28 ANTI-AGING


Assay (Promega Corporation, Madison, WI). Treatment of UVB and ATP inflammasome- activated NHEKs with topical ingredients through a 20-hour activation response was done with sub-cytotoxic doses of three blended ingredients including: 1) a blend of botanically-derived polysaccharides in water [StrataPhix™ POLY, BotanicalsPlus®


n 3 hours post stress n 20 hours post stress , Fairfield,


NJ] 2) a blend of botanically-derived antioxidants in water [StrataPhix AWS, BotanicalsPlus, Fairfield, NJ] and 3) a blend of botanically-derived antioxidants in Caprylic/Capric Triglyceride [StrataPhix AOS, BotanicalsPlus, Fairfield, NJ]. Expression of active Caspase-1 was measured again after a single treatment with the botanical blends. A known NLRP3 inhibitor, MCC950, was employed as a positive control.


Results Activation of Caspase-1 release via NLRP inflammasomes in NHEKs Treatment of Normal Human Epidermal Keratinocytes [NHEKs] with UVB radiation and with ATP, an extracellular marker of cellular microbial damage, caused significant upregulation of expression of active Caspase-1 in Normal Human Epidermal Keratinocytes exposed to these exogenous threats, (Figs 1 and 2). What was interesting from these initial findings was the time lag that occurred between the exposure of the skin cells to the exogenous threat and the upregulation of measurable and statistically significant amounts of active Caspase-1. While the skin cells do respond with an innate immune response, there is a time delay between the exposure moment and the release of the active protease, Caspase-1. The reasons for this time delay are presently unknown but likely have much to do with the time it takes for the NLRP Inflammasome Complexes to form and activate Pro-Caspase- 1. In addition, we also examined a well-known innate immune response activator, Nigericin, a microbial lipopolysaccharide (LPS) known to activate the NLRP cascades in immune cells. Though we tested the activator in two different ways, i.e., direct application of Nigericin (Fig 2), and application of Nigericin prior to follow up activation with ATP (Fig 3), we were unable to detect a direct upregulation of Caspase-1 release due to Nigericin. Again, this was a curious finding suggesting that the NHEKs were not responding to a threat from a microbial signaling molecule known to activate inflammasomes in immune cells. We also examined a single concentration of Urban Dust (PM10


) and did not note a significant


upregulation of Caspase-1 release. We continue to examine these cutaneous threats, but if the data continues to hold, these known skin health hazards may not function to cause inflammation through the skin’s innate immune response system in keratinocytes.


PERSONAL CARE NORTH AMERICA


0.1600 0.1400 0.1200 0.1000 0.0800 0.0600 0.0400 0.0200 0.0000


Untreated * *


5 mM ATP


1 mM ATP


Nigericin Treatment


Figure 2: Influence of various exogenous treatments including ATP, Nigericin and Urban Dust on Caspase-1 expression in NHEKs. Asterisks indicate statistical significance (p≤0.05).


120.0 100.0 80.0 60.0 40.0 20.0 0.0


20 µm


Nigericin


40 µm


100 µg/ml Urban Dust


*


Treatment


Figure 3: Influence of Nigericin pre-treatment on NHEKs followed by ATP activation. Asterisks indicate statistical significance (p≤0.05).


Influence of botanical blends on expression of active Caspase-1 in NHEKs After activation of the NLRP inflammasomes in keratinocytes, treatment of activated cells with unique, botanically-derived blends of known anti-inflammatory or antioxidant ingredients could inhibit the expression of active Caspase-1, potentially slowing the inflammaging process as described by Mejias.11


It was found that a water-soluble


polysaccharide extract mixture (StrataPhix POLY) comprised of Opuntia Ficus cladode polysaccharides, Schyzophillanmushroom mycelium glucans, Western Larch tree galactoarabinan and brown algae leaf polysaccharides showed a benefit at reducing active Caspase-1 expression from both UVB and ATP-activated cells (Figs 4 and 7). The blended antioxidants, which included an oil-soluble blend of antioxidants derived from Lithospermum erythrorhizon root extract, Haematococcus pluvialis algae extract, Camellia Sinensis leaf extract and Glycyrrhiza glabra root extract and a water- soluble antioxidant blend comprised of


extracts from Lycium Barbarumfruit, Opuntia Ficus-Indica Stem, Camellia Sinensis Leaf and Citrus Aurantium Dulcis (Orange) Fruit Extract, showed benefits at reducing active Caspase-1 release from UVB-activated cells only (Figs 5 and 6). This likely reflects on the mechanisms that


drive the two different pathways of formation of the NLRP Inflammasomes. In the case of the UVB energy, the NLR protein is responding to a Danger-Associated Molecular Pattern or DAMP. When the keratinocytes respond to excessive ATP as might occur, for example, during an infection, they are responding to a Pathogen- Associated Molecular Pattern or PAMP. The NLR inflammasome proteins simply respond to the threat via molecular structural change and assembly of the Inflammasome Complex. They do not need to be previously imprinted the way the downstream immune cells, like macrophages, must. In this way, the inflammasome could be classified as the guardians of the body because they respond to all types of exogenous threats.


May 2020


Caspase-1 Activation (Percent of Untreated)


Caspase-1 Activation (LUM)


Non-Stimulated Untreated 100 µM MCC950 100 µM Nigericin 2 hrs 10 µM Nigericin 2 hrs 1 µM Nigericin 2 hrs 0.1 µM Nigericin 2 hrs 00.1 µM Nigericin 2 hrs


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