Search Results (49)

Background/Objectives: Psychological stress triggers excessive melanin deposition via neuroendocrine pathways, yet targeted interventions for stress-induced hyperpigmentation remain limited. Salidroside (SAL) exhibits established depigmenting effects in UV-induced models and possesses neuroprotective properties. This study investigated SAL’s efficacy in psychological stress-induced hyperpigmentation and elucidated its underlying mechanisms. Methods: B16F10 melanocytes, C57BL/6J mice, zebrafish, and human foreskin organ cultures were subjected to stress factor (Substance P/cortisol) or α-MSH/IBMX stimulation to model psychological stress-induced and canonical cAMP-driven hyperpigmentation, respectively. Melanin content, tyrosinase activity, melanosome maturation (transmission electron microscopy/HMB45 staining), and melanogenic protein/mRNA expression were assessed. Drug Affinity Responsive Target Stability (DARTS) assays, molecular docking, and SEC23A siRNA knockdown were employed to identify and validate SAL’s molecular target and downstream signaling pathways. Results: SAL dose-dependently reduced melanin content, tyrosinase activity, and TYR/TRP-1/DCT expression in SP/Cort-stimulated melanocytes, exhibiting greater potency (200 μM) than in IBMX-induced models (400 μM). SAL reversed SP/Cort-induced hyperpigmentation in human skin explants, zebrafish, and C57BL/6J mice, and normalized melanosome number/maturation. DARTS and molecular docking identified SEC23A as a direct SAL-binding target. SP/Cort specifically upregulated SEC23A, which SAL suppressed. SAL concurrently activated the SEC23A-p-ERK-MITF axis and inhibited the NK1R-p38-MITF axis in the stress model. SEC23A knockdown potentiated SAL’s anti-melanogenic effects specifically in SP/Cort-stimulated cells. Conversely, in IBMX-induced models, SEC23A remained unchanged, and SAL acted via PKA/CREB, PI3K/AKT, and Wnt/β-catenin pathways. Conclusions: SEC23A is a novel core target in psychological stress-induced hyperpigmentation. SAL selectively binds SEC23A to inhibit stress-induced melanogenesis via dual ERK and p38 MAPK signaling axes, demonstrating etiological specificity distinct from canonical cAMP pathway inhibition. Full article

Background/Objectives: Individual responses to CFTR modulators vary widely among people with cystic fibrosis (pwCF), underscoring the need for functional approaches that provide biological context alongside genotype-based therapy selection. Nasal epithelial cultures provide an individual-specific model for theratyping, but most studies rely on freshly isolated cells, restricting repeated testing and long-term sample use. In this study, we tested whether CFTR modulator responses measured in biobanked nasal cells were associated with real-world clinical outcomes. Methods: Cryopreserved nasal epithelial cells from 23 pwCF were differentiated at the air–liquid interface and assessed for CFTR modulator-responsive ion transport using Ussing chambers. In vitro responses were correlated with 6-month changes in sweat chloride concentration (SCC), FEV₁, and BMI. Results: Cryopreserved cultures retained donor-specific CFTR modulator responsiveness. Modulator-induced forskolin/IBMX-stimulated currents correlated with changes in SCC (R = −0.512). CFTR inhibitor-sensitive currents correlated with FEV₁ (R = 0.564). Associations between forskolin/IBMX-stimulated currents and FEV₁ were positive but did not reach statistical significance using two-tailed analysis. BMI changes showed no significant association. Conclusions: Biobanked nasal epithelial cultures preserve clinically relevant CFTR modulator responses at the cohort level, supporting their use as functional assays for population-level assessment in cystic fibrosis. This cryopreservation-based strategy enables repeated testing and may expand access to theratyping beyond freshly obtained samples. Full article

