Inhibitory Effects of Naringenin on LPS-Induced Skin Inflammation by NF-κB Regulation in Human Dermal Fibroblasts.

Flavonoids are important natural compounds characterized by their extensive biological activities. Citrus flavonoids represent a significant segment of the broader flavonoid category. Naringenin, an integral part of this series, is recognized for its powerful anti-inflammatory and antioxidant properties. In addition, considering the lack of existing research on naringenin's potential effectiveness and intracellular mechanisms of action in skin-related applications, especially as a cosmetic ingredient, this study aimed to explore naringenin's role in reducing the fundamental generation of reactive oxygen species. This was achieved by examining its inhibitory effects on the expression levels of NADPH oxidase and iNOS, ultimately leading to a reduction in NO production. This research examined the anti-inflammatory and antioxidant capacities of naringenin by employing a cellular senescence model of LPS-induced HDFs. The evaluation of naringenin's efficacy was validated through several investigative procedures, including the NF-κB luciferase assay, ELISA assay, and qRT-PCR. To verify the anti-inflammatory effectiveness of naringenin, we measured the responsive elements of NF-κB using a luciferase reporter assay. This assessment revealed that naringenin could decrease the concentration of genes activated by NF-κB. Moreover, we found that naringenin inhibited the transcriptional expression of known NF-κB-regulated inflammatory cytokines, including IL-1ß, IL-6, and IL-8. In addition, results from the qRT-PCR analysis indicated that naringenin facilitated a reduction in iNOS expression. Based on the data gathered and analyzed in this study, it can be conclusively inferred that naringenin possesses promising potential as a cosmetic ingredient, offering both anti-inflammatory and antioxidant benefits.

Effects of pulsed electrical stimulation on α-smooth muscle actin and type I collagen expression in human dermal fibroblasts.

Pulsed electrical stimulation (PES) is known to affect cellular activities. We previously found PES to human dermal fibroblasts (HFs) promoted platelet-derived growth factor subunit A (PDGFA) gene expression, which enhanced proliferation. In this study, we investigated PES effects on fibroblast collagen production and differentiation into myofibroblasts. HFs were electrically stimulated at 4800 Hz and 5 V for 60 min. Imatinib, a specific inhibitor of PDGF receptors, was treated before PES. After 6 h of PES, PDGFA, α-smooth muscle actin (α-SMA), and collagen type I α1 chain gene expressions were upregulated in PES group. Imatinib suppressed the promoted expression except for PDGFA. Immunofluorescence staining and enzyme-linked immunosorbent assay showed the production of α-SMA and collagen I was enhanced in PES group but suppressed in PES + imatinib group at 48 h after PES. Therefore, PES promotes the production of α-SMA and collagen I in fibroblasts, which is triggered by PDGFA that is upregulated early after PES.

Antioxidant, Apoptotic, and Wound Healing Effects of Xantolis cambodiana Extracts in Normal Human Dermal Fibroblasts.

Xantolis cambodiana has demonstrated significant antioxidant properties; however, the mechanisms underlying its protective effects against oxidative stress in cellular systems remain unexplored. This work investigated the efficacy of methanolic extracts in exhibiting oxidative damage and examined their mechanisms. The methanolic extract had a high phenolic content (116.89 ± 29.01 mg GAE/g FW) and exhibited scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals with an IC50 value of 42.35 ± 9.20 µg/ml. In addition, it had the highest antioxidant activity based on ferric-reducing antioxidant power (467.45 ± 50.74 mg AA/100 g). Normal human dermal fibroblast (NHDF) cells were pretreated with the methanolic extract in a hydrogen peroxide (H2O2; 500 mM)-induced oxidative stress model, which resulted in a significant decrease in apoptosis and autophagy. Not only did the methanolic extract reduce mitochondrial membrane potential, it also stimulated NHDF cell migration and reduced reactive oxygen species production through mitochondrial dysfunction in the H2O-induced stress model. These findings suggested that the methanolic extract (25 µg/ml) attenuated H2O2-induced oxidative stress in NHDF cells, significantly reducing apoptosis, autophagy, and mitochondrial dysfunction. Thus, this extract has the potential to support the wound healing process due to its antioxidant activity.

