Snail slime (SS), a secretion produced by Helix aspersa, is a complex biological matrix rich in macromolecules has gained considerable interest due to its biologically relevant components and potential applications in medicine, cosmetics, and biotechnology. This study focuses on the chemical characterization of SS, comparing stabilized commercial slime with preservatives to a non-stabilized natural, preservative-free variant. Advanced analytical techniques, such as Attenuated Total Reflection Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, Solid-Phase Microextraction Gas Chromatography-Mass Spectrometry, Raman spectroscopy, and Thermal analysis were employed to identify key metabolites and bioactive compounds. Moreover, quantitative assays were performed to evaluate the antioxidant, metal chelation and enzyme inhibition activities. Analytical techniques identified mucins, glycosaminoglycans, antimicrobial peptides, and antioxidants, with variations in composition influenced by processing methods. Quantitative assays revealed that SS possesses strong antioxidant properties, significant metal chelation capacity, and inhibitory activity against cholinesterases, tyrosinase, amylase, and glucosidase. Cellular assays further demonstrated its non-toxic nature and capacity to enhance dermal fibroblast viability. Furthermore, the stabilized commercially used SS has better composition, stability and activities compared to non-stabilized SS. Additionally, this is the first direct comparison of stabilized and non-stabilized SS, using multimethod analytical approach, and correlation of chemical composition with bioactivity. These findings underscore SS's complex composition and potential in biomedical and cosmetic applications, particularly in wound healing, antimicrobial, antioxidants, anti-aging formulations, and enzyme inhibitory therapies.

Gastric cancer (GC) remains a major global health challenge, ranking among the most lethal malignancies due to late diagnosis, high tumor heterogeneity, and limited treatment efficacy. The search for safer, nutritionally based adjuncts to conventional therapies is therefore a research priority. Donkey milk (DM), traditionally used as a hypoallergenic substitute for infants, is emerging as a functional food with remarkable bioactivity. Its composition closely resembles human milk, with high levels of bioactive proteins, a favorable polyunsaturated lipid profile, antioxidant vitamins, and immune-supportive minerals. Despite its growing nutraceutical appeal, the anticancer potential of DM in GC has not yet been explored. This study represents the first investigation of DM in human gastric adenocarcinoma (AGS) cells. Using increasing concentrations of whole DM (25-100%), a dose-dependent inhibition of cell viability and migration was observed. Mechanistic insights reveal that DM induces mitochondrial oxidative stress, disrupts cell cycle progression (S/G2 accumulation at 75%, G2 arrest at 100%), and unexpectedly triggers a pro-inflammatory gene signature suggesting stress-driven immunostimulation rather than canonical apoptosis. These findings highlight a non-classical, context-dependent cytotoxic mechanism that distinguishes DM from conventional pro-apoptotic agents. DM may represent a promising nutraceutical candidate for GC management, bridging traditional food resources with modern oncology. By inhibiting hallmark cancer traits while engaging unique immunological pathways, DM offers a sustainable, low-toxicity approach with translational potential. Future studies will focus on the characterization of active components, validation in organoid and animal models, and exploring clinical applications of DM-derived bioactive components in cancer prevention and therapy.

Human carbonic anhydrases (hCAs) IX and XII have emerged as promising therapeutic targets and are overexpressed in hypoxic tumors. Leveraging the chemotype of umbelliferon (UMB), as a selective hCAs IX and XII inhibitor, we designed and synthesized several hybrids (7-33) connecting UMB natural scaffold with vanilloids by using methylene spacers or triazole linkers. These hybrids demonstrated nanomolar inhibitory activity against the tumor-associated hCAs IX and XII. Molecular modeling and dynamics simulations revealed stable hydrogen bonding and hydrophobic interactions. In vitro evaluation of human bronchial epithelial (BEAS-2B) and lung adenocarcinoma (A549) cell lines showed selective cytotoxicity against cancer cells. Selected compounds induced G1 cell cycle arrest, reduced expression of the metastasis-associated markers CD9 and epithelial cell adhesion molecule, and exhibited cytoprotective and anti-inflammatory effects in BEAS-2B cells. Collectively, these findings identify UMB-vanilloid hybrids as promising candidates for the development of novel therapeutics for nonsmall cell lung cancer.

