ROS Formation Research Articles

Investigation of the effect of serotonin-activated semen washing medium on sperm motility at the molecular level: a pilot study.

In Assisted Reproductive Technologies (ART), efficient sperm preparation is vital for successful fertilization, with washing media enhancing the process. This pilot study examines the molecular-level impact of a new serotonin-containing sperm-washing medium (Prototype) on sperm motility and ROS metabolism, comparing it with commercially available media (Origio and Irvine). Semen samples from thirty-one individuals underwent preparation using the swim-up method post-semen analysis. Each sample was separately washed with the Prototype, Origio and Irvine mediums. ROS formation was determined through flow cytometric, and AT2R and PRDX2 protein levels, associated with sperm motility, were assessed via Western blot. Statistical evaluation compared the findings among the three outlined media. Significant differences were found among three washing media in terms of total and progressive motility. The Prototype medium showed the highest increase in both total (66%) and progressive motility (59%), while the control group exhibited the lowest increases (41% and 27.7%, respectively). Regarding ROS levels, the prototype (11.5%) and Origio (10.7%) groups demonstrated a notable decrease, contrasting with Irvine (25.8%). Molecular assessment revealed a significant elevation in AT2R protein levels in the prototype medium (59%), compared to other media. Additionally, an increase in PRDX2 protein levels was observed in the prototype medium, although this didn't reach statistical significance. Serotonin-activated washing media for sperm preparation can be a suitable choice for selecting high-quality sperm in ART. A broader molecular analysis with a larger sample size is required to explore the mechanisms and effectiveness of using a serotonin-containing sperm-washing medium in routine ART.

1,4-Benzodioxane Substituted Aza-BODIPY: Towards Photostable yet Efficient Triplet Photosensitizer.

We report herein the synthesis of aza-BODIPY substituted with 1,4-benzodioxane-6-yl substituents at 3,5 positions of the chromophore system. Both pyrrole rings of the aza-BODIPY in question were substituted with bromine atoms in order to induce highly desirable photophysical properties, such as highly populated excited triplet state (T1) and long excited triplet-state lifetime (τT) of 21 μs. The photosensitized oxygenation of a model compounds, viz. DPBF, points to a high singlet oxygen and/or other ROS formation quantum yield of 0.42. The photosensitizer studied exhibited an absorption band within the so-called "therapeutic window", with λabs 678 nm. As estimated by CV/DPV measurements the 1,4-benzodioxane-6-yl substituted aza-BODIPYs studied exhibited a multi-electron oxidations at a relatively low potentials (Eox), pointing to the very good electron-donating properties of these molecules. High photostability and thermal stability was observed for all compounds studied. The good singlet oxygen quantum yield measured combined with an exceptional photostability makes this aza-BODIPY a promising candidate for applications such as photocatalysis and photodynamic therapy (PDT).

Long non-coding RNA PANDA promotes endothelial senescence and oxidative damage through the interaction with NRF2

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. LncRNAs are emerging as important players in the pathogenesis of cardiovascular disease. PANDA, a newly identified lncRNA, is a key regulator of cellular senescence, apoptosis and oxidative stress. Purpose To investigate the role and molecular mechanism of PANDA in diabetic vascular disease. Methods RNAseq was performed to aorta specimen from diabetic and no diabetic patients as well to HAECs exposed to 5 mM and 25 mM concentrations (Normal -NG- and High -HG-, respectively). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled siRNA wass used as a negative control. RNAseq and network perturbation analysis (NPA) of treated HAECs unveiled transcriptional changes upon PANDA depletion. Expression of PANDA was assessed by real time PCR. PANDA RNA-IP was performed to check its binding to relevant transcriptional factor (such as NRF2) as well Ch-IP was performed to check the NRF2 binding on oxidative gene promoters. Cellular localization of NRF2 was investigated by Immunofluorescence. Beta-galactosidase and superoxide staining was used to detect endothelial senescence and ROS formation; while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Results PANDA expression was significantly increased in diabetic human aorta as well in HG-treated HAECs (Figure1 A-B). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG cells (Figure1 C-D). Western blot analysis reveals the restored level of HMOX1 and TFRC1 upon PANDA depletion (Figure1 E-G). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. In HG, NFR2 is downregulated while PANDA silencing restores its level (Figure1 I-J). We found that in HG condition PANDA binds the transcription factor NRF2 and blocks its nuclear translocation thus impeding its binding on HMOX1 and TFRC promoters (Figure1 K-M). Silencing of PANDA in HG-treated HAECs reduced genes and cellular features of senescence (Figure2 A-B), restored the expression of anti-apoptotic genes and decreases caspase 3 activity (Figure2 C-D), improved endothelial migration and tube formation (Figure2 E and G), and reduced ROS formation (Figure2 F). Conclusions In hyperglycemia PANDA upregulation drives endothelial senescence while impairing angiogenic properties. On the other hand, PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes through the release of NRF2 that in turn translocate into the nucleus enhancing the expression of the antioxidant gene HMOX1. The results indicate PANDA as a putative novel molecular target in the setting of diabetic vascular disease (Figure2 H).

