Intracellular Reactive Oxygen Species Production Research Articles

Unveiling the antifungal mechanisms of CTP, a new copper(II)-theophylline/1,10-phenanthroline complex, on drug-resistant non-albicans Candida species.

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo)2phen(H2O).5H2O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.

Persistent organic pollutants disrupt the oxidant/antioxidant balance of INS-1E pancreatic β-cells causing their physiological dysfunctions

BackgroundPersistent organic pollutants (POPs) have emerged as potent diabetogenic agents, but their mechanisms of action remain poorly identified. ObjectivesIn this study, we aim to determine the mechanisms regulating the damaging effects of POPs in pancreatic β-cells, which have a central role in the development of diabetes. MethodsWe treated INS-1E pancreatic β-cells with PCB-153, p,p’-DDE, PCB-126, or TCDD at doses ranging from 1 × 10-15to 5 × 10-6M. We measured insulin content and secretion, cell viability and assessed the mRNA expression of the xenobiotic nuclear receptors Nr1i2 and Nr1i3, and the aryl hydrocarbon receptor (Ahr). In addition, we evaluated the antioxidant defense and production of reactive oxygen species (ROS). Finally, we studied the ability of the antioxidant N-acetyl-L-cysteine (NAC) to counteract the effects of POPs in INS-1E cells. ResultsWhen exposed to environmental POP levels, INS-1E cells had impaired production and secretion of insulin. These defects were observed for all tested POPs and were paralleled by reduced Ins1 and Ins2 mRNA expression. While POP treatment for 3 days did not affect INS-1E cell viability, longer treatment progressively killed the cells. Furthermore, we found that the xenobiotic detoxification machinery is poorly expressed in the INS-1E cells, as characterized by the absence of Nr1i2 and Nr1i3 and their respective downstream targets Cyp3a1/Cyp3a2 and Cyp2b1/Cyp2b3, and the weak functionality of the Ahr/Cyp1a1 signaling. Interestingly, POPs dysregulated key antioxidant enzymes such as glutathione peroxidases, peroxiredoxins, thioredoxins, and catalases. In parallel, the production of intracellular ROS, including superoxide anion (O2•−) and hydrogen peroxide (H2O2), was increased by POP exposure. Improving the oxidant scavenging capacity of INS-1E cells by NAC treatment restored the production and secretion of insulin. ConclusionBy promoting oxidative stress and impairing the ability of INS-1E cells to produce and secrete insulin, this study reveals how POPs can mechanistically act as diabetogenic agents, and provides new scientific evidence supporting the concept that POPs are fueling the diabetes epidemics.

The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

ObjectivesInterleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process related to inflammatory signals, intracellular reactive oxygen species (ROS) production, and lipid peroxidation, have been proposed as potential mechanisms that contribute to the development and progression of EMs. IL-33 is highly upregulated in the ectopic milieu. Moreover, ectopic endometrial cells constitutively express interleukin-33 receptor ST2 (IL-33R). However, the role of IL-33/ST2 in the EMT of EMs remains largely unknown. In this study, we aimed to mechanistically determine the role of IL-33/ST2 in EMs-associated fibrosis.Materials and methodsWe established a non-lethal oxidative stress model to explore the conditions that trigger IL-33 induction. We performed α-smooth muscle actin (α-SMA) protein detection, cell counting kit-8 (CCK-8) assays, and scratch assays to analyze the impact of IL-33 on primary endometrial stromal cells (ESCs) proliferation and invasion. Clinical samples from patients with or without EMs were subjected to immunohistochemical (IHC) and and immunofluorescence(IF) staining to assess the clinical relevance of IL-33 receptor ST2 and EMT-related proteins. Furthermore, we used the ectopic human endometrial epithelial cell line 12Z and normal human epithelial cell line EEC to evaluate the effects of IL-33 on Wnt/β-catenin signaling. The effect of IL-33 on EMT-associated fibrosis was validated in vivo by intraperitoneal injections of IL-33 and antiST2.ResultsWe observed that ectopic milieu, characterized by ROS, TGF-β1, and high level of estrogen, triggers the secretion of IL-33 from ectopic ESCs. Ectopic endometrial lesions exhibited higher level of fibrotic characteristics and ST2 expression than that in the normal endometrium. Exogenous recombinant human (rhIL-33) enhanced ESC migration and survival. Similarly, 12Z cells displayed a higher degree of EMT characteristics with elevated expression of CCN4 and Fra-1, downstream target genes of the WNT/β-catenin pathway, than that observed in EECs. Conversely, blocking IL-33 with neutralizing antibodies, knocking down ST2 or β-catenin with siRNA, and β-catenin dephosphorylation abolished its effects on EMT promotion. In vivo validation demonstrated that IL-33 significantly promotes EMs-related fibrosis through the activation of Wnt/β-catenin signaling.ConclusionOur data strongly support the vital role of the IL-33/ST2 pathway in EMs-associated fibrosis and emphasize the importance of the EMT in the pathophysiology of fibrosis. Targeting the IL-33/ST2/Wnt/β-catenin axis may hold promise as a feasible therapeutic approach for controlling fibrosis in EMs.