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The dysregulation of inducible nitric oxide synthase (iNOS) is linked to various diseases, including psoriasis, where it contributes to imbalanced nitro-oxidative stress. iNOS is primarily produced in the skin's epidermal layer and can be activated by cytokines elevated in psoriatic lesions, such as TNF-α and IL-17. Macrophages also play a role in psoriasis by producing cytokines and iNOS, and patients show increased levels of these immune cells in lesions. Given the association of iNOS with psoriasis severity, it is seen as a potential therapeutic target. However, no specific iNOS inhibitor has been reported as a treatment for psoriasis. The study describes the synthesis of new compounds based on prior iNOS inhibitors, their potency and selectivity of action, and the evaluation of the most interesting compounds in different in vitro and ex vivo cell models of psoriasis. Moreover, a computational analysis was performed that sheds light on the binding mode of the most promising molecule into both the iNOS and the constitutive endothelial NOS (eNOS). Compound 10 demonstrated significant effectiveness with respect to known iNOS inhibitors, reducing nitric oxide release, cytokine-induced inflammation, and cell necrosis, also shifting macrophages from a pro-inflammatory to a resolving phenotype. Its reasonable metabolic stability, along with the absence of significant in vivo toxicity, supports its further evaluation as a promising candidate for antipsoriatic drug development.

Alveolar bone resorption after tooth extraction complicates subsequent dental implant placement. OsteoBiol® GTO® (Tecnoss®, Giaveno, Italy) is an innovative pre-hydrated heterologous collagenated bone mix blended with a thermosensitive copolymer (OsteoBiol® TSV Gel) that has demonstrated osteoconductive properties. Despite direct contact with blood vessels upon socket filling, its pro-angiogenic potential has not been directly investigated on endothelial cells. Thus, in this study, an in vitro model, consisting of the EA.hy926 endothelial cell line exposed to different OsteoBiol® GTO® soaking preparations [original soaking (OS), centrifuged soaking (CS), and diluted soaking (DS)] at multiple concentrations (1, 5, 10, and 20 mg/mL) was established to identify optimal experimental conditions and characterize the underlying molecular mechanisms. Cell viability and collagen release quantification led to the selection of 10 mg/mL OS as the most suitable condition. Under this condition, OsteoBiol® GTO® induces an early increase in Cyclooxygenase-2 (COX-2) protein expression and Prostaglandin E-2 (PGE2) secretion, followed by upregulation of Vascular Endothelial Growth Factor (VEGF) protein expression and phosphorylation of Endothelial Nitric Oxide Synthase (eNOS) at serine 1177. The tube formation assay confirmed the pro-angiogenic functional outcome. These results suggest an association between the pro-angiogenic response of endothelial cells to OsteoBiol® GTO® and modulation of the COX-2/PGE2/VEGF axis. This effect could be attributed to collagen accumulation in the OS, thereby representing a novel and promising pro-angiogenic mechanism with potential implications for wound healing and guided bone regeneration following tooth extraction.

Dental inflammatory diseases remain a challenging clinical issue, whose causes and development are still not fully understood. During dental caries, bacteria penetrate the tooth pulp, causing pulpitis. To prevent pulp necrosis, it is crucial to promote tissue repair by recruiting immune cells, such as macrophages, able to secrete signal molecules for the pulp microenvironment and thus to recruit dental pulp stem cells (DPSCs) in the damaged site. To date, root canal therapy is the standard for dental caries, but alternative regenerative treatments are gaining attention. Complex Multifrequency Magnetoelectric Fields (CMFs) represent an interesting tool due to their potential anti-inflammatory activity. Against this background, the present work aims at investigating whether the CMF treatment might restore redox balance in a co-culture model of DPSCs and inflamed macrophages mimicking an inflammatory condition, like pulpitis. Results show that superoxide anion levels and markers related to the polarization of macrophages are modulated by the CMF treatment. In parallel, the use of CMFs discloses an impact on the odontogenic commitment of DPSCs, their immunophenotype being considerably modified. In conclusion, CMFs, by modulating the odontogenic commitment and the anti-inflammatory response of DPSCs, might represent a suitable therapeutic tool against pulpitis and, in general, towards dental inflammatory diseases.

