Halting Premature Sking Aging by Improving Emotional Resilience: An Innovative Lactobacillus Extract as a Neuro-cosmetic Ingredient

From exams to personal relationships and work situations, stress is not avoidable. When we face a stressful situation, a “fight or flight” reaction starts in our body and results in cortisol release to regulate blood pressure, blood sugar levels, immune system etc., therefore making our body alert to respond to the external stressful situation. However, prolonged cortisol production due tero chronic stress negatively impacts our health, well-being and sleep^1,2.

In skin, cortisol disrupts both the dermis and epidermis, causing skin atrophy, wound healing disruption, epidermal thinning and skin dryness. As a result skin resilience is lost and wrinkles appear^1,3. This process, also known as premature aging, impacts self-esteem and well-being. 

Fighting stress-related premature ageing is therefore an important target in the beauty industry. We developed a protocol to improve skin condition and holistic well-being by playing on autonomous skin circadian rhythm and by increasing well-being mediators locally in skin. 

Every tissue in our body, including skin, has its own circadian clock. These clocks are synchronized by the brain and help tissue recovery. ROR-α, PER and CRY are members of circadian clocks^4,5. In skin, ROR-α links circadian rhythm and epidermal regeneration. Indeed, besides being a member of circadian rhythm, ROR-α is a transcription factor responsible for expression of epidermal differentiation makers⁶. Furthermore, melatonin, a potent antioxidant and circadian rhythm regulator that is closely related to ROR-α, is also expressed locally in skin⁷.

Melatonin improves skin barrier functions and reduces the effects of aging through its antioxidant action potentially via Sirtuin 1^8,9,10. Sirtuin 1 controls different cellular processes, including mitochondrial biogenesis, inflammation, cellular metabolism and epigenetic modulation. In order to be active Sirtuin 1 needs to bind its coenzyme NAD+. With age, levels of Sirtuin1 and other NAD+ dependent enzymes decrease^11,12. Together Sirtuin1 and NAD+ form a skin longevity complex. 

The skin is a highly innervated organ and, as a result, is able to locally produce cortisol and other well-being mediators (dopamine, oxytocin and β-endorphin). These well-being factors are involved in skin regeneration and can therefore be a strategy to protect skin against cortisol^13,14,15.

Psychological well-being is highly impacted by skin conditions. Indeed, skin conditions of people with acne episodes, psoriasis or atopic dermatitis are linked to psychological distress¹⁶. In addition, stressful life events can themselves have an impact on the skin, which affects one’s self-esteem, sleep and well-being.

We implemented a holisitic approach to evaluate effects of a specific Lactobacillus rhamnosus GG postbiotic on skin well-being conditions.

Materials and Methods

Keratinocyte Treatment: Human primary keratinocytes were cultivated in 12 well-plates (Falcon) and kept in culture until they reached confluence. They were then treated with 3% L. rhamnosus GG postbiotic. For circadian rhythm assays, cortisol was then applied for 8 hours.

Gene Expressions: Expression levels of target genes (Per1, Per2, Cry2, Sirt1, Ror-a, Foxn1, Filaggrin, Involucrin, Corneodesmosin) were monitored by q-RT-PCR. RNA extraction was performed using an RNA extraction kit (Maxwell). RNA quantity was assessed using Nanodrop and 1μg RNA was reverse transcribed using iScript cDNA Synthesis kit (Biorad). Gene expression was assessed using SYBRGreen (Biorad, USA) and CFX technology (Biorad). Primers targeting PER1, PER2, CRY2, ROR-α, FOXN1, Filaggrin, Involucrin, Corneodesmosin and Sirtuin 1  were purchased from Biorad’s library of validated primers. Fold-change of gene expression was calculated using DDCt method.

NAD+/ NADH Dosage in Keratinocytes: 3 days post-treatment cells were lysed and NAD+/ NADH levels were quantified using a bioluminescence kit (Promega). NAD+ and NADH levels were reported to total protein levels (BCA assay, Interchim)

ATP Production in Keratincoytes: HaCaTs were seeded in 24-well plates (Falcon) in DMEM medium (Gibco). Cells were then treated with 3% L. rhamnosus GG postbiotic for 2 hours. ATP was extracted and quantified using a bioluminescence kit (Sigma) according to manufacturer’s instruction. Cells were fixed in histochoice (Interchim) and nuclei number was determined by Hoechst 33258 staining. ATP levels were normalized to cell number.

