The Effects of Nutrient Signaling Regulators in Combination with Phytocannabinoids on the Senescence-Associated Phenotype in Human Dermal Fibroblasts

Abstract

1. Introduction

The life expectancy in most developed countries almost doubled within the last century [1]. Longevity is a double-edged sword, as people live longer, life quality can be adversely affected by age-related diseases (ARDs). Research teams around the globe are focusing on investigating aging and treatment strategies to delay or prevent the development of ARDs to improve quality of life [2,3]. A significant challenge of aging research is distinguishing the pathogenesis of cell and tissue senescence from the myriad of changes that accompany it. Substantial research details the causes and mechanisms of aging; however, we still cannot stop aging, but only alleviate or delay some ARDs and their symptoms.

Few drugs and nutrients directed at the elimination of detrimental effects of aging have been discovered, including nutrient signaling regulators (NSRs) [3,4]. Nutrient signaling regulators maintain nutrient-sensitive signaling pathways and are vital for biological processes [3]. NSRs were shown to regulate processes related to age-related diseases such as organismal survival, growth, metabolism, signaling systems, including sirtuin, mammalian target of the rapamycin (mTOR), and AMP-activated protein kinase (AMPK) [5,6]. Among the most studied in this group are sirtuin regulators [7,8,9], mTOR inhibitors [10,11,12,13], and AMPK activators [1,14,15,16]. NSRs have been tested on eukaryotic organisms from yeast to humans, showing beneficial results and extending lifespans in model organisms by mainly targeting the master regulators of aging that affect autophagy, inflammation, and oxidative stress. In clinical trials, these drugs have been shown to prevent cardiovascular diseases, reduce inflammation, and potentially inhibit carcinogenesis [17]. Current studies in the field of aging are focused on the most popular anti-aging therapeutics: rapamycin, 1,1-Dimethylbiguanide (metformin), and 3,5,4′-trihydroxy-trans-stilbene (resveratrol).

Rapamycin is a central modulator of the mTOR pathway; it regulates transcription and protein synthesis by integrating with various signal transductions pathways to control growth, apoptosis, and autophagy in cells. The mTOR pathway is the crucial controller of growth in animals and is the fundamental link between the availability of nutrients in the environment and the control of most anabolic and catabolic processes [18]. Furthermore, mTOR drives growth-like conversion from quiescence to senescence in response to cell-cycle arrest [19]. Alterations in mTOR pathway regulation are commonly involved in several genetic diseases, including aging and ARDs in humans [20,21]. In addition, recent reports have shown deregulation of mTOR signaling in ARDs and aging in multiple model organisms [18,22,23].

Rapamycin and its analogs are currently approved by the United States Food and Drug Administration for the treatment of a number of conditions, as they have been demonstrated, both in vitro and in vivo, to slow aging and extend lifespan in different species [21] and prevent ARDs [19].

Apart from different diseases, rapamycin was also recommended in cosmetic practice for anti-aging and skin rejuvenation purposes. Rapamycin-containing cream was effective when applied to selected areas, like the hands and face, especially skin affected by age-related spots and dermatopathies [19]. Topical usage is considered safer and might spare the patients from the adverse effects of systemic treatment with rapamycin [24]; however, some evidence suggests that solely topical use of rapamycin may be insufficient to improve skin conditions [19]. Hence, use of rapamycin combined with other anti-aging compounds or natural products like phytocannabinoids for topical application might open new perspectives in the aging abatement and rejuvenation strategies.

Metformin is a widely used anti-hyperglycemic drug [25]. Mechanistically, metformin affects different cellular targets. Of those, it causes inhibition of the respiratory complex I of the electron transport chain [26,27], directly affecting reactions requiring adenosine triphosphate, and indirectly acting on the activation of AMPK [27,28]. As a result, this causes elevated accumulation of reduced nicotinamide adenine dinucleotide (NADH) compared to the oxidized form (NAD+) and inhibits production of NADH-ubiquinone oxidoreductase, which is localized on the mitochondrial membrane, thus activating AMPK and suppressing gluconeogenesis [26,29]. Subsequently, this leads to fatty acid synthesis and mTOR signaling network suppression [30], causing reduced cellular energy consumption. Moreover, studies have shown that long-term use of metformin alleviates oxidative damage and chronic inflammation, reduces cognitive deterioration, and prolongs health and life [31]. As a biguanide, metformin acts in a dietary restriction manner and on oxidative stress pathways in a lifespan-extending effect [25]. It reduces advanced glycation end-product (AGEs) accumulation in response to massive reactive oxygen species (ROS) generation in high glucose conditions. Accelerated dermal aging is directly correlated with increased protein deterioration in the existing collagen because of crosslinking. The progressive increase in AGEs affects cellular macromolecules during aging through oxidative damage and leads to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Metformin has been shown to protect T3 fibroblasts in vitro under high glucose conditions inducing cell proliferation, collagen I and III production, apoptosis inhibition, and reducing NF-κB (p65) activity [15]. In response to hypoglycemic conditions, metformin inhibits senescence-associated secretory phenotypes by repressing NF-κB pathway activation [32]. Thus, metformin may be a promising pharmacological dermal anti-aging remedy with a complex beneficial impact on the physiology of the skin and entire organism.