Background/Objectives: The term ‘cystic fibrosis transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID)’ refers to patients with positive screening tests but without a final diagnosis of Cystic Fibrosis (CF). Intestinal Current Measurement (ICM) is a novel diagnostic technique that may document the abnormal function of the cystic fibrosis transmembrane conductance regulator. Our study aims to compare the cumulative chloride secretory response in the ICM study in the Polish population of CF patients, CRMS/CFSPID, and in a control group. Methods: Forceps rectal biopsies were taken from 40 patients (CF; n = 17 mean age 9.10 ± 4.18 (0.7–17.20); CRMS/CFSPID: n = 16, mean age 6.66 ± 4.83 (0.6–18.0); healthy controls (HC): n = 7, mean age 23.7 ± 9.5 (7.8–34.6). ICM tests were performed in the Ussing Chamber according to standard protocol version 2.7 of the European Cystic Fibrosis Society Diagnostic Network Working Group. Delta short circuit-current (ΔIsc) was measured after carbachol (ΔIsc_carbachol), 3-isobutyl-1-methylxanthine with forskolin (ΔIsc_{IBMX/forskolin}), and histamine (Δisc_histamine) stimulation. Cumulative secretion was calculated for each study group. Results: We obtained statistically significant differences in cumulative chloride secretory response between CF and CRMS/CFSPID (CF ΔIsc_{carbachol+IBMX/forskolin+histamine} 15.32 ± 15.47 µA/cm² vs. CRMS/CFSPID ΔIsc_{carbachol+IBMX/forskolin+histamine} 86.84 ± 37.84 µA/cm²; p < 0.001), and between CF and healthy controls (CF ΔIsc_{carbachol+IBMX/forskolin+histamine} 15.32 ± 15.47 µA/cm² vs. HC ΔIsc_{carbachol+IBMX/forskolin+histamine} 80.16 ± 48.54 µA/cm²; p = 0.005). No differences in cumulative chloride secretion were observed between the CRMS/CFSPID and HC groups. Conclusions: The conducted study suggests that ICM may offer diagnostic value, especially in cases where sweat test results are equivocal. Full article

Background: Cucurbitacin E (CuE), a natural tetracyclic triterpenoid compound extracted from the melon stems of Cucurbitaceae plants, has been reported to exhibit anti-inflammatory and anti-cancer properties, along with the ability to enhance cellular immunity. However, its role and molecular mechanism in regulating lipid metabolism and adipogenesis remain unclear. This study aims to investigate the potential anti-adipogenic and anti-obesity effects of CuE in 3T3-L1 adipocytes. Materials and Methods: 3T3-L1 preadipocytes were cultured and induced to differentiate using a standard adipogenic cocktail containing dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), and insulin (DMI). CuE was administered during the differentiation process at various concentrations. Lipid accumulation was assessed using Oil Red O staining, and cell viability was evaluated via the MTT assay. To determine whether CuE induced apoptosis or necrosis, flow cytometry was performed using annexin V/PI staining. Additional molecular analyses, such as Western blotting and RT-PCR, were used to examine the expression of key adipogenic markers. Results: Treatment with CuE significantly reduced lipid droplet formation in DMI-induced 3T3-L1 adipocytes in a dose-dependent manner, as shown by decreased Oil Red O staining. Importantly, CuE did not induce apoptosis or necrosis in 3T3-L1 cells at effective concentrations, indicating its safety toward normal adipocytes. Moreover, CuE treatment downregulated the expression of adipogenic markers such as PPARγ and C/EBPα at both mRNA and protein levels. Discussion: Our findings suggest that CuE exerts a non-cytotoxic inhibitory effect on adipocyte differentiation and lipid accumulation. This anti-adipogenic effect is likely mediated through the suppression of key transcription factors involved in adipogenesis. The absence of cytotoxicity supports the potential application of CuE as a safe bioactive compound for obesity management. Further investigation is warranted to elucidate the upstream signaling pathways and in vivo efficacy of CuE. Conclusions: Cucurbitacin E effectively inhibits adipogenesis in 3T3-L1 adipocytes without inducing cytotoxic effects, making it a promising candidate for the development of functional foods or therapeutic agents aimed at preventing or treating obesity. This study provides new insights into the molecular basis of CuE’s anti-obesity action and highlights its potential as a natural lipogenesis inhibitor. Full article