LncRNA PVT1 delays skin photoaging by sequestering miR-551b-3p to release AQP3 expression via ceRNA mechanism.

Understanding human skin photoaging requires in-depth knowledge of the molecular and functional mechanisms. Human dermal fibroblasts (HDFs) gradually lose their ability to produce collagen and renew intercellular matrix with aging. Therefore, our study aims to reveal the mechanistic actions of a novel ceRNA network in the skin photoaging by regulating HDF activities. Photoaging-related genes were obtained in silico, followed by GO and KEGG enrichment analyses. Differentially expressed lncRNAs and miRNAs were screened from the GEO database to construct the ceRNA co-expression network. In skin photoaging samples, PVT1 and AQP3 were poorly expressed, while miR-551b-3p was highly expressed. The relationships among the lncRNA, miRNA and mRNA were explored through the ENCORI database and dual luciferase reporter assay. Mechanistically, PVT1 could sequester miR-551b-3p to upregulate the expression of AQP3, which further inactivated the ERK/p38 MAPK signaling pathway. HDFs were selected to construct an in vitro cell skin photoaging model, where the senescence, cell cycle distribution and viability of young and senescent HDFs were detected by SA-ß-gal staining, flow cytometry and CCK-8 assay. In vitro cell experiments confirmed that overexpression of PVT1 or AQP3 enhanced viability of young and senescent HDFs and inhibited HDF senescence, while miR-551b-3p upregulation counteracted the effect of PVT1. In conclusion, PVT1-driven suppression of miR-551b-3p induces AQP3 expression to inactivate the ERK/p38 MAPK signaling pathway, thereby inhibiting HDF senescence and ultimately delaying the skin photoaging.

Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function.

BACKGROUND: The function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it's expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated. METHODS: ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR). RESULTS: Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles. CONCLUSIONS: Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays. Video Abstract.

Preventive effect of andrographolide against ultraviolet-B radiation-induced oxidative stress and apoptotic signaling in human dermal fibroblasts.

Ultraviolet radiation induces oxidative photoaging in the skin cells. In this study, we investigated the ability of andrographolide (ADP) to protect human dermal fibroblasts (HDFa) from UVB radiation-induced oxidative stress and apoptosis. The HDFa cells were exposed to UVB (19.8 mJ/cm2 ) radiation in the presence or absence of ADP (7 µM) and then oxidative stress and apoptotic protein expression were analyzed. UVB exposure resulted in a significant decline in the activity of antioxidant enzymes and altered mitochondrial membrane potential (MMP). Furthermore, UVB-irradiation causes increased intracellular reactive oxygen species (ROS) production, apoptotic morphological changes, and lipid peroxidation levels in the HDFa. Moreover, the pretreatment with ADP reduced the UVB-induced cytotoxicity, ROS production, and increased antioxidant enzymes activity. Further, the ADP pretreatment prevents the UVB-induced loss of MMP and apoptotic signaling in HDFa cells. Therefore, the present results suggest that ADP protects HDFa cells from UVB-induced oxidative stress and apoptotic damage.

The Effect of Hydroxytyrosol in Type II Epithelial-Mesenchymal Transition in Human Skin Wound Healing.