Managing bone defects is challenging, with autologous grafts being the most effective treatment. Biomaterials like alginate/hydroxyapatite (Alg/Hap) composites are increasingly used due to their biocompatibility and osteoconductive properties. Graft implantation in the oral cavity may trigger inflammatory responses, such as periodontitis, pulpitis, or caries, due to biomaterial recognition as non-self and the presence of pathogens like Streptococcus mutans. Conjugating Alg/Hap composites with antimicrobial silver nanoparticles (nAg) offers a strategy to counteract oral inflammation caused by microbial biofilms. This study explores the anti-inflammatory and antibiofilm activities of these biomaterials during early implantation (24-72 h), as well as DPSC viability and collagen expression. A co-culture model of dental pulp stem cells (DPSCs) and Streptococcus mutans UA 159 strains was established. Streptococcus mutans viability and biofilm formation on scaffolds were evaluated through the live/dead assay and confocal microscopy. Lactate dehydrogenase (LDH), interleukin-6 (IL-6), and collagen type 1 from DPSCs were measured via ELISA assays. Nrf2 and COX-2 protein expression was evaluated by western blotting. Alg/Hap/nAg composites reduce S. mutans-derived biofilm formation, preserving biocompatibility toward DPSCs. Decreased IL-6 levels, restored collagen type 1 secretion (5.98 pg/mL in DPSCs-MOI 0.1-Alg/Hap/Ag vs. 3.04 pg/mL in DPSCs-Alg/Hap/Ag at 72 h), and modulation of antioxidant and inflammatory proteins were observed, including a two-fold increase of Nrf2 expression in cells seeded onto scaffolds in the presence of nAg. These findings highlight the potential of smart biomaterials to promote DPSC osteogenic and odontogenic differentiation, advancing oral tissue regeneration strategies.

PURPOSE:To investigate the interplay between inflammation and differentiation upon implantation, dental pulp stem cells (DPSCs) were cultured on 3D-printed titanium owning an internal open cell form, administering osteogenic factors by a liposomal formulation (LipoMix) compared to traditional delivery of differentiation medium (DM). MATERIALS AND METHODS:Osteogenic differentiation was evaluated via western blot by measuring β1 integrin expression and real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as measuring SP7 and type 1 collagen gene expression. In addition, angiogenesis was characterized by measuring vascular endothelial growth factor (VEGF) secretion levels. Matrix mineralization was assessed by means of Alizarin red staining, cell adhesion, and inflammation responses through western blot, enzymatic, and enzyme-linked immunosorbent assays (ELISA) that evaluated Nrf2 expression, catalase activity, and prostaglandin E2 (PGE2) secretion, respectively. RESULTS:LipoMix enhances cell proliferation and adhesion, as revealed by increased β1 integrin expression. Mineralized matrix deposition, SP7 gene expression, type 1 collagen release, and alkaline phosphatase activity appeared to increase in the LipoMix condition. Additionally, the redox-sensitive transcription factor Nrf2 was overexpressed at the earliest experimental times, triggering the catalase activity. CONCLUSIONS:The data reported confirmed that internal topography and post-production treatments on Ti surfaces dynamically and positively conditioned the DPSC progress toward the osteogenic phenotype; moreover, the combination with LipoMix quickened the positive modulation of inflammation under osteogenic conditions. Therefore, the development of customized surfaces along with the administration of differentiating factors enclosed in a liposomal delivery system could represent a promising and innovative tool in regenerative dentistry.

