HaCaT Research Articles

Milk exosome-infused fibrous matrix for treatment of acute wound

To provide an advanced therapy for wound recovery, in this study, pasteurized bovine milk-derived exosomes (mEXO) are immobilized onto a polydopamine (PDA)-coated hyaluronic acid (HA)-based electrospun nanofibrous matrix (mEXO@PMAT) via a simple dip-coating method to formulate an mEXO-immobilized mesh as a wound-healing biomaterial. Purified mEXOs (∼82 nm) contain various anti-inflammatory, cell proliferation, and collagen synthesis-related microRNAs (miRNAs), including let-7b, miR-184, and miR-181a, which elicit elevated mRNA expression of keratin5, keratin14, and collagen1 in human keratinocytes (HaCaT) and fibroblasts (HDF). The mEXOs immobilized onto the PDA-coated meshes are gradually released from the meshes over 14 days without burst-out effect. After treatment with HaCaT and HDF, the degree of in vitro cell migration increases significantly in the mEXO@PMAT-treated HaCaT and HDF cells compared to the unmodified or PDA-coated meshes-treated cells. Additionally, the mEXO@PMAT provides significantly faster wound closure in vivo without notable toxicity. Thus, the sustained liberation of bioactive mEXO from the meshes can effectively enhance cell proliferation in vitro and accelerate wound closure in vivo, which could be harnessed mEXO@PMAT as a promising wound-healing biomaterial.

Skin-permeable gold nanoparticles with modifications azelamide monoethanolamine ameliorate inflammatory skin diseases

BackgroundTraditional topical drug delivery for treating inflammatory skin diseases suffers from poor skin penetration and long-term side effects. Metal nanoparticles show promising application in topical drug delivery for inflammatory skin diseases.MethodsHere, we synthesized a new type of nanoparticles, azelamide monoethanolamine-functionalized gold nanoparticles (Au-MEA NPs), based on citrate-capped gold nanoparticles (Au-CA NPs) via the ligand exchange method. The physical and chemical properties of Au-CA NPs and Au-MEA NPs were characterized. In vivo studies were performed using imiquimod-induced psoriasis and LL37-induced rosacea animal models, respectively. For in vitro studies, a model of cellular inflammation was established using HaCaT cells stimulated with TNF-α. In addition, proteomics, gelatin zymography, and other techniques were used to investigate the possible therapeutic mechanisms of the Au-MEA NPs.ResultsWe found that Au-MEA NPs exhibited better stability and permeation properties compared to conventional Au-CA NPs. Transcutaneously administered Au-MEA NPs exerted potent therapeutic efficacy against both rosacea-like and psoriasiform skin dermatitis in vivo without overt signs of toxicity. Mechanistically, Au-MEA NPs reduced the production of pro-inflammatory mediators in keratinocytes by promoting SOD activity and inhibiting the activity of MMP9.ConclusionAu-MEA NPs have the potential to be a topical nanomedicine for the effective and safe treatment of inflammatory skin diseases.

The Effects of Marine Fungal Asterripeptides A-C on In Vitro and In Vivo Staphylococcus aureus Skin Infection.

Objectives: This study aimed to investigate the in vitro and in vivo antibacterial and cytoprotective activities of marine fungal tripeptide derivatives with cinnamic acid moiety asterripeptides A-C (1-3). Methods: The antimicrobial and antibiofilm activities of asterripeptides A-C were tested using the Staphylococcus aureus ATCC 21027 strain. Human HaCaT keratinocytes infected with S. aureus were used for the in vitro investigation of the various aspects of the influence of asterripeptides A-C by lumino- and fluorospectrometry, ELISA, flow cytometry, Western blotting, and microscopy techniques. In the in vivo experiments, mice with burns and scalped S. aureus-infected wounds were used according to ethical committee resolution. Results: Asterripeptides A-C (10 µM) inhibited S. aureus growth and biofilm formation. Asterripeptides A-C increased the viability, proliferation, and migration of S. aureus-infected HaCaT cells and reduced the release of reactive oxygen species (ROS), NO, TNF-α, and IL-18. Asterripeptides A-C protected HaCaT cells against TNF-α-induced inflammation, decreased the transcriptional level of NF-κB in JB6 Cl41 cells, and increased the protein levels of Nrf2 and glutathione synthetase in HaCaT cells. More active asterripeptide C was tested in in vivo burn wounds and S. aureus-infected incised wounds. Asterripeptide C significantly enhanced wound healing, normalized cytokine levels and profiles of peripheral blood samples, and decreased S. aureus contamination of wounds and blood in mice with infected incised wounds. Conclusions: Taken together, these results confirm the dual antibacterial and Nrf2-dependent anti-inflammatory activities of asterripeptides A-C in in vitro and in vivo assays.

