Skin ageing is a multifaceted biological process influenced by both intrinsic factors like genetic programming (chronological ageing) and extrinsic factors such as environmental pollution, UV radiation, and lifestyle habits (biological ageing). These factors contribute to cellular damage, oxidative stress, and inflammation, leading to premature skin ageing. The dermis, the main layer associated with skin ageing, relies heavily on fibroblasts, which are the primary producers of extracellular matrix (ECM) components like collagen and elastic fibres, essential for skin elasticity, strength, and resilience. With age, fibroblast function declines, they can become senescent, contributing to reduced collagen synthesis, matrix degradation, and visible signs of ageing such as wrinkles and loss of firmness.
Traditional anti-ageing therapies often utilize well-known molecules like retinoids, which can promote fibroblast activity, increase collagen production, and reduce wrinkles. However, retinoids are frequently associated with significant side effects, including topical irritation like dryness, itching, redness, and peeling, as well as potential systemic issues. This has led the cosmetics industry to actively seek safer and more biocompatible alternatives for addressing skin ageing.
A modern trend in cosmetology involves the use of naturally derived bioactive molecules, particularly polyphenols, known for their anti-inflammatory, antioxidant, and ECM-preserving properties. Recent advancements highlight the potential of whole plant in vitro cultures and plant-derived extracellular vesicles (EVs) as sustainable sources of such bioactives. While plant cell cultures have been incorporated into cosmetic products, the potential of EVs derived specifically from in vitro plant cultures remains largely unexplored. EVs are nano-sized, lipid bilayer-enclosed structures involved in cellular communication and various biological processes. They are highly tolerated, easily absorbed by cells, and have shown therapeutic potential in improving skin health. This study investigates the impact of extracts derived from Haberlea rhodopensis in vitro culture, including those enriched with EVs, on human dermal fibroblasts as a potential novel and safer approach to combat skin ageing. H. rhodopensis is a well-known resurrection plant endemic to the Balkan Peninsula, recognized for its resilience and diverse metabolic profile containing beneficial molecules like phenolic acids and flavonoids, along with demonstrated antioxidant and ECM-stimulating properties.
Methods
This study utilized human dermal fibroblasts (BJ cell line) to investigate the effects of Haberlea rhodopensis in vitro culture extracts and isolated extracellular vesicles (EVs). Two types of extracts (Extract 1, Extract 2) and an EV fraction were prepared and tested at 2% and 10% concentrations for 24 and 72 hours. The effects on cell morphology, mitochondrial dynamics, lysosomal activity, actin cytoskeleton organization, cell cycle progression, and genotoxicity were assessed using techniques including phase-contrast and fluorescence microscopy, FACS, and the neutral comet assay. Gene expression analysis for key skin ageing markers (COL1A1, COL3A1, CDKN1A, NFE2L2) was performed using RT-qPCR.
Key Findings
•Haberlea rhodopensis in vitro culture extracts (Extract 1, Extract 2) and EVs were biocompatible with BJ fibroblasts, maintaining typical elongated morphology similar to untreated cells at both 2% and 10% concentrations after 24 and 72 hours. No significant signs of stress or cellular damage were observed.
•The tested compounds enhanced mitochondrial mass and integrity. At 24 hours, 10% Extract 1 showed a pronounced 2.27-fold increase, while at 72 hours, 10% EVs resulted in a substantial 1.46-fold enhancement, suggesting stimulation of mitochondrial biogenesis or activity.
•The compounds influenced lysosomal mass and function. Higher concentrations of Extract 1 consistently promoted lysosomal activity, while EVs showed a stabilizing effect and a trend towards improved function, particularly at higher concentrations.
•Gene expression analysis showed that treatments upregulated genes associated with collagen synthesis, including COL1A1 and COL3A1, particularly after 72 hours, indicating stimulation of ECM production.
•NFE2L2, a key gene in the antioxidant response, was consistently upregulated across all treatments, supporting the potential of these compounds to protect fibroblasts against oxidative stress.
•Genotoxicity assessment using the neutral comet assay indicated minimal genotoxic effects. While some variations in DNA damage were observed, the overall findings suggest biocompatibility and do not appear to compromise genomic stability.
•Cell cycle analysis showed that while lower concentrations might support progression, prolonged exposure to higher concentrations (especially Extract 1) could induce a protective cell cycle arrest.
This research presents a novel exploration into the cosmetic potential of Haberlea rhodopensis derived from in vitro culture, specifically highlighting the benefits of both the culture extracts and, uniquely, the extracellular vesicles (EVs) isolated from the conditioned medium. The study successfully demonstrates that these compounds positively influence crucial cellular processes in human dermal fibroblasts relevant to skin ageing.
The findings indicate that H. rhodopensis in vitro culture extracts and EVs preserve fibroblast morphology, enhance mitochondrial mass, support lysosomal activity, improve cellular responses to oxidative stress, and significantly upregulate genes associated with collagen synthesis. These effects were shown to be concentration-dependent, suggesting that optimal concentrations may be needed to maximize benefits. Crucially, the compounds exhibited biocompatibility with minimal genotoxic effects, indicating a favourable safety profile for potential application in skin health formulations.
Utilizing advanced techniques, the study validates the presence and beneficial effects of both the extracts and EVs. The results position Haberlea rhodopensis in vitro culture extracts and EVs as promising candidates for the development of cosmetic and dermatological products targeting skin ageing, owing to their demonstrated regenerative and protective properties. Future implications involve further elucidating the precise molecular mechanisms responsible for these effects and optimizing formulation strategies to pave the way for clinical translation.
Link to study: https://www.mdpi.com/2079-9284/12/3/90