Limosilactobacillus reuteri (formerly Lactobacillus reuteri) is a probiotic bacterium involved in maintaining gut microbiota balance and modulating immune response. In this study, for the first time, we report the recombinant production and kinetic characterization of its γ-class carbonic anhydrase (CA, EC 4.2.1.1), referred to as LreCAγ. The enzyme catalyzes CO2 hydration with an efficiency comparable to that of other bacterial γ-CAs, although lower than that of α-hCA II. X-ray crystallization studies shed light on the enzyme structure, and inhibition studies with anions, sulfonamides, and related compounds revealed that LreCAγ is less susceptible to inhibition compared to the γ-class CA from Vibrio cholerae, used for comparison. Otherwise, activation assays with selected amines and amino acids identified the two enantiomers of His (25 and 26) as the most potent LreCAγ activators. Stereochemistry had a minimal impact on activity, except for L-Phe (27), which was twice as potent as its d-enantiomer (28). To assess the biological effects of CA modulation, E. coli DH5α, which expresses several CAs, was used as a model organism. CA activators were tested alone and in combination with the pan-CA inhibitor acetazolamide (AAZ), revealing CA-dependent effects on bacterial growth. Additionally, selected CA activators were evaluated for their effects on human macrophages and intestinal epithelial cells, with L-Trp (31) attenuating LPS-induced activation and exhibiting good biocompatibility in normal intestinal cells. Taken together, these results underscore the feasibility of targeting LreCAγ activation as a strategy to enhance probiotic efficacy.

Endothelial function plays a key role in tissue repair. Reactive Oxygen Species (ROS) production impairs tissue renewal and homeostasis. Complex Magnetic Fields (CMFs) have been attracting attention as a non-invasive tool to promote tissue regeneration, especially through angiogenic stimulation. The present study aims to investigate CMF effect in an in vitro model of oxidative stress-stimulated Endothelial Cells (ECs). Cells were pre-treated with H2O2 to mimic an oxidative environment, followed by the application of three CMF programs repeated in two experimental sets: two consecutive cycles (two cycles) or two cycles spaced 24 h apart (T0+T24). Flow cytometry investigation shows that both CMF applications reduce ROS production, presumably promoting SODs proteins expression. Specifically, two cycles affect mitochondrial SOD-2 expression, which may promote cellular turnover by upregulating pro-apoptotic proteins, leading to mild cell death balanced with increased cell viability. T0+T24 application promotes cytosolic SOD-1 expression, which may influence the expression and release of antioxidant molecules, as evidenced by the increased protein levels of Akt/Nrf2 and the overall antioxidant activity measured post-treatment. In conclusion, ROS-induced EC dysfunction can be reverted by CMF application: 2 cycles could be applied when cellular renewal is required (such as in pathological wounds) while T0+T24 could be useful when an antioxidant and anti-inflammatory effect is needed (e.g., in edema or muscular lesions).

This study explores whether hyaluronic acid (HA) of different molecular weights and collagen, given their role in tendon extracellular matrix maintenance, have a synergistic effect on human tendon-derived cells, with the aim to improve the treatment of tendinopathy. Human monocytes (CRL-9855™) and primary Achilles tendon-derived cells. The collagen/HA ratio was based on the formulation of the commercial food supplement TendoGenIAL™. The anti-inflammatory activity was evaluated on human lipopolysaccharide (LPS)-stimulated macrophages (24 h) or human Achilles tendon-derived cells under basal and pro-inflammatory conditions (24 and 72 h) and on a co-culture model of tenocytes and inflamed macrophages (24 h). These data show that the combination of high molecular weight hyaluronic acid and collagen reduces inflammation in macrophages by downregulating CD14, with a mean fluorescence intensity (MFI) of 36,274 (± 1780) (p < 0.0001). Additionally, this combination enhances the modulation of extracellular matrix remodeling proteins, such as CD44 whose MFI is 675,435 (± 28109) (p < 0.00001) and collagen type I at 17.838 µg/mL (± 6,259) (p < 0.00001). The observation of the ultrastructure of tenocytes through electron microscopy reveals increased cell membrane vesiculation and cell size, indicating that tenocytes start to proliferate and create abundant extracellular matrix proteins, important for the matrix remodeling. Our data confirm the anti-inflammatory activity of both hyaluronic acids and collagen, highlighting a synergistic effect of their combination in a determined ratio.

