Dichlorofluorescein Diacetate Research Articles

Antifungal and antiovarian cancer properties of α Fe2O3 and α Fe2O3/ZnO nanostructures synthesised by Spirulina platensis.

Candida albicans (C. albicans) infection shows a growing burden on human health, and it has become challenging to search for treatment. Therefore, this work focused on the antifungal activity, and cytotoxic effect of biosynthesised nanostructures on human ovarian tetracarcinoma cells PA1 and their corresponding mechanism of cell death. Herein, the authors fabricated advanced biosynthesis of uncoated α-Fe2O3 and coated α-Fe2O3 nanostructures by using the carbohydrate of Spirulina platensis. The physicochemical features of nanostructures were characterised by UV-visible, high resolution transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The antifungal activity of these nanostructures against C. albicans was studied by the broth dilution method, and examined by 2', 7'-dichlorofluorescein diacetate staining. However, their cytotoxic effects against PA1 cell lines were evaluated by MTT and comet assays. Results indicated characteristic rod-shaped nanostructures, and increasing the average size of α-Fe2O3@ZnO nanocomposite (105.2 nm × 29.1 nm) to five times as compared to α-Fe2O3 nanoparticles (20.73nm × 5.25 nm). The surface coating of α-Fe2O3 by ZnO has increased its antifungal efficiency against C. albicans. Moreover, the MTT results revealed that α-Fe2O3@ZnO nanocomposite reduces PA1 cell proliferation due to DNA fragmentation (IC50 18.5 μg/ml). Continual advances of green nanotechnology and promising findings of this study are in favour of using the construction of rod-shaped nanostructures for therapeutic applications.

Cu (II) and Co (II/III) complexes of N,O‐chelated Schiff base ligands: DNA interaction, protein binding, cytotoxicity, cell death mechanism and reactive oxygen species generation studies

A series of copper (II) (1and3) and cobalt (II/III) (2,4and5) complexes comprising different imino‐phenolate ligandsDCH,DTHandDBH2(whereDCH= 2,4‐dichloro‐6‐((mesitylimino)methyl)phenol,DTH= 2,4‐di‐tert‐butyl‐6‐((mesitylimino)methyl) phenol andDBH2= 2,4‐dibromo‐6‐((mesitylimino)methyl)phenol) have been prepared with excellent yield and high purity. By utilizing different spectroscopic tools such as UV–visible, electrospray ionization (ESI)‐mass, Fourier‐transform infrared (FTIR) spectrometry and elemental analysis, the prepared complexes (1–5) were thoroughly characterized. The molecular structure of the synthesized complexes was ascertained by using single‐crystal X‐ray diffraction studies (SCXRDs). The experiment reveals that Complexes1–5bind to calf thymus DNA (CT‐DNA) through non‐intercalative way with good interacting abilities. However,1–5are excellent quenchers of the fluorescence intensity of bovine serum albumin (BSA) following the static pathway. Additionally, they had shown remarkable cytotoxic potential against MCF‐7 (mammary gland adenocarcinoma) and A549 (lung adenocarcinoma) cell lines. The IC50values associated with these complexes were much lower than the conventional drug cisplatin. Apoptosis‐induced cell death was confirmed from the DNA fragmentation studies and Hoechst 33342 staining. The 2′,7′‐dichlorofluorescein diacetate (DCFDA) assay indicates that the complex mediated reactive oxygen species (ROS) generation is accountable for governing the apoptosis mechanism via oxidative cell distress. Apart from these studies, by carrying out density functional theory (DFT) method, highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) energy gap calculations and optimized structures of the synthesized complexes were accomplished.

The sp3/sp2 carbon ratio as a modulator of in vivo and in vitro toxicity of the chemically purified detonation-synthesized nanodiamond via the reactive oxygen species generation