Resveratrol is a type of natural phenol and a phytoalexin that can be obtained from several sources, including red wine, grapes, and certain medicinal plants [33,34,35]. Nevertheless, resveratrol is often used as a popular dietary supplement [26]. Resveratrol’s anti-aging characteristics have been investigated in different species [9]. It can alleviate the inflammatory phenotype in senescent-induced human fibroblasts through upregulation of autophagic pathways [36]. Resveratrol is known to prevent ARDs and extend the lifespan of mice and rats through sirtuin (SIRT) 1 pathway modulation [37,38,39,40]. SIRT1 is a NAD(+)-dependent deacetylase involved in gene silencing, anti-oxidative stress, anti-apoptosis, and inhibition of inflammation [34,41]. Resveratrol can increase SIRT1 during aging and chronic inflammation, processes that are associated with reduced SIRT1 levels and activity in response to oxidative stress [3]. A recent study showed that resveratrol significantly elevates the SIRT1 activity, inhibits the NF-κB pathway, and prevents the loss of intestinal stem cells [34]. Nonetheless, resveratrol has not been shown to prolong the life expectancy of mice [42]. Additionally, Luz et al. (2012) reported that low resveratrol doses and red wine supplementation improved vascular function and aerobic capacity, reduced p53 and p16 senescence biomarkers in rats, but did not extend their lifespan either [43]. Furthermore, resveratrol demonstrates a typical dose-dependent response with a small therapeutic window at low doses, whereas at higher doses (50 or 100 μM), resveratrol can reduce cellular quantity and augment apoptosis or necrosis [44].

There are numerous limitations in resveratrol effectiveness towards longevity and anti-aging because of its limited bioavailability [35,44,45]. Alternatively, the acetylated analog triacetylresveratrol (TRSV) has higher bioavailability [46,47]. Due to its higher hydrophobic nature, TRSV, compared to resveratrol, has been shown to be more effective in interacting with and crossing phospholipid bilayers [48]. Moreover, findings show TRSV might be a more potent therapeutic agent than resveratrol and can affect proliferation and apoptosis by decreasing NF-κB phosphorylation [46], and attenuate inflammation via the p38 mitogen-activated protein kinase/SIRT1 pathway [49]. Conversely, no studies have tested the anti-aging or rejuvenation properties of TRSV.

Some of the beneficial effects of rapamycin, metformin, and resveratrol have been achieved through cutaneous application. It was shown that locally applied metformin and TRSV, but not rapamycin, improved epidermis wound healing and collagen deposition in rodents [50]. Recent clinical studies have supported the efficacy of resveratrol and its analogs (i.e., resveratryl triacetate and resveratryl triglycolate) in human skin lightening and anti-aging because of its anti-inflammatory, anti-proliferative, and anti-pigmentation properties [51].

Similar to TRSV, information on anti-aging properties of phytocannabinoids is also scarce. Recent studies showed that topical application of hydrogels based on cannabis extracts have a beneficial effect on skin hydration, and inhibit the activity of matrix metalloproteinases (MMPs), collagenase, and elastase via reduction of oxidative stress markers; the extracts were shown to inhibit skin aging processes and positively affect the viability of skin cells [52]. Martinelli et al. (2022) noted photoprotective, antioxidant, and anti-inflammatory mechanisms of phytocannabinoids, emphasizing the possible impact of CBD on cell differentiation in the skin, especially in the case of dermatological disorders like psoriasis [53].

We hypothesize that the combination of NSRs and phytocannabinoids (pCBs) may have a beneficial anti-aging and potential rejuvenation effect. While the notion of using metformin, rapamycin, or resveratrol for skin rejuvenation is not novel, combining them with phytocannabinoids and using TRSV instead of resveratrol is a novel approach in anti-aging and rejuvenation strategies for aging skin.