Homotherapy for heteropathy: therapeutic effect of Butein in NLRP3-driven diseases

BackgroundAberrant inflammatory responses drive the initiation and progression of various diseases, and hyperactivation of NLRP3 inflammasome is a key pathogenetic mechanism. Pharmacological inhibitors of NLRP3 represent a potential therapy for treating these diseases but are not yet clinically available. The natural product butein has excellent anti-inflammatory activity, but its potential mechanisms remain to be investigated. In this study, we aimed to evaluate the ability of butein to block NLRP3 inflammasome activation and the ameliorative effects of butein on NLRP3-driven diseases.MethodsLipopolysaccharide (LPS)-primed bone-marrow-derived macrophages were pretreated with butein and various inflammasome stimuli. Intracellular potassium levels, ASC oligomerization and reactive oxygen species production were also detected to evaluate the regulatory mechanisms of butein. Moreover, mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis were used to test whether butein has protective effects on these NLRP3-driven diseases.ResultsButein blocks NLRP3 inflammasome activation in mouse macrophages by inhibiting ASC oligomerization, suppressing reactive oxygen species production, and upregulating the expression of the antioxidant pathway nuclear factor erythroid 2-related factor 2 (Nrf2). Importantly, in vivo experiments demonstrated that butein administration has a significant protective effect on the mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis.ConclusionOur study illustrates the connotation of homotherapy for heteropathy, i.e., the application of butein to broaden therapeutic approaches and treat multiple inflammatory diseases driven by NLRP3.

Effects of parity and week after calving on the metabolic, redox, and immune status of dairy cows

At the onset of lactation in dairy cows, inflammation and oxidative stress may occur and result in a risk of pathologies and lower milk yield. To propose an innovative management strategy for cows during this period, it is essential to better understand these physiological variations. Our objective was to evaluate the metabolic, redox and immune status of 7 primiparous and 8 multiparous Holstein cows during late gestation and the first months of lactation. Blood samples were collected between 3 weeks before calving until 12 weeks postpartum. Milk samples were also collected, but only at the time points after calving. The metabolic (nonesterified fatty acids (NEFA), BHB, glucose, urea, calcium) and redox (reactive oxygen metabolites (ROM), oxidative stress index (OSI), glutathione peroxidase activity, vitamin E) statuses were analyzed in plasma or erythrocytes. The expression of genes related to antioxidant functions was determined in leukocytes collected from milk. For immune status, plasma cytokine levels and the production of reactive oxygen species (ROS) in classical and regulatory neutrophils were measured in 2 whole blood ex vivo challenges. The data were analyzed using a mixed model that included the fixed effects of parity and week and their interaction. Milk yield, plasma NEFA and BHB in wk 2 and 4 after calving were higher in multiparous cows than in primiparous cows, whereas glucose and calcium tended to be lower. Plasma ROM and OSI levels in wk 8 were higher in multiparous than in primiparous cows. Multiparous cows also displayed higher glutathione peroxidase activity in erythrocytes, and antioxidant transcription factor and superoxide dismutase-1 expression levels in milk leukocytes. Moreover, multiparous cows had higher plasma concentrations of vitamin E but lower plasma levels of cytokines CXCL10, CCL2, IL1Rα and IFNγ. Following ex vivo whole blood stimulation with Escherichia coli, lower IL1α and TNFα levels were measured in multiparous than in primiparous cows. Intracellular ROS production by neutrophils was lower in multiparous than in primiparous cows. These results thus indicated marked physiological changes in wk 8 compared with wk 2 and 4 of lactation. These differences in the physiological status of primiparous and multiparous cows offer interesting perspectives for potential dietary strategies to prevent pathologies which take account of parity and week relative to calving.