Activation, regulation, and effects of the mitochondrial unfolded protein response in endothelial cells

Abstract Introduction Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) play significant roles in endothelial dysfunction and atherosclerosis. However, the understanding of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction is limited. This study aims to characterize the activation, regulation, and effects of the UPRMito to provide a comprehensive understanding of endothelial UPRMito. Methods and Results RNA sequencing and quantitative proteomic analysis were employed to investigate the impact of stressors on mitochondrial (MitoBlock-6, CDDO) and endoplasmic (Tunicamycin) protein homeostasis on the transcriptome and proteome of Human Coronary Artery Endothelial cells (HCAEC, Fig.1). The analysis identified 12,745 unique transcripts and 7,891 unique proteins. Mitocarta annotation highlighted the regulatory effect induced by these stressors, particularly on mitochondrial proteins and transcripts (Fig. 2). Geneset enrichment analyses revealed upregulation of pathways regulated by activating transcription factors 4/5 (ATF4/5) and the integrated stress response marker CHOP. Analysis of protein expression changes unveiled that 21 proteins were upregulated by both CDDO and MitoBlock-6, including essential mitochondrial chaperones (HSP10 and HSP60). In contrast, the UPRER stressor Tunicamycin did not induce canonical UPRMito activation. Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression, and ribosome biogenesis. Importantly, NAD+ADP-ribosyltransferase activity was highly downregulated, involving PARP proteins with roles in DNA damage repair. On a functional level, mitochondrial stressors led to dose-dependent alterations in HCAEC viability, apoptosis, and reactive oxygen species formation (ROS) measured by resazurin-based assays, caspase 3/7 activity, and MitoSox staining, respectively. Low doses and short incubations with mitochondrial stressors promoted endothelial cell health through cytoprotective UPRMito pathways, while prolonged incubation led to unresolvable mitochondrial stress (exemplarily shown in Fig. 3). SiRNA-mediated gene silencing of the UPRMito regulators ATF4 and ATF5 promoted mitochondrial ROS formation in cells treated with mitochondrial stressors. These effects were particularly more pronounced after ATF5-Knockdown, suggesting that ATF5 is the main regulator of the UPRMito in HCAEC (Fig. 4). Moreover, high-dose UPRMito stressors led to an impairment in mitochondrial membrane integrity with cytosolic release of pro-inflammatory mtDNA. Conclusion UPRMito and UPRER lead to a different stress response. Depending on the underlying conditions, both stress responses can exert both cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPRMito in endothelial dysfunction and atherosclerosis.Figures

Zinc Influences the Efficacy of Betulinic Acid Treatment and Radiotherapy in Breast Cancer Cells

The trace element zinc influences a number of biological reactions, including cell growth, apoptosis, and DNA damage, which affect tumor therapy. The natural compound betulinic acid (BA) and its derivatives are known for their antiviral, antibacterial, and antitumor effects. Previous studies show that BA and 3-acetyl-28-sulfamoyloxybetulin (CAI3) have high cytotoxicity and induce radiosensitization in breast cancer cells. This study investigates the effects of zinc supplementation on treatment with BA or CAI3 and radiotherapy of breast cancer cell lines MDA-MB-231 and HS578T. Expression analysis shows that BA and CAI3 lead to altered expression of genes involved in zinc metabolism. Zinc supplementation affects cell survival and cell death alone and in combination with BA or CAI3 in both breast cancer cell lines. In MDA-MB-231 cells, zinc excess protects against ROS formation by BA or CAI3 and exhibits radioprotective effects compared to the single agent treatment. In contrast, in HS578T cells, zinc induces ROS formation but does not affect radiosensitivity. The variable effects of zinc on radiosensitivity highlight the importance of individualized treatment approaches. Although zinc has cytotoxic, pro-apoptotic, and anti-clonogenic effects, it seems worthwhile to consider its radioprotective properties when making treatment decisions in the case of adjuvant radiotherapy of breast cancer.