In this study, we characterize the anti-aging effects of NSRs, metformin, rapamycin, and TRSV alone or in combination with delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on functional assays and molecular targets involved in pathogenesis of cellular senescence. We used the stress-induced premature senescence (SIPS) model of human dermal fibroblasts, which we have previously shown to be a reliable and effective model of SIPS [54], to assess anti-aging effects of phytocannabinoids and NSRs.

2. Results

3. Discussion

Despite immense research being performed, countless questions remain regarding the role of fibroblasts in dermal aging. Previously, we have shown fibroblasts demonstrate aging by displaying morphological alterations, elevations of biomolecular markers of senescence, changes of gene expression, changes in functionality, and metabolic reprogramming [54]. Our aims were to target the senescence-associated changes in dermal fibroblasts for the development of potential anti-aging and rejuvenation tools.

The current study established the effects of NSRs (metformin, TRSV, and rapamycin) alone and combined with pCBs on the healthy and senescent skin fibroblasts. Based on the cellular viability test, we determined the concentrations of 500 µM for metformin, 10 µM for TRSV, and 5 µM for rapamycin were the optimal doses for anti-aging. The efficacy of NSRs alone and combined with pCBs were tested by cellular viability MTT and NR assays, wound healing assay, RT-qPCR, and nuclear DAPI staining. The results demonstrated that NSRs differentially affected the senescence-associated phenotypes in dermal fibroblasts.

During routine microscopical examinations of prematurely aged fibroblasts, we revealed fibroblast enlargement with evident loss of their elongated spindle-like features, with fibroblasts turning from bipolar into amorphous-like shape [54]. These findings were consistent with the data from other cultures of senescent fibroblasts [71,72].

Although numerous studies showed a significant positive influence of metformin, resveratrol, and rapamycin on the viability of senescent cells [46,50,73,74,75,76,77,78], our results have not revealed significant improvements in cellular viability induced by NSRs in combinations with or without pCBs (Figure 2 and Figure 3). Furthermore, we discovered that rapamycin alone and combined with THC or CBD, and a mixed treatment that encompassed metformin, TRSV, rapamycin, THC, and CBD exerted a detrimental effect in both cellular viability assays in healthy and senescent dermal fibroblasts (Figure 2 and Figure 3). This is in contrast to published studies that demonstrated topical rapamycin application reduced senescence and age-related features in human skin [79].

Zhao et al. (2017) also noted that metformin and, to a lesser extent, resveratrol improved the healing process through stimulation of the AMPK pathway, which has been documented to inhibit wound healing [80], probably because of its immunosuppressive capability upon systemic administration [50,76,80]. Our results were generally in line with these findings, suggesting TRSV and metformin could be beneficial for therapeutics in dermal healing.

Wound-healing assays revealed that typically wound closure completed within 72 h (Figure 4 and Figure 5). In healthy fibroblasts, we detected rapamycin delayed wound healing in CCD-1064Sk fibroblasts. These results suggest rapamycin might be considered in treating hyperproliferative disorders like psoriasis and cosmetic manipulations, but not for wound healing or anti-aging therapeutics.

Remarkably, in the senescent CCD-1064Sk fibroblast cultures, we discovered a common trend to potentiate wound healing with the addition of THC or CBD to NSRs. Metformin with THC or CBD produced significantly improved wound healing compared to just metformin, while rapamycin + THC had significantly improved wound healing compared to just rapamycin (Figure 5). TRSV had the best wound healing out of any NSRs in either healthy or senescent fibroblasts (Figure 5B,D), while also not appearing to be improved by the addition of pCBs (Figure 5B,D). In fact, TRSV + THC had decreased wound healing ability compared to TRSV alone in senescent fibroblasts (Figure 5D). However, metformin + THC + CBD substantially delayed wound closure compared to the vehicle (Figure 5D).

