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Nonmelanoma skin cancers (NMSC) are the most common skin cancers, and about 5.4 million people are diagnosed each year in the United States. A newly developed T-lymphokine-activated killer cell-originated protein kinase (TOPK) inhibitor, HI-TOPK-032, is effective in suppressing colon cancer cell growth, inducing the apoptosis of colon cancer cells and ultraviolet (UV) light-induced squamous cell carcinoma (SCC). This study aimed to investigate the physicochemical properties, permeation behavior, and cytotoxicity potential of HI-TOPK-032 prior to the development of a suitable topical formulation for targeted skin drug delivery. Techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), X-ray powder diffraction (XRPD), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and Fourier transform infrared microscopy were used to characterize HI-TOPK-032. The dose effect of HI-TOPK-032 on in vitro cell viability was evaluated using a 2D cell culture of the human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEKs). Transepithelial electrical resistance (TEER) at the air–liquid interface as a function of dose and time was measured on the HaCAT human skin cell line. The membrane permeation behavior of HI-TOPK-032 was tested using the Strat-M® synthetic biomimetic membrane with an in vitro Franz cell diffusion system. The physicochemical evaluation results confirmed the amorphous nature of the drug and the homogeneity of the sample with all characteristic chemical peaks. The in vitro cell viability assay results confirmed 100% cell viability up to 10 µM of HI-TOPK-032. Further, a rapid, specific, precise, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for the quantitative estimation of HI-TOPK-032 was developed. This is the first systematic and comprehensive characterization of HI-TOPK-032 and a report of these findings. [ABSTRACT FROM AUTHOR]

• vera gel, B. frutescens aqueous and gel extracts showed wound healing effects. • Combining A. vera gel and B. frutescens aqueous extract increased wound healing. • Combining A. vera gel and B. frutescens gel extract did not increase wound healing. Many skin conditions, including wounds, have been treated traditionally with plant-based medicines such as Aloe vera and Bulbine frutescens. The gel from the leaves of A. vera has shown potential in burn wound treatment and other skin conditions, while B. frutescens exhibited accelerated wound healing properties in previous in vitro and in vivo studies. This study investigated whether the skin wound healing properties of A. vera gel could be enhanced by combining it with each of two different B. frutescens extracts (aqueous and gel). Firstly, the in vitro cytotoxicity of the selected plant materials was measured on HaCaT and 84BR cells by means of methylthiazol tetrazolium (MTT) assays. The in vitro re-epithelialization potential or wound healing properties were determined by means of the scratch assay on HaCaT cell layers. The wound closure percentage and migration rate values were measured in an untreated control group as well as after 24 h treatment with the individual plant materials as well as combinations thereof. The scratch assay results showed that the wound closure percentage and migration rate values for each of the selected plant materials individually were higher than that of the untreated control group. When A. vera gel (0.25 mg/ml) was combined with B. frutescens aqueous extract in three different concentrations (0.25, 0.50 and 0.75 mg/ml), all the combination ratios exhibited higher wound closure percentage and migration rate values than that of the control group and it was also higher than the effects of each individual plant material alone. On the other hand, combination of A. vera gel with B. frutescens gel extract did not show increased wound healing effects as compared to that of the control group nor the individual plant materials. It can be concluded that the wound healing properties of A. vera gel can be increased by combining it with B. frutescens aqueous extract. [ABSTRACT FROM AUTHOR]

Lavandula austroapennina N.G. Passal., Tundis and Upon has recently been described as a new species endemic to the southern Apennines (Italy). Locally, this species has a long ethnobotanical tradition of use for curative and decoration purposes and has been the protagonist of a flourishing essential oil production chain. Currently, while this tradition has long since ended, attention to the species is necessary, with a view to enhancing marginal and rural areas, as a recovery of a precious resource to (i) get insights into its (poly)phenolic fraction and (ii) address new and innovative uses of all its organs in various application fields (e.g., cosmeceutical sector). Therefore, after field sampling and dissection of its organs (i.e., corolla, calyx, leaf, stem and root), the latter, previously deterpenated and defatted, were subjected to accelerated ultrasound extraction and the related alcoholic extracts were obtained. Chemical composition, explored by UHPLC-QqTOF-MS/MS, and the following multivariate data analysis showed that the hydroxycinnamoyl derivatives are abundant in the leaf, stem and root, while flavonoids are more present in corolla and calyx. In particular, coumaroyl flavonoids with glyconic portion containing also hexuronyl moieties differentiated corolla organ, while yunnaneic acid D isomers and esculin distinguished root. When antiradical and reducing properties were evaluated (by means of ABTS, DPPH and PFRAP tests), a similar clustering of organs was achieved and the marked antioxidant efficacy of leaf, stem and root extracts was found. Thus, following cytotoxicity screening by MTT test on HaCaT keratinocytes, the protective effects of the organ extracts were assessed by wound closure observed after the scratch test. In addition, the extracts from corolla, leaf and stem were particularly active at low doses inducing rapid wound closure on HaCaT cells at a concentration of 1 μg/mL. The diversity in (poly)phenols of each organ and the promising bioactivity preliminarily assessed suggest further investigation to be carried out to fully recover and valorize this precious endemic vascular plant. [ABSTRACT FROM AUTHOR]