Anti-inflammatory effects of phytosphingosine-regulated cytokines and NF-kB and MAPK mechanism.

Phytosphingosine (PHS) is a major component of the skin barrier and a multifunctional physiologically active substance. This study aimed to investigate the types of cytokines regulated by PHS, their anti-skin inflammatory effects, and their anti-inflammatory mechanisms. RAW264.7 cells stimulated with Lipopolysaccharides (LPS) were treated with PHS to measure inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2), and gene expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX2) were confirmed by q-PCR. Cytokines regulated by PHS against LPS-induced inflammation were found through cytokine array, and each factor was reconfirmed through ELISA. Western blot was performed to confirm anti-inflammatory mechanism of Iκbα and MAPK. To confirm anti-skin inflammatory efficacy, HaCaT cells stimulated with TNF-α/IFN-γ were treated with PHS, and TARC, IL-6, and IL-8 were detected by ELISA. PHS suppressed the gene expression of iNOS and COX2, which were increased by LPS, and suppressed NO and PGE2 production. Through cytokine array, it was confirmed that IL-6, IL-10, IL-27 p28/IL-30, IP-10, I-TAC, MCP-5, and TIMP-1 increased by LPS were decreased by PHS. PHS inhibited NF-κB signaling by inhibiting LPS-induced NF-κB nuclear migration and p-Iκbα-mediated Iκbα degradation, and inhibited p38, ERK, and JNK signaling pathways. PHS reduced the production of TARC, IL-6, and IL-8 increased by TNF-α/IFN-γ. These results indicate PHS has anti-inflammatory effects via the suppression of inflammatory factors and pro-inflammatory cytokines through the NF-κB and MAPK pathways. Moreover, these results may explain beneficial effects of PHS in the treatment of skin inflammatory conditions induced by TNF-α/IFN-γ.

Enhancement of Growth and Lipid Production in Microalgae Using Aggregation-Induced Emission Based Luminescent Material for Sustainable Food and Fuel.

AIEbased nanomaterials are progressively gaining momentum owing to their evolvement into an interdisciplinary field ranging from biomass and biomolecule yield to image-guided photodynamic therapy. This study focuses on a novel strategy to enhance growth, lipid accumulation, and in vivo fluorescence visualisation in green microalgae Chlamydomonas reinhardtii using AIE nanoparticles to quantify radical changes. The absorption of AIE photosensitiser, TTMN was recorded from 420 to 570 nm with a peak at 500 nm, and the emission ranged from 550 to 800 nm with a peak at 650 nm. As a ROSmolecule, H2O2 generation of TTMN in C. reinhardtii cells was detected with AIE nanoprobes TPE-BO. H2O2 accumulation increased with the increase of TTMN concentrations. The maximum growth was observed at 10 µM TTMN-exposed C. reinhardtii cells. Significant lipid accumulation was found in both 10 and 15 µM TTMN-treated cells. For lipid visualisation, an AIE nanoprobe, 2-DPAN was used, and superior fluorescence was determined and compared with the traditional BODIPY dye. Cytotoxicity analysis of 10 µM TTMN on the HaCat cell line with 86.2% cell viability revealed its high biocompatibility on living cells. This AIE-based nanotechnology provides a novel approach for microalgae-derived sustainable biomass and eco-friendly biofuel production.