Most anti-aging nuclei studies were focused on genome organization and nuclear structure in young, proliferating, and often transformed cells. However, for the analysis of functional activity in replicative and stress-induced senescence, it would be more relevant to analyze the nucleus of non-proliferating quiescent or senescent cells [81]. Our previous results determined significant nuclear alterations in SIPS fibroblasts compared to healthy fibroblasts [54], while, in this study, we showed NSRs with or without pCBs altered nuclear architecture as well (Figure 8, Figure 9, Figure 10 and Figure 11). For instance, the nuclear area, a parameter that allows identifying size changes, was 149.5 µm² in healthy young cells, while it was 194.2 µm² in the SIPS CCD-1064Sk fibroblasts (Figure 8). Other studies reported similar alterations on nuclear architecture of different senescent fibroblast cell cultures. For example, Bridger and Kill (2004) studied nuclear abnormalities in normal dermal fibroblast cultures 1BR and NB1 and compared them with prematurely senescent ones from Hutchinson–Gilford progeria syndrome fibroblasts [82]. They revealed that cellular aging is characterized by a shift in the nuclear area towards larger nuclei. Moreover, it was noted that naturally aged nuclei exerted the same features as Hutchinson–Gilford progeria syndrome fibroblasts [82]. In line with these observations, we also found that senescent fibroblasts have larger nuclei.

We found metformin with and without pCBs altered nuclear architecture in healthy fibroblast cells. Changes included increased area (Figure 8A) and perimeter (Figure 9A) after 1 day of exposure, and increased circularity (Figure 10A) and decreased eccentricity (Figure 11A) after 5 days of exposure. These changes can help explain the nonsignificant trend of inhibited wound healing in healthy fibroblasts (Figure 5B). In contrast, metformin with or without pCBs generally did not affect nuclear architecture after 1 day of exposure. After 5 days of exposure, metformin with and without pCBs significantly increased area (Figure 8A) and perimeter (Figure 9A), while metformin decreased eccentricity (Figure 11A) in senescent fibroblasts. Since metformin with pCBs had significantly better wound healing than metformin alone (Figure 5D), it is difficult to understand why no major nuclear changes were seen between these groups unless nuclear architecture is not an important factor in the functional activity of metformin with pCBs.

A recent study reported dose-dependent resveratrol-induced premature senescence in dermal fibroblasts, based on increased β-Gal staining, increased expression of p16, p21, and p53 cell-cycle inhibitors, and accumulated senescence-associated heterochromatic foci (SAHF), known as areas of condensed and transcriptionally silenced DNA [83]. They found that the decline in proliferative activity was associated with resveratrol doses higher than 10 μM and that resveratrol induced apoptosis at 100 μM. The percentage of apoptotic cells was increased to 8.3 ± 1.5 at 100 μM and reached to 37 ± 1.5 after usage of 200 μM and 300 μM of resveratrol, while directly correlating with elevated SAHF appearance. These findings are essential considering the safety and efficacy of resveratrol. In contrast, our studies used 10 μM TRSV, an acetylated form of resveratrol with reportedly higher bioavailability [46,47].

Differences in nuclear architecture were apparent between TRSV and TRSV with pCBs after 1 and 5 days of exposure in healthy fibroblasts; however, no noticeable differences in healthy fibroblast wound healing were seen between treatments (Figure 5B). In contrast, nuclear architecture was significantly altered in senescent fibroblasts for TRSV, TRSV + THC and TRSV + CBD treatments after 5 days of exposure. Since TRSV with or without pCBs had the best wound healing out of all NSRs (Figure 5B,D), the data suggest the increased area and perimeter induced by TRSV, TRSV + THC, and TRSV + CBD in senescent fibroblasts may be compensatory mechanisms, while decreased eccentricity and increased circularity suggests amelioration of the nuclear architecture by reducing elongated phenotype that was previously shown to be induced by hydrogen peroxide [54]. Thus, we can speculate that TRSV possesses geroprotective activity toward senescent nuclei by ameliorating nuclear architecture.

Last, rapamycin exposure alone generally altered nuclear architecture, while the addition of pCBs generally returned nuclear architecture parameters to those observed in the vehicle (Figure 9, Figure 10 and Figure 11). These changes induced by rapamycin are detrimental as shown by the corresponding wound healing data (Figure 5B,D), while addition of THC or CBD appear to ameliorate these changes induced by rapamycin (Figure 8 and Figure 10 and Figure 11) and further support the use of THC or CBD in addition to NSRs.

The structural components of the nucleus, nuclear envelope, nucleoli, nuclear bodies, and the nuclear matrix, are intimately linked to the genome, maintaining signaling and ultimately control of the genome functionality [82]. Correspondingly, disorganization or defects in nuclear structures and/or architecture are associated with alterations of normal genome regulation and are commonly found in cancer and severe premature aging diseases, like progeroid syndromes and laminopathies [84]. This is also supported by the observation of changes in the expression of cell-cycle regulators involved in potentiating senescence phenotypes such as p16, p21, and p53. They are engaged in two well-known theoretical signaling transduction mechanisms responsible for cellular senescence, the p53-p21-pRb pathway and the p16-pRb pathway [85].