The study was aimed at developing topical gel containing papain as the drug and assessing its antipsoriatic activity. Carbopol was used as the polymer and drug-excipient compatibility was ascertained by infrared spectroscopy (Fourier transform) and differential scanning calorimetry. All prepared formulations were evaluated for viscosity, pH, homogeneity, extrudability, spreadability, in vitro diffusion and release kinetics. Anti-proliferative activity was determined on HaCaT cell line. The optimized formulation, F2 exhibited pH 6.8, optimum viscosity, extrudability and spreadability for convenient use. The optimized formulation followed Higuchi kinetic model with release mechanism being super-case two transport. The anti-proliferative activity of optimized formulation on HaCaT cell line showed only 3.9 % cells to be viable with respect to control confirming its efficacy in treating psoriasis. The gel exhibited stability under accelerated stability conditions. It can be inferred from the study that papain gel can be effectively used for managing psoriasis and its single application would give prolonged action. [ABSTRACT FROM AUTHOR]

Cell migration is a process that underlies the development and maintenance of multicellular organisms, with profound implications in various pathologies. The study of cell migration is fundamental in various fields of basic biology and pharmaceutical development. Wound healing assay is an indirect way to assess cell migration. Conventional methods, such as the scratch test, are inexpensive and easy to execute but have the disadvantages of being poorly reproducible and difficult to perform on a high-throughput scale. Meanwhile, commercial strategies are expensive. In the present work, we developed a lab-made wound healing assay device that is inexpensive, easy to handle, and reproducible. We designed 3D-printed stoppers compatible with cell culture in 96-well plates. These stoppers did not affect HaCaT cells viability. The stopper-produced initial wound size was reproducible on a high-throughput scale. Also, stoppers demonstrated their effectiveness to evaluate cell migration and allowed differentiating treatments with and without fetal bovine serum. Finally, proliferation assay was determined in this wound healing model. In conclusion, our lab-made 3D-printed stopper-based assay is a more economical alternative to currently available strategies for developing reproducible, high-throughput assays to assess cell migration and proliferation.

In this study, waste biomass of industrial enzyme producer Rhizomucor miehei was used as an alternative source for fungal chitosan production. Fungal chitosan was obtained from mycelial waste at the deproteinization and deacetylation stages at different temperatures and times of alkali and acid treatment with different concentrations of sodium hydroxide (1–3 N) and acetic acid (2–4 %), respectively. High chitosan yield (70.6 mg/g) was reached at the optimized conditions when the concentration of NaOH (3 N), the deproteinization time (20 mins) and temperature (95℃), the concentration of acetic acid (4 %), the deacetylation time (6 hrs) and temperature (85℃). The fungal chitosan with a molecular weight of 14 kDa showed a deacetylation degree of 78 %. The XRD pattern showed two peaks at 2θ values of 9° and 19°. The antimicrobial activity of the fungal chitosan showed broad spectrum at low concentrations (125–250 µg/mL) against the test microorganisms. As wound healing activity, after five days post-scratching, the HaCaT cells cultured in the presence of fungal chitosan demonstrated a notable 57 % increase in migration rate. These findings underscored the efficacy of the produced fungal chitosan, highlighting its promising biological activities. This is the first report to evaluate the use of waste Rhizomucor miehei biomass as alternative source for chitosan. [Display omitted] • The mycelial biomass (R. miehei) as an industrial waste was used as an alternative chitosan source. • The fungal chitosan extraction conditions from the waste were statistically optimized. • The obtained chitosan showed a broad-spectrum antimicrobial activity at low concentrations. • The fungal chitosan extracted from the waste may be used as a safe, eco-friendly natural antimicrobial and wound healing bioactive compound in various industries. [ABSTRACT FROM AUTHOR]

[Display omitted] • There is paucity of available preclinical psoriasis models that exactly mimic the state of human psoriasis. • The imiquimod-induced psoriasis is a widely used and acceptable animal model for psoriasis. • The newer and more trustworthy techniques such as 3D skin models, and zebrafish models for future purposes have also been discussed in this article. Psoriasis is a chronic, inflammatory, papulosquamous, noncontagious disease characterized by scaly, demarcated erythematous plaque, affecting skin, nails, and scalp. The IL-23/Th17 axis is the main operator in the development of psoriasis. Psoriasis is affecting worldwide, and new treatment options are urgently needed. Various local and systemic treatments are available for psoriasis but they only provide symptomatic relief because of numerous unknown mechanisms. Clinical trials demand overwhelming resources; therefore, drug development predominantly depends on the in-vivo , in-vitro, and ex-vivo techniques. Immediate attention is required to develop experimental techniques that completely imitate human psoriasis to assist drug development. This review portrays the various in-vivo, in-vitro, and ex-vivo techniques used in psoriasis research. It describes these techniques' characteristics, pathological presentations, and mechanisms. The experimental techniques of psoriasis provide significant information on disease progression mechanisms and possible therapeutic targets. However, until now, it has been challenging to invent a timely, affordable model that precisely imitates a human disease. Only the xenotransplantation model is reckoned as the closer, that mimics the complete genetic, and immunopathogenic event. Imiquimod-induced psoriasis and HaCat cell lines are popular among researchers because of their convenience, ease of use, and cost-effectiveness. There need to further improve the experimental techniques to best serve the disease imitation and meet the research goal. [ABSTRACT FROM AUTHOR]