Nanodiamonds have been suggested as biocompatible materials and are suitable for various biomedical applications, but little is known about how to synthesize safer nanodiamonds. Herein, seven different detonation-synthesized nanodiamonds (DNDs) with sequential sp3/sp2 carbon ratios were assembled by controlling the chemical purification parameters and the role of sp3/sp2 carbon ratio on the toxicity of DNDs was investigated. Carbon black and nickel oxide nanoparticles were used as reference particles. The intrinsic reactive oxygen species (ROS) generation potential of DNDs was estimated by a 2′7′-dichlorofluorescein diacetate (DCFH-DA) assay, and these values showed a good negative correlation with the sp3/sp2 carbon ratios, which implies that ROS generation increased as the sp3/sp2 carbon ratio decreased. As a model to investigate inflammogenic potential of DND samples, a rat intratracheal instillation model was used as the lung is very sensitive to nanoparticle exposures. The sp3/sp2 carbon ratios or the estimated values of ROS generation potential showed excellent linear correlations with the number of neutrophils and pro-inflammatory cytokines in bronchoalveolar lavage fluid at 24 h after instillation. Treatment of DND samples to THP-1 derived macrophages also showed that the sp3/sp2 carbon ratios or the estimated values of ROS generation potential were closely related with the toxicity endpoints such as cell viability and pro-inflammatory cytokines. Taken together, these data demonstrate that the sp3/sp2 carbon ratio is the key determinant for the toxicity of DNDs, which can be a useful tool for the safer-by-design approach of DNDs and the safety assessment of carbon nanoparticles.

Yuzu and Hesperidin Ameliorate Blood-Brain Barrier Disruption during Hypoxia via Antioxidant Activity.

Yuzu and its main component, hesperidin (HSP), have several health benefits owing to their anti-inflammatory and antioxidant properties. We examined the effects of yuzu and HSP on blood–brain barrier (BBB) dysfunction during ischemia/hypoxia in an in vivo animal model and an in vitro BBB endothelial cell model, and also investigated the underlying mechanisms. In an in vitro BBB endothelial cell model, BBB permeability was determined by measurement of Evans blue extravasation in vivo and in vitro. The expression of tight junction proteins, such as claudin-5 and zonula occludens-1 (ZO-1), was detected by immunochemistry and western blotting, and the reactive oxygen species (ROS) level was measured by 2′7′-dichlorofluorescein diacetate intensity. Yuzu and HSP significantly ameliorated the increase in BBB permeability and the disruption of claudin-5 and ZO-1 in both in vivo and in vitro models. In bEnd.3 cells, yuzu and HSP were shown to inhibit the disruption of claudin-5 and ZO-1 during hypoxia, and the protective effects of yuzu and HSP on claudin-5 degradation seemed to be mediated by Forkhead box O 3a (FoxO3a) and matrix metalloproteinase (MMP)-3/9. In addition, well-known antioxidants, trolox and N-acetyl cysteine, significantly attenuated the BBB permeability increase, disruption of claudin-5 and ZO-1, and FoxO3a activation during hypoxia, suggesting that ROS are important mediators of BBB dysfunction during hypoxia. Collectively, these results indicate that yuzu and HSP protect the BBB against dysfunction via maintaining integrity of claudin-5 and ZO-1, and these effects of yuzu and HSP appear to be a facet of their antioxidant properties. Our findings may contribute to therapeutic strategies for BBB-associated neurodegenerative diseases.

Anti-fibrosis potential of pirarubicin via inducing apoptotic and autophagic cell death in rabbit conjunctiva

This study investigated the potential efficacy of pirarubicin (THP) in modulating rabbit conjunctival fibrosis both in vitro and in vivo and characterized the underlying mechanisms. Primary rabbit conjunctival fibroblasts (RCF) were cultured and treated with THP or mitomycin C (MMC) for 5 min, followed by assaying for cell viability, cell cycle distribution, apoptotic and autophagic pathways. The production of reactive oxygen species (ROS) and chemotaxis of macrophages by RCF were evaluated using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) labeling and transwell migration assay, respectively. Limbal stem cell excision in combination with alkali burn was performed on the rabbits to establish a model of limbal deficiency and conjunctival fibro-vascular invasion. After three months, the modeled fibro-vascular tissue was excised combined with topical subconjunctival 5-min exposure to THP compared with MMC intraoperatively. The recurrence of postoperative fibrosis and the expression of apoptosis, autophagy, and inflammation markers were evaluated by immunohistochemistry. All modeled rabbits developed conjunctival fibro-vascular lesions, which were similar to human recurrent pterygium (HRP). Both THP and MMC inhibited RCF proliferation and arrested cell cycle at the G0/G1 phase. In particular, 7.5 μmol/L THP remarkably promoted RCF autophagy by upregulating the levels of Beclin 1, Atg 5/12 conjugate, and LC3B, whereas, 15 μmol/L THP significantly triggered a cascade of mitochondrial-associated RCF apoptosis. THP induced the production of ROS and enhanced the chemoattraction of macrophages by RCF. Similar to 600 μmol/L MMC, both 7.5 μmol/L and 15 μmol/L THP attenuated postoperative conjunctival fibrosis in the models; 7.5 μmol/L THP preferentially enhanced autophagy while causing fewer side effects. THP exerted its antifibrotic action by modulating autophagy in RCF, inducing cell cycle arrest, and mitochondrial-mediated apoptosis. THP at the dose of 7.5 μmol/L prevented postoperative conjunctival fibrosis in an animal model.