Protective Effects of the Postbiotic Levilactobacillus brevis BK3 against H2O2-Induced Oxidative Damage in Skin Cells.

Postbiotics have various functional effects, such as antioxidant, anti-inflammatory, and anti-obesity. Levilactobacillus brevis BK3, the subject of this study, was derived from lactic acid bacteria isolated from Kimchi, a traditional Korean fermented food. The antioxidant activity of BK3 was confirmed through the measurements of 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and total antioxidant capacity (TAC). The wrinkle improvement effect was validated by assessing elastase inhibitory activity and collagenase inhibitory activity. The intracellular activity was confirmed using human keratinocytes (HaCaT) and human fibroblasts (HFF-1). BK3 protects skin cells from oxidative stress induced by H2O2 and reduces intracellular reactive oxygen species (ROS) production. In addition, the expressions of the antioxidant genes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were upregulated. Meanwhile, matrix metalloproteinase-1 (MMP-1) and collagen type I alpha 1 (COL1A1), involved in collagen degradation and synthesis, were significantly regulated. These results suggest the possibility of utilizing BK3 as a functional ingredient with antioxidant and wrinkle-improving effects.

Knockdown of iPLA2γ enhances cisplatin-induced apoptosis by increasing ROS-dependent peroxidation of mitochondrial phospholipids in bladder cancer cells

Cisplatin (CDDP) is a platinum-based drug with anti-cancer activity and is widely used as a standard therapy for bladder cancer. It is well known that CDDP causes cell death by increasing the generation of reactive oxygen species (ROS) and lipid peroxidation, but the mechanism of its anti-cancer effects has not been fully elucidated. There are still some problems such as chemoresistance in CDDP therapy. In the present study, we found the expression of Ca2+-independent phospholipase A2γ (iPLA2γ), which has been reported to regulate cellular redox homeostasis by inhibiting lipid peroxide accumulation, in human bladder cancer tissues. Thus, we investigated the effect of iPLA2γ knockdown on CDDP-induced bladder cancer cell death. As a result, we found that iPLA2γ knockdown significantly enhanced CDDP-induced apoptosis, intracellular and mitochondrial ROS production, cytochrome c release and caspase activation in bladder cancer cells. Moreover, mitochondrial membrane potential was decreased and peroxidation of mitochondrial phospholipids was increased by iPLA2γ knockdown. It was also shown that co-treatment of bromoenol lactone, an iPLA2 inhibitor, increased CDDP-induced apoptosis. These results indicated that iPLA2γ plays an important role in protecting bladder cancer cells from CDDP-induced apoptosis, and that iPLA2γ inhibitors might represent a novel strategy in CDDP-based multi-drug therapy.

The Power of Synergism: A Novel Drug Combination for Improved Therapy of Glioblastoma

AbstractOne of the biggest obstacles in treating glioblastoma multiforme (GBM) is the plasticity and adaptability of GBM cells, which renders monotherapy ineffective. Novel drug combinations that target multiple pathways are required to overcome this challenge. In the current study, the PI3K/AKT/mTOR pathway is identified as a crucial component in GBM cell survival using the KEGG database and literature search. Hence, it is hypothesized that a potential synergistic therapy can be achieved by targeting different stages of this pathway by combining disulfiram (DSF) and 7‐ethyl‐10‐hydroxycamptothecin (SN‐38). The efficacy of this drug combination is evaluated using 2D and 3D in vitro assays, which exhibited a significant synergism with 5 × 104 times lower IC50 than that of temozolomide (TMZ), the gold‐standard drug. The combination treatment increases intracellular ROS (reactive oxygen species) production, and ROS inhibition by using N‐Acetyl Cysteine (NAC) reduced the cytotoxicity substantially, indicating that ROS played a crucial role in the synergistic cytotoxicity. The combination treatment inhibited GBM cell proliferation, migration, and stem cell marker expression. Mechanistically, the DSF+SN‐38 combination is found to increase p53 expression, inhibit PI3K signaling, and activate caspase 9. Altogether, this study demonstrates that the DSF+SN‐38 combination can present a promising therapeutic strategy for treating GBM.