Nonmelanoma skin cancers (NMSC) are the most common skin cancers, and about 5.4 million people are diagnosed each year in the United States. A newly developed T-lymphokine-activated killer cell-originated protein kinase (TOPK) inhibitor, HI-TOPK-032, is effective in suppressing colon cancer cell growth, inducing the apoptosis of colon cancer cells and ultraviolet (UV) light-induced squamous cell carcinoma (SCC). This study aimed to investigate the physicochemical properties, permeation behavior, and cytotoxicity potential of HI-TOPK-032 prior to the development of a suitable topical formulation for targeted skin drug delivery. Techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), X-ray powder diffraction (XRPD), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and Fourier transform infrared microscopy were used to characterize HI-TOPK-032. The dose effect of HI-TOPK-032 on in vitro cell viability was evaluated using a 2D cell culture of the human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEKs). Transepithelial electrical resistance (TEER) at the air–liquid interface as a function of dose and time was measured on the HaCAT human skin cell line. The membrane permeation behavior of HI-TOPK-032 was tested using the Strat-M® synthetic biomimetic membrane with an in vitro Franz cell diffusion system. The physicochemical evaluation results confirmed the amorphous nature of the drug and the homogeneity of the sample with all characteristic chemical peaks. The in vitro cell viability assay results confirmed 100% cell viability up to 10 µM of HI-TOPK-032. Further, a rapid, specific, precise, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for the quantitative estimation of HI-TOPK-032 was developed. This is the first systematic and comprehensive characterization of HI-TOPK-032 and a report of these findings.

The abundance of cerium in natural resources, its ability to absorb UV light while being transparent to visible light, as well as low photocatalytic activity make ceria (CeO2) a promising candidate for UV filter material in sunscreens. Doping with different elements can further decrease ceria catalytic and photocatalytic activity, thus preventing the degradation of other sunscreen ingredients. In this work, pure and zinc-doped ceria nanoparticles were prepared by a simple and environmentally benign hydrothermal synthesis, and characterized using various techniques. Fine ceria and doped ceria nanoparticles with particle sizes of 6.1±0.9 and 4.2±0.4 nm were prepared. In both samples, cubic ceria was the only crystalline phase, but the homogeneous distribution of zinc in the doped sample was confirmed by energy dispersive X-ray spectrometry. Nanoparticles exhibited transparency in the visible region and absorbance in the UV region with band gap of 3.23 to 3.14 eV for pure and doped sample, respectively. The oxidation stability time, determined through Castor oil oxidation process, was 23 hours for the pure and 15 hours for the doped sample, which is quite satisfactory. In vitro cytotoxicity study showed that the prepared nanoparticles were well tolerated by human skin keratinocytes (HaCaT cell line) with no significant differences in skin cells viability. However, further investigations on in vivo systems are necessary to reach a firm conclusion regarding the toxicity of ceria and doped ceria nanoparticles, and other potential dopants should be considered for improvement of ceria properties for sunscreen application.

Natural polymer-based scaffolds are generally considered as favourable matrices for the adhesion and growth of cells in tissue repair. One of the most popular materials in this respect is silk fibroin, known for its wide usage in biomedical applications. This work focuses on the development of electrospun scaffolds based on poly(ε-caprolactone) (PCL) and silk fibroin (SF) evaluated regarding the SF effect on their morphology, surface wetting ability, thermal properties, and HaCaT model cell line biocompatibility. The study revealed that the lowest PCL/SF concentration resulted in highest bead-like morphology formation, relatively thick fibers with the presence of random beads in the case of PCL, while uniform and thinner fibers in the case of increasing PCL/SF content scaffolds. The addition of SF reduced the degree of crystallinity in the PCL due to the less organized crystal structure, and decreased its thermal stability. Both SEM and MTT analyses showed cell presence on all scaffolds three days after cell seeding. Although SF improved PCL hydrophilicity, as shown quantitatively by the MTT assay for improved cytocompatibility properties, more structured electrospun PCL/SF scaffold strategies are required.

Lavandula austroapennina N.G. Passal., Tundis and Upon has recently been described as a new species endemic to the southern Apennines (Italy). Locally, this species has a long ethnobotanical tradition of use for curative and decoration purposes and has been the protagonist of a flourishing essential oil production chain. Currently, while this tradition has long since ended, attention to the species is necessary, with a view to enhancing marginal and rural areas, as a recovery of a precious resource to (i) get insights into its (poly)phenolic fraction and (ii) address new and innovative uses of all its organs in various application fields (e.g., cosmeceutical sector). Therefore, after field sampling and dissection of its organs (i.e., corolla, calyx, leaf, stem and root), the latter, previously deterpenated and defatted, were subjected to accelerated ultrasound extraction and the related alcoholic extracts were obtained. Chemical composition, explored by UHPLC-QqTOF-MS/MS, and the following multivariate data analysis showed that the hydroxycinnamoyl derivatives are abundant in the leaf, stem and root, while flavonoids are more present in corolla and calyx. In particular, coumaroyl flavonoids with glyconic portion containing also hexuronyl moieties differentiated corolla organ, while yunnaneic acid D isomers and esculin distinguished root. When antiradical and reducing properties were evaluated (by means of ABTS, DPPH and PFRAP tests), a similar clustering of organs was achieved and the marked antioxidant efficacy of leaf, stem and root extracts was found. Thus, following cytotoxicity screening by MTT test on HaCaT keratinocytes, the protective effects of the organ extracts were assessed by wound closure observed after the scratch test. In addition, the extracts from corolla, leaf and stem were particularly active at low doses inducing rapid wound closure on HaCaT cells at a concentration of 1 μg/mL. The diversity in (poly)phenols of each organ and the promising bioactivity preliminarily assessed suggest further investigation to be carried out to fully recover and valorize this precious endemic vascular plant.

