ANTI-AGEING 59 *p<0.05 versus initial conditions 10 n Placebo n 2% IceAwakeTM *p<0.05 versus initial conditions and placebo 10 * 8 0 6 n Placebo n 2% IceAwakeTM
4 -10 2 * -20 Figure 5: Decrease of wrinkle depth upon treatment with IceAwakeTM
Materials and methods Gene expression in aged fibroblasts To mimic the ageing process, normal human dermal fibroblasts were cultured for 17 passages prior to the experiment. Aged fibroblasts were subsequently treated or not (control) with 1 % Iodobacter ssp. extract for 24 h. All experimental conditions were performed in n=3. Following treatment, cells were harvested, and total RNA was extracted from the pooled sample of each condition using TriPure Isolation Reagent®
(Roche, Mannheim) according to
the supplier’s instructions. Complementary DNA (cDNA) was synthesised from total RNA in the presence of oligo(dT) primer and Transcriptor Reverse Transcriptase (Roche). RT-qPCR for the target genes was performed in n=2 using the LightCycler® system (Roche).
Determination of ATP production in a cellular model of sleep deprivation Fibroblasts isolated from a patient with Alzheimer’s disease (AD) combine impairment of mitochondrial function and increased ER-stress and were used as a cellular model for sleep deprivation. AD- fibroblasts were grown in assay medium which inhibited TCA cycle activity, thereby inducing additional mitochondrial stress. Following a wash, cells were incubated with 0.01% Iodobacter ssp. extract for 120 minutes or left untreated prior to measurement of ATP levels by chemiluminescence. All measurements were performed in quadruplicates.
Fluorescent staining of mitochondria- associated membrane (MAM) contact points AD-fibroblasts were treated with 0.01% Iodobacter ssp. extract or left untreated for 120 minutes, healthy cells were grown in parallel and left untreated. Cells were subsequently fixed and permeabilised, and
November 2019 . 0 Figure 6: Reduction of tired appearance upon treatment with IceAwakeTM
II in situ red proximity ligation assay (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Cell nuclei were counterstained with 4’,6’-diamin-2- phenylindol (DAPI). Fluorescent signals resulting from protein interactions (<40 nm) between GPR75 and SERCA2, as well as nuclear stain were visualised by immunofluorescence microscopy (Zeiss, Germany) and quantified. All measurements were performed in quadruplicates.
MAM contact points were stained using the DuoLink®
Skin adaptation in clinical studies with overworked volunteers The anti-tiredness effect of lodobacter ssp. extract was evaluated in two randomised, double-blind, placebo-controlled clinical studies involving twenty-three Asian women (41-57 years, mean age: 50.7 years) and twenty-one Caucasian men and women (44-66 years, mean age: 53.7 years), respectively. For inclusion into the study populations, volunteers were required to present with a lack of sleep and/or tired appearance with dark circles around the eyes and medium-deep crow’s feet wrinkles. After a wash-out phase of three to seven days, volunteers applied a cream containing 2% lodobacter-derived active ingredient on one half of the face and a corresponding placebo cream on the other half of the face twice daily for 14 days. The wrinkle depth of crow’s feet was determined using a PRIMOS Premium (GF Messtechnik, Germany) or PRIMOSlite® (Canfield, Germany). Additionally, a clinical expert grading of skin radiance and visible tiredness was performed on photographs taken of the volunteers using a Visia®
CR
camera (Canfield, USA) or a ColorFace® camera (Newtone Technologies, France).
Results and discussion Iodobacter ssp. extract induces chaperone
.
expression in aged fibroblasts The effect of Iodobacter ssp. on the expression of several chaperones involved in protein folding in the ER was analysed in aged fibroblasts treated with an extract of Iodobacter ssp. and compared to an untreated control. Upon treatment with 1% Iodobacter ssp. extract for 24 h, the expression levels of key chaperones involved in the UPR, namely BiP, endoplasmin, calnexin and calreticulin, were increased by up to 100 % in aged fibroblasts compared to untreated cells (Fig 1).
It has previously been shown that various ER chaperones are upregulated upon cellular stress caused for example by sleep deprivation.5,6
However, recent research
revealed that aged cells lose the capacity to activate the UPR, and the cells consequently fail to prevent protein misfolding and aggregation.3,7
Moreover,
the basal expression of BiP, the main ER chaperone responsible for assistance in protein folding and prevention of protein aggregation, has been shown to decline in aged cells.7
This causes further stress to the ER, initiating a vicious cycle which leads to insufficient recovery of the cells during sleep. Treatment with an extract of Iodobacter ssp. reverses these ageing effects and supports protein folding in the ER by upregulating chaperone expression.
Reduction of ER stress by Iodobacter ssp. extract in a cellular model of sleep deprivation
Sleep deprivation causes cellular stress which results in impaired mitochondrial and ER function. Both the decrease in ATP levels, as well as the formation of mitochondria-associated membrane (MAM) contact points, were shown to be indicators of these impairments.8.9
In order to assess
the efficacy of Iodobacter ssp. extract to alleviate these signs of stress, cells of an
PERSONAL CARE ASIA PACIFIC
Change in wrinkle depth compared to initial conditions in %
Improvement of skin radiance compared to initial conditions in %
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