the possibility that leghemoglobin may mimic the function of cytoglobin by shuttling oxygen to prolyl-4-hydroxylase, the enzyme responsible for oxidizing proline residues in procollagen bundles. This hypothesis is supported by DNA microarray sequencing data that demonstrate that treatment of normal human dermal fibroblasts (NHDF) with highly purified cytoglobin or leghemoglobin upregulates a number of key collagen-related genes including COL1A1 and COL1A2.