Microplastics are detected in bull epididymal sperm and polystyrene microparticles impair sperm fertilization.

An increase in global infertility has coincided with the accumulation of microplastics (MPs) in the environment. This trend is particularly troubling because only 10% of male infertility cases can be attributed to identifiable causes, leaving a knowledge gap in our understanding of their underlying factors. To bridge this, it is important to explore the connection between the accumulation of MPs and the observed decline in male fertility. We assessed the presence of microplastics in epididymal sperm from bulls and used it as baseline concentrations for sperm exposure. MPs were detected in all epidydimal sperm (ES) samples, with a mean concentration of 0.37μgmL-1. Next, to investigate the effects of MPs on fertility, bovine sperm was exposed to three different concentrations of a mixture of 1.1, 0.5, and 0.3μm polystyrene (PS) beads: (1) 0.7μgmL-1, blood concentration of PS in cows (bPS); (2) 0.37μgmL-1, based on the concentration of total MPs found in ES (esMP); and (3) 0.026μgmL-1, based on the concentration of PS found in ES (esPS). All sperm samples incubated with PS exhibited reduced motility compared with the control at 0.5h. However, PS exposure did not affect acrosome integrity or induced oxidative stress. Embryos produced from sperm exposed to PS had reduced blastocyst rates, in addition to increased ROS formation and apoptosis. By employing physiological exposure, this research provided evidence of MPs in bovine epididymal sperm and demonstrated the detrimental effect of PS on sperm functionality.

Comparison of Dietary Supplementation with Krill Oil, Fish Oil, and Astaxanthin on an Experimental Ethanol-Induced Gastric Ulcer Model: A Biochemical and Histological Study

Background/Objectives: Despite advances in ulcer treatment research, the search for new, safe, and effective strategies for preventing and treating ulcer diseases persists. Methods: In this study, the protective effects of dietary supplementation with krill oil (KO), fish oil (FO), and astaxanthin (ASX) on an ethanol-induced gastric ulcer model were compared during biochemical and histological observations. Sprague–Dawley (n = 64) rats randomly divided into four groups—normal control (vehicle), KO, FO, and ASX groups—received the supplements via the orogastric route at a rate of 2.5% (v/w) of their daily feed consumption for 4 weeks. Then, ulcer induction was performed with ethanol. Results: The ulcer group showed increased levels of malondialdehyde (MDA), chemiluminescence (CL), and myeloperoxidase (MPO) activity and decreased levels of glutathione in the gastric tissues. While KO, FO, and ASX supplementation decreased chemiluminescence levels in the ulcer group, only ASX supplementation decreased MDA levels and MPO activity. Conclusions: In conclusion, supplementation with KO or FO has a similar protective effect against ethanol-induced ulcer damage, as it inhibits ROS formation and reduces lipid peroxidation. However, ASX supplementation has a higher protective effect than KO or FO supplementations against experimental ethanol-induced gastric lesions in rats, as it inhibits ROS formation and reduces neutrophil infiltration and lipid peroxidation.

Protective Effects of Keratinocyte-Derived GCSF and CCL20 on UVB-Induced Melanocyte Damage.

The skin microenvironment created by keratinocytes (KC) influences the stress responses of melanocytes (MC) to UVB insults. This study employed RNA sequencing analysis as well as in vitro and in vivo models to elucidate the underlying mechanisms. Our RNA-Seq analysis revealed a statistically significant upregulation of GCSF and CCL20 genes in UVB-irradiated KC, correlating with the protective effects of KC on MC responses to UVB exposure. Recombinant GCSF and CCL20 exhibited the most pronounced modulation of UVB-induced MC responses. These effects included the attenuation of apoptosis and reduction of ROS formation, along with the upregulation of tyrosinase and tyrosinase-related protein-1, which are involved in the melanogenic pathway. ELISA was also used to confirm that UVB could induce the secretion of GCSF and CCL20 from KC. A similar correlation between GCSF and CCL20 expression in KC and tyrosinase levels in MC was observed in UVB-irradiated mouse skin. Our study provides novel insights into the protective role of GCSF and CCL20 in the paracrine effects of KC on UVB-induced MC damage through the modulation of stress response pathways, the MITF-tyrosinase axis, and the regulation of p53. These findings have implications for the development of pharmacological strategies targeting KC-derived paracrine factors for the prevention of skin photodamage.