Skin wound healing is a multiphase physiological process that involves the activation of numerous types of cells and is characterized by four phases, namely haemostasis, inflammatory, proliferative, and remodeling. However, on some occasions this healing becomes pathological, resulting in fibrosis. Epithelial mesenchymal transition (EMT) is an important process in which epithelial cells acquire mesenchymal fibroblast-like characteristics. Hydroxytyrosol (HT) is a phenolic compound extracted from olive oil and has been proven to have several health benefits. The aim of this study was to determine the effect of HT in type II EMT in human skin wound healing via cell viability, proliferation, migration, and proteins expression. Human dermal fibroblasts (HDF) isolated from skin samples were cultured in different concentrations of HT and EMT model, induced by adding 5 ng/mL of transforming growth factor-beta (TGF-ß) to the cells. HT concentrations were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells' migrations were evaluated using scratch and transwell migration assay. Protein expressions were evaluated via immunocytochemistry. The result showed that HT at 0.2% and 0.4% significantly increased the proliferation rate of HDF (p < 0.05) compared to control. Scratch assay after 24 h showed increased cell migration in cells treated with 0.4% HT (p < 0.05) compared to the other groups. After 48 h, both concentrations of HT showed increased cell migration (p < 0.05) compared to the TGF-ß group. Transwell migration revealed that HT enhanced the migration capacity of cells significantly (p < 0.05) as compared to TGF-ß and the control group. In addition, HT supplemented cells upregulate the expression of epithelial marker E-cadherin while downregulating the expression of mesenchymal marker vimentin in comparison to TGF-ß group and control group. This study showed that HT has the ability to inhibit EMT, which has potential in the inhibition of fibrosis and persistent inflammation related to skin wound healing.

Oleuropein-Rich Gellan Gum/Alginate Films as Innovative Treatments against Photo-Induced Skin Aging.

Olea europaea L. leaf extracts (OLEs) represent highly value-added agro-industrial byproducts, being promising sources of significant antioxidant compounds, such as their main component, oleuropein. In this work, hydrogel films based on low-acyl gellan gum (GG) blended with sodium alginate (NaALG) were loaded with OLE and crosslinked with tartaric acid (TA). The films' ability to act as an antioxidant and photoprotectant against UVA-induced photoaging, thanks to their capability to convey oleuropein to the skin, were examined with the aim of a potential application as facial masks. Biological in vitro performances of the proposed materials were tested on normal human dermal fibroblasts (NhDFs), both under normal conditions and after aging-induced UVA treatment. Overall, our results clearly show the intriguing properties of the proposed hydrogels as effective and fully naturally formulated anti-photoaging smart materials for potential use as facial masks.

Protective effects of Capsicum fruits and their constituents on damage in TNF-α-stimulated human dermal fibroblasts.

BACKGROUND: Antioxidant and anti-inflammatory effects of natural products on skin cells have been proved to be effective in improving skin damage. Capsicum species contain capsaicinoids that have antioxidant and anti-inflammatory properties, and various subspecies are cultivated. In this study, the effects of four Capsicum fruits and major constituents on oxidative stress and inflammatory reactions were measured using human dermal fibroblasts (HDFs) to verify their effects on skin damage. RESULTS: The inhibitory effects of nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E2 (PGE2 ) by cucumber hot pepper, red pepper (RDP), Shishito pepper (SSP), and Cheongyang pepper were determined in HDFs. RDP and SSP inhibited the production of NO, ROS, and PGE2 in tumor necrosis factor-alpha-stimulated HDFs. Additionally, SSP seeds restored tumor necrosis factor-alpha-induced increase in matrix metalloproteinase-1 and decreased procollagen I α1 (COLIA1). In high-performance liquid chromatography analysis of the capsaicinoids capsaicin (CAP) and dihydrocapsaicin (DHC), CAP was detected at a higher level than DHC in the peel and seeds of all four types of Capsicum fruits, and the total amount of capsaicinoids was the highest in SSP. CAP and DHC, which are major constituents of Capsicum fruits, also inhibited NO, ROS, and PGE2 and restored matrix metalloproteinase-1 and procollagen I α1. CONCLUSION: RDP and SSP were shown to have a significant protective effect on skin damage, including oxidative stress, inflammatory reactions, and reduction of collagens. Capsaicinoids CAP and DHC were proved as active constituents. This research may provide basic data for developing Capsicum fruits as ingredients to improve skin damage, such as inflammation and skin aging. © 2022 Society of Chemical Industry.