NSRs and pCBs significantly affected both senescence pathways: the telomere-based p53-p21-pRb, which typically represents replicative senescence (RS), and the stress-based p16-pRB pathway, which represents SIPS [86]. The inhibitory effects of TRSV and its combinations with pCBs on TP53, CDKN2A (p16), and CDKN1A (p21) discovered in our studies are in line with recent studies on the anti-aging properties of resveratrol [85,87]. For example, adding 30 µM and 60 µM of resveratrol partly downregulated the expression of two senescence markers (p16 and p53) in a concentration-dependent manner, suggesting that resveratrol can suppress mechanical overloading-induced nucleus pulposus cell senescence [85]. Furthermore, 4 weeks application of 5 µM of resveratrol on an in vitro senescence model of human MRC5 lung fibroblasts lowered the levels of p53 acetylation by about 35% in RS cells (PDL > 50) [87]. Our data show TRSV similarly downregulates CDKN1A, CDKN2A, and TP53 (Figure 6). The addition of pCBs generally prevented these changes or upregulated mRNA expression (Figure 6). Whether or not these changes are reflected in the encoded proteins remains to be investigated.

Furthermore, Zhao et al. (2017) showed metformin-induced mRNA upregulation of cyclin D1 (CCND1), a proliferative promoter, and downregulated cell cycle inhibitors P53, P21, and P16, in wounds of aged mice [50]. In parallel, they noted resveratrol was less potent in CCND1 promotion and inhibition of P53, P21, and P16 than metformin. Moreover, the authors admitted paradoxical suppressive effects of rapamycin on P53 and P21 but stimulatory effects on P16, which might cause the increased CCND1 levels. Both metformin and resveratrol demonstrated an increase in the viability of proliferative cells alongside the epidermis and around hair follicles, ameliorated aging in cutaneous wound healing, with metformin having a more profound anti-aging effect [50]. Our data on metformin and pCBs’ effect on cell cycle regulators is not uniform with metformin, generally decreasing CDKN2A, increasing CDKN1A, and decreasing TP53 mRNA expression.

Based on the free-radical theory of aging, oxidative stress caused by ROS accumulation can potentiate cellular senescence [54]. In the present study, we also analyzed the effects of NSRs and pCBs on NF-κB activation induced by ROS in dermal fibroblasts exposed to hydrogen peroxide to induce senescence [88].

In healthy dermal fibroblasts, NF-κB was downregulated in all treatment groups following 5 days of exposure (Figure 6D). We also detected increased mRNA levels of NF-κB in the rapamycin treatment in SIPS fibroblasts compared to the vehicle (Figure 6D). However, NF-κB transcripts in senescent fibroblasts were lower than the corresponding levels in healthy cells. There is a substantial body of evidence showing the transcriptional activity of NF-κB is increased in aged tissues and associated with multiple age-related degenerative diseases, including Alzheimer’s, diabetes, and osteoporosis [40]. Furthermore, Tilstra and colleagues reported that NF-κB inhibition leads to delayed onset of age-related symptoms and pathologies in murine models [89]. They also pointed out that stimulation of NF-κB is involved in the supervision of numerous known lifespan regulators, including insulin/IGF-1, FOXO, SIRT, mTOR, and DNA damage. Thus, the inhibitory effect of all treatments on healthy cells might provide anti-aging effects of healthy skin (Figure 6D); however, in senescent cells, no treatments significantly reduced NF-κB expression. Furthermore, NF-κB expression was strongly upregulated in the rapamycin exposure of senescent cells. This could be one mechanism that prevents rapamycin from providing any potential beneficial anti-aging effects in skin (Figure 5D).

There is a large body of data supporting the involvement of NF-κB in regulating metabolic pathways directly linked to sirtuin modulation [34,49,50,89]. The leading role of sirtuins on the protection from cellular senescence has mainly been intimately associated with SIRT1 and SIRT6 [90]. SIRT1 is predominately found in the nucleus and shuttles between the cytoplasm and nucleus and is highly involved in cellular senescence and aging via enzymatic activity of deacetylase resulting in adenosine-diphosphate-ribosyl-transferase triggered DNA repair [6,38,91,92]. SIRT6 is primarily localized in the heterochromatic regions and modulates lifespan extension via adenosine-diphosphate-ribosylation activities, while its deacetylase activity is focused on DNA repair, genome stability, and telomere maintenance [92].