Antioxidant, anti-photoaging, anti-inflammatory, and skin-barrier-protective effects of Gleichenia japonica extract

Ferns native to Korea, such as Davallia mariesii, Dicranopteris pedata, and Gleichenia japonica, possess antioxidant and antibacterial properties. However, their inhibitory effects on skin photoaging have not been demonstrated. Measurement and comparison of the antioxidant activity of three types of ferns revealed that the extract from G. japonica had the best effect. This study evaluates the potential of G. japonica extract as a new functional material for preventing skin damage caused by ultraviolet radiation. G. japonica extracts showed protective effects against ultraviolet B (UVB) radiation in human epidermal keratinocyte cells; the extracts inhibited intracellular reactive oxygen species production. In addition, collagen biosynthesis increased, and matrix metalloproteinase-1 activity and protein expression level decreased in human primary dermal fibroblast irradiated with UVB. The main peak (compound 1) of the extract was separated through high-performance liquid chromatography analysis and preparative liquid chromatography. Compound 1 is strongly inferred to be the main active ingredient because it showed better antioxidant activity and UVB protection effect than G. japonica extract. These results demonstrate the physiological effects of G. japonica extract and suggest its applicability as a new functional substance for preventing skin damage caused by ultraviolet radiation.

Intracellular Oxygen Transient Quantification in Vivo During Ultra-High Dose Rate FLASH Radiation Therapy

Large, rapid extracellular oxygen transients (ΔpO2) have been measured in vivo during ultra-high dose rate radiation therapy; however, it has been unclear if they match intracellular oxygen levels. Here, the endogenously produced protoporphyrin IX (PpIX) delayed fluorescence signal was measured as an intracellular in-vivo oxygen sensor to quantify these transients, with direct comparison to extracellular pO2. Intracellular ΔpO2 is closer to the cellular DNA, the site of major radiobiological damage, and therefore should help elucidate radiochemical mechanisms of the FLASH effect and potentially be translated to human tissue measurement. PpIX was induced in mouse skin through intraperitoneal injection of 250 mg/kg of aminolevulinic acid. The animals were also administered a 50 µL intradermal injection of 10 µM oxyphor G4 (PdG4) for phosphorescence lifetime pO2 measurement. Paired oxygen transients were quantified in leg or flank tissues while delivering 10 MeV electrons in 3 µs pulses at 360 Hz for a total dose of 10 to 28 Gy. Transient reductions in pO2 were quantifiable in both PpIX delayed fluorescence and oxyphor phosphorescence, corresponding to intracellular and extracellular pO2 values, respectively. Reponses were quantified for 10, 22, and 28 Gy doses, with ΔpO2 found to be proportional to the dose on average. The ΔpO2 values were dependent on initial pO2 in a logistic function. The average and standard deviations in ΔpO2 per dose were 0.56 ± 0.18 mm Hg/Gy and 0.43 ± 0.06 mm Hg/Gy for PpIX and oxyphor, respectively, for initial pO2 > 20 mm Hg. Although there was large variability in the individual animal measurements of ΔpO2, the average values demonstrated a direct and proportional correlation between intracellular and extracellular pO2 changes, following a linear 1:1 relationship. A fundamentally new approach to measuring intracellular oxygen depletion in living tissue showed that ΔpO2 transients seen during ultra-high dose rate radiation therapy matched those quantified using extracellular oxygen measurement. This approach could be translated to humans to quantify intracellular ΔpO2. The measurement of these transients could potentially allow the estimation of intracellular reactive oxygen species production.

Aging Is Associated with a Circulating Endothelial Cell-Derived Microvesicle Phenotype that Increases Cerebral Oxidative Stress