The abundance of cerium in natural resources, its ability to absorb UV light while being transparent to visible light, as well as low photocatalytic activity make ceria (CeO2) a promising candidate for UV filter material in sunscreens. Doping with different elements can further decrease ceria catalytic and photocatalytic activity, thus preventing the degradation of other sunscreen ingredients. In this work, pure and zinc-doped ceria nanoparticles were prepared by a simple and environmentally benign hydrothermal synthesis, and characterized using various techniques. Fine ceria and doped ceria nanoparticles with particle sizes of 6.1±0.9 and 4.2±0.4 nm were prepared. In both samples, cubic ceria was the only crystalline phase, but the homogeneous distribution of zinc in the doped sample was confirmed by energy dispersive X-ray spectrometry. Nanoparticles exhibited transparency in the visible region and absorbance in the UV region with band gap of 3.23 to 3.14 eV for pure and doped sample, respectively. The oxidation stability time, determined through Castor oil oxidation process, was 23 hours for the pure and 15 hours for the doped sample, which is quite satisfactory. In vitro cytotoxicity study showed that the prepared nanoparticles were well tolerated by human skin keratinocytes (HaCaT cell line) with no significant differences in skin cells viability. However, further investigations on in vivo systems are necessary to reach a firm conclusion regarding the toxicity of ceria and doped ceria nanoparticles, and other potential dopants should be considered for improvement of ceria properties for sunscreen application. [ABSTRACT FROM AUTHOR]

Natural polymer-based scaffolds are generally considered as favourable matrices for the adhesion and growth of cells in tissue repair. One of the most popular materials in this respect is silk fibroin, known for its wide usage in biomedical applications. This work focuses on the development of electrospun scaffolds based on poly(e-caprolactone) (PCL) and silk fibroin (SF) evaluated regarding the SF effect on their morphology, surface wetting ability, thermal properties, and HaCaT model cell line biocompatibility. The study revealed that the lowest PCL/SF concentration resulted in highest bead-like morphology formation, relatively thick fibers with the presence of random beads in the case of PCL, while uniform and thinner fibers in the case of increasing PCL/SF content scaffolds. The addition of SF reduced the degree of crystallinity in the PCL due to the less organized crystal structure, and decreased its thermal stability. Both SEM and MTT analyses showed cell presence on all scaffolds three days after cell seeding. Although SF improved PCL hydrophilicity, as shown quantitatively by the MTT assay for improved cytocompatibility properties, more structured electrospun PCL/SF scaffold strategies are required. [ABSTRACT FROM AUTHOR]

The study deals with the formulation of biological macromolecule chitosan-based Tween 80-coated nanoparticles to deliver gallic acid and rutin into the skin for psoriasis treatment. To optimize the nanoformulations for minimum particle size and maximum possible entrapment efficiency (dependent variables), 32 full factorial design was used to optimize the formulation. Concentration of Tween 80 and chitosan were independent variables. The optimized chitosan nanoformulation of gallic acid, rutin and their combination was explored against psoriasis using in vitro methods and HaCaT cell line. The results indicated excellent entrapment of drug in the chitosan polymer and Higuchi model of drug release. The optimized encapsulated Tween 80-coated chitosan nanoparticles containing combination of gallic acid and rutin showed the reduced keratinocyte hyperproliferation, antioxidant, anti-inflammatory and antimicrobial activity even better than the chitosan nanoparticles containing gallic acid and rutin individually in the nanoparticles. Anti-psoriasis like activity of Tween 80-coated nanoformulations of gallic acid may be attributed to faster penetration of the drug by increased permeation and fusion of drug molecules. Thus, the present in vitro investigation indicates that chitosan-based nanoformulations hold promising potential in the treatment of psoriasis. The activity was increased once we combine gallic acid and rutin. [ABSTRACT FROM AUTHOR]

In this work, we developed a solid lipid nanoparticle (SLN) formulation with (+)-limonene 1,2-epoxide and glycerol monostearate (Lim-SLNs), stabilized with Poloxamer® 188 in aqueous dispersion to modify the release profile of the loaded monoterpene derivative. We also evaluated the role of SLNs in lipid peroxidation and cytotoxicity in a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (the HaCaT cell line). For the cell viability assay, the colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. Lim-SLNs with a loading capacity and encapsulation efficiency of 0.39% and 63%, respectively, were produced by high pressure homogenization. A mean particle size of 194 ± 3.4 nm and polydispersity index of 0.244 were recorded for the loaded Lim-SLNs, as compared to 203 ± 1.5 nm (PI 0.213) for the non-loaded (blank) SLNs. The loading of the monoterpene derivative into glycerol monostearate SLNs fitted into the zero-order kinetics, and ameliorated both lipid peroxidation and cytotoxicity in a keratinocyte cell line. A promising formulation for antioxidant and anti-tumoral activities is here proposed.