BACKGROUND:Snail slime (SS), a complex biological substance produced by various snail species, has garnered significant attention in recent years due to its diverse applications in health, cosmetics, and biotechnology. AIMS:Our previous review focused on the biological activities of SS, while the current one explores the science behind SS with a special focus on environmental factors affecting its quality and quantity, non-lethal extraction methods, its composition, current applications in health and cosmetics followed by its emerging applications, and future prospects while achieving sustainability. METHODS:A literature review on background, uses in health and cosmetics, and future prospects of SS was conducted. PubMed and Google Scholar were used to find the key articles exploring SS and the data is summarized and described here. RESULTS:Extraction methods range from traditional farming practices to advanced, non-invasive techniques aimed at minimizing stress on snails. Emerging applications include potential use in sustained and targeted drug delivery systems, tissue engineering, and as components in advanced biomaterials. Future perspectives involve technological advancements in production, such as precision farming and biotechnology-enhanced mucin production. The development of synthetic alternatives and sustainable practices is crucial for the industry's long-term viability. CONCLUSION:As research continues to uncover new properties and applications, SS is poised to play an increasingly important role in the health and beauty industries, highlighting the need for balanced exploitation, standardization, quality control, and regulatory compliance in its production and use.

Snail slime (SS) is a natural secretion rich in bioactive components such as glycoproteins, hyaluronic acid, glycolic acid (GA), and antimicrobial peptides. GA, a key component of SS, is known for its exfoliative properties. This study investigates SS’s effects on keratinocytes (HaCaT) and endothelial cells (ECs), comparing its properties to those of GA. HaCaT cell viability and cytotoxicity, ROS release, and inflammation-related signaling (PI3K/Akt/NF-κB and COX-2 gene expression) were assessed. Extracellular matrix (ECM) remodeling was evaluated by gene expression of MMPs. In ECs, a preliminary evaluation of SS’s effect was conducted in terms of cell viability and migration. Results demonstrated that SS is well tolerated by keratinocytes whereas GA exhibits cytotoxicity, suggesting that SS’s natural composition mitigates GA’s adverse effects. SS induced a controlled, brief inflammatory response, via the PI3K/Akt/NF-κB pathway, unlike GA, responsible for stronger and sustained pro-inflammatory events. Additionally, SS, through the upregulation of MMPs, contributes to ECM remodeling. In ECs, SS preserves viability and also enhances migration, thus supporting wound healing. These findings highlight SS’s ability to balance pro-inflammatory events, making it a promising candidate for advanced dermatological applications, underscoring SS’s potential in modulating key cellular signaling pathways, and supporting its future therapeutic prospects in wound healing.

Objectives: Bone defects, resulting from many causes, represent a challenge in maxillofacial and orthopedic surgery. Regenerative medicine offers promising strategies by introducing exogenous materials to modify the tissue environment and modulate the body’s natural healing mechanisms. Dental pulp stem cells (DPSCs) are considered an effective source for tissue repair. Small molecules such as caffeic acid phenethyl ester (CAPE), although having promising effects in promoting bone regeneration, are characterized by low chemical stability, which impairs their clinical application. This study aimed to investigate the bone regenerative capability of four CAPE derivatives, recently synthesized in our laboratory and selected based on previous studies. Methods: DPSCs were induced to osteogenic differentiation in the presence of these compounds (0–5 μM), and cell viability, matrix deposition, alkaline phosphatase activity, and osteogenic marker gene expression were evaluated. In addition, bone biomaterials composed of a chitosan/agarose matrix reinforced with nanohydroxyapatite and enriched with these CAPE derivatives were fabricated and assessed for cytotoxicity and cell adhesion. Results: Two of the tested compounds effectively enhanced DPSC differentiation toward the osteogenic lineage. The fabricated bone biomaterials showed no cytotoxicity and supported cell adhesion. Furthermore, these compounds demonstrated stability under various conditions, confirming their suitability for incorporation into bone biomaterials. Conclusions: The tested CAPE derivatives exhibit promising osteoinductive properties and stability, offering a valid alternative to traditional therapeutic strategies in regenerative medicine.

