In Silico and In Vitro Studies on an Asymmetrical Porphyrin Derivative with Therapeutic Potential in Skin Disorders.

For developing novel photosensitizers with therapeutic potential in non-malignant and malignant cutaneous disorders, the unsymmetrical porphyrin, 5-(2-hydroxy-3-methoxyphenyl)-10, 15, 20-tris-(4-carboxymethylphenyl) porphyrin, was evaluated in silico and in vitro. The cellular uptake of the investigated porphyrin and its ability to perform photodynamic therapy were investigated in terms of the viability, proliferation, and necrosis of human HaCaT keratinocytes and human Hs27 skin fibroblasts, in correlation with the predictions regarding diffusion through cell membranes, ADMET profile (absorption, distribution, metabolism, elimination, toxicity), and potential pharmacological mechanism. Molecular docking and 250 ns molecular dynamics simulations revealed that P5.2 has the potential to form a relatively stable complex with the carbonic anhydrase IX catalytic site, the lowest predicted free energy of binding (MM/PBSA) being -39.097 kcal/mol. The results of the in vitro study showed that P5.2 is incorporated within 24 h in the investigated cells, especially in HaCaT keratinocytes, indicating its photosensitizing ability. Nevertheless, P5.2 does not exert significant cytotoxicity in "dark" conditions. In turn, PDT induced a decrease in the number of metabolically active HaCaT keratinocytes within 24 h, accompanied by a 4-fold increase in lactate dehydrogenase release, indicating its ability to perform PDT in human skin cells. The experimental results suggest that the asymmetrical porphyrin is a promising candidate theranostics agent for skin disorders.

Anti-Inflammatory Response of New Postbiotics in TNF-α/IFN-γ-Induced Atopic Dermatitis-like HaCaT Keratinocytes.

This study examines the synergistic interaction between the immunomodulatory functions of lactic acid bacteria postbiotics and the anti-inflammatory properties of Smilax china L. extract through a combined fermentation process. Using atopic dermatitis (AD) as a model, characterized by an immune imbalance that leads to skin inflammation, we developed a fermented product, MB-2006, and compared its effects to those of the heat-killed probiotics Lactobacillus acidophilus (LAC) and Lactobacillus rhamnosus (LRH). Our experiments focused on elucidating the mechanism of action of MB-2006 in AD-like HaCaT keratinocyte cells, particularly its impact on the NF-κB pathway, a pivotal regulator of inflammation. MB-2006 proved more effective in reducing inflammation markers, such as IL-4 and thymic stromal lymphopoietin (TSLP), and in inhibiting NF-κB activation compared to LAC and LRH. Significantly, MB-2006 also reduced the expression of thymus- and activation-regulated chemokine (TARC), highlighting a synergistic effect that enhances its therapeutic potential. These results suggest that the combined fermentation of Smilax china L. extract with lactic acid bacteria enhanced both the anti-inflammatory and immunomodulatory effects, presenting a promising integrative approach to treating conditions like AD. Further studies are needed to validate these results in clinical settings and fully explore the potential of this synergistic fermentation process.

Identification of protein components of the transformation system in the cell line of immortalized human keratinocytes HaCaT exposed to surfactants.

Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/ml and 25.0 µg/ml). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

Cyanobacterial bloom-associated lipopolysaccharides induce pro-inflammatory processes in keratinocytes in vitro.

Our previous studies have shown that CyanoHAB LPS (lipopolysaccharides) and LPS from cyanobacterial cultures induce pro-inflammatory effects on intestinal epithelial and immune cells in vitro. To expand our understanding, we investigated their impact on human keratinocytes, which are targeted during water recreational activities. LPS samples were isolated from CyanoHAB biomasses dominated by Microcystis, Aphanizomenon, Planktothrix, and Dolichospermum, or from axenic cultures of these genera. We identified two CyanoHAB biomasses containing a high proportion of Gram-negative bacteria, including potentially pathogenic genera. These biomasses showed the highest induction of interleukin (IL) 8, IL-6, C-C motif chemokine ligand (CCL) 2 (also known as MCP-1), and CCL20 production by HaCaT cells. Interestingly, all CyanoHAB-derived LPS and LPS from axenic cultures (except for Microcystis) accelerated cell proliferation and migration. Our findings highlight the role of G- bacteria composition and LPS structural disparities in influencing these effects, with implications for skin health during recreational activities.