In this paper, the bioactive compound artemisinin (ART) was evaluated by instrumental techniques, regarding its thermal stability and mechanism of decomposition, but for the biological activity as well, using in vitro techniques, namely the cytotoxic activity evaluation on two cell types (HaCaT and A375) employing the MTT proliferation method, and the antioxidant activity (AOA) using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The results of the isoconversional kinetic study first lead to an indication of complex decomposition mechanism, which was later confirmed by the modified nonparametric kinetics (NPK) method. According to the NPK method, it was shown that ART is mainly degraded by the involvement of two parallel processes, with different energetic contributions that are due to chemical and physical transformations. However, the estimated mean apparent activation energy yields similar results with all four kinetic methods, ranging between 61.3 and 68.7 kJ mol−1, confirming the low thermal stability of the compound, which can be explained by the presence of reactive functional groups, such as the peroxide one. Regarding the biological activity of ART, it was shown that it can be used to induce cell growth arrest on human melanoma cell line A375, but affecting in a smaller measure the viability of HaCaT cell line, while the AOA is negligible, in comparison with standard antioxidants. [ABSTRACT FROM AUTHOR]

Loss of p53 function via mutation is a very common cause of human cancers. Recent studies have provided evidence on presence of self aggregated p53 in cancer cells leading to its altered functions towards cause of cancer. The general notion has been that mutated p53 exposes adhesive sites that promote self aggregation, however a complete mechanistic understanding to this has been lacking. We embarked on the present study towards exploring the differential aggregation pattern in cells expressing mutated TP53 (HaCaT keratinocytes) vs those expressing the wild type copy of the p53 protein (A549 lung cancer cell line). The studies led us to interesting observation that formation of p53 protein aggregates is not always associated with TP53 mutation. The A549 lung cancer cells, having wild type TP53, showed the appearance of p53 protein aggregates, while no protein aggregates were observed in normal HaCaT keratinocytes carrying mutant TP53. We went on to study the effect of blocking protein aggregation by emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) and figured that inhibiting p53 protein aggregation can elevate the level of autophagy in A549 lung cancer cell line while there is no significant effect on autophagy in normal non-cancerous HaCaT cells. Moreover, ATG5 was found to be coaggregated with p53 aggregates which dissociated after emodin treatment, indicating further induction of autophagy in A549 cells only. From these observations, we conclude that the increased level of autophagy might be the mechanism for the removal of p53 protein aggregates which restores p53 function in A549 cells after emodin treatment .This encourages further studies towards deciphering related mechanistic aspects vis-à-vis potential therapeutic strategies against cancer. [ABSTRACT FROM AUTHOR]

Earthworm has a long history of being used for medical purposes in many countries. This study investigated the therapeutic effects of earthworm extract (G-90′) from head-regenerating tissue of Eisenia fetida on wound healing process in vitro and in vivo . Among three salting out parts (ES1–ES3) of G-90′, ES2 displayed the significant wound healing ability via promoting proliferation of fibroblast and keratinocyte as well as stimulating the expression of fibroblast growth factor and vascular endothelial growth factor in wound area. The wound healing-specific proteins in ES2 were further analysed by “bottom-up” proteomic analytic method. Two proteins in ES2 were identified through “bottom-up” analysis, but their effects on wound healing process remains enigmatic. The bioactive proteins (ES2) in G-90′ enhance the proliferative phase in acute wound healing process, providing a new concept for transforming this natural material for use in wound therapy. [ABSTRACT FROM AUTHOR]

Bacterial infection is an important factor that can trigger and exacerbate psoriasis. The protein triggering receptor expressed on myeloid cells type-1 (TREM-1) is overexpressed in psoriasis and decreased after a successful treatment. Hypoxia inducible factor-1α (HIF-1α), subunit of the transcription factor HIF-1, has participated in angiogenesis and inflammation in psoriasis. Increased expressions of TREM-1 and HIF-1α are associated with the infection of microbial pathogens. However, the association between TREM-1 and HIF-1α still needs to be elucidated. Results of immunofluorescence showed an overexpression of TREM-1 and HIF-1α in HaCaT keratinocytes exposed to 1 µg/mL of lipopolysaccharide (LPS). Particularly, silencing of TREM-1 expression by siRNA suppressed the inducible effect of LPS on phosphoinositide 3-kinase (PI3 K)/Akt, the critical transduction mediator, and HIF-1α. Furthermore, the PI3 K inhibitor wortmannin effectively blocked the increased level of HIF-1α induced by LPS. However, there was no significant change in LPS-induced expression of TREM-1. Expressions of TREM-1, HIF-1α, and phosphorylated Akt proteins were further examined by real-time PCR and Western blot, respectively. Our data suggest that TREM-1 and HIF-1α are expressed on keratinocytes and could be upregulated by bacterial infection. Moreover, LPS-induced TREM-1 has an ability to mediate the expression of HIF-1α in HaCaT cells through the PI3 K/Akt pathway. Our study provides new insights into the possible mechanism of TREM-1 and HIF-1α in psoriasis. [ABSTRACT FROM AUTHOR]

The leaf essential oils of Cymbopogon citratus L. and Cymbopogon nardus L.(Poaceae) from Togo were steam-distilled, analyzed for percentage composition and investigated in vitro for their potential cytotoxic activity on human epidermic cell line HaCat. The percentage composition showed that the main constituents of essential oils samples were respectively geranial (45.2%), neral(32.4%) and myrcène (10.2%) for C. citratus essential oil and citronellal (35.5%), geraniol (27.9%) and citronellol (10.7%) for that of C. nardus. The in vitro cytotoxicity bioassays on human epidermic cell line HaCaT revealed that the toxicityof the essential oil from C. citratus (IC50: 150 μL.mL-1) was higher than that of the essential oil from C. nardus (IC50: 450 μL.mL-1). Pure commercial neral, geranial, and citronellal standards showed respectively the following IC50 values: 100, 250 and 300 μL.mL-1). Conversely, pure citronellol standard appeared almost non-toxic (IC50>1000 μL.mL-1), proving the major role played in synergyby neral and geranial in the overall toxicity showed by the citratus oil sample tested in this work.