Melatonin release: keratinocytes and fibroblasts

Keratinocytes: human primary keratinocytes were treated with 3% L. rhamnosus GG postbiotic and melatonin levels were assessed using LC-MS/MS.

Fibroblasts: Human primary fibroblasts were seeded in 24-well plates (Falcon) and treated for 72h with 3% L. rhamnosus GG postbiotic. Melatonin production was measured using Elisa Kit (R&D). Cell number was assessed using Hoechst 33342 and melatonin levels were reported to cell number.

Keratinocyte-neuron Co-culture (Oxytocin, Dopamine and MOR): Sensory neurons issued from hiPSC (human induced pluripotent stem cells) were incubated with 0.3% L. rhamnosus GG postbiotic. The next day cortisol was added to the culture. Keratinocytes when then seeded and the treatment was pursued for 5 days. Oxytocin and dopamine levels were measured by Elisa, while mu-opioid receptor (MOR) levels were quantified by immunofluorescent staining. Data were reported to cell number in order to allow comparison.

Ex vivo assay: Skin explants from a 24 years -old donor (Female, Caucasian, phenotype II) were treated with cortisol (in the media) and with 3% L. rhamnosus GG postbiotic (topically) for 6 days. Skin explants were frozen and sectioned (7µm). ZO-1 staining was performed and quantified using ImageJ. 

Clinical Assays: Women aged from 30 to 60 years old (mean age 49 years old) applied a face cream containing 3% L. rhamnosus GG postbiotic (24 volunteers) or placebo (20 volunteers) twice a day for 1 month. Skin regeneration was measured using Easystiff instrument (Biomeca). For skin resilience, volunteers were asked to frown their forehead. Pictures were taken immediately after frowning. Measurements were performed to determine the ability of skin to recover after facial expression. Volunteers also passed a well established well-being questionnaire ¹⁷.

Emotional Impact via Electroencephalography (EEG) and Galvanic Skin Response (GSR): Before evaluating the emotional impact of L. rhamnosus GG creams, we compared creams containing placebo and postbiotic to ensure there was no perceived sensory difference that could bias our study. Then, a panel composed of 13 volunteers aged from 21 to 55 years old (mean age 48 years old) applied creams at the forearm. Neuronal activity was measured using an EEG EPOC headset (Emotiv) while arousal was determined via GSR using Shimmer3 device (Shimmer).

Sleep monitoring in volunteers: Twenty-one women aged from 30 to 60 years old (mean age 50 years old) applied a face cream containing 3% L. rhamnosus GG postbiotic twice a day for 1 month. Sleep quality and duration were measured at home using an under-mattress sensor EMFIT QS (Emfit).

Statistical analysis: Stastistical analysis was performed using Student’s t test or non parametric Wilcoxon test or Mann-Whitney test.

P-values p<0.05 or p<0.01 were considered statistically significant.

Results

Protecting Epidermal Barier Against Cortisol

Prolonged cortisol exposure due to chronic stress is known to disrupt skin barrier. Keratinocytes were therefore treated with cortisol +/- 3% L. rhamnosus GG postibiotics. Expression of epidermal differentiation markers was decreased by cortisol (see Figure 1). ROR-α positively regulates FOXN1, the master regulator of epidermal differentiation genes. L. rhamnosus GG postibiotic treatment counteracts cortisol by inducing expression of epidermal differentiation markers (see Figure 1). L. rhamnosus GG postbiotic has therefore an impact in protecting skin barrier against cortisol in 2D.

Figure 1 - Percentage change of skin barrier markers vs. untreated control, cortisol or cortisol + 3% L. rhamnosus postbiotic.Courtesy of Croda Beauty Actives

Skin explants were treated with cortisol and/or 3% L. rhamnosus GG postbiotic. To mimic systemic cortisol, cortisol was added to the media, while postbiotic was applied topically on the skin explant surface. ZO-1 was decreased by 13% (p<0.01) in explants treated with cortisol. In skin treated with both cortisol and postbiotic, ZO-1 levels increased by about 50% (p<0.01) compared to cortisol treatment alone ( see Figure 2).

Figure 2 - Representative images of ZO-1 staining in skin explants: untreated (left), treated with cortisol (middle) or treated with cortisol + 3% L. rhamnosus postbiotic (right).