Insights into the wound-healing properties of medicinally important South African Bulbine species – A comparative study

Ethnopharmacological relevanceSouth Africa harbours a large number of Bulbine (Xanthorrhoeaceae) species, which includes ethnobotanically important indigenous species. Traditionally, Bulbine leaves are used by several ethnic groups in South Africa to treat dermatological conditions including wounds, which led to the development of Bulbine-containing cosmetic products. However, scientific evidence is needed to support the claims in treating skin conditions and wound-healing. Aim of the studyThis comparative study was undertaken to investigate the wound-healing properties of five Bulbine species indigenous to South Africa, using in vitro and in vivo models. Materials and methodsFive Bulbine species, B. abyssinica, B. asphodeloides, B. frutescens, B. latifolia and B. narcissifolia were collected from natural populations in the Eastern Cape Province of South Africa. The chemical profiles of the methanol leaf extracts were acquired using ultra-performance liquid chromatography with photodiode array detection in tandem with quadrupole time-of-flight mass spectrometry. The methyl thiazolyl tetrazolium (MTT) assay and maximum tolerated concentration (MTC) assay were used to assess the in vitro and in vivo toxicity of the extracts, respectively. The in vitro scratch assay was employed to monitor cell migration and wound-closure in a HaCaT cell monolayer, following treatment with the plant extracts for 48 h. In vivo wound-healing potential was determined using the zebrafish larvae caudal fin amputation assay, assessed in three-days post fertilization larvae and various concentrations of the plant extracts were tested in both assays to determine the concentration-response effect. Data were analysed using MS Excel® enhanced with the Real Statistics add-in. Results and discussionUsing UPLC-MS, 11 major compounds were tentatively identified in the five Bulbine species. Although the compounds varied between species, all five Bulbine species contained the phenylanthraquinone, knipholone. Kaempferol glucoside was identified in four species, but not in B. abyssinica. The five Bulbine species were non-cytotoxic (cell viability > 80%) towards keratinocytes at all three tested concentrations. However, B. latifolia was toxic towards zebrafish larvae at all the tested concentrations, while the other four species were non-toxic at low concentrations. The results of the scratch assay revealed that B. abyssinica was the most active extract at 100 μg/mL. Compared to the untreated control, wound-closure notably increased by 28% (p < 0.05), 44% (p < 0.01) and 34% (p < 0.05) after 12 h, 24 h and 36 h post-treatment, respectively. Although none of the species achieved 100% caudal fin regeneration by the end of the treatment period, B. frutescens demonstrated the highest regeneration (90%) and most significant difference (p < 0.01) compared to the untreated control. ConclusionThe results revealed that the five Bulbine species have complex chemical profiles, however, they share major compound classes (i.e. phenylanthroquinones and flavonoid analogues) across the species. The study highlights the wound-healing properties of the five species, which is consistent with their traditional use.

Development of phyto-compound-based nanoformulation for skin regeneration and its in vitro evaluation

Skin regeneration is a process that regenerates damaged or aged skin cells, resulting in a healthier and younger appearance. The demand for nano dosage forms containing phyto-compounds in the cosmetics industry is increasing daily to control or prevent this process. Phyto-compounds with cell regenerative properties often show limited effectiveness in conventional use due to their low hydrophilicity and molecular weight, as well as their low absorption from the skin. Glycyrrhizic acid (GA), a phyto-compound obtained from licorice root, has many pharmacological properties, including skin regeneration properties. The aim of this study is to develop a highly stable GA-containing nanoemulsion (GA-NE) formulation for topical application, determine its anti-collagenase activity, and evaluate its safety and effectiveness by in vitro cell culture methods. In this context, GA-NEs were synthesized with sixteen production procedures using independent variables (oil, water percentage, surfactant, and homogenization time). The average droplet size, polydispersity index (PdI), and zeta potential (ZP) of the synthesized GA-NEs were determined by dynamic light scattering (DLS) analysis. The heating-cooling cycle (H-C) test result showed that the F4P3 formulation was the most stable formulation. Therefore, 90-day physicochemical stability tests were performed with the F4P3 formulation. In vitro release results revealed that GA was released from NEs at a rate of 93.15 ± 0.61 % within 24 h. An ex vivo permeation and penetration study was performed, and the permeation and penetration profile of F4P3 was determined. In vitro cell culture studies showed that the F4P3 formulation had higher cell viability and regeneration than GA in the HaCaT cell line. Anti-collagenase activity results also revealed a better activity of the F4P3 formulation compared to GA. Consequently, characterization, efficacy, and safety studies showed that the F4P3 formulation could be an innovative cell regeneration product candidate suitable for topical use with high stability.