On-chip real-time detection and quantification of reactive oxygen species in MCF-7 cells through an in-house built fluorescence microscope

Reactive oxygen species (ROS) are formed by several different reactions and play a major role in cell cycling and as a messenger in normal cell transduction and have diverse biological effects when high in concentration. ROS may trigger DNA damage, cell mutation, inflammation, cell proliferation, and ultimately they may induce carcinogenesis. This study aims to design, fabricate, and develop a novel on-chip ROS detection based on mounted in-house built fluorescence microscope in microfluidics, which has not been reported previously. An in-house 3D printed built digital portable fluorescence type microscope for on-chip ROS detection has been developed. The combined use of the fluorescence microscope and a glass-based microfluidic chip both enabled to provide high-quality images of the living cells with ease of controlling and handling of the biological sample. As an evidence of the fluorescence microscope, breast cancer cells were loaded with 2′,7′-dichlorofluorescein diacetate (DCFDA, 50 μM) for 45 min on-chip which produces green fluorescence upon reaction with intracellular ROS. Fluorescence of the MCF-7 cells has been captured to obtain a profile of ROS generation for 1 day. LabVIEW 2019 was used along with its high-performance image analysis and IMAQ Vision tools. The viability of the cells was performed by LIVE/DEAD assay and Luna cell counter. The developed platform provides real-time monitoring and quantification of ROS. A strong linear correlation between concentrations versus fluorescence has been recorded. This result recommends that the current microfluidic chip provides an in-vitro platform for the assessment of cell cycle progression and evaluation of the variety of drugs.

The aspect ratio of gold nanorods as a cytotoxicity factor on Raphidocelis subcaptata

Gold nanorods (AuNRs) are promising nanoscale materials for several technological and biomedical applications. The physicochemical properties of AuNRs, including size, shape and surface features, are crucial factors affecting their cytotoxicity. In this study, we investigated the effects of different aspect ratios of AuNRs (1.90, 2.35, 3.25 and 3.50) at concentrations of 2 and 10 μg mL−1 on their cytotoxicity and cellular uptake in green algae Raphidocelis subcaptata. The experiment was performed in oligotrophic freshwater medium in a growth chamber with constant agitation of 80 rpm under controlled conditions (120 μEm−2s−1 illumination; 12:12h light dark cycle and constant temperature of 22 ± 2 °C). The algal growth was monitored daily for 96 h via electronic absorbance scanning at 600–750 nm. Oxidative stress, cell viability and autofluorescence were evaluated using a flow cytometer. Oxidative stress quantified by loading cultures with the fluorescent dye 2′, 7′-dichlorofluorescein diacetate. To assess algal cell viability, propidium iodide was selected as the fluorescent probe. Our results indicated that the aspect ratio of AuNRs mediates their biological effects in green algae R. subcaptata. A positive correlation between oxidative stress and increase of aspect ratio was found at concentration of 10 μg mL−1. Higher cytotoxicity and mortality were observed for algae incubated with higher aspect ratios AuNRs (3.50). These findings may be useful to understand the impact of the AuNRs in aquatic environments, contributing to ecosystem management and nanomaterials regulation.

The protective effects of polyamines on salinity stress tolerance in foxtail millet (Setaria italica L.), an important C4 model crop.

