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29 January 2026

14 Pages

Triticum vulgare Extract Treatment in UVB-Exposed Human Dermal Fibroblasts Modulates Inflammation, Fibrosis and Oxidative Stress Markers

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1
Department of Clinical Medicine and Surgery, University of Naples “Federico II”, 80131 Naples, Italy
2
Unit of Dermatology, University of Campania Luigi Vanvitelli, 80131 Naples, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pharmaceuticals2026, 19(2), 232;https://doi.org/10.3390/ph19020232
This article belongs to the Special Issue Plant Extracts with Biological Activity and Potential Antioxidant Action
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Abstract

Background/Objectives: UVB radiation triggers oxidative stress, inflammation and extracellular matrix (ECM) remodeling in dermal fibroblasts, contributing to skin aging and fibrosis. Plant-derived extracts with antioxidant and anti-inflammatory activity may counteract these effects. This study evaluated the protective role of Damor Triticum vulgare Aqueous Extract (DTVE) in human dermal fibroblasts (HDFs) exposed to UVB. Methods: Primary HDFs were irradiated with UVB (1.50 J/m2) and treated with DTVE either after irradiation (post-ir) or before and after irradiation (pre-ir). Cell viability was assessed by Trypan Blue and MTT assays. Inflammatory cytokines, fibrosis-related genes, p21 expression, mitochondrial ROS (MitoSOX) and αSMA accumulation were quantified by qRT-PCR, ELISA and immunofluorescence. Results: DTVE was not cytotoxic and preserved HDF viability under UVB exposure. UVB significantly increased pro-inflammatory cytokines, profibrotic markers, αSMA, mitochondrial ROS and p21. DTVE reduced all these UVB-induced alterations, with the pre-ir regimen providing the strongest protection. The extract attenuated early inflammatory activation, limited fibroblast-to-myofibroblast transition and decreased mitochondrial oxidative stress while reducing p21 upregulation. Conclusions: DTVE exerts protective antioxidant, anti-inflammatory and antifibrotic effects in UVB-exposed fibroblasts, particularly when used as pretreatment. These findings support DTVE as a promising candidate to mitigate UVB-induced dermal damage and warrant further investigation for potential therapeutic and cosmetic applications.

1. Introduction

Chronic exposure to ultraviolet (UV) radiation, encompassing both UVA and UVB wavelengths, represents a major environmental factor contributing to cutaneous damage and skin aging. UVA rays penetrate deeply into the dermis and generate reactive oxygen species (ROS) through indirect photochemical processes, while UVB carries higher photon energy and is more directly absorbed by DNA and chromophores, causing damage at different levels [1,2]. Chronic exposure to UVA and UVB leads to aberrant production or degradation of collagen and elastin, matrix disorganization and accelerated skin aging through oxidative stress, inflammation and extracellular matrix (ECM) remodeling [3]. Fibroblasts play a central role in maintaining ECM homeostasis through the synthesis of structural proteins such as collagen I, fibronectin and the secreted protein acidic and rich in cysteine (SPARC) and can differentiate into myofibroblasts under profibrotic stimuli. Upon UVB irradiation, dermal fibroblasts undergo oxidative damage, DNA strand breaks and altered gene expression, and can secrete inflammatory cytokines and matrix metalloproteinases, rendering these cells active participants in UV-driven skin damage and recovery processes [4].
UVB irradiation induces increased ROS generation, mediating oxidative damage to DNA, proteins and lipids, and activating stress-responsive signaling pathways, eventually resulting in cell cycle arrest, apoptosis or senescence. As result, the regenerative capacity of the dermis and the ability of fibroblasts to maintain ECM integrity are negatively affected [5].
ROS generated by UVB exposure activate redox-sensitive signaling, such as NF-kB and MAPKs, and induce the release of pro-inflammatory cytokines, including IL-6, IL-1β and TNFα, further driving fibroblast activation, ECM dysregulation and myofibroblast conversion, leading to fibrosis [4].
Collectively, these mechanisms illustrate the convergence of oxidative stress, ECM degradation, inflammation and fibrogenesis in UV-induced fibroblast dysfunction.
There has been increasing interest in natural plant-derived extracts as therapeutic or preventive agents in skin protection. Specifically, natural compounds, including polyphenols, carotenoids, flavonoids and phenolic acids, have demonstrated protective effects in skin cells by scavenging ROS, inhibiting MAPK/AP-1/NF-κB pathways, suppressing cytokine release and modulating ECM degradation thanks to their antioxidant and regenerative ability, mitigating UV-induced dermal damage [6,7,8,9,10]. Among these, Damor Triticum vulgare Aqueous Extract (DTVE) stands out due to the distinctive features of its production process as a medical device and the resulting biological properties [11]. DTVE has been found to be a potent radical-scavenger and to possess the ability to attenuate oxidative stress markers in vitro [12,13]. In human dermal fibroblasts, DTVE stimulates cellular migration, cytoskeletal reorganization and fibronectin deposition, enhancing tissue repair and ECM restoration [14,15]. Moreover, DTVE exhibits anti-inflammatory activity, reducing nitric oxide, TNF-α and IL-6 production, thereby modulating the SASP response and decreasing pro-inflammatory signaling [12,16].
Despite these findings, the protective role of DTVE against UVB-induced fibroblast dysfunction remains uncharacterized.
The present study aims to investigate the effects of DTVE in human dermal fibroblasts subjected to UVB irradiation, as an in vitro model of skin aging [17,18], focusing on key features such as cell viability, mitochondrial ROS production, and early markers of inflammation and fibrotic remodeling to provide an evaluation of DTVE’s potential to mitigate UVB-induced dermal damage and preserve fibroblast functionality.