Liquiritin targeting Th17 cells differentiation and abnormal proliferation of keratinocytes alleviates psoriasis via NF-κB and AP-1 pathway.

Psoriasis is a common immune-mediated inflammatory skin disease, caused by disturbed interactions between keratinocytes and immune cells. Chinese medicine shows potential clinical application for its treatment. Liquiritin is a flavone compound extracted from licorice and shows potential antitussive, antioxidant and antiinflammatory effects, and therefore may have potential as a psoriasis therapeutic. The aim of this work was to examine the possible roles that liquiritin may have in treating psoriasis. HaCaT cells were stimulated by TNF-α with or without liquiritin, harvested for analysis by western blots and RT-qPCR, and the cellular supernatants were collected and analyzed by ELISA for cytokines. In addition, 4 groups of mice were examined: Normal, Vehicle, LQ-L and LQ-H. The mice were sacrificed after 6 days and analyzed using IHC, ELISA, RT-qPCR and flow cytometry. The results showed that liquiritin could significantly inhibit the progression of psoriasis both in vitro and in vivo. Liquiritin strongly suppressed the proliferation of HaCaT keratinocytes but did not affect cell viability. Moreover, liquiritin alleviated imiquimod-induced psoriasis-like skin inflammation and accumulation of Th17 cells and DCs in vivo. In TNF-α-induced HaCaT keratinocytes, both protein and mRNA expression levels of inflammatory cytokines were sharply decreased. In imiquimod-induced mice, the activation of NF-κB and AP-1 was reduced after treatment with liquiritin. Collectively, our results show that liquiritin might act as a pivotal regulator of psoriasis via modulating NF-κB and AP-1 signal pathways.

The anti-inflammation and skin-moisturizing effects of Boehmeria tricuspis-mediated biosynthesized gold nanoparticles in human keratinocytes.

Introduction: Recently, nanotechnology has emerged as a potential technique for skin generation, which has several treatment advantages, such as decreased drug cytotoxicity and enhanced skin penetration. Boehmeria tricuspis (BT) belongs to the Urticaceae family and is rich in phenolic and flavonoid compounds. In this study, we biosynthesized gold nanoparticles (BT-AuNPs) using BT extract to explore their anti-inflammatory and skin-moisturizing properties in keratinocytes. Methods: Field-emission transmission electron microscopy, energydispersive X-ray spectrometry, dynamic light scattering, and Fourier-transforminfrared spectroscopy were used to examine the synthesized BT-AuNPs. qRT-PCR, western blot, and ELISA were applied for investigating the effect of BT-AuNPs on anti-inflammation and moisturizing activity in HaCaT cells. Results: At concentrations below 200 µg/mL, BT-AuNPs had no cytotoxic effect on keratinocytes. BT-AuNPs dramatically alleviated the expression and secretion of inflammatory chemokines/cytokine, such as IL-6, IL-8, TARC, CTACK, and RANTES in keratinocytes stimulated by tumor necrosis factor-α/interferon-γ (T + I). These anti-inflammatory properties of BT-AuNPs were regulated by inhibiting the NF-κB and MAPKs signaling pathways. Furthermore, BT-AuNPs greatly promoted hyaluronic acid (HA) production by enhancing the expression of hyaluronic acid synthase genes (HAS1, HAS2, and HAS3) and suppressing the expression of hyaluronidase genes (HYAL1 and HYAL2) in HaCaT cells. Discussion: These results suggest that BT-AuNPs can be used as a promising therapeutic alternative for treating skin inflammation. Our findings provide a potential platform for the use of BT-AuNPs as candidates for treating inflammatory skin diseases and promoting skin health.

Exploring the Potential of Lapatinib, Fulvestrant, and Paclitaxel Conjugated with Glycidylated PAMAM G4 Dendrimers for Cancer and Parasite Treatment.