L. rhamnosus GG postbiotic is therefore able to protect epidermal barrier against cortisol. This makes it a good candidate to fight the impact of psychological stress on skin.

Protecting Circadian Rhythm Against Cortisol

Besides its role in epidermal marker expression and therefore regeneration, ROR-α is a member of the circadian clock. Since prolonged cortisol exposure is known to disrupt circadian rhythm, we decided to investigate the impact of L. rhamnosus GG postbiotic on the circadian rhythm of our keratinocytes in 2D.

In only 8h, cortisol induced overexpression of PER1, PER2 and CRY2, while treatment with 3% L. rhamnosus GG postbiotic partially inhibited effect of cortisol at this time point (see Table 1).

Interestingly, for CRY2, cortisol treatment completely abolished rhythmic expression, while addition of 3% L. rhamnosus GG postbiotic, restored rhythmicity in CRY2 expression (see Figure 3). This cyclic expression is crucial because it ensures that physiological processes are synchronized with the day-night cycle. 

Figure 3 - Expression of CRY2 during time in kertinocytes.Courtesy of Croda Beauty Actives

Altogether these data show that L. rhamnosus GG postbiotic protects keratinocyte autonomous circadian rhythm against cortisol.

Inducing Melatonin Production

 Melatonin is a key regulator of the circadian rhythm. Melatonin and cortisol follow opposing expression patterns throughout the day. Interestingly, melatonin levels are positively correlated with ROR-α. Given that the postbiotic derived from Lacticaseibacillus rhamnosus GG influences both ROR-α and circadian rhythm, we investigated whether it could also modulate melatonin production. 

Figure 4 - Melatonin production in fibroblasts (left) and keratinocytes (right) treated with L. rhamnosus GG postbiotic.Courtesy of Croda Beauty Actives

To assess this, melatonin levels were measured in fibroblasts (see Figure 4A) and keratinocytes (see Figure 4B). Treatment with 3% L. rhamnosus GG postbiotic induced production of melatonin in both fibroblasts and keratinocytes.

L. rhamnosus GG postbiotic: Sirtuin 1 – NAD+/NADH – ATP

Given the increase in melatonin production, we wondered how L. rhamnosus GG postbiotic could impact Sirtuin 1 and NAD+. In keratinocytes treated with 3% postbiotic, Sirtuin 1 expression was increased by 91% (p<0.01) and NAD+ production increased by 60% (p<0.01). In parallel NADH production was also measured, and a 28% (p<0.01) increase was observed.

NAD+ and NADH are essential for ATP production. Since they both increased, we measured ATP levels in keratinocytes. As expected, L. rhamnosus GG postbiotic induces ATP production (+83%, p<0.01).

Altogether these data show that besides its effect on melatonin and circadian rhythm, L. rhamnosus GG is involved with longevity markers and boosts cell metabolism.

Combatting Cortisol via Well-Being Mediators

Another method to block cortisol's negative impact on skin is by increasing local well-being mediators, such as oxytocin, dopamine and β-endorphin. Besides their impact in cell-cell communication, these markers are all involved in skin regeneration. Increasing their expression is a strategy to combat cortisol. 

Figure 5 - Keratinocyte-neuron co-culture model and measurement of well-being mediators.Courtesy of Croda Beauty Actives

In a keratinocyte-neuron co-culture treated with cortisol + 0.3% L. rhamnosus GG postbiotic, oxytocin production increased by 61% (p<0.05) and dopamine by 33% (p<0.05). We then monitored expression of MOR that has a high affinity for b-endorphin. Treatment of our model with β-endorphin induced MOR expression by 33% (p<0.05), while our postbiotic had a β-endorphin like effect on this target (see Figure 5).

Our pre-clinical data showed that L. rhamnosus GG postbiotic fights cortisol via different pathways: circadian rhythm, epidermal regeneration, melatonin increase, by boosting longevity markers and well-being mediators.

Increased Skin Barrier and Resilience 

Skin regeneration is correlated with its stiffness. Increased tissue density and cell-cell interaction result in stiffer and thus more resistant tissue. Easystiff applies a force to distort the skin and records its resistance. After 28 days of treatment with the cream containing the postbiotic, stiffness of stratum corneum and epidermis increased by 13% (p<0.05) and 14% (p<0.05) respectively, while placebo cream had no impact. These data are in line with those observed in-vitro on increase of epidermal differentiation markers and improved barrier.