2. Results

2.1. DTVE Is Not Cytotoxic and Preserves Fibroblast Viability

The cytotoxic effects of DTVE on the HDF cells were assessed using 5%, 10% and 20% dilutions of the drug. Cell viability tests, such as the Trypan Blue dye exclusion counting and MTT assay (Figure 1A,B), showed that the compound had no significant cytotoxic effect, either under basal conditions or after exposure to UVB rays. The cells remained viable under all experimental conditions, indicating that the treatment was well tolerated. Therefore, a concentration of 20% DTVE was selected as the optimal dose for subsequent analyses, as it preserved the viability of fibroblasts while allowing for a clear assessment of biological effects. Specifically, based on the dry matter content of the extract, which is 0.6% w/v, the 20% (v/v) experimental concentration corresponds to 0.120% w/v.
Figure 1. DTVE does not alter cell viability in UVB-exposed HDFs: (A) assessment of cell viability by Trypan Blue counting in HDFs treated with different concentrations of DTVE (5%, 10%, 20%) with or without UVB exposure (n = 3); (B) MTT assay for the determination of metabolic viability under the same experimental conditions (n = 3).

2.2. DTVE Treatment Reduces UV-Driven Inflammation

After UVB irradiation, HDF cells developed an inflammatory response, with a marked increase in the transcription of the main pro-inflammatory cytokines considered as early markers of inflammation, including IL-6, TNFα and IL-1β as compared to controls (Figure 2A–C). This transcriptional activation was confirmed at the protein level by ELISA testing, which revealed an increase in the secretion of the same cytokines in the culture medium of UVB-treated cells (Figure 2D–F). Treatment with DTVE significantly attenuated the inflammatory profile and, interestingly, we observed that the anti-inflammatory effect was evident both when the compound was administered immediately after irradiation and when the cells were pre-incubated with DTVE. The latter condition proved particularly effective, limiting the onset of inflammation from the early stages of the stress response (Supplementary Tables S1 and S2).
Figure 2. DTVE reduces pro-inflammatory markers upon UVB exposure: (A–C) analysis of IL-6, IL-1β and TNF-α gene expression in non-irradiated HDFs (NO UVB), treated with DTVE (NO UVB + DTVE) and irradiated with UVB in the presence or absence of post- or pre-DTVE treatment; (D–F) ELISA assays of the same cytokines in the culture medium. n = 3. * p < 0.05; *** p < 0.001.