Background: Pectin is a heterogeneous polysaccharide mainly present in citrus fruits and has different biological activities. Objective: High molecular weight Citrus Pectin and modified citrus pectin (MCP) were tested for their cytotoxic, anti-proliferative, and anti-oxidant activity. Methods: The cytotoxicity of pectin was studied against HaCaT cell line (human keratinocyte cell line) using Trypan blue method and LDH-cytotoxicity assay. Anti-proliferative activity was assayed using a WST-1 proliferation kit. Antioxidant activity was determined using the DPPH scavenging assay. Results: MCP and Pectin both reduced the viability of HaCaT cells in a dose dependent manner; however, MCP was found to be more cytotoxic than high molecular weight citrus pectin since it had a lower IC50 (300ug/ul). At non-cytotoxic concentrations, the viability of cells decreased with increase of concentration of MCP as determined by the WST-1. MCP exhibited a higher antioxidant effect than pectin (SC50 at a concentration range between 2 and 4mg/ml). Conclusion: This study suggests that MCP exhibits a stronger cytotoxic and anti-proliferative effect on HaCaT cell line than pectin. The most probable explanation of this observation is the different effects due to the variable molecular weight and exposed side-chains of MCP and high molecular weight citrus pectin. [ABSTRACT FROM AUTHOR]

Bioremediation has proven to be the most desirable and cost effective method to counter textile dye pollution. Hairy roots (HRs) of Ipomoea carnea J. were tested for decolourization of 25 textile azo dyes, out of which >90% decolourization was observed in 15 dyes. A diazo dye, Acid Red 114 was decolourized to >98% and hence, was chosen as the model dye. A significant increase in the activities of oxidoreductive enzymes was observed during decolourization of AR114. The phytodegradation of AR114 was confirmed by HPLC, UV–vis and FTIR spectroscopy. The possible metabolites were identified by GCMS as 4- aminobenzene sulfonic acid 2-methylaniline and 4- aminophenyl 4-ethyl benzene sulfonate and a probable pathway for the biodegradation of AR114 has been proposed. The nontoxic nature of the metabolites and toxicity of AR114 was confirmed by cytotoxicity tests on human keratinocyte cell line (HaCaT). When HaCaT cells were treated separately with 150 μg mL −1 of AR114 and metabolites, MTT assay showed 50% and ≈100% viability respectively. Furthermore, flow cytometry data showed that, as compared to control, the cells in G2-M and death phase increased by 2.4 and 3.6 folds respectively on treatment with AR114 but remained unaltered in cells treated with metabolites. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S -transferases (GST). Here, we describe an LC–MS (liquid chromatography–mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24 h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization. [ABSTRACT FROM AUTHOR]

The leaf essential oils of Cymbopogon citratus L. and Cymbopogon nardus L. (Poaceae) from Togo were steam-distilled, analyzed for percentage composition and investigated in vitro for their potential cytotoxic activity on human epidermic cell line HaCat. The percentage composition showed that the main constituents of essential oils samples were respectively geranial (45.2%), neral (32.4%) and myrc¨ne (10.2%) for C. citratus essential oil and citronellal (35.5%), geraniol (27.9%) and citronellol (10.7%) for that of C. nardus. The in vitro cytotoxicity bioassays on human epidermic cell line HaCaT revealed that the toxicity of the essential oil from C.citratus (IC50: 150 µL.mL-1) was higher than that of the essential oil from C.nardus (IC50: 450 µL.mL-1). Pure commercial neral, geranial, and citronellal standards showed respectively the following IC50 values: 100, 250 and 300 µL.mL-1). Conversely, pure citronellol standard appeared almost non-toxic (IC50>1000 µL.mL-1), proving the major role played in synergy by neral and geranial in the overall toxicity showed by the citratus oil sample tested in this work.

Arsenic toxicity may lead to skin manifestations and arsenic accumulation in keratinised tissue. Thus human keratinocytes has been extensively used to study dermal effects of arsenic exposure. The present study was aimed to investigate time and dose-dependent effects of arsenic using HaCaT cell line. Another major focus of the study was to evaluate if treatment with monoisoamyl dimercaptosuccinic acid (MiADMSA) offers protection against arsenic-induced oxidative stress and apoptotic cell death using HaCaT cells. HaCaT cell lines were incubated to three different concentrations of arsenic (10, 30 and 50 μM) for 24 h to identify the toxic dose by measuring oxidative stress variables. Later, MiADMSA pre-incubation for an hour preceded arsenic exposure (30 μM). We evaluated cell morphology, lactate dehydrogenase, glutathione linked enzyme and antioxidant enzyme activities to measure oxidative stress status, while MTT assay and caspase 9 and 3 levels were determined for cell viability and apoptotic status. The present study suggests arsenic-induced toxicity in a concentration-dependant manner. Arsenic also caused a significant increase in lactate dehydrogenase accompanied by an elevated antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and caspase activity). Interestingly, pre-treatment of cell with MiADMSA elicited significant protection against arsenic-induced oxidative stress and apoptotic cell death. The present findings are of clinical relevance and suggest MiADMSA to be a promising candidate in protecting skin against arsenic-induced toxic effects, which need further exploration using in vivo experimental models. [ABSTRACT FROM AUTHOR]