Aim: Myricetin, a natural bioflavonoid, is reported as an anti-diabetic agent since it possesses the ability to inhibit α-glucosidase activity, stimulate insulin action and secretion, manage ROS, and prevent diabetes complications. Myricetin was identified as a new insulin secretagogue that enhances glucose-stimulated insulin secretion and seems like a better antidiabetic drug candidate. Here, we explored the insulinotropic mechanism(s) of myricetin in vitro in mice islets and in silico. Methods: Size-matched pancreatic islets were divided into groups and incubated in the presence or absence of myricetin and agonists/antagonists of major insulin signaling pathways. The secreted insulin was measured by ELISA. Molecular docking studies were performed with the key player of insulin secretory pathways. Results: Myricetin dose-dependently enhanced insulin secretion in isolated mice islets, and its insulinotropic effect was exerted at high glucose concentrations distinctly different from glibenclamide. Myricetin-induced insulin secretion was significantly inhibited using the diazoxide. Furthermore, myricetin amplified glucose-induced insulin secretion in depolarized and glibenclamide-treated islets. Myricetin showed an additive effect with forskolin- and IBMX-induced insulin secretion. Interestingly, H89, a PKA inhibitor, and MAY0132, an Epac-2 inhibitor, significantly inhibited myricetin-induced insulin secretion. The in silico molecular docking studies further validated these in vitro findings in isolated pancreatic islets. Conclusions: Myricetin, a potential natural insulin secretagogue, amplifies glucose-induced insulin secretion via the cAMP-PKA-Epac-2 signaling pathway. Full article

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that is dysfunctional in individuals with cystic fibrosis (CF). The permeability of CFTR can be experimentally manipulated though different mechanisms, including activation via inducing the phosphorylation of residues in the regulatory domain as well as altering the gating/open probability of the channel. Phosphorylation/activation of the channel is achieved by exposure to compounds that increase intracellular cAMP, with forskolin and IBMX commonly used for this purpose. C_act-A1 is a unique CFTR activator that does not increase intracellular cAMP, and VX-770 (ivacaftor) is a CFTR potentiator that is used experimentally and therapeutically to increase the open probability of the channel. Using primary human nasal epithelial cell (HNEC) cultures and Fischer rat thyroid (FRT) epithelial cells exogenously expressing functional CFTR, we examined the impact of VX-770, C_act-A1, and forskolin/IBMX on CFTR activity during analysis in an Ussing chamber. Relative contributions of these compounds to maximal CFTR activity were dependent on order of exposure, the presence of chemical and electrical gradients, the level of constitutive CFTR function, and the cell model tested. Increasing intracellular cAMP appeared to change cellular functions outside of CFTR activity that resulted in alterations in the drive for chloride through CFTR. These results demonstrate that one can utilize combinations of small-molecule CFTR activators and potentiators to provide detailed characterization of CFTR-mediated ion transport in primary HNECs and properties of these modulators in both primary HNECs and FRT cells. Future studies using these approaches may assist in the identification of novel defects in CFTR function and the identification of modulators with unique impacts on CFTR-mediated ion transport. Full article