Aging is associated with increased risk and prevalence of ischemic stroke. Vascular oxidative stress impairs cell function and is considered to be an important mechanism underlying the age-related increase in cerebrovascular accidents (CVA). However, the direct, and indirect, effects of aging on endothelial cell oxidative stress are complex and not completely understood. Increase in reactive oxygen species coupled with reduced anti-oxidative capacity creates a prooxidative endothelial phenotype resulting in impaired cell dysfunction, accelerated unprogrammed cell death and, in turn, CVAs. Several recent seminal studies have demonstrated that circulating endothelial cell- derived microvesicles (EMVs) are etiologically involved in the development and progression of endothelial dysfunction and CVA. We have previously demonstrated that aging is associated with increased circulating levels of EMVs. The aim of this study was to determine, in vitro, the effect of circulating EMVs isolated from young and older adults on brain endothelial cell oxidative stress. We hypothesized that EMVs from older adults would induce a prooxidative endothelial phenotype. As part of an ongoing study, 16, non-obese, normotensive, sedentary adults were studied: 8 young (5M/3F; age: 25±1 yr) and 8 older (4M/4F; 69±2 yr). EMV identification (CD144+) and isolation from peripheral blood was performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject for 24 hours. Intracellular reactive oxygen species (ROS) production was determined using CellROX Deep Red Reagent. Circulating EMVs were ~75% higher (P=0.002) in older (140±13 EMV/μL) vs young (82±8 EMV/μL) adults. Cellular ROS was significantly higher (~40%) in hCMECs treated with EMVs from older (141±14%) compared with young (101±5%) adults. In addition, intracellular expression of key anti-oxidant proteins were significantly lower in cells treated with EMVs from older adults. Cell expression of both superoxide dismutase 1 (SOD-1: 20.6±2.3 vs 36.3±3.1 AU) and catalase (17.1±1.9 vs 27.5±3.0 AU) were ~35% lower in hCMECs treated with EMVs from older adults. Moreover, intracellular ROS was inversely and significantly correlated with SOD-1 (r=-0.54; P=0.03) and catalase (r=-0.62; P=0.01) expression reflective of the cellular oxidative milieu. In conclusion, circulating EMVs may contribute to the increased risk of ischemic stroke with aging by increasing brain endothelial cell oxidative stress and reducing oxidative defense mechanisms. EMVs may represent a viable therapeutic target for mitigating the risk of CVA with aging. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elevated Glucose Levels Increase Oxidative Stress in Human Cerebral Microvascular Endothelial Cells

Although glucose is the primary fuel source for cerebral metabolism and function, chronic exposure to high glucose levels impairs cerebral endothelial cell function and increases the risk of cerebrovascular accidents (CVA). Indeed, hyperglycemia is linked to an increased risk and prevalence of ischemic stroke, primarily attributed to the detrimental impact of glucose on cerebral endothelial cell function. The direct and indirect effects of glucose on brain endothelial cells are complex and not well understood. It is known that hyperglycemia can induce a profound pro-oxidative state with deleterious effects on peripheral vascular function resulting in pathologic outcome. The experimental aim of this study was to determine, in vitro, the effect of elevated glucose concentrations on cerebral endothelial cell oxidative stress. Human cerebral microvascular endothelial cells (hCMECs) were cultured (3rd passage) and incubated with culture media containing 5 mM D-glucose (normal glucose condition [NG]), 6.5 mM glucose (concentration representative of impaired fasting glucose [IFG]), and 8 mM (concentration representative of type 2 diabetes [T2D]) for 3 hours. Cells were harvested and intracellular reactive oxygen species (ROS) production was determined using CellROX Deep Red Reagent and expression of key anti-oxidant defense proteins, superoxide dismutase 1 (SOD-1) and catalase, were determined by capillary electrophoresis immunoassay. In separate experiments, the effect of each glucose condition was determined in the presence of the ROS scavenger, vitamin C (200 μmol). Cells treated with glucose concentration representing IFG (137+8 %) and T2D (162+12%) significantly increased intracellular ROS compared with NG (98+3 %). Concordantly, expression of SOD-1 and catalase were significantly higher in hCMECs treated with glucose concentration representing IFG (190.2+17.8 AU; 134.7+15.4 AU, respectively) and T2D (186.7+19.6 AU; 137.2+17.5 AU, respectively) compared with NG (24.4+1.8 AU; 65.6+4.3 AU, respectively). Pretreatment of cells with vitamin C prevented the increase in ROS in both the IFG and T2D conditions. Of note, there were no significant differences in ROS or the expression of SOD-1 and catalase in hCMECs between the IFG and T2D glucose conditions. In conclusion, glucose concentrations associated with IFG and T2D markedly increased ROS in hCMECs. In addition, there was a compensatory increase in SOD-1 and catalase to counteract the increase in cellular oxidative burden. Similar effects between the IFG and T2D glucose conditions on the oxidative milieu of hCMECs is consistent with the similar clinical cerebrovascular risk burden between these pathologic conditions. Glucose-induced increased oxidative stress in cerebral endothelial cells may contribute to the increased risk of CVA, such as ischemic stroke, associated with IFG and T2D. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cytotoxic steroidal compounds, enriched fraction and extract of Trillium govanianum against HCT‐116 cell line