Fulvestrant (F), lapatinib (L), and paclitaxel (P) are hydrophobic, anticancer drugs used in the treatment of estrogen receptor (ER) and epidermal growth factor receptor (EGFR)-positive breast cancer. In this study, glycidylated PAMAM G4 dendrimers, substituted with F, L, and/or P and targeting tumor cells, were synthesized and characterized, and their antitumor activity against glioma U-118 MG and non-small cell lung cancer A549 cells was tested comparatively with human non-tumorogenic keratinocytes (HaCaT). All cell lines were ER+ and EGFR+. In addition, the described drugs were tested in the context of antinematode therapy on C. elegans. The results show that the water-soluble conjugates of G4P, G4F, G4L, and G4PFL actively entered the tested cells via endocytosis due to the positive zeta potential (between 13.57-40.29 mV) and the nanoparticle diameter of 99-138 nm. The conjugates of G4P and G4PFL at nanomolar concentrations were the most active, and the least active conjugate was G4F. The tested conjugates inhibited the proliferation of HaCaT and A549 cells; in glioma cells, cytotoxicity was associated mainly with cell damage (mitochondria and membrane transport). The toxicity of the conjugates was proportional to the number of drug residues attached, with the exception of G4L; its action was two- and eight-fold stronger against glioma and keratinocytes, respectively, than the equivalent of lapatinib alone. Unfortunately, non-cancer HaCaT cells were the most sensitive to the tested constructs, which forced a change in the approach to the use of ER and EGFR receptors as a goal in cancer therapy. In vivo studies on C. elegans have shown that all compounds, most notably G4PFL, may be potentially useful in anthelmintic therapy.

In Vitro Characterization of an Anodized Surface of a Dental Implant Collar and Dental Abutment on Peri-Implant Cellular Response.

The purpose of this paper was to determine the effect of anodization on the in vitro proliferation and adhesion of immortalized human keratinocytes (HaCats) and mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) in Titanium Grade 23 (Ti6Al4V ELI) discs and to describe the surface topography, roughness, and composition of dental implants (body and collar) and abutments submitted to an area-specific anodization process. HaCat cells and BM-MSCs were seeded onto discs with three different surface treatments: machined, area-specific anodization for abutments, and area-specific anodization for implant collars. Cell proliferation was assessed using a resazurin-based fluorescent dye on days 1, 3, and 7, while cell adhesion was examined using scanning electron microscopy (SEM). Surface topography, roughness, and composition were evaluated for six implant bodies with an anodized rough surface, six anodized implant smooth collars, and six anodized prosthetic abutments. Both HaCats and BM-MSCs showed increased viability over time (p < 0.001) with no statistically significant differences among the different surfaces (p = 0.447 HaCats and p = 0.631 BM-MSCs). SEM analysis revealed an enhanced presence and adhesion of HaCat cells on the anodized surface for the implant collars and an increased adhesion of BM-MSCs on both the anodized and machined surface abutments. The topography characteristics of the treated implants and abutments varied depending on the specific implant region. Chemical analysis confirmed the presence of oxygen, calcium, phosphorus, and sodium on the anodized surfaces. The area-specific anodization process can be utilized to create variable topography, increase the specific surface area, and introduce oxygen, calcium, phosphorus, and sodium to dental implants and abutments. While BM-MSCs and HaCat cells showed similar adhesion and proliferation on anodized and machined surfaces, a positive interaction between anodized Ti6Al4V ELI surfaces and these two cell lines present in the peri-implant mucosa was observed. Due to the limitations of the present study, further research is necessary to confirm these findings.

Human Keratinocytes and Fibroblasts Co-Cultured on Silk Fibroin Scaffolds Exosomally Overrelease Angiogenic and Growth Factors.