Changes in miroRNA-103 expression in wound margin tissue are related to wound healing of diabetes foot ulcers.

To investigate the relationship between small noncoding microRNA-103 (miR-103) and wound healing of diabetic foot ulcers (DFU) and the underlying molecular mechanism, forty type 2 diabetes mellitus with DFU (DFU group), and 20 patients with a chronic skin ulcer of lower limbs and normal glucose tolerance (SUC group) were included. Quantitative real-time PCR method was used to determine miR-103 expression levels in the wound margin tissue of subjects, and to analyse the relationship between the expression of miR-103 and DFU wound healing. In vitro experiments were also performed to understand the effect of miR-103 on the high glucose-induced injury of normal human dermal fibroblasts (NHDFs) cells. The results showed that the miR-103 expression level in the DFU group was significantly higher than that in the SUC group [5.81 (2.25-9.36) vs 2.08 (1.15-5.72)] (P < 0.05). The expression level of miR-103 in the wound margin tissue of DFU was negatively correlated with the healing rate of foot ulcers after four weeks (P = 0.037). In vitro experiments revealed that miR-103 could inhibit the proliferation and migration of NHDF cells and promote the apoptosis of NHDF cells by targeted regulation of regulator of calcineurin 1 (RCAN1) gene expression in a high glucose environment. Down-regulation of miR-103 could alleviate high glucose-induced NHDF cell injury by promoting RCAN1 expression. Therefore, the increased expression of miR-103 is involved in the functional damage of NHDF cells induced by high-glucose conditions, which is related to poor wound healing of DFU. These research findings will provide potential targets for the diagnosis and treatment of chronic skin wounds in diabetes.

Parvovirus B19 induces cellular senescence in human dermal fibroblasts: putative role in systemic sclerosis-associated fibrosis.

OBJECTIVE: Emerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in ageing or in SSc. Recently we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/pro-inflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis. METHODS: Primary cultures of fibroblasts were infected with B19V and analysed for the acquisition of senescence markers, such as morphological modifications, senescence-associated ß-galactosidase (SA-ß-gal) activity, DNA damage response and expression of senescence-associated secretory phenotype (SASP)-related factors. RESULTS: We demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-ß-gal activity similar to that observed in SSc skin fibroblasts. They also developed an SASP-like phenotype characterized by mRNA expression and release of some pro-inflammatory cytokines, along with activation of the transcription factor nuclear factor κB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significantly higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. An increased level and nuclear localization of γH2AX, a hallmark of DNA damage response, were also found. CONCLUSIONS: B19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process.

PINK1 phosphorylates Drp1S616 to regulate mitophagy-independent mitochondrial dynamics.

Impairment of PINK1/parkin-mediated mitophagy is currently proposed to be the molecular basis of mitochondrial abnormality in Parkinson's disease (PD). We here demonstrate that PINK1 directly phosphorylates Drp1 on S616. Drp1S616 phosphorylation is significantly reduced in cells and mouse tissues deficient for PINK1, but unaffected by parkin inactivation. PINK1-mediated mitochondrial fission is Drp1S616 phosphorylation dependent. Overexpression of either wild-type Drp1 or of the phosphomimetic mutant Drp1S616D , but not a dephosphorylation-mimic mutant Drp1S616A , rescues PINK1 deficiency-associated phenotypes in Drosophila. Moreover, Drp1 restores PINK1-dependent mitochondrial fission in ATG5-null cells and ATG7-null Drosophila. Reduced Drp1S616 phosphorylation is detected in fibroblasts derived from 4 PD patients harboring PINK1 mutations and in 4 out of 7 sporadic PD cases. Taken together, we have identified Drp1 as a substrate of PINK1 and a novel mechanism how PINK1 regulates mitochondrial fission independent of parkin and autophagy. Our results further link impaired PINK1-mediated Drp1S616 phosphorylation with the pathogenesis of both familial and sporadic PD.

