1. IntroductionThe link between anxiety, psychological stress, and aging (as neurogenic aging, resulting from chronic neurogenic inflammation) is now well documented, though some underlying mechanisms are not yet fully understood. Evidence suggests that chronic psychological stress stimulates the hypothalamic–pituitary–adrenal (HPA) axis, inter alia, when the body attempts to resolve perceived threats. Prolonged activation of these pathways can result in chronic immune dysfunction, chronic inflammation, increased production of reactive oxygen species, and DNA damage, which contribute to the accelerated aging of skin and other tissues [1].Anxiety and stress are conditions characteristic of modern life, characterized by feelings of emotional or physical tension, fear, worry, or unease. A wide range has been reported for the prevalence of anxiety in the general population, partly due to variations in methodology. Baxter et al. [2] quote a prevalence of 7.3% among the general population, and 10.4% among Western nations. Remes et al. [3] quote a range of up to 25% in the general population, and emphasize subgroups including women, young adults, people with chronic disease, and Western cultures. Bryant et al. [4], focusing on adults aged over 60, quotes a prevalence of up to 15%, but notes that the prevalence of anxiety symptoms is up to 52%.Recent research has confirmed the skin as both a target of stress mediators and a local source for these factors. Stress conditions exert their effects on skin mainly through the HPA axis. Neurons in the hypothalamus secrete corticotropin-releasing hormone (CRH), which is transported to the pituitary gland, where it binds to the CRH receptor type-1 (CRH-R1) and stimulates the secretion of adrenocorticotropin (ACTH) (Figure 1). In turn, ACTH travels to the outer layer of the adrenal cortex and stimulates the production of cortisol. Cortisol is the primary stress hormone in humans and regulates a wide range of stress responses, inducing various immune and inflammation responses. Cortisol circulates through the blood stream to the skin and activates skin mast cells, which produce stress hormones locally, including CRH. This leads to a stress-induced inflammatory cascade [5,6]. CRH produced by activated mast cells binds to CRH-R1-expressing keratinocytes, inducing NF-kB activation [7,8]. The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions, serving as a pivotal inflammatory response mediator. NF-κB induces the expression of pro-inflammatory genes, including those encoding cytokines and chemokines such as IL1, IL6, and TNF-α [9]. The inflammatory response increases blood flow to the inflamed site, to supply more nutrients and immune cells. Blood vessels dilate, resulting in redness and edema [10]. In addition, chronic inflammation is recognized as an important factor in the aging process (sometimes referred to as “inflammaging”). One of the features of this process is the release of matrix metalloproteinases (MMPs) that degrade the extracellular matrix, resulting in accelerated loss of skin elasticity and firmness, and the appearance of wrinkles [11]. Adverse effects of psychological stress on skin have been documented in rats, where immobilization stress resulted in skin mast cell degranulation [12]; and, in humans, where stress (Trier Social Stress Test) was shown to correlate with delayed skin barrier recovery [13].

The above suggests that blocking the CRH-R1 receptor locally in the skin should alleviate the effects of chronic states of stress on skin, by preventing NF-κB activation and related pro-inflammatory cytokine production—thus potentially representing a valuable strategy to address neurogenic skin inflammation and aging.