FmRbohH Mediates ROS Generation and Enhances Pollen Tube Growth in Fraxinus mandshurica

Flowering plants require normal pollen germination and growth to be fertilized, but studies on the mechanism regulating pollen tube growth in Fraxinus mandshurica are limited. Here, we used transcriptomic data to study the oxidative phosphorylation pathway during pollen tube growth in Fraxinus mandshurica. Our study identified 8,734 differentially expressed genes during the stages S1 to S3 of pollen tube growth. Significant enrichment of the oxidative phosphorylation pathway, amino acid synthesis, protein processing in the ER, carbon metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis were examined using the Kyoto Encyclopedia of Genes and Genomes, and 58 genes linked to ROS synthesis and scavenging during the S1–S3 stages were identified. Also, H2DCFDA staining confirmed ROS formation in the pollen and the pollen tubes, and treatment with copper (II) chloride (CuCl2) and diphenyleneiodonium (DPI) was shown to reduce ROS in the pollen tube. Reduction in ROS content caused decreased pollen germination and pollen tube length. Furthermore, FmRbohH (respiratory burst oxidase homolog H) expression was detected in the pollen and pollen tube, and an antisense oligodeoxynucleotide assay demonstrated reduced ROS and pollen tube growth in Fraxinus mandshurica. This study shed more light on the RbohH gene functions during pollen tube growth.

Calcitriol/vitamin D receptor system alleviates PM2.5-induced human bronchial epithelial damage through upregulating mitochondrial bioenergetics in association with regulation of HIF-1α/PGC-1α signaling

PM2.5 exposure causes lung injury by triggering oxidative stress, mitochondrial dysfunction, and modulating HIF-1α signaling. Calcitriol activates VDR, which regulates cellular homeostasis. This study evaluated the protective role of the calcitriol/VDR system in PM2.5-induced damage to BEAS-2B bronchial epithelial cells by reducing oxidative stress, upregulating mitochondrial bioenergetics, and downregulating HIF-1α. We found that the calcitriol/VDR system decreased ROS formation and restored mitochondrial bioenergetics in PM2.5-treated cells. This improvement correlated with reduced HIF-1α nuclear translocation and increased PGC-1α protein and mitochondrial gene expressions. This study is the first to suggest that targeting the calcitriol/VDR system could be a promising pharmacological strategy for mitigating PM2.5-induced lung epithelial damage by promoting mitochondrial bioenergetics and regulating PGC-1α and HIF-1α signaling.

Cobalt stress enhanced dendrobine-type total alkaloids biosynthesis of Trichoderma longibrachiatum UN32 through reactive oxygen species formation.

Trichoderma longibrachiatum UN32 is a well-documented mutant strain known to produce dendrobine-type total alkaloids (DTTAs). It was serendipitously observed that the addition of Co2+ to the medium resulted in a notable enhancement in DTTAs production in the T. longibrachiatum UN32 strain, accompanied by an upregulating effect on the expression of antioxidase-related genes. Hence, the objective of the present work was to ascertain whether ROS (intracellular levels of hydrogen peroxide) induced by Co2+ treatment has a beneficial or detrimental impact on DTTAs biosynthesis. A comparison of the intracellular levels of hydrogen peroxide (H2O2) and DTTAs treated with CoCl2 and CH3COOH revealed that CoCl2 was the optimal inducer for investigating the relationship between ROS formation and DTTAs production. This was due to the observation that ROS formation was reduced by approximately 4% and DTTAs production was increased by 12.55% in comparison to the CH3COOH treatment. The physiological results revealed that the introduction of Co2+ resulted in the oxidative damage and activation of the expression of intracellular superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Furthermore, it was confirmed that ROS induced by Co2+ was beneficial to DTTAs production by adding exogenous ROS scavengers. The inclusion of all ROS scavengers, including vitamin C, tocopherol, melatonin, mannitol, and sesamol, resulted in a reduction in ROS accumulation and a concomitant decrease in DTTAs production. Specifically, the addition of melatonin at a concentration of 0.4mg/L demonstrated significant effects, resulting in a 32.53% (P < 0.01) decrease in ROS accumulation and a 45.22% (P < 0.01) reduction in DTTAs production. Subsequently, the timelines of accumulation of intracellular H2O2 and DTTAs content indicated that ROS are also crucial for normal fermentation without CoCl2 addition. Specifically, the proper H2O2 dose for DTTAs accumulation is between 8.82 and 18.86μmol/g. The present study offers the initial experimental evidence indicating that CoCl2 enhance DTTAs production during the culture of T. longibrachiatum UN32 via leading an increase in intracellular ROS, which is conductive to DTTAs production and can be inhibited by the ROS scavengers. Our results provide insights into the mechanistic study of DTTAs biosynthesis.