Sulfur mustard single-dose exposure triggers senescence in primary human dermal fibroblasts.

Chronic wounds, skin blisters, and ulcers are the result of skin exposure to the alkylating agent sulfur mustard (SM). One potential pathomechanism is senescence, which causes permanent growth arrest with a pro-inflammatory environment and may be associated with a chronic wound healing disorder. SM is known to induce chronic senescence in human mesenchymal stem cells which are subsequently unable to fulfill their regenerative function in the wound healing process. As dermal fibroblasts are crucial for cutaneous wound healing by being responsible for granulation tissue formation and synthesis of the extracellular matrix, SM exposure might also impair their function in a similar way. This study, therefore, investigated the SM sensitivity of primary human dermal fibroblasts (HDF) by determining the dose-response curve. Non-lethal concentrations LC1 (3 µM) to LC25 (65 µM) were used to examine the induction of senescence. HDF were exposed once to 3 µM, 13 µM, 24 µM, 40 µM or 65 µM SM, and were then cultured for 31 days. Changes in morphology as well as at the genetic and protein level were investigated. For the first time, HDF were shown to undergo senescence in a time- and concentration-dependent manner after SM exposure. They developed a characteristic senescence phenotype and expressed various senescence markers. Proinflammatory cytokines and chemokines were significantly altered in SM-exposed HDF as part of a senescence-associated secretory phenotype. The senescent fibroblasts can thus be considered a contributor to the SM-induced chronic wound healing disorder and might serve as a new therapeutic target in the future.

Chlorogenic Acid Prevents UVA-Induced Skin Photoaging through Regulating Collagen Metabolism and Apoptosis in Human Dermal Fibroblasts.

Skin aging is categorized as chronological aging and photo-aging that affected by intrinsic and extrinsic factors. The present study aimed to investigate the anti-aging ability and its underlying mechanism of chlorogenic acid (CGA) on human dermal fibroblasts (HDFs). In this study, CGA specifically up-regulated collagen I (Col1) mRNA and protein expressions and increased the collagen secretion in the supernatant of HDFs without affecting the cell viability, the latter was also demonstrated in BioMAP HDF3CGF system. Under ultraviolet A (UVA)-induced photoaging, CGA regulated collagen metabolism by increasing Col1 expression and decreasing matrix metalloproteinase 1 (MMP1) and MMP3 levels in UVA-irradiated HDFs. The activation of transforming growth factor-ß (TGF-ß)-mediated Smad2/3 molecules, which is crucial in Col1 synthesis, was suppressed by UVA irradiation and but enhanced at the presence of CGA. In addition, CGA reduced the accumulation of UVA-induced reactive oxygen species (ROS), attenuated the DNA damage and promoted cell repair, resulting in reducing the apoptosis of UVA-irradiated HDFs. In conclusion, our study, for the first time, demonstrate that CGA has protective effects during skin photoaging, especially triggered by UVA-irradiation, and provide rationales for further investigation of CGA being used to prevent or treat skin aging.

2-O-ß-d-Glucopyranosyl-4,6-dihydroxybenzaldehyde Isolated from Morus alba (Mulberry) Fruits Suppresses Damage by Regulating Oxidative and Inflammatory Responses in TNF-α-Induced Human Dermal Fibroblasts.