Objectives: The optimal healing of skin wounds, deep burns, and chronic ulcers is an important clinical problem. Attempts to solve it have been driving the search for skin equivalents based on synthetic or natural polymers. Methods: Consistent with this endeavor, we used regenerated silk fibroin (SF) from Bombyx mori to produce a novel compound scaffold by welding a 3D carded/hydroentangled SF-microfiber-based nonwoven layer (C/H-3D-SFnw; to support dermis engineering) to an electrospun 2D SF nanofiber layer (ESFN; a basal lamina surrogate). Next, we assessed-via scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, mono- and co-cultures of HaCaT keratinocytes and adult human dermal fibroblasts (HDFs), dsDNA assays, exosome isolation, double-antibody arrays, and angiogenesis assays-whether the C/H-3D-SFnws/ESFNs would allow the reconstitution of a functional human skin analog in vitro. Results: Physical analyses proved that the C/H-3D-SFnws/ESFNs met the requirements for human soft-tissue-like implants. dsDNA assays revealed that co-cultures of HaCaTs (on the 2D ESFN surface) and HDFs (inside the 3D C/H-3D-SFnws) grew more intensely than did the respective monocultures. Double-antibody arrays showed that the CD9+/CD81+ exosomes isolated from the 14-day pooled growth media of HDF and/or HaCaT mono- or co-cultures conveyed 35 distinct angiogenic/growth factors (AGFs). However, versus monocultures' exosomes, HaCaT/HDF co-cultures' exosomes (i) transported larger amounts of 15 AGFs, i.e., PIGF, ANGPT-1, bFGF, Tie-2, Angiogenin, VEGF-A, VEGF-D, TIMP-1/-2, GRO-α/-ß/-γ, IL-1ß, IL-6, IL-8, MMP-9, and MCP-1, and (ii) significantly more strongly stimulated human dermal microvascular endothelial cells to migrate and assemble tubes/nodes in vitro. Conclusions: Our results showed that both cell-cell and cell-SF interactions boosted the exosomal release of AGFs from HaCaTs/HDFs co-cultured on C/H-3D-SFnws/ESFNs. Hence, such exosomes are an asset for prospective clinical applications as they advance cell growth and neoangiogenesis and consequently graft take and skin healing. Moreover, this new integument analog could be instrumental in preclinical and translational studies on human skin pathophysiology and regeneration.

Benzoylaconitine Alleviates Progression of Psoriasis via Suppressing STAT3 Phosphorylation in Keratinocytes.

Psoriasis is a chronic and multifactorial skin disease which is caused by inflammatory infiltrates, keratinocyte hyperproliferation, and accumulation of immune cells. As part of the Aconitum species, Benzoylaconitine (BAC) shows potential antiviral, anti-tumor, and anti-inflammatory effects. In this study, we investigated the effects and mechanisms of BAC on tumor necrosis factor-alpha (TNF-α)/LPS-induced HaCaT keratinocytes in a imiquimod(IMQ)-induced mice model. The results showed that BAC could relieve the symptoms of psoriasis by inhibiting cell proliferation, the release of inflammatory factors, and the accumulation of Th17 cells, while no obvious effect on cell viability and safety was observed both in vitro and in vivo. Additionally, BAC can markedly inhibit the protein and mRNA levels of inflammatory cytokines in TNF-α/LPS-induced HaCaT keratinocytes by inhibiting the phosphorylation of STAT3. In brief, our data indicated that BAC could alleviate the progression of psoriasis and may be a potential therapeutic agent for treating psoriasis in clinical practice.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes.

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

The Cytoprotective Effects of Baicalein on H2O2-Induced ROS by Maintaining Mitochondrial Homeostasis and Cellular Tight Junction in HaCaT Keratinocytes.

Reactive oxygen species (ROS) promote oxidative stress, which directly causes molecular damage and disrupts cellular homeostasis, leading to skin aging. Baicalein, a flavonoid compound isolated from the root of Scutellaria baicalensis Georgi has antioxidant, anticancer, anti-inflammatory, and other medicinal properties. We aimed to investigate the protective effect of baicalein on the disruption of tight junctions and mitochondrial dysfunction caused by H2O2-induced oxidative stress in HaCaT keratinocytes. The cells were pretreated with 20 and 40 µM baicalein followed by treatment with 500 µM H2O2. The results revealed that baicalein exerted antioxidant effects by reducing intracellular ROS production. Baicalein attenuated the degradation of the ECM (MMP-1 and Col1A1) and the disruption of tight junctions (ZO-1, occludin, and claudin-4). In addition, baicalein prevented mitochondrial dysfunction (PGC-1α, PINK1, and Parkin) and restored mitochondrial respiration. Furthermore, baicalein regulated the expression of antioxidant enzymes, including NQO-1 and HO-1, via the Nrf2 signaling pathway. Our data suggest that the cytoprotective effects of baicalein against H2O2-induced oxidative stress may be mediated through the Nrf2/NQO-1/HO-1 signaling pathway. In conclusion, baicalein exerts potent antioxidant effects against H2O2-induced oxidative stress in HaCaT keratinocytes by maintaining mitochondrial homeostasis and cellular tight junctions.