Basella alba has been used in Thai remedies to treat skin disorders, but scientific evidence supporting its efficacy is currently limited. In this study, we investigated the inhibitory effects of B. alba extracts on melanin production using melanoma cells, as well as their impact on oxidative stress and inflammation in keratinocytes. The results demonstrate that B. alba extracts inhibited melanin content and cellular tyrosinase activity in 3-isobutyl-1-methylxanthine (IBMX)-induced melanoma cells by downregulating MITF and the pigmentary genes TYR, TRP-1, and DCT. Interestingly, the MITF regulator gene was inhibited by both the 50% and 95% ethanolic extracts of B. alba with levels of 0.97 ± 0.19 and 0.92 ± 0.09 of the control, respectively, which are comparable to those observed in the arbutin treatment group at 0.84 ± 0.05 of the control. Moreover, after hydrogen peroxide (H₂O₂) exposure, pretreatment with B. alba reduced lipid peroxidation byproducts and increased the levels of antioxidant-related genes, including SOD-1, GPX-1, and NRF2. Notably, the suppression of the POMC promoter gene in keratinocytes was observed, which may disrupt melanogenesis in melanocytes involving the MC1R signaling pathway. MC1R mRNA expression decreased in the treatments with 50% and 95% ethanolic extracts of B. alba, with relative levels of 0.97 ± 0.18 and 0.90 ± 0.10 of the control, respectively, similar to the arbutin-treated group (0.88 ± 0.25 of control). A significant reduction in nitric oxide was also observed in the B. alba-treated groups, along with a decrease in genes associated with pro-inflammatory cytokines, including IL-1β, IL-6, and COX-2. These findings suggest that B. alba has potential in the prevention of skin-related problems. Full article

Cultivated meat, also known as cell-based or clean meat, utilizes mesenchymal stem cells to cultivate mature cell types like adipocytes, which are pivotal for imparting the desired taste and texture. The delivery of differentiated cells, crucial in cultivated meat production, is facilitated through extensive exploration of 3D culturing techniques mimicking physiological environments. In this study, we investigated the adipogenic differentiation potential of bovine umbilical cord stem cells (BUSCs), sourced from discarded birth tissue, and assessed the feasibility of delivering differentiated cells for cultivated meat using gelatin methacrylate (GelMA) as a carrier for adipose tissue. Various adipogenic inducers, previously reported to be effective for human mesenchymal stem cells (hMSCs), were evaluated individually or in combination for their efficacy in promoting the adipogenesis of BUSCs. Surprisingly, while the traditional adipogenic inducers, including insulin, dexamethasone, isobutylmethylxantine (IBMX), indomethacin, and rosiglitazone, showed no significant effect on the adipogenic differentiation of BUSCs, efficient differentiation was achieved in the presence of a fatty acid cocktail. Furthermore, we explored methods for the delivery of BUSCs. Differentiated cells were delivered either encapsulated in GelMA hydrogel or populated on the surface of GelMA microparticles (MPs) as the adipose component of cultivated meat. Our findings reveal that after adipogenic induction, the lipid production per cell was comparable when cultured either within hydrogel or on MPs. However, GelMA-MPs supported better cell growth compared to hydrogel encapsulation. Consequently, the overall lipid production is higher when BUSCs are delivered via GelMA-MPs rather than encapsulation. This study not only systematically evaluated the impact of common adipogenic inducers on BUSCs, but also identified GelMA-MPs as a promising carrier for delivering bovine adipocytes for cultivated meat production. Full article

The infusion of Santolina impressa, an endemic Portuguese plant, is traditionally used to treat various infections and disorders. This study aimed to assess its chemical profile by HPLC-DAD-ESI-MSⁿ and validate its anti-inflammatory potential. In addition, the antioxidant capacity and effects on wound healing, lipogenesis, melanogenesis, and cellular senescence, all processes in which a dysregulated inflammatory response plays a pivotal role, were unveiled. The anti-inflammatory potential was assessed in lipopolysaccharide (LPS)-stimulated macrophages, cell migration was determined using a scratch wound assay, lipogenesis was assessed on T0901317-stimulated keratinocytes and melanogenesis on 3-isobutyl-1-methylxanthine (IBMX)-activated melanocytes. Etoposide was used to induce senescence in fibroblasts. Our results point out a chemical composition predominantly characterized by dicaffeoylquinic acids and low amounts of flavonols. Regarding the infusion’s bioactive potential, an anti-inflammatory effect was evident through a decrease in nitric oxide production and inducible nitric oxide synthase and pro-interleukin-1β protein levels. Moreover, a decrease in fibroblast migration was observed, as well as an inhibition in both intracellular lipid accumulation and melanogenesis. Furthermore, the infusion decreased senescence-associated β-galactosidase activity, γH2AX nuclear accumulation and both p53 and p21 protein levels. Overall, this study confirms the traditional uses of S. impressa and ascribes additional properties of interest in the pharmaceutical and dermocosmetics industries. Full article