AbstractTrillium govanianum is a source of steroidal compounds and is used for the treatment of sexual disorders, dysentery, cancer, and inflammation. In the current investigation, steroidal compounds 1–5 (20‐hydroxyecdysone [1], pennogenin‐3‐O‐β‐chacotrioside [2], dioscin [3], trillin [4], diosgenin [5]) were isolated, including dioscin and trillin, as a new report from this species. Further, extract (TGE), steroids enriched fraction (TGEF) and 1–5 were evaluated for cytotoxicity against the HCT‐116 cell line. Cytotoxicity activity was found to be significant in samples and might be due to the generation of intracellular reactive oxygen species production. TGE and TGEF showed maximum inhibition (>92%) at 25 and 5 µg/mL in 72 h, while 2 and 3 showed better activity (>95%) at 5.65 and 11.51 µM in 72 h than the vinblastine drug (20 µM). Furthermore, the ultra‐performance liquid chromatography‐photo diode array method was validated for a determination of 1–5. Compounds 1–3, and 5 were quantified in TGE and TGEF while 4 was only identified. The findings revealed that activity of TGE and TGEF, might be due to compounds 2 (49.04 ± 2.22 and 77.40 ± 0.72 mg/g) and 3 (230.88 ± 7.07 and 584.67 ± 5.34 mg/g), respectively. The study also suggested that 2 and 3 can be a drug molecule as vinblastine while TGEF may be developed as a phytopharmaceutical drug for colon cancer treatment.

Extraction, purification and in vitro assessment of the antioxidant and anti-inflammatory activity of policosanols from non-psychoactive Cannabis sativa L.

Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans-esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity.HPLC-ELSED analysis of the purified mixture from hemp wax revealed C26OH and C28OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy

PurposeUremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. MethodsUCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). ResultsUCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. ConclusionThis study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.

ROS formation, mitochondrial potential and osmotic stability of the lamprey red blood cells: effect of adrenergic stimulation and hypoosmotic stress.

Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 μM) and forskolin (Forsk, 20 μM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by β-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration.

Neuroprotective and anti-inflammatory effects of eicosane on glutamate and NMDA-induced retinal ganglion cell injury.

To investigate the protective effects, antioxidant potential, and anti-inflammatory mechanisms of eicosane on glutamate-induced cell damage and on N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) injury in a mouse model of glaucoma. The protective effects of eicosane on the rat R28 retinal precursor cell line were assessed using cell counting kit-8 assays and Hoechst-propidium iodide staining. Intracellular reactive oxygen species (ROS) production was measured using the fluorescent probe 2'-7'-dichlorofluorescin diacetate and flow cytometry. The protective role of eicosane on NMDA-induced RGC injury in a mouse glaucoma model was determined by immunostaining of frozen sections of retina. The effects of eicosane on the metabolome of the retina in mice with NMDA-induced RGC damage were evaluated by liquid chromatography-mass spectroscopy (LC-MS) and untargeted metabolomics analyses. Eicosane treatment significantly attenuated glutamate-induced damage to R28 cells in vitro. Eicosane also protected RGCs against NMDA-induced injury in a mouse glaucoma model. Untargeted metabolomics analyses showed that eicosane increased multiple metabolites, including L-arginine and L-carnitine, in the retina. Eicosane has protective effects, antioxidant potential, and anti-inflammatory properties in an in vitro model of glutamate-induced cell damage and in an in vivo model of NMDA-induced RGC injury in mouse glaucoma through modulation of L-arginine and/or L-carnitine metabolism.

18β-glycyrrhetinic acid alleviates radiation-induced skin injury by activating the Nrf2/HO-1 signaling pathway.

Radiation-induced skin injury is a common side effect of radiotherapy, but there are few therapeutic drugs available for prevention or treatment. In this study, we demonstrate that 18β-Glycyrrhetinic acid (18β-GA), a bioactive component derived from Glycyrrhiza glabra, substantially reduces the accumulation of reactive oxygen species (ROS) and inhibits apoptosis in HaCaTcells after ionizing radiation (IR), thereby mitigating radiation-induced skin injury. Mechanistically, 18β-GA promotes the nuclear import of Nrf2, leading to activation of the Nrf2/HO-1 signaling pathway in response to IR. Importantly, Nrf2 silencing increases cell apoptosis and reverse the protective effect of 18β-GA on radiation-induced skin injury. Furthermore, 18β-GA preserves skin tissue structure after irradiation, inhibits inflammatory cell infiltration, and alleviates radiation dermatitis. In conclusion, our results suggest that 18β-GA reduces intracellular ROS production and apoptosis by activating the Nrf2/HO-1 signaling pathway, leading to amelioration of radiation dermatitis.

Both hemoglobin and hemin cause damage to retinal pigment epithelium through the iron ion accumulation

Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2′-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1β (IL-1β) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.