Photoprotective Effects of Dendrobium nobile Lindl. Polysaccharides against UVB-Induced Oxidative Stress and Apoptosis in HaCaT Cells.

Acute ultraviolet (UV)-B radiation is the major external factor causing photodamage. In this study, we aimed to determine the effects of Dendrobium nobile Lindl. polysaccharides (DNPs) on photodamage in HaCaT keratinocytes after UVB irradiation and the underlying mechanisms. We found that DNPs significantly attenuated the decline in the viability and proliferation of HaCaT cells after UVB irradiation. Moreover, DNPs scavenged reactive oxygen species (ROS), improved the activities of endogenous antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, and reduced the levels of malondialdehyde, while partially attenuating cell cycle arrest, suggesting their antioxidant and anti-apoptotic properties. The mitogen-activated protein kinase (MAPK) pathway was found to be important for the attenuation of UVB-induced photodamage in the HaCaT cells. Furthermore, DNPs exerted cytoprotective effects by downregulating UVB-induced ROS-mediated phosphorylation of MAPKs, including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, and by inhibiting p53 expression as well as the apoptotic cascade response. Therefore, DNPs ameliorated UVB-induced oxidative damage and apoptosis in HaCaT cells via the regulation of MAPKs. Our findings thus highlight the Dendrobium nobile Lindl polysaccharides as promising therapeutic candidates for UVB-induced photodamage.

Torilis japonica Extract Suppresses the Induction of Atopic Inflammation.

As one of the major intractable allergic disorders, atopic inflammation is commonly accompanied by itching, dry skin, and inflammation. Atopic inflammation deteriorates the quality of life and has no fundamental cure, so it is crucial to urgently explore and develop natural resources for long-term treatment without any side effects. This study aimed to verify Torilis japonica extract (TJE)'s relieving effect and mechanism against atopic inflammation using skin cells and skin equivalent models, as well as to investigate torilin's effect (obtained from TJE) and other unknown components as marker compounds. Torilin concentration was verified in TJE using high-performance liquid chromatography and analyzed the unknown components using nuclear magnetic resonance spectroscopy. Furthermore, TJE's cytotoxicity, regenerative effect, and cell cycle regulation effects were confirmed using skin cells with atopic inflammation (human dermal fibroblasts and HaCaT keratinocytes) by using TNF-α and IFN-γ treatments. Consequently, TJE was demonstrated to regulate TARC and CTACK expressions as chemokines and those of interleukin-4, -5, and -13 as cytokines related to atopic inflammation. TJE was further confirmed to affect the matrix metalloproteinase-1, -2, and -9 expressions, which are essential in skin damage. Lastly, this study confirmed TJE's relieving effect against atopic inflammation through a 3D skin model and RhCE model using human dermal fibroblasts and HaCaT keratinocytes. These findings on atopic inflammation verified torilin's relieving effects and TJE's other components.

Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.

The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.

Assessment of Some Unsymmetrical Porphyrins as Promising Molecules for Photodynamic Therapy of Cutaneous Disorders.