Epidermal melanin synthesis determines an individual’s skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen^® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable. Full article

Gap junctional connection (GJC) in the cumulus–oocyte complex (COC) provides necessary support for message communication and nutrient transmission required for mammalian oocyte maturation. Cyclic adenosine monophosphate (cAMP) is not only a prerequisite for regulating oocyte meiosis, but also the key intercellular factor for affecting GJC function in COCs. However, there are no reports on whether cAMP regulates connexin 37 (Cx37) expression, one of the main connexin proteins, in sheep COCs. In this study, the expression of Cx37 protein and gene in immature sheep COC was detected using immunohistochemistry and PCR. Subsequently, the effect of cAMP on Cx37 expression in sheep COCs cultured in a gonadotropin-free culture system for 10 min or 60 min was evaluated using competitive ELISA, real-time fluorescent quantitative PCR (RT-qPCR), and Western blot. The results showed that the Cx37 protein was present in sheep oocytes and cumulus cells; the same results were found with respect to GJA4 gene expression. In the gonadotropin-free culture system, compared to the control, significantly higher levels of cAMP as well as Cx37 gene and protein expression were found in sheep COCs following treatment in vitro with Forskolin and IBMX (100 μM and 500 μM)) for 10 min (p < 0.05). Compared to the controls (at 10 or 60 min), cAMP levels in sheep COCs were significantly elevated as a result of Forskolin and IBMX treatment (p < 0.05). Following culturing in vitro for 10 min or 60 min, Forskolin and IBMX treatment can significantly promote Cx37 expression in sheep COCs (p < 0.05), a phenomenon which can be counteracted when the culture media is supplemented with RP-cAMP, a cAMP-specific competitive inhibitor operating through suppression of the protein kinase A (PKA). In summary, this study reports the preliminary regulatory mechanism of cAMP involved in Cx37 expression for the first time, and provides a novel explanation for the interaction between cAMP and GJC communication during sheep COC culturing in vitro. Full article

With the increase in global life expectancy, maintaining health into old age becomes a challenge, and research has thus concentrated on various strategies which aimed to mitigate the effects of skin aging. Aromatic plants stand out as promising sources of anti-aging compounds due to their secondary metabolites, particularly essential oils (EOs). The aim of this study was to ascribe to Ferulago lutea EO several biological activities that could be useful in the context of skin aging. The EO was obtained using hydrodistillation and characterized by gas chromatography–mass spectrometry (GC/MS). The anti-inflammatory potential was assessed using lipopolysaccharide (LPS)-stimulated macrophages. The effect on cell migration was disclosed using scratch wound assay. Lipogenesis was induced using T0901317, hyperpigmentation with 3-isobutyl-1-methylxantine (IBMX) and senescence with etoposide. Our results show that the EO was characterized mainly by α-pinene and limonene. The EO was able to decrease nitric oxide (NO) release as well as iNOS and pro-IL-1β protein levels. The EO promoted wound healing while decreasing lipogenesis and having depigmenting effects. The EO also reduced senescence-associated β-galactosidase, p21/p53 protein levels and the nuclear accumulation of γH2AX. Overall, our study highlights the properties of F. lutea EO that make it a compelling candidate for dermocosmetics applications. Full article