Sirtuins modulate the organismal lifespan by interacting with several lifespan regulating signaling pathways, including the insulin/IGF-1 signaling pathway, TOR, AMPK, FOXO, and the NAD+-dependent sirtuin deacetylases that are commonly dysregulated in aging and ARDs [90,93,94]. Furthermore, the promotion of premature senescence-like phenotypes in endothelial cells was induced by the reduction of SIRT1 and SIRT6 through siRNA or miRNA targeting via pharmacological inhibitors [2]. On the other hand, overexpression of SIRT1 [95,96] or inhibition of SIRT6 [97,98] reduces senescence biomarkers in multiple different cell models.

Our previous studies identified the dysregulation of multiple SIRT family members in SIPS dermal fibroblasts [54]. During this study, we found SIRT1 was downregulated by metformin, metformin + THC, and metformin + CBD but upregulated by TRSV + CBD after 5 days of exposure in senescent fibroblasts (Figure 6). In addition, SIRT6 was upregulated for TRSV, TRSV + THC, TRSV + CBD, and rapamycin after 5 days of exposure. These results are somewhat contradictory as SIRT1, the ‘longevity’ biomarker, mRNA expression would suggest TRSV + CBD are best for anti-aging treatments; however, increased SIRT6 expression would suggest otherwise. In contrast, in healthy cells, SIRT1 was decreased in all treatments, while SIRT6 was decreased in all treatments except rapamycin, which upregulated SIRT6 (Figure 6). Further work should be performed on protein expression to confirm the results demonstrated here.

Resveratrol was reported to exert neuroprotective effects by modulation of mitochondrial function, and the elimination of mutant proteins associated with neurodegenerative diseases by enhancement of autophagic and lysosomal clearance of Aβ-amyloid. These effects were found to occur by induction of SIRT1 and AMPK activation leading to mTOR suppression [9,74,86]. Moreover, NSRs were found to reduce the concentrations of the hormonal effectors such as insulin, IGF1, and growth hormone, which stimulated or inhibited the activity of numerous metabolic sensors (i.e., sirtuins, AMPK, TOR). Bonkowski and Sinclair (2016) showed that sirtuin-activating compounds such as SRT1720 and SRT2104 can directly activate SIRT1, whereas rapamycin is a direct inhibitor of mTOR, and metformin indirectly activates AMPK. In addition, these metabolic regulators mediate downstream activities such as DNA repair, mitochondrial biogenesis and function, stress resistance, stem cell and telomere maintenance, autophagy, chromatin modifications, reduced inflammation, and translation fidelity [93]. In addition, the inhibitory effects of resveratrol on the mTOR regulation, accompanied by potentiation of autophagic and lysosomal activities in senescent cells, might explain our findings related to increased lysosomal NR accumulation and enhanced cellular viability in prematurely senescent fibroblasts exposed to TRSV and pCBs (Figure 3). Altogether, these activities improved cellular homeostasis, reduced morbidity, and presented a more youthful-like state [77,93].

These results supported the idea that NSRs in combination with pCBs exerted protective activity in cellular senescence and might be used as anti-aging remedies by improving the metabolic functionality in prematurely aged dermal fibroblasts. Specifically, our data suggest TRSV or metformin would likely provide more beneficial effects than rapamycin.

Apart from the metabolic regulation, the AMPK pathway also modulates fibroblasts’ functional activity linked to the generation of collagen and other ECM components [50]. The UV-induced elevation of MMP1 expression in human dermal fibroblasts was related to the potentiation of mTOR phosphorylation. In contrast, augmented phosphorylation of AMPK and liver kinase B1, which are mTOR inhibitors, led to suppression of MMP1-initiated activity towards ECM degradation [99]. Metformin was reported to ameliorate age-related thinning of the epidermis, reduction in hair follicle number, decreased collagen deposition, and downregulation of p-acetyl-CoA carboxylase and p-AMPK in the epidermis [50].