Unveiling Lobophytum sp. the neuroprotective potential of Parkinson's disease through multifaceted mechanisms, supported by metabolomic analysis and network pharmacology

A main feature of neurodegenerative diseases is the loss of neurons. One of the most prevalent neurodegenerative illnesses is Parkinson disease (PD). Although several medications are already approved to treat neurodegenerative disorders, most of them only address associated symptoms. The main aim of the current study was to examine the neuroprotective efficacy and underlying mechanism of Lobophytum sp. crude extract in a rotenone-induced rat model of neurodegeneration mimicking PD in humans. The influence of the treatment on antioxidant, inflammatory, and apoptotic markers was assessed in addition to the investigation of TH (tyrosine hydroxylase) immunochemistry, histopathological changes, and α-synuclein. Metabolomic profiling of Lobophytum sp. crude extract was done by using High-Resolution Liquid Chromatography coupled with Mass Spectrometry (HR-LC–ESI–MS), which revealed the presence of 20 compounds (1–20) belonging to several classes of secondary metabolites including diterpenoids, sesquiterpenoids, steroids, and steroid glycosides. From our experimental results, we report that Lobophytum sp. extract conferred neuroprotection against rotenone-induced PD by inhibiting ROS formation, apoptosis, and inflammatory mediators including IL-6, IL-1β, and TNF-α, NF-кB, and subsequent neurodegeneration as evidenced by decreased α-synuclein deposition and enhanced tyrosine hydroxylase immunoreactivity. Moreover, a computational network pharmacology study was performed for the dereplicated compounds from Lobophytum sp. using PubChem, SwissTarget Prediction, STRING, DisGeNET, and ShinyGO databases. Among the studied genes, CYP19A1 was the top gene related to Parkinson’s disease. Dendrinolide compounds annotated a high number of parkinsonism genes. The vascular endothelial growth factor (VEGF) pathway was the top signaling pathway related to the studied genes. Therefore, we speculate that Lobophytum sp. extract, owing to its pleiotropic mechanisms, could be further developed as a possible therapeutic drug for treating Parkinson's disease.

Fucoxanthin Attenuates Angiogenesis by Blocking the VEGFR2-Mediated Signaling Pathway through Binding the Vascular Endothelial Growth Factor.

Fucoxanthin, a dietary carotenoid, is predominantly found in edible brown algae and is commonly consumed worldwide. Fucoxanthin has been shown to possess beneficial health activities such as antidiabetic, anti-inflammatory, antimutagenic, and antiobesity; however, the effects of fucoxanthin on VEGF-mediated angiogenesis and its possible binding with VEGF are unknown. Here, different lines of evidence supported the suppressive roles of fucoxanthin in VEGF-mediated angiogenesis. In human umbilical vein endothelial cells, fucoxanthin remarkedly suppressed VEGF-mediated cell proliferative, migration, and invasive abilities, as well as tube formation, without cytotoxicity. In addition, fucoxanthin inhibited the subintestinal vessel formation of zebrafish in vivo. In signaling cascades, fucoxanthin was proposed to interact with VEGF, thus attenuating VEGF's functions in activating the VEGF receptor and its related downstream signaling, i.e., phosphorylations of MEK and Erk. Fucoxanthin also significantly blocked VEGF-triggered ROS formation. Furthermore, the outcomes of applying fucoxanthin in cancer cells were identified, which included (i) inhibiting VEGF-mediated cell proliferation and migration and (ii) inhibiting NF-κB translocation via limiting MMP2 expression. These lines of investigations supported the antiangiogenic roles of fucoxanthin, as well as reviewing its signaling mechanisms, in blocking the VEGF-triggered responses. The results would benefit the potential development of fucoxanthin for the prevention and treatment of angiogenesis-related diseases.