2.3. DTVE Modulates Fibrosis Markers upon UVB Irradiation

We then investigated whether DTVE might exert antifibrotic properties assessing the modulation of genes and proteins, referred to as early makers of fibrosis, associated with fibroblast activation and extracellular matrix remodeling. Exposure to UVB rays induced a higher expression of genes associated with fibroblast activation and extracellular matrix remodeling, as compared to controls, such as COL1A1, FN1, SPARC and ACTA2. Treatment with DTVE reversed this trend by reducing the profibrotic markers. This effect was particularly evident in pretreated cells, which showed a more balanced expression profile even after UVB exposure (Figure 3A–D). Immunofluorescence analysis provided further evidence of the antifibrotic activity of DTVE. In irradiated cells, the fibrotic marker αSMA was upregulated compared to controls, and treatment with DTVE reduced its accumulation, thus counteracting UVB-induced fibrotic remodeling (Figure 3E,F) (Supplementary Tables S1 and S3).
Figure 3. DTVE modulates fibrotic markers in UVB-exposed HDFs: (A–D) analysis of COL1A1, FN1, SPARC and ACTA2 gene expression in non-irradiated and irradiated HDFs, treated or not treated with DTVE before or after irradiation; (E,F) representative immunofluorescence images for αSMA (green) and DAPI (blue) in cells exposed to UVB and treated with DTVE. Scale bar = 50 μm. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

2.4. DTVE Attenuates Mitochondrial ROS Production Induced by UV Exposure

To investigate whether DTVE treatment might exert antioxidant effects, we evaluated mitochondrial reactive oxygen species (ROS) production as a marker of oxidative stress, and the MitoSOX probe analysis revealed that UVB irradiation led to a significant accumulation of ROS compared to non-irradiated cells. Treatment with DTVE significantly reduced ROS production, thereby protecting cells from oxidative stress (Figure 4A,B). DTVE pretreatment provided the most consistent protection, suggesting that DTVE is capable of not only counteracting but also preventing mitochondrial oxidative stress. To link the inflammatory response, the fibrosis activation and the oxidative stress, we also evaluated the expression of the p21 gene (Figure 4C), which resulted in elevation upon UVB exposure compared to controls. DTVE treatment induced a reduction of p21 mRNA levels in both treatment regimens, showing a more protective effect when pre-incubated with UVB-exposed HDFs (Supplementary Tables S1 and S3).
Figure 4. DTVE prevents mitochondrial oxidative stress: (A) representative fluorescence images and (B) analysis of mitochondrial ROS production using MitoSOX probe in HDFs treated with or without UVB and DTVE (scale bar = 50 μm); (C) gene expression of p21 (CDKN1A) in non-irradiated and irradiated HDFs, treated or not treated with DTVE before or after irradiation. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001.