In order to select for further development novel photosensitizers for photodynamic therapy in cutaneous disorders, three unsymmetrical porphyrins, namely 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.2), 5-(2-hydroxy-5-methoxyphenyl)-10,15,20-tris-(4-carboxymethylphenyl) porphyrin (P3.2), and 5-(2,4-dihydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P4.2), along with their fully symmetrical counterparts 5,10,15,20-tetrakis-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.1) and 5,10,15,20-tetrakis-(4-carboxymethylphenyl) porphyrin (P3.1) were comparatively evaluated. The absorption and fluorescence properties, as well as atomic force microscopy measurements were performed to evaluate the photophysical characteristics as well as morphological and textural properties of the mentioned porphyrins. The cellular uptake of compounds and the effect of photodynamic therapy on the viability, proliferation, and necrosis of human HaCaT keratinocytes, human Hs27 skin fibroblasts, human skin SCL II squamous cell carcinoma, and B16F10 melanoma cells were assessed in vitro, in correlation with the structural and photophysical properties of the investigated porphyrins, and with the predictions regarding diffusion through cell membranes and ADMET properties. All samples were found to be isotropic and self-similar, with slightly different degrees of aggregability, had a relatively low predicted toxicity (class V), and a predicted long half-life after systemic administration. The in vitro study performed on non-malignant and malignant skin-relevant cells highlighted that the asymmetric P2.2 porphyrin qualified among the five investigated porphyrins to be a promising photosensitizer candidate for PDT in skin disorders. P2.2 was shown to accumulate well within cells, and induced by PDT a massive decrease in the number of metabolically active skin cells, partly due to cell death by necrosis. P2.2 had in this respect a better behavior than the symmetric P.2.1 compound and the related asymmetric compound P4.2. The strong action of P2.2-mediated PDT on normal skin cells might be an important drawback for further development of this compound. Meanwhile, the P3.1 and P3.2 compounds were not able to accumulate well in skin cells, and did not elicit significant PDT in vitro. Taken together, our experiments suggest that P2.2 can be a promising candidate for the development of novel photosensitizers for PDT in skin disorders.

Singlet oxygen from endoperoxide initiates an intracellular reactive oxygen species release in HaCaT keratinocytes.

Singlet oxygen (|1O2) is a selective intermediate reactive oxygen species generated naturally in biological systems by light- and non-light mediated processes. Although |1O2 plays an important role in cell signaling and in maintaining homeostasis, it can be toxic due to its ability to diffuse across considerable distances. Several in vitro studies have investigated the pathways by which |1O2 mediates oxidation of biological molecules and potential pathogenesis. However, understanding how singlet oxygen exerts cell injury through the production of subsequent reactive oxygen species remains unexplored. To study this, we used a hydrophobic endoperoxide as a source of |1O2. Endoperoxides are reagents that quantitatively generate singlet oxygen in solution at 35°C by thermal decomposition. Our chemiluminescence and cell viability assay data revealed that |1O2 stimulated a secondary intracellular reactive oxygen species production in a very short time. To determine the source of these reactive oxygen species with endo-peroxide exposure, cells were treated with inhibitors targeting NADPH oxidases and platelet activating factor receptors. Our results showed that addition of the platelet activating factor receptor antagonist, Apafant (WEB2086), alleviated cell injury and hydrogen peroxide levels following endoperoxide stimulation. Furthermore, intracellular calcium assay data demonstrated a potential calcium sensitive production of intracellular reactive oxygen species.

In vitro assessment of the anti-inflammatory and skin-moisturizing effects of Filipendula palmata (Pall.) Maxim. On human keratinocytes and identification of its bioactive phytochemicals.

ETHNOPHARMACOLOGICAL RELEVANCE: The meadowsweet family (genus Filipendula) includes about 30 species, which have been traditionally used in folk medicine to treat various inflammatory diseases. Particularily, F. palmata (Pall.) Maxim. (Siberian meadowsweet) were traditionally and widely used as an ethnic herb in the Oroqen application. AIM OF THE STUDY: Limited studies have been documented on most species, except for two main species, F. ulmaria (L.) Maxim. and F. vulgaris Moench. Thus, this study aimed to investigate the anti-inflammatory and skin-moisturizing effects of 70% ethanolic extract (FPE) of F. palmata on human epidermal keratinocytes. MATERIALS AND METHODS: HaCaT keratinocytes were treated with FPE under different conditions. Quantitative real time-PCR, enzyme-linked immunosorbent assay, western blotting methods were used to evaluate the effect and molecular mechanism of the cells treated with FPE. The bioactive compounds in FPE, which are responsible for biological activities, was explored using mass spectrometric analysis. RESULTS: FPE did not show a cytotoxic effect on the cells at concentrations below 200 µg/mL. FPE significantly suppressed the intracellular reactive oxygen species and mitochondrial superoxide of inflamed HaCaT cells induced by tumor necrosis factor-α and interferon-γ (T + I) and inflammatory chemokine genes and proteins, such as CC chemokine ligands (CCL5, CCL17, and CCL27) and CXC chemokine ligand (CXCL8). These anti-inflammatory activities of FPE were mediated by the downregulation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. In normal HaCaT cells, FPE significantly promoted the production of hyaluronic acid (HA) via the downregulation of hyaluronidase (HYAL1 and HYAL2) and upregulation of hyaluronic acid synthase (HAS1, HAS2, and HAS3) genes, and these effects seemed to be associated with the PI3K/Akt/NF-κB signaling. Ultraperformance liquid chromatography-tandem mass spectrometry indicated that FPE contains four flavonoids, including (+)-catechin, miquelianin, scutellarin, and quercitrin, as its major phytochemicals. Finally, we demonstrated that miquelianin and quercitrin contribute partially to the anti-inflammatory and HA-producing activity of FPE without cytotoxic effects on HaCaT cells. CONCLUSIONS: Our findings suggest that topical applications of FPE can be utilized as an alternative therapy for treating skin inflammation. Additionally, our findings serve as a reference in applying FPE as a functional ingredient to treat inflammatory skin diseases and promote skin health.