Soil salinity is a major abiotic stress that adversely affects crop growth, development and productivity worldwide. In this study, the individual and synergistic roles of putrescine (Put) and spermidine (Spd) in salinity stress tolerance of foxtail millet (Setaria italica L.) was assessed. In the present study, plants treated with combined biogenic amines Put + Spd possess very efficient antioxidant enzyme systems which help to control the uninhibited oxidation and protect the plants from oxidative damage by ROS scavenging. Additionally, lower concentration of Put + Spd under NaCl stress showed reduced hydrogen peroxide, electrolyte leakage and caspase-like activity than control. FTIR analysis underlying the ability of PAs induced tolerance and the chemical bonds of Put + Spd treated plants were reminiscent of control plants. Moreover, histochemical analysis with 2',7'-dichlorofluorescein diacetate (DCF-DA), 3,3'-Diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) revealed that ROS accumulation was inhibited by combined PAs under salt stress condition. These results showed that Put + Spd significantly improve the endogenous PAs, which enhance high-salinity stress tolerance by detoxifying ROS. For the first time, the synergistic ROS scavenging ability of Put along with Spd was investigated upon salinity tolerance in C4 model foxtail millet crop. Overall, our findings illustrated the implication for improving salinity tolerance of agronomically important crop species.

In vitro model using cytokine cocktail to evaluate apoptosis in Min6 pancreatic beta cells

IntroductionDevelopment of therapy options for treatment of type 1 diabetes mellitus is hampered by non-availability of appropriate experimental models that can exactly mimic the in vivo situation. Apoptosis of beta cells by T cells and cytokine action leads to loss of beta cells. We propose a simple and elegant model using cytokine cocktail of TNF-α, IFN-γ and IL-1β, the major cytokines responsible for apoptosis in Min6 beta cell line. MethodsA cocktail of TNF-α, IFN-γ and IL-1β was used to induce apoptosis in Min6 beta cell line. Apoptosis was assessed by flow cytometry using CytoFLEX (Beckman Coulter). The destruction of beta cells is through production of nitric oxide (NO), oxidative stress and change in mitochondrial membrane permeability. NO was measured using Griess reagent. Oxidative stress was assessed using 2',7'-dichlorofluorescein diacetate, a cell-permeable fluorogenic dye and mitochondrial membrane potential was determined on the basis of retention of rhodamine 123 using flow cytometer. Results and discussionVery low concentration of the cocktail viz. TNF-α 25 ng/ml, IFN-γ 25 ng/ml and IL-1β 50 ng/ml has demonstrated effective early and late apoptosis in as short a time period as 6 h. The experimental model used demonstrated 1.5 fold higher production of NO, 1.2 fold increased oxidative stress and lower mitochondrial membrane potential as compared to the positive control used. Hence the above model can be easily used for assessment and screening of drugs that can prevent apoptosis of beta cells and stop progression of type 1 diabetes.

New Selenonapthaquinone-Based Copper (II) Complexes as the Next-Generation Photochemotherapeutic Agents.

Photoactive transition metal complexes like copper complexes find great interest in promoting metal-based photochemotherapeutic agents. In the present study, we explored the photocytotoxic efficacy of new selenylnaphthoquinone-based copper (II) complexes that provide a phenomenal platform in making an effective photo-chemotherapeutic agent via PDT in the clinical field of cancer therapy. Three new copper(II) complexes (1-3) were synthesized in 40-60% yield and characterized analytically/ spectroscopically. ATCC® Normal Adult Human Primary Epidermal Keratinocytes were grown in Dermal Cell Basal Media supplemented with Keratinocyte Growth Kit components, to propagate keratinocytes in serum- free (not animal free) conditions. Anticancer activity of the complexes was studied using MTT (3- [4,5- dimethyltiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. The intracellular ROS (1O2) generation was studied by using Flow Cytometric Analysis (FACS) on HaCaT cells using cell accessible non-polar 2',7'- Dichlorofluorescein Diacetate (DCFH-DA) dye. The Acridine Orange/Ethidium Bromide (AO/EB) dual staining assay was performed for detecting apoptosis in HaCaT cells. Several photophysical studies probing the generation of singlet oxygen was also carried out. We have performed Time-Dependent Density Functional Theory (TD-DFT) calculations using unrestricted B3LYP to understand the mechanism of type-II process. All the complexes were remarkably cytotoxic in HaCaT cells with IC50, 1-4μM under visible light with comparing lower dark toxicity. The presence of low-lying and long-lived triplet excited state allowed effective intersystem crossing and subsequent generation of singlet oxygen, which was the primary cytotoxic species responsible for oxidative stress and apoptosis. The experimental findings are in good agrrement with the computational analysis (TD-DFT). The remarkably enhanced cytotoxicity of the new selenyl copper (II) complexes under the visible light probed the role of Se in photosensitized generation of singlet oxygen which was responsible for apoptosis in HaCaT cells. The results in the present work are of paramount importance in developing next generation copper(II)-based PDT agents.