3. Discussion

Our study aimed to evaluate the DTVE effects on UVB-exposed human dermal fibroblasts (HDFs), as an in vitro model of skin damage, as they display key features such as oxidative stress, early inflammatory response and extracellular matrix remodeling [17]. It is worth mentioning that although UVA radiation penetrates deeper into the dermis than UVB, the latter can cross the epidermal barrier and reach the upper dermal compartment, interacting with papillary dermal fibroblasts. It has been shown that UVB irradiation induces oxidative stress, DNA damage and senescent phenotypes in HDFs, rendering HDFs a reliable experimental model for investigating UV-induced aging [5,19,20]. Also, our experimental design included a pre-irradiation setting to assess the ability of DTVE to modulate early stress and inflammatory responses triggered by UVB, while the post-irradiation treatment allowed evaluation of DTVE capacity to attenuate already-established UVB-induced molecular alterations. These approaches were tested to assess the modulatory potential of DTVE under different exposure conditions. We showed that DTVE exerts protective effects on HDFs exposed to UVB, preserving cell survival and attenuating inflammation, oxidative stress and fibrotic markers, representing the canonical UVB-driven response. DTVE did not exert cytotoxicity when used at the working concentrations (5–20% dilution), both under basal conditions and after UVB exposure, highlighting the safety of the extract, as also reported in previous studies in different cell models [12,13,15]. The choice to use 20% as the optimal concentration allows the biological effects to be evaluated without compromising cell survival. The pro-inflammatory response of HDFs to UVB irradiation was attenuated upon cell treatment with DTVE, in line with the anti-inflammatory properties of the extract already observed in other models [16]. Notably, DTVE mostly exerted a greater effect when used as pretreatment compared to the post-ir condition, suggesting that earlier modulation of inflammatory pathways might limit inflammation, senescent phenotype activation and the progression of cellular damage. The pro-inflammatory environment is usually associated with fibrosis activation in UV-exposed skin, with structural deterioration and dysregulated matrix generation [13,21]. Indeed, we found that UVB exposure promoted the expression of fibrotic genes. DTVE treatment reversed this pattern, inducing the coordinated reduction in fibrotic markers (COL1A1, FN1, SPARC, ACTA2), pointing to a restoration of the balance between ECM deposition and degradation, particularly in the pretreatment condition, indicating that DTVE restrains fibroblast activation and ECM remodeling after UVB exposure. These results are in line with previous studies showing UV induced a pro-inflammatory environment and impairment of the matrix degradation [12]. In UV-damaged skin, pro-inflammatory cytokines such as IL-6 and IL-1β induce profibrotic programs [22]; thus, possibly, the anti-inflammatory response we observed in DTVE-treated cells led to a concomitant antifibrotic effect. The more pronounced effect in pretreated cells suggests that DTVE is able to balance this regulatory connection between inflammation and fibrosis. This aspect is further confirmed by the modulation of fibroblast-to-myofibroblast differentiation [23,24], a typical feature of HDF fibrosis. The reduction in αSMA protein accumulation upon DTVE treatment or pretreatment supports the ability of the extract to limit the fibroblast transition towards myofibroblast in UV-induced fibrosis. These results are consistent with the literature linking inflammation, through TGF-β activation, to myofibroblastic differentiation and dermal fibrosis. Also, fibrosis and inflammation are associated with ROS production in skin cells, as a response to DNA damage. UVB-induced mitochondrial ROS production was reduced by DTVE. On the other hand, DTVE markedly reduces mitochondrial ROS accumulation in pretreatment conditions. Notably, it has been demonstrated that DTVE exhibits significant radical scavenging and cytoprotective activity, showing comparable or even superior efficacy to classical antioxidants such as ascorbic acid [12]. It is worth noting that mitochondrial ROS represent mediators of UVB injury that are able to activate NF-κB/MAPK signaling, thereby increasing pro-inflammatory cytokine production in skin cells [3]. Our data support the antioxidant role of DTVE that, in turn, blunted inflammation and protected from fibrosis induced by UVB exposure. In this context, the antifibrotic effects observed upon DTVE treatment cannot be attributed to a direct modulation of fibrotic pathways alone. However, they are likely due to the concomitant attenuation of oxidative stress and inflammation. We also investigated the gene expression of p21 (CDKN1A) molecule, since it is at the crossroads between oxidative damage, inflammation and fibrosis-induced senescence [25] and represents a downstream effector of the DNA damage response and cell cycle control [26,27]. UVB irradiation upregulated p21 transcription, while DTVE reduced its expression, with a stronger effect as pretreatment. p21 is a p53-responsive effector that mediates oxidative DNA damage-induced cell cycle arrest, and if elevated it might contribute to senescence and the senescence-associated secretory phenotype (SASP), typically present in fibrosis, together with IL-6 and IL-1 promoting inflammation and matrix remodeling [28]. Therefore, the reduction in p21 observed upon DTVE treatment in HDFs supports the modulation of inflammation and profibrotic signaling in our system.
Our results align with other reports showing that plant-derived molecules such as carotenoids, flavonoids and plant antioxidants exert antioxidant properties and protect dermal fibroblasts from UVB-induced DNA damage, pro-inflammatory cytokine release and ECM remodeling [14,29].
Moreover, a comparative study, showing that DTVE was more efficient in inducing wound healing of skin lesions as compared to hyaluronic acid-based formulation [14], strongly suggests that DTVE might provide high fibroblast protection, potentially due to its dual antioxidant and anti-inflammatory effects [30]. Unlike many conventional botanical extracts, often crude, heterogeneous mixtures with variable bioactivity [31], this extract is predominantly oligosaccharide-rich. This distinguishes it from preparations whose activity is mainly attributed to proteins, growth factors or polyphenols, and suggests mechanisms extending beyond classical polyphenol-driven redox effects. Rather, its activity may relate to modulation of dermal hydration and barrier-associated processes. Although DTVE is already commercially available in formulations with regenerative activity, the present study represents an initial in vitro step aimed at exploring its potential role in UVB-induced aging-related pathways. Thus, the readouts of our in vitro work, although informative, have several limitations. The use of one commercial HDF line derived from one donor does not address donor-to-donor variability, limiting the generalizability of the findings. However, our data across different experimental readouts support the observed effects. Future studies are needed to expand these findings using fibroblasts from multiple donors. Although we focused our work on DTVE effects on dermal fibroblasts, this does not account for crosstalk between keratinocytes and fibroblasts during UVB damage. A co-culture system might be a more relevant model to study DTVE biological effects in a more complex system, and this will deserve further investigation. Another limitation is that here we did not directly measure classical markers such as cyclobutane pyrimidine dimers (CPDs) or γH2AX to assess DNA damage induced by UVB, which will be included in future investigations to further support our evidence. Also, further investigation is needed to strengthen the observed effects of DTVE treatment, linking the p21/p53, NF-κB and TGF-β pathways, at the protein level, and the oxidative damage driving inflammation, also expanding the timeframe of molecular change analysis. Also, functional senescence/fibrosis assays and mitochondrial activity are envisaged to reinforce the DTVE protective activity on UVB-exposed HDFs. Moreover, since plant extracts are not necessarily interchangeable, as differences in composition and standardization may translate into distinct efficacy and reproducibility, future studies could further benefit from the inclusion of additional comparators, such as other plant extracts or synthetic antioxidants to broaden the comparative framework. To better understand DTVE bioavailability, effective dermal concentration and possible preventive or therapeutic applications that use 3D skin models, in vivo approaches and clinical studies are envisaged.