Se-incorporated polycaprolactone spherical polyhedron enhanced vitamin B2 loading and prolonged release for potential application in proliferative skin disorders

Development of novel drug vehicles for vitamin B2 (VitB2) delivery is very important for designing controllable release system to improve epidermal growth and bone metabolism. In this work, selenium (Se)-incorporated polycaprolactone (PCL) spherical polyhedrons are successfully synthesized via a single emulsion solvent evaporation method which is utilized to load VitB2 to fabricate cell-responsive Se-PCL@VitB2 delivery systems. Their physicochemical properties are characterized by DLS, SEM, XRD, FTIR, and TGA-DSC. The release kinetics of VitB2 or Se from the samples are investigated in PBS solution (pH = 2.0, 5.0, 7.4, 8.0 and 12.0). The cytocompatibilities are also evaluated with normal BMSC and epidermal HaCat cells. Results exhibit that Se-PCL@VitB2 particles presents spherical polyhedral morphology (approximately (3.25 ± 0.46) μm), negative surface charge (-(54.03 ± 2.94) mV), reduced crystallinity and good degradability. Stability experiments imply that both VitB2 and Se might be uniformly dispersed in PCL matrix. And the incorporation of Se facilely promotes the loading of VitB2. The encapsulation efficiency and loading capacity are (98.42 ± 1.06)% and (76.25 ± 1.27) for Se-PCL@VitB2 sample. Importantly, it exhibits more prolonged release of both VitB2 and Se in neutral PBS solution (pH = 7.4) than other pH conditions. Presumably, the electrostatic interaction between Se, VitB2 and PCL contribute to its release mode. Cell experiments show that Se-PCL@VitB2 presents strong cytotoxicity to HaCat cells mainly due to the cytotoxic effect of Se anions and PCL degradation products. However, it exhibits weak inhibitory effect on BMSC cells. These note that the synthesized Se-PCL@VitB2 particles can be promising drug vehicles for potential application in epidermal proliferative disorders.

In-vitro anti-acne activity of Teucrium oliverianum methanolic extract against Cutibacterium acnes.

Acne vulgaris is a skin infection widely seen in adolescents between 10-19years with males affected more than females. It mainly affects the face but may also affect the back and chest. The symptoms vary with mild acne manifesting as comedones and moderate acne as inflammatory lesions (papulopustular), nodules, and mild scarring while severe acne has the same symptoms that have not subsided within 6months of treatment. Various treatments including topical medications containing different antibiotics are used to treat acne. Recently, herbal treatments have been shown as better alternatives to conventional treatment. Teucrium oliverianum Ging. ex Benth (Lamiaceae) is traditionally used for skin infections such as wound healing and biofilm formation. Methanolic extract of T. oliverianum was subjected to liquid chromatography-mass spectrometry (LC-MS) analysis, and its antibacterial effect against Cutibacterium acnes. The anti-acne, anti-inflammatory, and antioxidant effects were also assessed using HaCaT cells infected with C. acnes. The cytotoxicity of the extract was evaluated using a neutral red uptake assay, and anti-inflammatory effects were determined by measuring TNF-α, IL-1β, INF-γ, and COX2 inhibition. The antioxidant action was assessed by ROS generation in HaCaT cells infected with C. acnes. LC-MS analysis of the extract showed the presence of 16 different metabolites with L-carnitine, esculin sesquihydrate, and gamma-linoleic acid as major metabolites. The methanolic extract of T. oliverianum showed an antibacterial effect against C. acnes with an IC50 value of 263.2μg/mL. The extract attenuated the cytotoxicity of C. acnes on the HaCaT cell and the IC50 was found to be 676.2μg/mL. It also decreased dose-dependently the expression of TNF-α, IL-1β, INF-γ, and inhibited COX2 in the HaCaT cells infected with C. acnes. It also decreased the generation of reactive oxygen species. The results support the use of T. oliverianum as an anti-acne agent but it possesses mild antibacterial action. It showed anti-inflammatory effects in HaCaT cells infected with C. acnes. It is also an effective antioxidant and decreased the generation of reactive oxygen species. Comparison of the anti-acne effects and adverse reactions of extract with other treatments will provide more insight into its clinical efficacy and toxicity.

Betulin-NLC-hydrogel for the Treatment of Psoriasis-like Skin Inflammation: Optimization, Characterisation, and In vitro and In vivo Evaluation.