Local industrial pollution induces astrocyte cytoskeleton rearrangement in the dice snake brain: GFAP as a biomarker

The present study was designed to evaluate the responsiveness of modulation of glial fibrillary acidic protein (GFAP) content and its fragmentation in the snake brain as a biomarker of local industrial pollution of aquatic ecosystems. Despite GFAP being a well known cytoskeleton marker of astrocytes’ reactivity in the brain of vertebrates, its expression in the snake brain remains insufficiently described. The GFAP expression and its fragmentation were detected using the immunoblot method in the snake brain. ROS level was determined with dichlorofluorescein diacetate fluorescence. The content of the glial fibrillary acidic protein (GFAP) of filament (cytoskeleton) and soluble (cytosol) fractions in the brain of dice snake Natrix tessellata from three ecosystems with different rates of industrial pollution were studied (two polluted and one clean control site). Characteristic increase in GFAP fragmentation was noted for the snakes from both the researched polluted sites. Significant increase in the content of the GFAP cleaved polypeptide fragments induced by industrial pollution exposure was confirmed in the snakes’ brains. Meaningful GFAP fragmentation was determined in snake brain astrocytes as an increase in cleaved fragments of 47–35 kDa molecular weight for both soluble and cytoskeletal GFAP fractions. We found significant abnormality in the ratio of the GFAP soluble fraction to the cytoskeletal one in contaminant-exposed dice snakes. It should testify to significant metabolic disturbance in nerve cells of the dice snakes. Furthermore, growth of reactive oxygen species level as the main cause of oxidative stress was determined in brains of the snakes exposed to environmental toxicity. Thus, astrocyte cytoskeleton disorders are associated with pollutant-induced redox imbalance in the snake brain. Despite the limited data on glial cell biology in the reptilian brain, the observed results prove that snake astrocytes can respond to the environmental toxicity using typical astroglial response. The presented results evidence that monitoring of molecular characteristics of glial cytoskeleton in dice snakes could be used as reliable biomarker of neurotoxicity and adverse effects of industrial pollution. Further studies are required to elucidate the role of astrocyte cytoskeleton in the response against neurotoxic contaminants.

Triggering of Apoptosis in Osteosarcoma 143B Cell Line by Carbon Quantum Dots via the Mitochondrial Apoptotic Signal Pathway.

Objectives Carbon-based nanomaterials have gained attention in the field of biomedicine in recent years, especially for the treatment of complicated diseases such as cancer. Here, we report a novel carbon-based nanomaterial, named carbon quantum dots (CQDs), which has potential for cancer therapy. We performed a systematic study on the effects of CQDs on the osteosarcoma 143B cell line in vitro and in vivo. Methods Cell counting assay, the neutral red assay, lactic dehydrogenase assay, and fluorescein isothiocyanate (FITC) Annexin V/Propidium iodide (PI) were used to detect the cytotoxicity and apoptosis of CQDs on the 143B cell line. Intracellular reactive oxygen species (ROS) were detected by the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate. The JC-10 assay was used to detect the mitochondrial membrane potential (MMP) of 143B cells incubated with CQDs. The effects of CQDs on the 143B cell line were evaluated by Western blot and immunofluorescence analysis of apoptosis-related proteins Bax, Bcl-2, cytochrome-C, caspase-3, cleaved-caspase-3, PARP1, and cleaved-PARP1. Male tumor-bearing BALB/c nude mice were used to investigate the antitumor effects of CQDs, and the biosafety of CQDs in vivo was tested in male BALB/c mice by measuring weight changes, hematology tests, and histological analyses of major organs. Results CQDs exhibited a high cytotoxicity and induced apoptosis toward the 143B cell line. CQDs can also significantly increase the intracellular level of ROS and lower the mitochondrial membrane potential levels of 143B cells. CQDs increase apoptotic protein expression to induce apoptosis of 143B cells by triggering the mitochondrial apoptotic signaling pathway. The tumor volume in the CQD-treated mice was smaller than that in the control group, the tumor volume inhibition rate was 38.9%, and the inhibitory rate by tumor weight was 30.1%. All biosafety test indexes were within reference ranges, and neither necrosis nor inflammation was observed in major organs. Conclusions CQDs induced cytotoxicity in the 143B cell line through the mitochondrial apoptotic signaling pathway. CQDs not only showed an antitumor effect but also high biocompatibility in vivo. As a new carbon-based nanomaterial, CQDs usage is a promising method for novel cancer treatments.