4. Materials and Methods

4.1. Cell Cultures and Treatments

Primary human dermal fibroblasts (HDF, PCS-201-012, ATCC—University Blvd, Manassas, VA, USA), derived from one donor, was cultured in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, GIBCO), 2 mM glutamine and 1% P/S (GIBCO). Cells were cultured and expanded by incubation at 37 °C and 5% CO2 until reaching ~80% confluence. The cells were used for subsequent experiments up to passage 4.
The Damor Triticum vulgare Aqueous Extract (DTVE) was obtained from Farmaceutici Damor S.p.A. (Napoli, Italy) through a sophisticated, proprietary and globally patented standardized process of tissue vegetal extraction, designed to isolate the specific active fraction responsible for its unique biological properties. The extract originated from a controlled aqueous extraction and filtration protocol designed to selectively concentrate bioactive components from a complex plant matrix (predominantly oligosaccharide-rich composition fractions with defined molecular ranges). This approach enables the enrichment of fractions associated with antioxidant and moisturizing functions while reducing the presence of inactive or interfering constituents, thereby providing a more consistent and reproducible biological profile [12]. Cells underwent DTVE treatments (from a single batch preparation) and UV-B irradiation according to the following scheme: DTVE post-ir, in which HDF were irradiated and immediately incubated with DTVE for 24 h; DTVE pre-ir, in which HDF were pre-incubated with DTVE for 2 h, then irradiated and re-incubated with the compound for 24 h. Control groups included untreated cells (NO UVB) and untreated cells + DTVE (NO UVB + DTVE).
Irradiation dosage was set at 1.50 J/m2, established by preliminary observation in HDF cells exposed to a UVB range from 0.200 to 2.00 J/m2. The selected UVB dosage (substantially lower as compared to UVB dosing in patients defined at the skin surface and influenced by barrier-dependent attenuation) was used to avoid overt cytotoxicity in order to evaluate molecular changes in the subsequent experiments. UVB irradiation using a Philips TL/01 lamp. During irradiation, the medium was replaced with PBS 1X (GIBCO) to prevent absorption and subsequently restored after treatment. After setting the UVB dosage, different concentrations of DTVE were tested at 5%, 10% and 20% of a 100% pharmaceutical formulation (dissolved in water), based on previous reports [32,33]. For the cell viability assay, cells in control conditions (CTR) were treated with the same amount of water used for 20% DTVE treatments, while for all the other experiments control conditions such as NO UVB and UVB were treated with the same amount of water used for DTVE treatments.