Psoriasis is a chronic inflammatory skin disorder that poses significant challenges regarding effective and targeted drug delivery. Bioactive substances like betulin have shown tremendous utility in treating these conditions; however, they pose limited utility owing to their physicochemical characteristics. Here, we aimed to develop a novel topical dosage form for treating psoriasis, utilising betulin-loaded solid lipid nanoparticles (NLCs) incorporated into a hydrogel matrix. The optimization of the formulation was meticulously conducted using a design of experiments methodology, and its diverse physicochemical attributes were thoroughly examined. Evaluating betulin's in vitro release pattern from the NLC-hydrogel demonstrated consistent and regulated drug release properties. Additionally, the formulation demonstrated improved skin penetration abilities as determined by in vitro skin permeation experiments employing Franz diffusion cells- furthermore, the therapeutic effectiveness of the betulin-NLC-hydrogel was assessed by an in vivo experiment carried out using an imiquimod-induced psoriasis-like skin inflammation model in BALB/c female mice. The NLCs exhibited a pH of 5.67±0.86, particle size of 148.16±12.66 nm, and zeta potential of -22.84±2.37 mV, ensuring stability and suitability for topical use. The gel, with a pH of 6.05±0.43 and viscosity of 17550±120 cPs, showed enhanced skin hydration and lipid restoration. Drug release studies indicated a slower release from NLCs and gel, improving skin retention. Stability tests revealed that the formulations were stable at room temperature but not at elevated temperatures. The in vitro safety profile of the formulation revealed no significant adverse effects on HaCaT cell lines. The NLC gel demonstrated significant anti-psoriatic activity, reducing inflammation and cytokine levels. The betulin-NLC-hydrogel formulation exhibited promising characteristics for the topical treatment of psoriasis, showcasing optimised drug delivery, sustained release, and notable therapeutic efficacy. The findings from this study provide a foundation for the potential clinical translation of this innovative topical dosage form for improved psoriasis management.

FUNDC1-mediated mitophagy regulates photodamage independently of the PINK1/Parkin-dependent pathway

BackgroundUltraviolet B(UVB) triggers a pro-survival response through mitophagy, but the role of FUNDC1-mediated mitophagy in photodamaged skin remains unexplored. ObjectivesTo clarify the function of mitophagy in UVB-induced photodamaged skin. MethodsTo investigate the role of FUNDC1-mediated mitophagy in UVB-induced mitochondrial damage and cell apoptosis, FUNDC1 knockdown in C57BL/6 mice was performed using adeno-associated virus. Additionally, FUNDC1 overexpression and knockdown in HaCaT cells were conducted using lentivirus. A comprehensive analysis was conducted on a panel of human sun-exposed skin samples, alongside control samples, to assess the expression levels of FUNDC1. ResultsIn UVB-induced C57BL/6 mice, the dorsal skin showed photodamage including erythema, scaling, erosion, and scabs. The expression levels of PINK1, Parkin, and BNIP3 did not show significant changes, while FUNDC1 expression consistently declined along with LC3B. Cytochrome C, Bax, and cleaved-caspase3 were upregulated, while Bcl2 was downregulated. UVB-induced HaCaT cells showed mitochondrial damage, accompanied by FUNDC1 downregulation and BNIP3 upregulation, while PINK1 and Parkin showed no significant changes. FUNDC1 overexpression led to an increase in mtROS and a decrease in mitochondrial membrane potential and ATP levels, indicating complete mitochondrial clearance and exacerbated cell death. FUNDC1 knockdown protected against UVB-induced photodamage in mice and mitigated mitochondrial damage and apoptosis in HaCaT cells by activating compensatory PINK1/Parkin-dependent mitophagy, which was evidenced by upregulation of PINK1 and Bcl2 and downregulation of Bax. In human sun-exposed skin samples, there was a decrease in the number of FUNDC1+ cells compared with non-sun-exposed controls. ConclusionsFUNDC1-mediated mitophagy regulates skin photodamage and provides a novel mechanism for resisting photodamage, presenting a potential target for future therapeutic interventions.

CAPE derivatives: Multifaceted agents for chronic wound healing.

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

Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

BackgroundHuman interleukin-22 (IL-22) is known as a “dual function” cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis.MethodsWe used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis.ResultsWe demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1β, IL-6, IL-10, and IL-17A.ConclusionsWe developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

Functional Properties and Components of Koenigia alpina Extract.