Theaflavin-3, 3'-Digallate Suppresses RANKL-Induced Osteoclastogenesis and Attenuates Ovariectomy-Induced Bone Loss in Mice.

Theaflavin-3, 3′-digallate (TF3) is extracted from black tea and has strong antioxidant capabilities. The aim of this study was to assess the influences of TF3 on osteoclastogenesis and explore the underlying mechanisms. TF3 efficiently decreased receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and reactive oxygen species (ROS) generation in a dose-dependent manner. Mechanistically, TF3 reduced ROS generation by activating nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream heme oxygenase-1 (HO-1) and also inhibited the mitogen-activated protein kinases (MAPK) pathway. Moreover, micro-computed tomography (CT) analysis, hematoxylin and eosin (H&E) staining, and TRAP staining of the femurs of C57BL/6J female mice showed that TF3 markedly attenuated bone loss and osteoclastogenesis in mice. Immunofluorescence staining, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining, and measurement of the levels of malonaldehyde (MDA) and superoxide dismutase (SOD) revealed that TF3 increased the expression of Nrf2 and decreased the intracellular ROS level in vivo. These findings indicated that TF3 may have the potential to treat osteoporosis and bone diseases related to excessive osteoclastogenesis via inhibiting the intracellular ROS level.

Melatonin inhibits high glucose-induced ox-LDL/LDL expression and apoptosis in human umbilical endothelial cells

Cardiovascular disease (CVD) is one of the major cause of mortality in diabetic patients. Evidence suggests that hyperglycemia in diabetic patients contributes to increased risk of CVD. This study is to investigate the therapeutic effects of melatonin on glucose-treated human umbilical vein endothelial cells (HUVEC) and provide insights on the underlying mechanisms. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and membrane potential was detected using 2',7'-dichlorofluorescein diacetate and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) dye staining, respectively. While, cell apoptosis was determined by Annexin-V staining and protein expression was measured using Western blot. Our results suggested that melatonin inhibited glucose-induced ROS elevation, mitochondria dysfunction and apoptosis on HUVEC. Melatonin inhibited glucose-induced HUVEC apoptosis via PI3K/Akt signaling pathway. Activation of Akt further activated BcL-2 pathway through upregulation of Mcl-1 expression and downregulation Bax expression in order to inhibit glucose-induced HUVEC apoptosis. Besides that, melatonin promoted downregulation of oxLDL/LOX-1 in order to inhibit glucose-induced HUVEC apoptosis. In conclusion, our results suggested that melatonin exerted vasculoprotective effects against glucose-induced apoptosis in HUVEC through PI3K/Akt, Bcl-2 and oxLDL/LOX-1 signaling pathways.

TGF-β1 Activates Nasal Fibroblasts through the Induction of Endoplasmic Reticulum Stress.

(1) Background: Tissue remodeling and extracellular matrix (ECM) accumulation contribute to the development of chronic inflammatory diseases of the upper airway. Endoplasmic reticulum (ER) stress is considered to be the key signal for triggering tissue remodeling in pathological conditions. The present study aimed to investigate the role of ER-stress in TGF-β1-stimulated nasal fibroblasts and inferior turbinate organ cultures; (2) Methods: Fibroblasts and organ cultures were pretreated with 4-phenylbutyric acid (PBA) and stimulated with TGF-β1 or thapsigargin (TG). Expression of ER-stress markers, myofibroblast marker, and ECM components was measured by Western blotting and real-time PCR. Reactive oxygen species (ROS) were quantified using 2′,7′-dichlorofluorescein diacetate. Cell migration was evaluated using Transwell assays. Contractile activity was measured by collagen contraction assay; (3) Results: 4-PBA inhibited TGF-β1 or TG-induced ER-stress marker expression, phenotypic changes, and ECM. Pre-treatment with ROS scavengers inhibited the expression of TGF-β1-induced ER-stress markers. Migration and collagen contraction of TGF-β1 or TG-stimulated fibroblasts were ameliorated by 4-PBA treatment. These findings were confirmed in ex vivo organ cultures; (4) Conclusions: 4-PBA downregulates TGF-β1-induced ER-stress marker expression, migration, and collagen contraction via ROS in fibroblasts and organ cultures. These results suggest that ER-stress may play an important role in progression of chronic upper airway inflammatory diseases by aiding pathological tissue remodeling.