4.2. Cell Viability Assay

The viability of HDF cells was assessed in the presence and absence of different doses of UVB and DTVE. A total of 2 × 104 cells were plated in 24-well plates in complete DMEM; after 16 h, the cells were treated for 24 h, as described in the previous section and counted using the Trypan blue exclusion method to assess cytotoxicity. Specifically, after trypsinization and washing in phosphate-buffered saline (PBS), the cells were resuspended in diluted Trypan blue and then immediately counted in a Bürker chamber. Viable (unstained) and non-viable (stained) cells were counted separately.
Cell viability under the conditions described above was also assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay (Sigma-Aldrich, St. Louis, MO, USA). This assay measures the activity of enzymes that reduce MTT to formazan, producing blue/purple crystals that precipitate within the cells. This reduction occurs mainly in mitochondria through the action of succinate dehydrogenase, an enzyme active in vital and metabolically active cells. The enzyme breaks down the tetrazolium ring of MTT (a yellow substance), resulting in the formation of formazan (a blue salt). Specifically, 1 × 104 cells were seeded in 96-well plates and incubated for 24 h in complete DMEM; after 24 h, the cells were treated for 24 h, as described above. Subsequently, the cells were treated with an MTT solution (0.5 mg/mL) for 2 h at 37 °C. After incubation, the formazan salts were dissolved in a DMSO/isopropanol solution (1:1, v/v) and the reaction product was quantified by spectrophotometric measurement at a wavelength of 595 nm.

4.3. Gene Expression Analysis (qRT-PCR)

HDF cells were cultured to semi-confluence in 12-well plates in complete medium and treated as described above. Total RNA was extracted using the PureZOL™ Reagent (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed using the All-In-One 5X RT MasterMix with gDNA Removal (Applied Biological Materials Inc., Richmond, BC, Canada). qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and gene-specific primers as reported in Table 1. The amplification was performed using the CFX Connect Real- Time PCR Detection System (Bio-Rad) with the following cycling conditions: cDNA denaturation and polymerase activation step at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 30 s; melting curve analysis step at 65 °C to 95 °C with 0.5 °C increment for 5 s/step. Gene expression analysis of target genes was compared with the 18s housekeeping reference gene [10,34] following the comparative 2−ΔCt method and the expression was reported as Arbitrary Units (A.U.).
Table 1. The forward and reverse primers of the genes.

4.4. Cytokine Detection in Culture Media

HDF cells were cultured to semi-confluence in 12-well plates in complete DMEM and after 16 h were treated as described above according to the experimental conditions. After 24 h of irradiation and treatments, the supernatants were collected. ELISA tests were performed to detect IL-6, IL-1β and TNFα using commercial kits, following the manufacturer’s instructions (Elabscience®, Wuhan, China). Optical densities (OD) at 450 nm were measured using the iMark™ microplate absorption reader (Bio-Rad, Hercules, CA, USA), and concentrations were determined by interpolation from the standard curve using non-linear regression (four-parameter logistic, 4PL) in GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

4.5. Fluorescence Microscopy Analysis

For immunofluorescence experiments, HDF cells (2 × 104) were cultured on glass coverslips in complete DMEM for 16 h and then irradiated and treated following the previously described scheme. On the following day, cells were then fixed in 4% PFA for 15 min at room temperature, permeabilized with 0.2% Triton™ X-100 (Sigma-Aldrich) in PBS, and blocked with PBS 5% FBS for 1 h at room temperature. The primary antibody anti-αSMA (Cell Signaling Technologies, Danvers, MA, USA) was diluted 1:250 in blocking buffer and incubated for 2 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Invitrogen Thermo Fisher Scientific, Carlsbad, CA, USA) for 45 min at room temperature. Nuclei were stained with DAPI 1 µg/mL (Sigma Aldrich).
For the reactive oxygen species (ROS) analysis, mitochondrial ROS were measured using the MitoSOX Red probe (1 µM; Invitrogen, Molecular Probes™, Thermo Fisher Scientific, Waltham, MA, USA), a specific indicator of mitochondrial superoxide (excitation/emission 510/580 nm). Briefly, HDF cells were cultured on glass coverslips in complete DMEM. On the following day, cells were treated as described above, probed with MitoSOX red for 20 min, fixed in 4.0% PFA for 15 min at room temperature. The nuclei were stained with 1 µg/mL DAPI (Sigma Aldrich, St. Louis, MO, USA). Images were acquired using a LEICA DMi8 fluorescence microscope equipped with Leica Application Suite LAS X Imaging software (v. 3.6.0.20104). ImageJ software (v. 2.3.0/1.53f51) was used to analyze fluorescence intensity, and data were represented as corrected total cell fluorescence (CTCF) calculated as integrated density—(Area of selected cell X Mean fluorescence of background readings).