Koenigia alpina (All.) T.M.Schust. & Reveal (alpine knotweed) is a perennial herb belonging to the Polygonaceae family. Several studies have examined Polygonaceae species' potential applications as cosmeceutical materials; however, the potential of K. alpina as a cosmeceutical has not yet been studied. Hydrogen peroxide (H2O2) and lipopolysaccharide were used to induce an inflammatory response in RAW 264.7 cells. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals and H2O2 were used to evaluate the free-radical scavenging activity of K. alpina extract and its protective effect against reactive oxygen species (ROS)-induced cell damage. The whitening, antiaging, and cell proliferation/migration effects of the extracts were evaluated via tyrosinase inhibition, collagenase/elastase inhibition, and wound healing assays, respectively. The anti-inflammatory effect was confirmed by evaluating nitric oxide (NO) production in RAW 264.7 cells. High-performance liquid chromatography (HPLC), UV, and MS/MS were used to determine the main components of the extract and fractions. The ethyl acetate (EA) fraction and its aglycone fraction showed very high free-radical scavenging activities (47.5 and 47.1µg/mL, respectively). The extract/fractions also showed significant tyrosinase inhibition (IC50 = 0.38mg/mL in EA fraction), collagenase inhibition (IC50 = 0.21mg/mL in EA fraction), and elastase inhibition (IC50 = 0.57mg/mL in aglycone fraction). NO production in lipopolysaccharide-induced RAW 264.7 cells was inhibited by the extract/fractions. The extract also promoted the closure of scratch wounds in HaCaT cells. The K. alpina extract/fractions contained cardamonin, quercetin, and quercitrin. K. alpina extracts/fractions showed antioxidant, antiaging, whitening, and anti-inflammatory activities, suggesting they may have potential as antiaging cosmeceuticals.

New quinoxaline-oxadiazole hybrids as tubulin inhibitors: Synthesis, cytotoxicity, and molecular dynamics simulations

This study describes the synthesis and biological evaluation of a series of novel quinoxaline-oxadiazole hybrid compounds. These compounds were designed to enhance anticancer activity through the incorporation of specific pharmacophoric groups. Structural characterization using NMR and HRMS spectroscopy confirmed the successful synthesis of the hybrids. Cytotoxicity assays revealed that several compounds exhibited significant activity against various cancer cell lines. Compounds 8a and 8f demonstrated potent activity against A549 cells, while 8a, 8b, and 8c were effective against HaCaT cells. Additionally, compounds 8i and 8a showed activity against HepG2 cells. Molecular docking and dynamics simulations suggested that compounds 8c and 8d bind strongly to the tubulin protein, a target for anticancer drugs. These compounds exhibited binding affinities comparable to known anticancer compounds. The results of this study highlight the potential of quinoxaline-oxadiazole hybrids, especially those with halogen substitutions, as promising candidates for the development of novel anticancer therapeutics.

Antibacterial and Antibiofilm potential of Thuja orientalis L. extract targeting cariogenic Enterococcus faecalis ATCC 29212: A combined in-vitro, in-silico study, and cytotoxicity screening

ObjectivesIn this study, we explored the efficacy of methanolic extract of Thuja orientalis (TOME) as a novel antibacterial and antibiofilm agent against a cariogenic bacterium, Enterococcus faecalis ATCC 29212. DesignAntibacterial susceptibility studies were conducted and surface morphology analysis was performed using field emission scanning electron microscopy (FESEM). Antibiofilm activity was evaluated through both qualitative and quantitative biofilm inhibition assays and validated by microscopic analysis. In-silico molecular docking studies were conducted using the EDock server. The effectiveness of TOME was substantiated by biofilm model on dentin discs and cytotoxicity towards the HaCaT cell line was assessed using the MTT assay. ResultsTOME exhibited significant bactericidal activity with minimum inhibitory concentration of 12.5mg/mL and additionally, it effectively compromised bacterial cell wall integrity. Qualitative, quantitative and microscopic studies depicted the inhibition of biofilm formation. TOME significantly impacted the production of extracellular polymeric substance and extracellular DNA. Molecular docking studies identified beta-caryophyllene as a potent inhibitor of the Enterococcal surface protein (Esp). Biofilm model depicted the reduction of bacterial load on dentin discs. Additionally, TOME showed reduced cytotoxicity on HaCaT cells, indicating its potential as a safe therapeutic agent. ConclusionThese findings highlight TOME's promise for developing novel treatments for dental infections and biofilm-associated diseases. Further research should focus on isolating and characterizing the active compounds within TOME, particularly beta-caryophyllene, to elucidate their precise mechanisms of action.