Fucoidan Isolated from Sargassum confusum Suppresses Inflammatory Responses and Oxidative Stress in TNF-α/IFN-γ- Stimulated HaCaT Keratinocytes by Activating Nrf2/HO-1 Signaling Pathway.

Recent studies have revealed that marine brown seaweeds contain numerous bioactive compounds which exhibit various bioactivities. The present study investigated the effect of low molecular weight fucoidan (SCF) isolated from Sargassum confusum, a brown alga, on inflammatory responses and oxidative stress in HaCaT keratinocytes stimulated by tumor necrosis factor (TNF)-α/interferon (IFN)-γ. SCF significantly increased the cell viability while decreasing the intracellular reactive oxygen species (ROS) production in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. In addition, SCF effectively reduced inflammatory cytokines (interleukin (IL)-1ß, IL-6, IL-8, IL-13, TNF-α, and IFN-γ) and chemokines (Eotaxin, macrophage-derived chemokine (MDC), regulated on activation, normal T cell expressed and secreted (RANTES), and thymus and activation-regulated chemokine (TARC)) expression, by down-regulating the expression of epithelial and epidermal innate cytokines (IL-25, IL-33, and thymic stromal lymphopoietin (TSLP)). Furthermore, SCF suppressed the activation of TNF-α/IFN-γ-stimulated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, while activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. The cytoprotective effect of SCF against TNF-α/IFN-γ stimulation was considerably reduced upon inhibition of HO-1 activity by ZnPP. Overall, these results suggest that SCF effectively suppressed inflammatory responses and oxidative stress in TNF-α/IFN-γ-stimulated HaCaT keratinocytes via activating the Nrf2/HO-1 signaling pathway.

In vitro biocompatibility and wound healing properties of latex proteins dressing.

The chronification of ulcers or sores may result in a dramatic outcome such as amputation. Currently, the search for plant based treatments of various diseases/disorders, including complicated ones, is getting the attention of researchers worldwide. The soluble latex protein fraction (CpLP) obtained from Calotropis procera (Apocynaceae) was previously demonstrated to accelerate wound healing by topical application or when incorporated in a polyvinyl alcohol biomembrane (BioMemCpLP). Here, in vitro assays were performed to investigate and characterize the biocompatibility and bioactivity of latex proteins dressing. Macrophages (RAW 264.7), fibroblasts (L929) and keratinocytes (HaCaT) cell lines were used to evaluate the effect of CpLP. These cell lines were exposed to concentrations of CpLP comparable to those found in BioMemCpLP during 24-72 h. The cytotoxicity, proliferation, release of wound healing mediators (TGF-ß, VEGF, IL-10, IL-6, IL-1ß, TNF-α and NO) and migration of cells (E-cadherin and ß-catenin) incubated with CpLP was assessed and the cell adhesion to BioMemCpLP as well. The results showed that CpLP has no cytotoxic effects. It induced a suitable balance between pro- and anti-inflammatory mediators, enhanced proliferation and re-epithelialization in all cell lines, but the intensity of each effect was different at various doses in all cell strains. The BioMemCpLP stimulated cell adhesion to PVA substrate. The CpLP-PVA based biomembrane can be a good option for healing of different wounds.