Notably, our results discovered effects of NSRs combined with pCBs on gene expression in healthy fibroblasts (Figure 7). A prominent increase in COL1A1 expression was induced by metformin, metformin + THC, TRSV + THC, TRSV + CBD, and rapamycin in both healthy and senescent cells (Figure 7A). Unfortunately, significantly decreased COL3A1 expression was observed in all treatments on healthy fibroblasts, and most treatments also decreased mRNA expression in senescent fibroblasts (Figure 7B). TRSV + CBD increased COL3A1 and COL1A1 expression in both the senescent and healthy fibroblasts, suggesting TRSV + CBD may be the best treatment option for collagen production. Once again, our data would suggest NSRs in combination with pCBs provide more therapeutic value than NSRs alone.

These changes are likely to be related to ROS-induced excessive protein glycation causing the generation of AGEs, which contribute to skin aging as it deteriorates the existing collagen by crosslinking. Besides, the progressive increase of AGEs during aging causes oxidative damage to cellular macromolecules and modulates the activation of transcription factor NF-κB resulting in apoptosis enhancement and ceased proliferative activity [15]. This assertion aligns with our experimental findings, where NF-κB was predominantly upregulated in SIPS fibroblasts and downregulated in multiple treatment groups (Figure 6D).

Another crucial part of the dermal extracellular matrix is ELN. Elastin helps to retain elasticity and regain ECM shape after stretching or contracting. In healthy fibroblasts, mRNA levels of ELN were upregulated by metformin + THC, TRVS + THC, and TRSV + CBD exposure, while TRVS, TRSV + THC also upregulated ELN in senescent cells (Figure 7C). For ELN mRNA expression, our results demonstrated TRSV alone or in combination with pCBs produced beneficial effects, especially TRSV + THC.

Meanwhile, matrix metalloproteinases degrade collagen, elastin, and other components of the extracellular matrix. While overactive MMPs can be detrimental, it is important to note MMPs have a physiological role for healthy ECM remodeling in tissue repair and wound healing. We noticed decreased MMP2 in healthy cells after exposure to all treatments (Figure 7D). As healthy cells do not need high levels of MMPs to remodel the ECM, this shows all treatments had beneficial effects. In senescent cells, we noticed metformin + THC, metformin + CBD, TRSV + THC, and TRSV + CBD upregulated MMP2 levels (Figure 7D). Upregulation of MMP2 levels is likely beneficial and help support ECM remodeling especially considering that most treatments greatly upregulated mRNA expression of COL1A1 and COL3A1 as well.

In recent studies, metformin is often mentioned as a collagen protector in aging and high glucose-associated conditions. For instance, 24 h of treatment with 500 μM of metformin significantly increased collagen I and III generation in aged 3T3 fibroblasts, while the activity of MMPs was decreased [15,16]. Similar results were reported by Shao et al. (2014)—metformin had a direct effect on osteoblast-like cells MG63 and attenuated the suppression of proliferation induced by high glucose with increased expression of collagen type I, osteocalcin, osteoprotegerin, while suppressing MMP1 and MMP2 [75]. In contrast, Benazzoug et al. (1998) have reported that glucose specifically increases collagen type III synthesis both at the mRNA and protein levels, without alteration of collagen type I production in young and replicative senescent cultured human skin fibroblasts [73]. Furthermore, ceramides have been found to prevent ECM atrophy and increase dermal functionality via ECM remodeling through upregulation of elastin and MMP2 through a TGF-β mechanism [100]. In conjunction with our findings, positive effects of TRSV might be mediated via the SIRT1 pathway component—peroxisome proliferator-activated receptor γ coactivator, accompanied by NAD+ elevation and AMPK pathway stimulation [78].

Senescence is inseparably accompanied by the inflammatory process, ROS-induced gradual damage of cellular components and deterioration of molecular signaling pathways, and alteration of cell-cycle and metabolic regulation that eventually leads to increased growth and cell death. Nevertheless, stimulation or inhibition of specific components of ECS, the influence of endocannabinoids (eCBs), or pCBs have the potential to delay or alleviate this process targeting multiple components of the vicious cycle of aging. The main targets of eCBs/pCBs are cannabinoid receptors CB1, which is associated with the analgesic effects of cannabinoids, and CB2, which modulates cytokine release from immune cells and reduces inflammation [101]. In the skin, cannabinoid receptor antagonists exacerbate allergic inflammation, whereas receptor agonists attenuate inflammation. Pharmacological inhibition or knockout of CB1/CB2 receptors in mice augmented the hapten dinitrofluorobenzene-induced dermatitis and was linked to the increased levels of eCBs, anandamide, and 2-arachidonoylglycerol. In addition, dinitrofluorobenzene treatment decreases CB1 mRNA and increases CB2 mRNA [102]. Moreover, reduced expression of cannabinoid receptors was observed in contact dermatitis [69]. At the same time, CB1 activation suppressed the expression of two damage-induced keratins, keratin 6 and keratin 16, which are highly upregulated in hyperproliferative disorder psoriasis [103]. Therefore, we measured mRNA levels of CB1/CB2. In healthy dermal fibroblasts, we identified metformin and rapamycin stimulated CB1 expression, while metformin + THC inhibited CB1 (Figure 7E). In contrast, metformin + THC, TRSV, TRSV + THC, TRSV + CBD, and rapamycin upregulated CB1 expression (Figure 7E). Furthermore, TRSV + CBD decreased CB2 expression in healthy fibroblasts while TRSV, TRSV + THC, and TRSV + CBD increased CB2 expression in senescent fibroblasts (Figure 7F). Alteration of expression of CB1 and CB2 would suggest altered activity of these receptors. As expression was increased in both receptors by TRSV, TRSV + THC, and TRSV + CBD in senescent cells, there would likely be higher receptor activity and attenuated inflammation as a result.