Cistus incanus is a Mediterranean shrub, rich in polyphenols and representing a source of valuable bioactive compounds. Secondary sources report that Cistus incanus has been used in traditional medicine, inter alia as an anti-inflammatory agent, to encourage wound healing, and in the treatment of some skin conditions [14,15,16,17,18,19]. Furthermore, Cistus incanus extracts containing polyphenolic compounds have been reported to possess antioxidant [20], antimicrobial [21], and antiviral [22,23] properties. Recently, aqueous extracts of the aerial parts of this plant have been demonstrated to possess in vivo antioxidant capacities, possibly attributable to their high polyphenol content [24]. Cistus species produce flavonoids, a class of polyphenolic secondary metabolites, and particularly flavanols (quercetin, kaempferol, and myricetin derivatives) [16,20,25,26,27,28]. In the extract reported herein, we identified the myricetin glycoside myricitrin, originally isolated from the bark of Myrica rubra (Lour.) [29], but previously unreported in aqueous Cistus incanus extract fractions.Myricitrin is a 3-O-α-L-rhamnopyranoside of myricetin, reported to have anxiolytic effects [30]. The underlying mode of action is unclear. Myricitrin has also been shown to block activation of the NF-κB signaling pathway, decrease the production of pro-inflammatory factors including IL1-β, IL6, and TNFα [29,31], downregulate the activation of Janus kinases (as well as that of downstream transcription factor STAT1), and reduce ROS production (in a NOX2-dependent way). These precedents suggested that a Cistus incanus extract containing myricitrin may be an interesting candidate for an attempt to mitigate psychological stress-induced neurogenic inflammation. We therefore set out to test the efficacy of such an extract in in vitro, ex vivo, and in vivo (clinical) models, focusing on effects in skin tissue. Given the crucial role of the CRH signaling pathway in neurogenic inflammation, we also planned to look into the extract’s ability to interfere with said pathway. The present work reports on these studies and their results, aiming to demonstrate a solution to premature neurogenic skin aging driven by psychological stress using an extract of Cistus incanus in topical application. 4. Discussion

In a functional receptor assay, we demonstrated that a Cistus incanus extract is an effective antagonist of the CRH-R1 receptor, indicating that the extract may have the capacity to interrupt the neurogenic inflammation signaling chain at this stage and thus potentially blocking the stress response chain.

This was corroborated in an ex-vivo skin explant model, stimulated with CRH. In this model, CRH stimulation significantly increased expression of the master inflammation regulator NF-kB, as expected. On the other hand, addition of Cistus incanus extract was able to block the effects of the CRH stimulation and effectively decrease NF-kB levels in the explants. This result seems to confirm that the Cistus incanus extract is able to suppress the CRH-induced activation of an inflammation signaling cascade, and thus may offer some protection from psychogenic stress-induced inflammation processes.

These indications are further supported by results in a human keratinocyte culture under stimulation by CRH. In this model, the CRH stimulation increased the expression of inflammation markers IL1-β, IL6, and TNF-α, also as expected. Conversely, addition of Cistus incanus extract effectively decreased the expression of the same markers, negating the effects of the CRH stimulation. Here also, these results reinforce the implication that the extract is able to block downstream inflammatory processes resulting from CRH signaling, and therefore may effectively block stress-induced inflammation.

The ability of the extract to inhibit downstream effects of CRH signaling, and therefore also prevent some of the eventual damage caused by psychological stress on living tissue, in this case skin, was finally confirmed in vivo in a double-blind, placebo-controlled clinical trial in a population sample selected for high levels of psychological stress. These volunteers were recruited on the basis of a dual evaluation, featuring a screening questionnaire as well as saliva cortisol measurements. In this trial, we were able to observe that the extract delivered significant baseline anti-inflammatory effects (observable through microcirculation measurements and analysis of cross-polarized visible light photographs). We observed significantly improved skin resilience to inflammation-inducing provocations, as evaluated by following the evolution of skin redness and microcirculation after a chemical insult with model irritant SLS, which showed that treatment with the extract significantly reduced peak irritation and increased the speed of recovery from insult. Finally, and perhaps most valuable where the ultimate objective is to reduce the signs of premature aging induced by chronic neurogenic inflammation, we observed that treatment with the extract resulted in significantly reduced skin wrinkling after 28 days, with a statistically significant advantage over the placebo product.

The potential for use of Cistus incanus extracts in cosmetic, skin care, or personal care applications has been reported, in particular by Gawel–Beben et al. [33], who report on these extracts’ contents of phenolic and flavonoid species, their activity as tyrosinase inhibitors (an effect linked to possible reduction of pigmentation in the skin), and in particular on their antioxidant potential, as evaluated by DPPH radical scavenging. A DNA-protective effect has also been reported for Cistus extracts by Vanella et al. [34], most likely resulting from the same antioxidant properties. Vanella et al. show that the Cistus extracts can scavenge oxidative species including the DPPH radical and the superoxide anion, inhibit lipoperoxidation in rat liver microsomes, and protect DNA from damage caused by hydroxyl radicals. These antioxidant effects could be seen as likely contributors to the anti-inflammatory and anti-aging benefits observed in our own work.