The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco’s Modified Eagle’s Medium, 10 μg/mL insulin, 0.55 μg/mL transferrin, 6.8 ng sodium selenite, 1 μM dexamethasone, and 1–5 μM of rosiglitazone, is compared to the 3-isobutyl-1-methylxantine (IBMX) protocol, where rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin. Cell viability, cytotoxicity, a morphometric analysis of the lipid, and the expression of adipogenic markers for 14 days were assessed. The data revealed that using 5 µM of rosiglitazone promotes the adipogenic differentiation capacity in horse, sheep, and dog cells compared to IBMX induction. Meanwhile, marked reductions in the cell viability and cell number with the IBMX protocol were detected, and rosiglitazone increased the cell number and lipid droplet size, prevented apoptosis, and upregulated FABP-4 and Leptin expression in the cells of most of the species. Our data revealed that the rosiglitazone protocol improves the adipogenesis of ASCs, together with having less toxicity, and should be considered for cell reproducibility and clinical applications targeting obesity. Full article

Glucocorticoid receptors (GRs) play a pivotal role in the stress response of the body, but overactivation can disrupt normal physiological functions. This study explores the role of cyclic adenosine monophosphate (cAMP) in GR activation and the associated mechanisms. We initially used the human embryonic kidney 293 cell line (HEK293) and found that cAMP enhancement, using forskolin and 3-isobutyl-1-methylxanthine (IBMX), did not alter glucocorticoid signaling under normal conditions, as evidenced by glucocorticoid response element (GRE) activity and the translocation of GR. However, in stressful conditions induced by dexamethasone, a synthetic glucocorticoid, cAMP was found to lessen glucocorticoid signaling within a short time frame but amplify it over an extended period in HEK293 cells. Bioinformatic analysis revealed that cAMP upregulation triggers the extracellular signal-regulated kinase (ERK) pathway, which influences GR translocation and ultimately regulates its activity. This stress-modulating function of cAMP was also investigated in the Hs68 dermal fibroblast line, known for its susceptibility to glucocorticoids. We found that cAMP enhancement via forskolin reduces GRE activity and reverses collagen loss in Hs68 cells exposed to dexamethasone. These findings underline the context-specific role of cAMP signaling in managing glucocorticoid signaling and its potential therapeutic application in treating stress-related pathological conditions like skin aging characterized by collagen reduction. Full article

Kaempferia parviflora Wall. ex Baker (Zingiberaceae), commonly known as Thai ginseng or black ginger, is a tropical medicinal plant in many regions. It has been traditionally used to treat various ailments, including ulcers, dysentery, gout, allergies, abscesses, and osteoarthritis. As part of our ongoing phytochemical study aimed at discovering bioactive natural products, we investigated potential bioactive methoxyflavones from K. parviflora rhizomes. Phytochemical analysis aided by liquid chromatography–mass spectrometry (LC-MS) led to the isolation of six methoxyflavones (1–6) from the n-hexane fraction of the methanolic extract of K. parviflora rhizomes. The isolated compounds were structurally determined to be 3,7-dimethoxy-5-hydroxyflavone (1), 5-hydroxy-7-methoxyflavone (2), 7,4′-dimethylapigenin (3), 3,5,7-trimethoxyflavone (4), 3,7,4′-trimethylkaempferol (5), and 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (6), based on NMR data and LC-MS analysis. All of the isolated compounds were evaluated for their anti-melanogenic activities. In the activity assay, 7,4′-dimethylapigenin (3) and 3,5,7-trimethoxyflavone (4) significantly inhibited tyrosinase activity and melanin content in IBMX-stimulated B16F10 cells. In addition, structure–activity relationship analysis revealed that the methoxy group at C-5 in methoxyflavones is key to their anti-melanogenic activity. This study experimentally demonstrated that K. parviflora rhizomes are rich in methoxyflavones and can be a valuable natural resource for anti-melanogenic compounds. Full article