Positive Impacts on Skin Resilience

When stressed and worried, our face is also submitted to mechanical stress. As a result, expression lines are formed. In order to test the impact of L. rhamnosus GG postbiotic on skin resilience, volunteers that applied the cream containing either the postbiotic or a placebo, were asked to frown their forehead. 

Figure 6 - Representation of measurement of skin resilience on volunteers.Courtesy of Croda Beauty Actives

Ability of skin to recover after expression was measured (see Figure 6). Data showed that skin resilience and tonicity were improved in 53% of volunteers that were treated with the cream containing the postbiotic and only in 17% of volunteers treated with placebo cream (p<0.05). L. rhamnosus GG postbiotic has therefore a significant impact on skin resilience.

Emotional Impact of L. rhamnosus GG Postbiotic

Positive emotions are associated with higher activity in the brain's left hemisphereFigure 7 - Representation of emotional impact of creams containing placebo and postbioticCourtesy of Croda Beauty Actives while negative emotions correlate with the right hemisphere¹⁸. Excitation status (arousal) on the other hand can be determined through skin conductance. To assess emotional impact of L. rhamnosus GG postbiotic, measurement of neuronal activity (valence) and skin conductivity (arousal) were measured immediately after cream application.

The Valence/Arousal representation shows that placebo and postbiotic position are different. Interestingly, while volunteers treated with placebo are in a more “annoyed” state, postbiotic-cream induced a feeling of relaxation and calm (see Figure 7).

Improvements in Well-Being and Sleep

Because stress deeply impacts our well-being and sleep, we decided to focus on these two parameters.

Volunteers were asked to fill a well-being questionnaire established by Massé et al.¹⁷ before D0 and at the end of treatment. These questionnaires showed that in volunteers that received cream containing the postbiotic, happiness, self-esteem and emotional balance were significantly improved when compared to placebo (see Figure 8A). 

Sleep was monitored by under-mattress contact-free sensors. Data showed that volunteers sleep longer, better and recover better after waking up (see Figure 8B).

Figure 8 - Impact of L. rhamnosus GG on well-being questionnaire (A) and sleep (B).Courtesy of Croda Beauty Actives

Discussion

Interest in neurocosmetics has been growing both from a market and a scientific perspective. Claiming an effect on holistic well-being from a cosmetic ingredient is a real challenge as well-being is the essence of cosmetics .

With this challenge in mind, we designed an in-vitro and in vivo approach to evaluate the impact of an active ingredient against stress.

We first implemented an in-vitro assay to mimic the impacts of psychological stress on skin. Keratinocytes, keratinocyte-neurons co-cultures and skin explants were treated with cortisol, known as the stress hormone. Impact of cortisol on skin barrier, circadian rhythm and well-being related mediators (oxytocin, dopamine and b-endorphin receptor) were evaluated in-vitro. 

Cortisol disrupted both skin barrier function and circadian rhythm in vitro. However, treatment with Lacticaseibacillus rhamnosus GG postbiotic protected against cortisol-induced skin damage and significantly increased levels of well-being mediators (oxytocin and dopamine), as well as β-endorphin receptor expression.

In our clinical trials, we focused on visible signs of stress and fatigue on the skin. The data showed that a cream containing L. rhamnosus GG postbiotic improved skin regeneration and resilience—findings that were consistent with our in vitro results. Given the strong link between skin appearance, stress, and well-being, we then adopted a holistic approach focusing on well-being, emotional status and sleep of our volunteers. Volunteers who applied the postbiotic-containing cream reported overall improvements in well-being. L.rhamnosus GG postbiotic had an immediate relaxing/calming effect on the brain. Finally, measurements on sleep duration and quality showed a significant improvement.

Our innovative and complete protocol allowed us to study the impact of psychological stress and that of a cosmetic ingredient with a holistic approach.

Conclusion

In this study we were able to show the positive impact of a postbiotic issued from L. rhamnosus GG, against stress-related signs on skin. Mechanisms of action include protection of skin circadian rhythm, increase of longevity markers, boost in metabolism and increase of well-being mediators. Significant impact of L. rhamnosus GG on skin was translated into improved self-esteem and overall wellbeing leading to better sleep. Sleep quality, in turn, affects skin quality. Our active ingredient therefore creates a true virtuous circle that benefits the skin.

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