Andrographis paniculata and Its Bioactive Diterpenoids Against Inflammation and Oxidative Stress in Keratinocytes.

Andrographis paniculata was widely used in traditional herbal medicine to treat various diseases. This study explored the potential anti-aging activity of Andrographis paniculata in cutaneous cells. Human, adult, low calcium, high temperature (HaCaT) cells were treated with methanolic extract (ME), andrographolide (ANDRO), neoandrographolide (NEO), 14-deoxyandrographolide (14DAP) and 14-deoxy-11,12-didehydroandrographolide (14DAP11-12). Oxidative stress and inflammation were induced by hydrogen peroxide and lipopolysaccharide/TNF-α, respectively. Reactive oxygen species (ROS) production was measured by fluorescence using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe and cytokines were quantified by ELISA for interleukin-8 (IL-8) or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for tumor necrosis factor-α (TNF-α). Hyaluronic acid (HA) secretion was determined by an ELISA. Our results show a decrease in ROS production and TNF-α expression by ME (5 µg/mL) in HaCaT under pro-oxidant and pro-inflammatory conditions, respectively. ME protected HaCaT against oxidative stress and inflammation. Our findings confirm that ME can be used for the development of bioactive compounds against epidermal damage.

Miconazole and terbinafine induced reactive oxygen species accumulation and topical toxicity in human keratinocytes

There are an estimated 1 billion cases of superficial fungal infection globally. Fungal pathogens form biofilms within wounds and delay the wound healing process. Miconazole and terbinafine are commonly used to treat fungal infections. They induce the accumulation of reactive oxygen species (ROS) in fungi, resulting in the death of fungal cells. ROS are highly reactive molecules, such as oxygen (O2), superoxide anion (O2•−), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH). Although ROS generation is useful for killing pathogenic fungi, it is cytotoxic to human keratinocytes. To the best of our knowledge, the effect of miconazole and terbinafine on HaCaT cells has not been studied with respect to intracellular ROS stimulation. We hypothesized that miconazole and terbinafine have anti-wound healing effects on skin cells when used in antifungal treatment because they generate ROS in fungal cells. We used sulforhodamine B protein staining to investigate cytotoxicity and 2′,7′-dichlorofluorescein diacetate to determine ROS accumulation at the 50% inhibitory concentrations of miconazole and terbinafine in HaCaT cells. Our preliminary results showed that topical treatment with miconazole and terbinafine induced cytotoxic responses, with miconazole showing higher cytotoxicity than terbinafine. Both the treatments stimulated ROS in keratinocytes, which may induce oxidative stress and cell death. This suggests a negative correlation between intracellular ROS accumulation in keratinocytes treated with miconazole or terbinafine and the healing of fungi-infected skin wounds.

Protein-Bound Polysaccharides from Coriolus Versicolor Induce RIPK1/RIPK3/MLKL-Mediated Necroptosis in ER-Positive Breast Cancer and Amelanotic Melanoma Cells.

The induction of necroptosis, a form of caspase-independent cell death, represents one of the most promising anticancer therapeutic modalities, as necroptosis serves as an alternative way to eliminate apoptosis-resistant tumor cells. Here, we investigated whether protein-bound polysaccharides (PBPs) derived from the fungus Coriolus versicolor (CV) induce the necroptotic death pathway in breast cancer and melanoma cells. MCF-7 and SKMel-188 cells were exposed to PBPs either alone or in combination with necrostatin-1 (Nec-1), GSK'872 or necrosulfonamide (NSA), pharmacological inhibitors of the kinases receptor-interacting protein 1 kinase (RIPK1), receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), respectively, which are involved in necroptotic processes. The effects of cellular treatment with these inhibitors were quantified by measuring cell viability and reactive oxygen species (ROS) generation via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein diacetate (DCF-DA) assays, respectively. The morphological changes induced in these cells were detected using holotomographic (HT) microscopy. Activation of the TNF-α/TNFR1 pathway in the PBP-stimulated cells was evaluated using TNF-α-neutralizing antibody, qRT-PCR and immunofluorescence-based assays. PBPs showed effective antitumor activity against MCF-7 and SKMel-188 cells. Cotreatment of the cells with Nec-1, GSK'872 or NSA abrogated PBP-induced cell death, and the cells were protected against membrane rupture. Moreover, breast cancer cell death caused by PBPs was mediated by induced activation of the TNF-α/TNFR1 pathway. Interestingly, the melanoma cells did not express TNF-α or TNFR1 after PBP stimulation; instead, PBPs triggered intracellular ROS generation, which was partially diminished by the inhibitors Nec-1, GSK'872 and NSA. These results suggest that PBPs from the fungus CV induce RIPK1/RIPK3/MLKL-mediated necroptosis in breast cancer and melanoma cells, providing novel insights into the molecular effects of PBPs on cancer cells.