CH25H Promotes Autophagy and Regulates the Malignant Progression of Laryngeal Squamous Cell Carcinoma Through the PI3K-AKT Pathway.

Laryngeal squamous cell carcinoma (LSCC) is a type of cancer of the respiratory tract that often presents with subtle symptoms at the early stage and is susceptible to recurrence and metastasis. To find out key regulatory genes involved in LSCC development, we downloaded LSCC-related sequencing datasets for bioinformatics analysis. WGCNA was performed on GSE142083 and differential analysis was conducted on GSE51985 and TCGA-HNSC. Intersectiongenes were taken from the above three datasets. To confirm the function of genes, we overexpressed and knocked down genes in cells and treated them with autophagy agonist Rapamycin and PI3K-AKT pathway inhibitor. At the cellular level, the expression of CH25H, autophagy-related proteins (LC3 I, LC3 II, p62, and Beclin 1), and PI3K-AKT pathway-related proteins (PI3K, AKT, and p-AKT) were assessed via Western blot; the mRNA level of CH25H was evaluated through qRT-PCR; the cell activity was examined by CCK8; the apoptosis was assessed through flow cytometry; and the cell migration and invasion were assessed through wound healing and Transwell assays. Through bioinformatics analysis, we screened 7 genes (CH25H, NELL2, STC2, TMEM158, ZIC2, HOXD11, and HOXD10). Ultimately, CH25H was selected for follow-up experiments. By detecting CH25H expression in human immortalized keratinocytes (HaCaT) and LSCC cells (Tu-686, SNU899, and AMC-HN-8), it was found out that CH25H expression was higher in HaCaT cells than in LSCC cells. To elucidate the role of CH25H in LSCC development, we overexpressed CH25H in Tu-686 cells and downregulated its expression in AMC-HN-8 cells. CH25H was revealed to reduce the proliferation, activity, invasion, and migration of LSCC cells while increasing their apoptosis levels. Significant changes were also observed in the expressions of autophagy- and PI3K-AKT pathway-related proteins. To further investigate the roles of autophagy and the PI3K-AKT pathway in LSCC development, we respectively employed autophagy agonists and inhibitors targeting the PI3K-AKT pathway to intervene the cells, and found that CH25H regulated the PI3K-AKT pathway to promote autophagy, thus enhancing the apoptosis of LSCC cells. We further investigated CH25H's impact on tumor growth, autophagy, and the PI3K-AKT pathway at the animal level and found that CH25H promoted autophagy of LSCC cells and inhibited the PI3K-AKT pathway, and ultimately inhibiting the progression of LSCC. In summary, CH25H promotes autophagy and affects the malignant progression of LSCC through the PI3K-AKT pathway.

P2X7R-primed keratinocytes are susceptible to apoptosis via GPCR-Gβγ-pERK signal pathways

BackgroundCell death constitutes a pivotal biological phenomenon essential for the preservation of homeostasis within living organisms. In the context of maintaining a functional skin barrier, keratinocytes exert positively and negatively control cell death signals. However, in patients with severe drug eruptions, anomalous overexpression of the formyl peptide receptor 1 (FPR1) in keratinocytes elicits a distinctive mode of cell death known as necroptosis, thereby suffering a loss of the skin barrier. The precise molecular mechanisms connecting FPR1 activation to this cell death remain unclear. ObjectiveWe have investigated the intracellular signal transduction cascade governing FPR1-mediated cell death in cultured keratinocytes. MethodsWe used HaCaT cells as a model keratinocyte. The expression of FPR1 was detected with qPCR. The presence of cell death events was monitored through live-cell fluorescent staining and LDH release assays. Furthermore, the phosphorylation of ERK was assessed via Western blot analysis. Intracellular signal pathways were investigated using specific inhibitors. ResultsLigand stimulation of an endogenous ion channel, purinergic receptor P2X7 (P2X7R), increased the FPR1 expression level. This upregulated FPR1 demonstrated functional competence in the phosphorylation of downstream MAP kinase and the initiation of cell death. Notably, this cell death was ameliorated upon the administration of inhibitors targeting Gβγ, ERK, and caspases. ConclusionThe induction and stimulation of FPR1 initiated apoptosis in keratinocytes via the Gβγ-pERK signaling pathway. Our findings postulate that the downstream components of FPR1 represent an alternative therapeutic target for preventing unintended keratinocyte cell death.