Nevertheless, to the best of our knowledge, the work described herein constitutes the first direct, controlled, in-vivo demonstration of the anti-inflammatory and anti-aging effects of such an extract to be published in the scientific literature. What is more, to the best of our knowledge no work has been published regarding the effect of a Cistus extract (or indeed, any other similar botanical extract) on the interruption of psychological stress signaling or neurogenic inflammation, especially via blockage of the CRH-R1 receptor.

As noted above, the flavonoid myricitrin, identified in the extract, has been associated with similar effects, including antioxidant and anti-inflammatory effects. Zhang et al. reported that myricitrin reduced the production of inflammatory markers including IL6 and TNF-α, as well as nitric oxide (NO) and the enzyme catalyzing its production (iNOS), in mouse macrophages [32]. Du et al. [29] also reported on anti-inflammatory effects, showing decreased inflammatory mediators including IL1-β, IL6, TNFα, as well as lowered COX-2 and iNOS expression in the nigrostriatum neurons of an LPS-stimulated mouse model of neuroinflammation. Of particular interest in context of our own results, the same authors also showed that myricitrin could block the activation of NF-kB (as well as TLR4 and MyD88, upstream regulators of the NF-kB signaling pathway), and concluded that this was a main pathway responsible for the neuroprotective effect against LPS-induced inflammation and injury observed in their work.

While other compounds present in the extract likely also play a role, the presence of myricitrin in our extract, in light of the pathways on which myricitrin has been shown to have an influence and their possible association to the effects we observe in our studies, seems to suggest that this compound may be linked to some or all these same effects.

Conversely, it is difficult to unequivocally attribute the entirety of the effects observed, especially those observed in vivo, to the single mechanism of CRH-R1 blockage. Other effects and biological mechanisms may be in play. For example, the extract’s anti-oxidant potential [33,34], remarked upon above, may play a direct role on improving tissue inflammation states, independently of the CRH stress signaling chain—and may also be behind part of the benefits observed in vivo. One could also imagine that other compounds in the extract could have an independent anti-inflammatory effect, once again bypassing neurogenic stress signaling or affecting a different level of the signaling chain. Nevertheless, blockage of the CRH receptor by the extract is clearly demonstrated here, as are several expected downstream effects—suggesting that this mechanism is indeed behind at least part of the extract’s observed efficacy.

Some further limitations of the work presented herein include the need for a better understanding of the mechanisms of the effects demonstrated here, in particular regarding their dose- and time-dependency, especially in connection with the influence on NF-kB and resulting downstream effects. This understanding could be expanded through further studies, using similar ex-vivo and/or clinical models. Additionally, our investigations have been limited to effects on skin, while effects on skin appendages and/or other types of tissue affected by neurogenic inflammation may also be of scientific and industrial interest.

5. Conclusions

We have demonstrated that an extract of Cistus incanus aerial parts may interrupt the psychological stress signaling cascade at the stage where this signaling chain is relayed by CRH, through blockage of the CRH-R1 receptor. We have also shown that this same extract can deliver significant anti-inflammatory effects, preventing inflammation triggered by CRH and thereby preventing or alleviating at least part of the inflammatory state triggered by psychological stress. Finally, we were able to confirm these effects in vivo, on a group of healthy volunteers selected for a high level of psychological stress; in these volunteers, we showed the anti-inflammatory benefits of treatment with the extract, as well as the extension of these effects into improvements in skin resilience to chemical (SLS) insult and improvements in aging signs (wrinkles).

Taken together, these results effectively indicate that an extract of Cistus incanus aerial parts may mitigate the effects of neurogenic stress on skin and therefore has significant potential for application as an active ingredient in a broad range of skin care applications, especially applications aimed at delivering anti-inflammatory and/or anti-aging benefits in populations suffering from chronic psychological states of stress.

It may be valuable to expand on the data presented herein through further studies, in order to shed further light on the dose- and time-dependency of the effects described, as well as on effects on other tissue types (e.g., hair and other skin appendages, but possibly also internal tissues such as soft joint tissues, cardiac muscle tissue, and more), and under different types of psychological stresses.