Physiochemical Characterization and Anti-colon Cancer Activity of Biosurfactant Produced from Marine Pseudomonas sp.

Background: Biosurfactants are the surface-active molecules produced by the living micro-organisms. They have wide application in the food, cosmetic, pharmaceutical and petrochemical industry due to its physiological and functional properties. Objectives: In the present study, biosurfactant produced from Pseudomonas sp. was used for its physiochemical characterizations and anti-tumergenic property against colon cancer cell line (SW480). Materials and Methods: The biosurfactant was produced in Bushnell Haas Broth media and extracted by solvent extraction method. The physiochemical characterizations such as water solubility index, water activity, foaming index, oil holding capacity of extracted biosurfactant were performed. The degree of toxicity of biosurfactant was measured using a conversion assay of 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) dye against SW480 and mouse L292 fibroblastic cell line (non-tumergenic). Furthermore, acridine orange-EtBr (ethidium bromide) staining, Dichlorofluorescein Diacetate (DCF-DA) staining, wound healing assay were performed to determine the anti-colon cancer activity of biosurfactant. Results: The oil holding capacity, water activity (aw), foaming capacity and water solubility index for biosurfactant were found to be 4.36 ± 0.05 g/g, 0.32 ± 0.03, 52.28 ± 0.10 % and 80.86 ± 0.20 % respectively. In the MTT assay, after 48 h of incubation, 84.7% of cell viability on L292 was observed at 250 μg/ml (maximum concentration) for biosurfactant. Whereas, only 50% cell viability was observed after 48 h of incubation on SW480 at 168.52 μg/ml biosurfactant concentration. Conclusion: The biosurfactant could be a bench-mark as a non-cytotoxic biosurfactant which may be used as a possible biological substance in the food industry as a stabilizing agent and in pharmaceutical industry as an anti-colon cancer agent.

Effects of Eurya japonica extracts on human corneal epithelial cells and experimental dry eye.

Eurya japonica (EJ) leaves have been indicated to exert anti-oxidative and anti-inflammatory effects. Dry eye disease (DED) is a chronic inflammatory disease and oxidative stress is closely associated with DED. The aim of the present study was to analyze the therapeutic efficacy of EJ in DED using human corneal epithelial (HCE) cells and a mouse model of experimental dry eye (EDE). EJ extracts (0.001, 0.01 and 0.1%) were used to treat HCE cells. Cell viability and mitochondrial function were detected using a EZ-Cytox cell viability assay kit and mitochondrial membrane potential assays. Dichlorofluorescein diacetate (DCF-DA) assay was used to measure cellular reactive oxygen species (ROS) levels. Subsequently, eye drops consisting of BSS or 0.001%, 0.01 and 0.1% EJ extracts were applied for treatment of EDE. At 7 days, conjunctival ROS production was measured using a DCF-DA assay. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, 10 kDa interferon gamma-induced protein 10 (IP-10) and monokine induced by interferon-γ (MIG) levels in the conjunctiva were analyzed using a multiplex immunobead assay. Tear film and ocular surface parameters were measured. Treatment with EJ extracts in HCE cells effectively improved cell viability, ROS levels and mitochondrial function. Mice treated with 0.01 and 0.1% EJ extracts indicated a significant decrease in ROS, TNF-α, IL-1β, IP-10 and MIG levels compared with the EDE or BSS groups. Furthermore, a significant improvement in all clinical parameters was observed in the 0.01 and 0.1% EJ extract groups. EJ extracts could decrease cytotoxicity and ROS production in HCE cells. Additionally, topical EJ extracts reduced oxidative damage and inflammation and improved clinical signs of EDE, suggesting that EJ extracts may be used as an adjunctive therapy for DED.