Abstract: For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products designed to come in direct contact with the skin. In this study we investigated the effects of Ag NPs on skin using the human-derived keratinocyte HaCaT cell line model. Ag NPs caused a concentration- and time-dependent decrease of cell viability, with IC50 values of 6.8±1.3μM (MTT assay) and 12±1.2μM (SRB assay) after 7days of contact. A 24h treatment, followed by a 6day recovery period in Ag NPs-free medium, reduced cell viability with almost the same potency (IC50s of 15.3±4.6 and 35±20μM, MTT and SRB assays, respectively). Under these conditions, no evidence of induction of necrotic events (propidium iodide assay) was found. Apocynin, NADPH-oxidase inhibitor, or N(G)-monomethyl-l-argynine, nitric oxide synthase inhibitor, did not prevent NPs-induced reduction of cell viability. TEM analysis of cells exposed to NPs for 24h revealed alteration of nuclear morphology but only a marginal presence of individual NPs inside the cells. These results demonstrate that on HaCaT keratinocytes a relatively short time of contact with Ag NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag NPs internalization. [Copyright &y& Elsevier]

Essential oil extracted (4.4% in yield) from air-dried fruits of Xylopia aethiopica harvested in Togo was investigated for percentage composition and in vitro cytotoxicity. The chemical composition of the essential oil was examined by GC and GC/MS. Thirty-five compounds were identified representing 89.9% of total oil. The major constituents were ß-pinene (23.6%), α-pinene (11%), sabinene (9.8%), germacrene D (8.3%) and 1,8 cineole (8.2%). The cytotoxicity of the volatile oil was evaluated in vitro on the human epidermal cell line HaCaT. The tested sample did not show any cytotoxicity (IC50 >3000 μg.ml-1) effect at concentrations around 3000 μg.ml-1. Further testing in bioassay would probably help in validating some of medicinal uses of X. aethiopica in topical drugs and/or in cosmetics as natural products. [ABSTRACT FROM AUTHOR]

We applied atomic force microscopy (AFM) to visualize ultrastructural changes of the keratinocyte morphology after narrow-band ultraviolet B (NB-UVB) irradiation. Immortalized human keratinocytes were cultured under standard conditions, irradiated with NB-UVB light at doses ranging from 50 to 800 mJ/cm2 and imaged by AFM mounted on an inverted optical microscope. It was observed, that NB-UVB irradiation provoked dose-dependent alterations of the keratinocyte morphology. While the surface of non-irradiated cells exhibited homogenously distributed crest-like shaped protrusions (height 0.16 ± 0.05 μm), cells irradiated with a dose of 800 mJ/cm2 in addition showed round shaped protrusions (height 0.14 ± 0.06 μm) distributed predominantly around the nucleus and bleb-like protrusions irregularly distributed on the cell surface (height 0.95 ± 0.29 μm). These irradiated cells easily detached from the supporting glass surface, showed impaired contact with adjacent keratinocytes and significantly rearranged their cytoskeleton network. We hypothesize that these structural and functional alterations reflect ongoing apoptosis in UVB treated cells. [ABSTRACT FROM AUTHOR]

Abstract: In view of the powerful inherent oncolytic activity exhibited by the vesicular stomatitis virus (VSV) in several tumor types, we set out to investigate the susceptibility of the immortalized HaCaT keratinocyte cell line to VSV, and analyzed the role of apoptosis in the VSV-mediated induction of cell death. Indirect immunofluorescence assays, Western blot analyses and plaque titrations demonstrated that the HaCaT cell line was permissive to VSV replication. The results of ELISA for detection of the enrichment of nucleosomes in the cytoplasm of apoptotic cells revealed that VSV infection elicits the apoptotic death of HaCaT cells. Mock-infected HaCaT cells displayed the endogenous expression of ΔNp63α, p53 mutated on UV hot spots (p53mt), Bcl-2 and p21 Bax. The levels of ΔNp63α and p53mt were decreased, Bcl-2 remained unaffected, while the expressions of p21Bax and p18 Bax were increased in VSV-infected HaCaT cells. Together, these data demonstrate that VSV replicates efficiently and triggers apoptosis in the immortalized HaCaT keratinocyte cell line. The VSV-mediated alterations in the expressions of ΔNp63α and Bax may be implicated in the apoptotic responses of infected cells and may also sensitize to other apoptotic stimuli. These findings may stimulate further studies with the goal of developing VSV-based virotherapy into an effective modality for the treatment of epithelial-derived malignant tumors of the skin. [Copyright &y& Elsevier]

Abstract: Matrine is a kind of alkaloid found in certain Sophora plants, which has been extensively used in China for the treatment of viral hepatitis, cancer, cardiac diseases and skin diseases (such as atopic dermatitis and eczema). It also has been confirmed that substance P (SP) and its receptor (neurokinin-1 receptor, NK-1R) are involved in the pathogenesis of inflammatory skin disorders. So the present study was designed to investigate the effect of matrine on the expression of NK-1R and cytokines production induced by SP in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. In addition, cell viability was also evaluated. The results showed that matrine inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of interleukin (IL)-1β, IL-8, interferon (IFN)-γ, and monocyte chemotactic protein (MCP)-1 in both cell types. Matrine 5–100 μg/mL had little effect on cell viability. It inhibited SP-induced IL-1β, IL-8 and MCP-1 production in HaCaT cells and fibroblasts, while it increased the production of IFN-γ in HaCaT cells. Both SP and matrine had no effect on the secretion of IL-6. These findings suggest that matrine may have potential treatment function on SP related cutaneous inflammation by inhibition of the expression of substance P receptor and regulation of the production of inflammatory cytokines. [Copyright &y& Elsevier]