We believe TRSV in combination with CBD provides the best potential anti-aging therapeutic because of increased cell viability, restored wound healing functional activity, reduced metabolic dysfunction, and ameliorated nuclear parameters. Although the mechanism of how TRSV + CBD achieves this has not been elucidated, this likely occurs through the CB1 receptor. Our data shows all TRSV groups significantly altered CB1 and CB2 mRNA expression in senescent fibroblasts, but not in healthy fibroblasts. Resveratrol has affinity for CB1 acting as a neutral antagonist [104]. It is possible that overactive CB1 signaling in the senescent state could be ameliorated with either TRSV alone or TRSV + CBD, but not TRSV + THC. Indeed, we show that TRSV + THC significantly inhibits wound healing compared to either TRSV alone or TRSV + CBD and is comparable to the vehicle. Although beneficial effects of CBD may be established through the CB2 receptor, it is unlikely TRSV’s beneficial effects occur through the CB2 because of resveratrol’s fivefold selectivity for CB1 over CB2 [104]; however, this cannot be excluded. Furthermore, TRSV, which has a bioavailability of 80% compared to 29.7% for resveratrol, may also be beneficial, as metabolites of resveratrol/TRSV appear to have no affinity for CB1 [104]. Further research to validate the mechanism of action should be performed.

Since many polyphenols derived from natural sources have low solubility, low bioavailability, and poor stability [105,106], there has been a growing trend to utilize nano-formulations and emulsions to bypass the limiting pharmacokinetics of each polyphenol and increase efficacy [107,108,109]. Although TRSV has 80% bioavailability when taken orally, these technologies could still help decrease metabolism and increase the half-life of TRSV, which is only 104 min [110]. Rapamycin could similarly benefit from nano-formulations and emulsions to prevent or minimize first-pass metabolism because of the limited bioavailability of 14% despite a long half-life of 68 h [111]. In addition, metformin is not metabolized by the liver and has a bioavailability of ~50% with a half-life between 4 and 8.7 h [112]. Furthermore, TRSV and CBD could be combined with nano-formulations or/and emulsions to produce one pill that simplifies administration. Future studies should test the efficacy of emulsions and nano-formulations of TRSV and CBD in vivo.

4. Materials and Methods

5. Conclusions

In summary, NSRs can act as potential anti-aging compounds but affects skin fibroblasts differently. We found differential effects of NSRs on senescence-associated gene regulation, metabolic and functional maintenance, and cutaneous wound healing. We, for the first time, show that application of TRSV in combination with CBD constitutes a very promising anti-aging and regenerative regimen that can potentially be used for treatment or/and prevention of appearance of aging spots and treating cutaneous wounds. Moreover, we found pCBs alone appeared to be highly efficacious as an anti-aging treatment. Further work should study and test pCBs alone, as well as TRSV in combination with CBD as anti-aging remedies.

Acknowledgments

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms23158804/s1.

Funding Statement

This study was supported by NSERC—RGPIN 2016-06555 to I.K. and MITACS Accelerate IT11447 to I.K. and O.K.

Author Contributions

M.G. performed all molecular techniques and statistical analysis; G.I.R. performed all QuPath analysis and assisted with immune fluorescence and statistical analysis; I.K. and O.K. provided supervision and guidance; all authors were involved in designing the study and writing/editing the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Neither humans nor animals were utilized in this study. CCD-1064Sk was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

References