Human skin is composed of three layers, of which the dermis is composed of an extracellular matrix (ECM) comprising collagen, elastin, and other proteins. These proteins are reduced due to skin aging caused by intrinsic and extrinsic factors. Among various internal and external factors related to aging, ultraviolet (UV) radiation is the main cause of photoaging of the skin. UV radiation stimulates DNA damage, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine production such as tumor necrosis factor-alpha (TNF-α), and promotes ECM degradation. Stimulation with ROS and TNF-α upregulates mitogen-activated protein kinases (MAPKs), nuclear factor kappa B (NF-κB), and activator protein 1 (AP-1) transcription factors that induce the expression of the collagenase matrix metalloproteinase-1 (MMP-1). Moreover, TNF-α induces intracellular ROS production and several molecular pathways. Skin aging progresses through various processes and can be prevented through ROS generation and TNF-α inhibition. In our previous study, 2-O-ß-d-glucopyranosyl-4,6-dihydroxybenzaldehyde (GDHBA) was isolated from the Morus alba (mulberry) fruits and its inhibitory effect on MMP-1 secretion was revealed. In this study, we focused on the effect of GDHBA on TNF-α-induced human dermal fibroblasts (HDFs). GDHBA (50 µM) inhibited ROS generation (18.8%) and decreased NO (58.4%) and PGE2 levels (53.8%), significantly. Moreover, it decreased MMP-1 secretion (55.3%) and increased pro-collagen type I secretion (207.7%). GDHBA (50 µM) decreased the expression of different MAPKs as per western blotting; p-38: 35.9%; ERK: 47.9%; JNK: 49.5%; c-Jun: 32.1%; NF-κB: 55.9%; and cyclooxygenase-2 (COX-2): 31%. This study elucidated a novel role of GDHBA in protecting against skin inflammation and damage through external stimuli, such as UV radiation.

Continuous Production of Highly Tuned Silk/Calcium-Based Composites: Exploring New Pathways for Skin Regeneration.

Calcium plays an important role in barrier function repair and skin homeostasis. In particular, calcium phosphates (CaPs) are well established materials for biomedical engineering due to their biocompatibility. To generate biomaterials with a more complete set of biological properties, previously discarded silk sericin (SS) has been recovered and used as a template to grow CaPs. Crucial characteristics for skin applications, such as antibacterial activity, can be further enhanced by doping CaPs with cerium (Ce) ions. The effectiveness of cell attachment and growth on the materials highly depends on their morphology, particle size distribution, and chemical composition. These characteristics can be tailored through the application of oscillatory flow technology, which provides precise mixing control of the reaction medium. Thus, in the present work, CaP/SS and CaP/SS/Ce particles were fabricated for the first time using a modular oscillatory flow plate reactor (MOFPR) in a continuous mode. Furthermore, the biological behavior of both these composites and of previously produced pure CaPs was assessed using human dermal fibroblasts (HDFs). It was demonstrated that both CaP based with plate-shaped nanoparticles and CaP-SS-based composites significantly improved cell viability and proliferation over time. The results obtained represent a first step towards the reinvention of CaPs for skin engineering.

Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF, TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines.

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-ß signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-ß signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients.

A link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

Atomoxetine and circadian gene expression in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder.

Atomoxetine (ATO) is a second line medication for attention-deficit hyperactivity disorder (ADHD). We proposed that part of the therapeutic profile of ATO may be through circadian rhythm modulation. Thus, the aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after ATO exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with a diagnosis of ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different ATO concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. No statistical significant effect of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, sleep WASO and total number of wake bouts was observed. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. ATO induced the rhythmicity of Clock in the ADHD group. This effect, however, was not observed in HDF cultures of healthy controls. Bmal1 and Per2 expression showed a significant ZT × group interaction via mixed ANOVA. Strong positive correlations for chronotype and circadian genes were observed for Bmal1, Cry1 and Per3 among the study participants. Statistical significant different Clock, Bmal1 and Per3 expressions were observed in HDFs exposed to ATO collected from ADHD participants exhibiting neutral and moderate evening preference, as well as healthy participants with morning preferences. The results of the present study illustrate that ATO impacts on circadian function, particularly on Clock, Bmal1 and Per2 gene expression.