The vascular system is primarily responsible for orchestrating the underlying healing processes to achieve tissue regeneration, thus the promotion of angiogenic events could be a useful strategy to repair injured tissues. Among several approaches to stimulate tissue regeneration, non-invasive devices are currently widely diffused. Complex Magnetic Fields (CMFs) are innovative pulsed multifrequency electromagnetic fields used for their promising results in clinical applications, such as diabetic foot treatment or edema resorption. Nevertheless, few papers are available demonstrating the biological mechanisms involved. In this paper, in order to understand CMFs’ capability to promote angiogenic events, Regenerative Tissue Program (RTP) was applied to an in vitro Endothelial Cells (ECs) model. ECs were stimulated with (I) 2 RTP consecutive cycles, (II) with an interval of 8 h (T0 + T8), or (III) 24 h (T0 + T24) from one cycle to another. Results demonstrate that (I) extracellular matrix degradation is promoted through matrix metalloproteinases 2 and 9 modulation, leading to an increased cell migratory capability; (II) CMFs support EC growth, activating Integrin β1-Erk-Cdk2 pathway and sustaining G1/S transition; (III) vessel morphogenesis is promoted when CMFs are applied. In conclusion, the promising clinical results are supported by in vitro analyses which evidence that main angiogenic events are stimulated by CMFs.

Gliomas are aggressive brain tumors with limited treatment options, often leading to poor patient outcomes despite surgery, radiation, and chemotherapy. Current therapies, such as temozolomide and radiation, provide only temporary control, as gliomas frequently develop resistance. Therefore, there is an urgent need for new therapeutics to improve survival and quality of life for patients. In the present study, we explore the hypothesis that the dual inhibition of both the neuronal and inducible nitric oxide synthases could represent a promising therapeutic approach, being these two enzymes often dysregulated in gliomas. To this end, the new quinoline-based compound 3 was synthetized by a simple, innovative and solvent-free procedure. The molecule was a potent dual inhibitor and demonstrated significant antitumor activity against glioma, both as a monotherapy and in combination with temozolomide. Graphical abstract The new quinoline-based compound 3 was synthetized by a simple, innovative and solvent-free procedure. The molecule was a potent dual nitric oxide synthase inhibitor and demonstrated significant antitumor activity against glioma, both as a monotherapy and in combination with temozolomide.

Inflammation, oxidative stress, and androgen activity are key features in benign prostate hyperplasia (BPH). Risks associated with the long-term use of 5α-reductase inhibitors have led to the search for alternative therapies, including food supplements. This study investigates the effectiveness of the combination of pollen extracts, namely Graminex®G96® (G) and Teupol 25P (T), towards oxidative stress and inflammation on human macrophages and benign prostate hyperplasia cells (BPH-1), both of which are LPS stimulated. The Nrf2-dependent antioxidant intracellular cascade as well as the NF-ĸB-driven inflammatory cascades were analyzed. The anti-proliferative effect of G and T, alone and in association, were evaluated on prostatic adenocarcinoma cells (PC-3) and BPH-1 cells. Finally, the inhibitory activity of GT on 5α-reductase was investigated in PC-3 cells by measuring epiandrosterone amounts, with the 5α-reductase inhibitor finasteride administered for comparison. All experiments were conducted in triplicate; data are presented as mean values ± standard deviations. Statistical analysis was performed using one-way analysis of variance. Our work demonstrates that GT promotes Nrf2-dependent antioxidant responses and counteracts the NF-ĸB-driven pathway in macrophages. GT is effective in counteracting the expression of pro-inflammatory cytokines and the generation of reactive oxygen species by promoting HO-1-dependent antioxidant responses in BPH-1 cells. GT reduces PC-3 and BPH-1 proliferation when associated with finasteride through a statistically significant inhibition of 5α-reductase activity. Data obtained in vitro and in silico demonstrate the potential efficacy of a multitargeted approach in the treatment of BPH.