4.6. Statistical Analysis

All data were expressed as mean ± standard error of the mean (SEM) from at least three independent experimental replicates (n = 3), each performed in technical triplicate. Statistical analysis was conducted using one-way or two-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons of more than two groups (GraphPad Prism 6.0; GraphPad Software, Inc., La Jolla, CA, USA). p values  <  0.05 were considered as statistically significant (* p < 0.05, ** p < 0 .01, *** p <  0.001, **** p < 0.0001).

5. Conclusions

Taken together, our data showed that DTVE is not cytotoxic and exerted a protective effect on HDFs by reducing inflammation, fibrosis marker and oxidative stress when applied after or, to a greater extent, before UVB irradiation. This indicates that the prevention of oxidative stress and early inflammation is a key mechanism of protection mediated by DTVE, representing a promising candidate to limit fibrosis in dermal fibroblasts and underscoring the importance of a preventive approach in protecting the skin from UV damage. These results pave the way for further in vitro and in vivo studies to evaluate the clinical efficacy and possible cosmetic or therapeutic application of the extract.

6. Patents

The results from this work will be part of a patenting process owned by Farmaceutici Damor S.p.A. (Napoli, Italy).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph19020232/s1, Table S1: Statistical analysis related to gene expression analysis showed in Figure 2A–C, Figure 3A–D and Figure 4C; Table S2: Statistical analysis related to inflammatory cytokine detection showed in Figure 2D–F; Table S3: Statistical analysis related to immunofluorescence evaluation showed in Figure 3F and Figure 4B.

Author Contributions

Conceptualization, A.B.; methodology, C.M. and C.S.; validation, A.B. and C.M.; formal analysis, S.C., E.V.D.B., M.A. and M.N.; investigation, C.S. and C.M.; resources, G.A.; data curation, A.B. and C.M.; writing—original draft preparation, C.S. and C.M.; writing—review and editing, A.B., C.M., G.A., C.S., S.C., E.V.D.B., M.A. and M.N.; supervision, A.B.; project administration, G.A.; funding acquisition, G.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Farmaceutici Damor S.p.A. This work was conducted as part of the Specific MIMIT-Funded Project, P.O.T.E.R.I.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
ACTA2Actin Alpha 2, Smooth Muscle
AP-1Activator Protein-1
COL1A1Collagen Type I Alpha 1 Chain
CTCFCorrected Total Cell Fluorescence
CO2Carbon Dioxide
cDNAComplementary DNA
DMEMDulbecco’s Modified Eagle Medium
DMSODimethyl Sulfoxide
DTVEDamor Triticum vulgare Aqueous Extract
DNADeoxyribonucleic Acid
ECMExtracellular Matrix
ELISAEnzyme-Linked Immunosorbent Assay
FBSFetal Bovine Serum
FN1Fibronectin 1
HDF/HDFsHuman Dermal Fibroblasts
IL-1βInterleukin-1 Beta
IL-6Interleukin-6
IRIrradiation
IFImmunofluorescence
MAPKsMitogen-Activated Protein Kinases
MitoSOXMitochondrial Superoxide Indicator
mRNAMessenger RNA
MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
µm/µL/µgMicrometer/Microliter/Microgram
NF-κBNuclear Factor kappa-B
NO UVBNon-Irradiated, No UVB
ODOptical Density
PBSPhosphate-Buffered Saline
PFAParaformaldehyde
P/SPenicillin–Streptomycin
p21 (CDKN1A)Cyclin-Dependent Kinase Inhibitor 1A
PCRPolymerase Chain Reaction
qRT-PCRQuantitative Reverse Transcription PCR
RNARibonucleic Acid
ROSReactive Oxygen Species
SEMStandard Error of the Mean
SPARCSecreted Protein Acidic and Rich in Cysteine
SASPSenescence-Associated Secretory Phenotype
SYBRCyanine-Based Fluorescent Dye Used in PCR
TGF-βTransforming Growth Factor Beta
TL/01Philips TL/01 UVB lamp
TLAThree-Letter Acronym
TNFαTumor Necrosis Factor Alpha
Triton X-100Non-Ionic surfactant (permeabilizing agent)
UV/UVA/UVBUltraviolet/Ultraviolet A/Ultraviolet B

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