Photoprotective properties of four structure propolis from Heterotrigona itama stingless beehive: Fractionation, bioactivity analysis, and chemical profiling

Heterotrigona itama stingless bee propolis extract is known for its diverse bioactive compounds, making it a potential natural shield against UV radiation. This research assesses the photoprotective potential of crude ethanol extract from H. itama propolis collected from four structures (involucrum, pillar, pot, and entrance) of five bee hives (H1-H5), totalling 20 samples. Initially, the samples underwent testing for SPF value and UV absorption spectra. The crude ethanol extract (E) from the involucrum (H4) with the highest SPF value and broadest spectrum was selected for fractionation using hexane and water. Subsequently, the extract (E) and its hexane (H) and water (W) fractions were subjected to SPF analysis, UVA/UVB absorption assessment, determination of total phenolic and flavonoid content, free radical scavenging capacity, anti-collagenase effects, and cytotoxicity assessment. Additionally, LC-MS/MS analysis was performed to identify chemical constituents in the active fraction (W). The extract E demonstrated an SPF of 8.23 ± 0.09 and UV absorption. Notably, its fraction W exhibited the highest SPF (16.55 ± 0.24) at 100 μg/mL, surpassing the H fraction (SPF 5.7 ± 0.45). Phenolic content was highest in the H fraction (388.95 ± 4.54 mg/g GAE DW), followed by the W fraction (286.76 ± 6.48 mg/g GAE DW) and crude E (91.83 ± 4.12 mg/g GAE DW) from the involucrum. Regarding flavonoids, the fraction W led with 79.82 ± 6.21 mg/g QE DW, followed by the H fraction (45.56 ± 0.05 mg/g QE DW) and E (34.57 ± 1.11 mg/g QE DW). The extract E also exhibited modest DPPH scavenging (EC50 = 120 μg/mL), while the H fraction demonstrated stronger activity (EC50 = 4.37 μg/mL), and the W fraction displayed moderate effects (EC50 = 17.55 μg/mL). Notably, the W fraction showed remarkable anti-collagenase activity, outperforming the positive control, EG. HaCaT cell cytotoxicity revealed that the extract E was cytotoxic, whereas the H and W fractions showed no toxicity. LC-MS/MS analysis identified bioactive flavonoids (e.g., pratensein, quercetin) in the W fraction. These findings highlight the superior photoprotective properties of the water fraction from the involucrum of H. itama stingless bee propolis extract, suggesting its potential as a natural and effective ingredient for sunscreen and skincare formulations.

The interaction of interferon gamma and gaillardin and the formation of a nanocomplex with improved anti-melanoma effects

This study presents a comprehensive analysis of IFN-γ-Gaillardin nanoparticles (NPs) using a combination of computational, biophysical and cell-based approaches. The molecular docking analysis revealed that both hydrogen and hydrophobic forces are involved in the formation of IFN-γ-Gaillardin complex The interaction between IFN-γ and Gaillardin was further characterized by a pronounced ANS fluorescence spectrum peak and a higher intensity for IFN-γ. The Langmuir, Scatchard, and Hill analyses revealed a higher affinity and lower dissociation constant for IFN-γ NPs compared to IFN-γ alone, suggesting enhanced complex stability. Thermal gravimetric analysis confirmed that the Gaillardin interaction might improve the thermal stability of the NPs. The NPs demonstrated robust stability in various media, highlighting their potential as a delivery system. However, size increase in deionized water suggests the need for formulation optimization. Cell-based assays revealed selective cytotoxicity towards A-375 melanoma cancer cells with minimal impact on non-cancerous HaCaT cells, indicating targeted antitumor effects. Real-time PCR showed gene expression changes consistent with antitumor activity and immune response modulation. The findings suggest that IFN-γ-Gaillardin NPs have potent antitumor properties and the ability to modulate the immune system, warranting further investigation into their therapeutic applications. The development of an IFN-γ-based nanocarrier system for Gaillardin delivery offers a promising approach to melanoma therapy, setting a new direction for NP-based cancer treatment strategies.