The present study aimed to investigate the rationale and efficacy of testing endogenous substances widely used as dietary supplement such as citicoline, coenzyme Q10 (CoQ10) and a fixed combination of them in countering the oxidative stress and neurotoxicity occurring in neurological diseases. Rat CTX-T NA2 astrocytes, which have considerable antioxidant potential and could represent a key target for neurotherapies were selected as in vitro model to conduct the experiments. The efficacy of citicoline and coenzyme Q10 (1 nM-10 μM), with their fixed combination, were assayed in rat astrocytes either in basal condition or after challenging the cells with hydrogen peroxide in order to evaluate the biocompatibility of treatments. The gene expression of B-Cell Lymphoma protein 2 (BCL-2), BCL-2 Associated X (BAX), Superoxide dismutase 2 (SOD2), Cardiolipin Synthase 1 (CRLS1), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) involved in neurodegenerative diseases and neuroinflammation were investigated in CTX-TNA2 cells. Furthermore, in the same condition, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in astrocytes. Neither citicoline, nor coenzyme Q10 significantly altered astrocytes cell viability, thus suggesting the biocompatibility of single ingredients and fixed combination in the concentration range considered for the study. Moreover, each compound tested alone or in combination were effective in inhibiting the hydrogen peroxide-induced gene expression of BAX, and SOD2, in inducing the gene expression of BCL-2 and CRLS1 and in reducing apoptosis. The blunting effects induced by the abovementioned treatments on hydrogen-peroxide induced apoptosis was also confirmed by TUNEL assay that demonstrated the capability of citicoline, COQ10, and their combination to reduce the ratio TUNEL positive nuclei/total nuclei, a reliable marker of apoptosis. Additionally, citicoline, CoQ10, and their association were effective in inhibiting the hydrogen peroxide-induced NFkB, TNFα, and IL-6 gene. In parallel, there was an inhibition of both TNFα and IL-6 gene expression in basal condition. The co-administration of citicoline/coenzyme Q10 was overall more effective than individual ingredients. The present findings support the beneficial and synergistic effects of citicoline and coenzyme Q10 in fixed combination in reducing oxidation, and in stimulating neuroprotection in rat CTX-TNA2 astrocytes.

Chronic wounds significantly impact the patients' quality of life, creating an urgent interdisciplinary clinical challenge. The development of novel agents capable of accelerating the healing process is essential. Caffeic acid phenethyl ester (CAPE) has demonstrated positive effects on skin regeneration. However, its susceptibility to degradation limits its pharmaceutical application. Chemical modification of the structure improves the pharmacokinetics of this bioactive phenol. Hence, two novel series of CAPE hybrids were designed, synthesized, and investigated as potential skin regenerative agents. To enhance the stability and therapeutic efficacy, a caffeic acid frame was combined with quinolines or isoquinolines by an ester (1a-f) or an amide linkage (2a-f). The effects on cell viability of human gingival fibroblasts (HGFs) and HaCaT cells were evaluated at different concentrations; they are not cytotoxic, and some proved to stimulate cell proliferation. The most promising compounds underwent a wound-healing assay in HGFs and HaCaT at the lowest concentrations. Antimicrobial antioxidant properties were also explored. The chemical and thermal stabilities of the best compounds were assessed. In silico predictions were employed to anticipate skin penetration capabilities. Our findings highlight the therapeutic potential of caffeic acid phenethyl ester (CAPE) derivatives 1a and 1d as skin regenerative agents, being able to stimulate cell proliferation, control bacterial growth, regulate ROS levels, and being thermally and chemically stable. An interesting structure-activity relationship was discussed to suggest a promising multitargeted approach for enhanced wound healing.