Background There is increasing evidence that neuropeptides, especially substance P (SP), may be involved in the pathogenesis of cutaneous allergic inflammation (CAI). Objectives To investigate expression of the SP receptor (neurokinin-1 receptor, NK-1R) in human epidermal keratinocytes and dermal fibroblasts and its potential influence in CAI. Methods HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts were cultured. The expression of NK-1R protein was examined by immunohistochemistry, and the mRNA level was detected by semiquantitative reverse transcriptase–polymerase chain reaction. The modulation of NK-1R expression in HaCaT cells and fibroblasts was detected by flow cytometry and Western blotting analysis. Results NK-1R expression was found in HaCaT cells and fibroblasts. The expression of NK-1R mRNA in fibroblasts was weaker than in HaCaT cells. SP and interferon (IFN)- γ significantly upregulated the expression of NK-1R. [d-Arg1,d-Trp7,9 Leu11]-SP (Spantide I), a panspecific NK-1R antagonist, reduced the expression of NK-1R stimulated by SP. Conclusions HaCaT cells and fibroblasts can express NK-1R at protein and transcription levels, and the expression was modulated by SP, IFN- γ and Spantide I. This indicates that keratinocytes and fibroblasts are involved in the regulation of skin immunity and that NK-1R may play an important role in the pathogenesis of CAI. [ABSTRACT FROM AUTHOR]

The bioeffects of silica nanoparticles (SiNP), phosphorylate-terminated nanoparticles (PO4-NP) and amino-terminated nanoparticles (NHO2NP) on HaCaT cell line have been studied in this paper. The effects of the three kinds of functionalized silica nanoparticles on adherence, proliferation and cycle of HaCaT cells have been investigated. And the cellular uptake of the three kinds of functionalized silica nanoparticles by HaCaT cells has also been examined. Results indicated that the bioeffects of the three kinds of functionalized nanoparticles on HaCaT cells were concentration-dependent. And the three kinds of functionalized nanoparticles all exhibited well biocompatibility if the concentration was below 0.2 µg/µL. While the cytotoxicities of the three kinds of functionalized nanoparticles on HaCaT cells would increase with the increasing of nanoparticles concentration, and the following order was observed: NH2NP > SiNP > PO4NP. In addition, the quantity and rapidity of cellular uptake of nanoparticles by HaCaT cells were diverse due to the different functional groups. Under the same conditions, NH2NP was most and fast internalized by HaCaT cells, followed by SiNP, and PO4NP was the least and slowest. These results provided theoretical foundation for the safe application and further modification of silica nanoparticles, which could broaden the application of silica nanoparticles in biomedicine. [ABSTRACT FROM AUTHOR]

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Cell migration is a process that underlies the development and maintenance of multicellular organisms, with profound implications in various pathologies. The study of cell migration is fundamental in various fields of basic biology and pharmaceutical development. Wound healing assay is an indirect way to assess cell migration. Conventional methods, such as the scratch test, are inexpensive and easy to execute but have the disadvantages of being poorly reproducible and difficult to perform on a high-throughput scale. Meanwhile, commercial strategies are expensive. In the present work, we developed a lab-made wound healing assay device that is inexpensive, easy to handle, and reproducible. We designed 3D-printed stoppers compatible with cell culture in 96-well plates. These stoppers did not affect HaCaT cells viability. The stopper-produced initial wound size was reproducible on a high-throughput scale. Also, stoppers demonstrated their effectiveness to evaluate cell migration and allowed differentiating treatments with and without fetal bovine serum. Finally, proliferation assay was determined in this wound healing model. In conclusion, our lab-made 3D-printed stopper-based assay is a more economical alternative to currently available strategies for developing reproducible, high-throughput assays to assess cell migration and proliferation., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial conflict of interest, (Copyright © 2021. Published by Elsevier Inc.)

In the present work, we establish novel "environmentally-friendly" oil-in-water nanoemulsions to enhance the transdermal delivery of bakuchiol, the so-called "bioretinol" obtained from powdered Psoralea corylifolia seeds via a sustainable process, i.e., using a supercritical fluid extraction approach with pure carbon dioxide (SC-CO2). According to Green Chemistry principles, five novel formulations were stabilized by "green" hybrid ionic surfactants such as coco-betaine—surfactin molecules obtained from coconut and fermented rapeseed meal. Preliminary optimization studies involving three dispersion stability tests, i.e., centrifugation, heating, and cooling cycles, indicated the most promising candidates for further physicochemical analysis. Finally, nanoemulsion colloidal characterization provided by scattering (dynamic and electrophoretic light scattering as well as backscattering), microscopic (transmission electron and confocal laser scanning microscopy), and spectroscopic (UV–Vis spectroscopy) methods revealed the most stable nanocarrier for transdermal biological investigation. In vitro, topical experiments provided on human skin cell line HaCaT keratinocytes and normal dermal NHDF fibroblasts indicated high cell viability upon treatment of the tested formulation with a final 0.02–0.2 mg/mL bakuchiol concentration. This excellent biocompatibility was confirmed by ex vivo and in vivo tests on animal and human skin tissue. The improved permeability and antiaging potential of the bakuchiol-encapsulated rich extract were observed, indicating that the obtained ecological nanoemulsions are competitive with commercial retinol formulations. [ABSTRACT FROM AUTHOR]