Computational Insights into the Radical Scavenging Activity and Xanthine Oxidase Inhibition of the Five Anthocyanins Derived from Grape Skin.

Anthocyanins, typical polyphenol compounds in grape skin, have attracted increasing interest due to their health-promoting properties. In this body of work, five representative anthocyanins (Cy-3-O-glc, Dp-3-O-glc, Pn-3-O-glc, Mv-3-O-glc, and Pt-3-O-glc) were studied using the density functional theory (DFT) to elucidate structure-radical scavenging activity in the relationship and the reaction path underlying the radical-trapping process. Based on thermodynamic parameters involved in HAT, SET-PT, and SPLET mechanisms, along with the structural attributes, it was found that the C4' hydroxyl group mainly contributes to the radical scavenging activities of the investigated compounds. Pt-3-O-glc exhibits a good antioxidant capacity among the five compounds. The preferred radical scavenging mechanisms vary in different phases. For the Pt-3-O-glc compound, the calculations indicate the thermodynamically favoured product is benzodioxole, rather than o-quinone, displaying considerably reduced energy in double HAT mechanisms. Additionally, the thermodynamic and kinetic calculations indicate that the reaction of •OH into the 4'-OH site of Pt-3-O-glc has a lower energy barrier (7.6 kcal/mol), a higher rate constant (5.72 × 109 M-1 s-1), and exhibits potent •OH radical scavenging properties. Molecular docking results have shown the strong affinity of the studied anthocyanins with the pro-oxidant enzyme xanthine oxidase, displaying their significant role in inhibiting ROS formation.

NoxO1 Determines the Level of ROS Formation by the Nox1-Centered NADPH Oxidase.

The Nox1-centered NADPH oxidase complex facilitates the transfer of electrons from intracellular NADPH across the cell membrane to extracellular molecular oxygen, resulting in the formation of superoxide. The complex is comprised of two membrane-bound subunits, namely Nox1 and p22phox, and the cytosolic subunits, namely NoxA1 and NoxO1. The presence of NoxO1 facilitates the proximity of all components, thereby enabling the complex to exhibit constitutive activity. Despite the theoretical sufficiency of all subunits in a 1:1 ratio, the precise composition of the Nox1-centered NADPH oxidase remains unknown. Analyses of mRNA expression in different cell lines revealed an unequal expression of the components, with an excess of NoxO1. Furthermore, plasmid-based overexpression of individual components of the Nox1-centered NADPH oxidase resulted in an excess of NoxO1 mRNA. The objective of this study was to analyze the ability of NoxO1 to control the level of ROS formation by the Nox1 complex. To this end, we generated Hek293 cells for constitutive expression of Nox1 and NoxA1, which were then transfected with increasing concentrations of NoxO1. The data presented herein suggests that ROS formation by the Nox1-centered NADPH oxidase is dependent on the concentration of NoxO1. A surplus of NoxO1 has been observed to exert control over the activity of the complex in accordance with a dose-dependent mechanism. We thus conclude that the ratio of Nox1, NoxA1, and NoxO1 complexes does not adhere to a 1:1 ratio. Conversely, the availability of NoxO1 serves to regulate the formation of ROS by the Nox1-centered NADPH oxidase.

Analysis of High-Dose Ascorbate-Induced Cytotoxicity in Human Glioblastoma Cells and the Role of Dehydroascorbic Acid and Iron.

Several studies have demonstrated, both in vitro and in animal models, the anti-tumor efficacy of high-dose ascorbate treatment against a variety of tumor entities, including glioblastoma, the most common and aggressive primary malignant brain tumor. The aim of this study was to investigate the effects of high-dose ascorbate as well as dehydroascorbic acid on human glioblastoma cell lines and to evaluate different treatment conditions for the combined administration of ascorbate with magnesium (Mg2+) and iron (Fe3+). Intracellular levels of reactive oxygen species and the induction of cell death following ascorbate treatment were also investigated. We demonstrated high cytotoxicity and antiproliferative efficacy of high-dose ascorbate in human glioblastoma cells, whereas much weaker effects were observed for dehydroascorbic acid. Ascorbate-induced cell death was independent of apoptosis. Both the reduction in cell viability and the ascorbate-induced generation of intracellular reactive oxygen species could be significantly increased by incubating the cells with Fe3+ before ascorbate treatment. This work demonstrates, for the first time, an increase in ascorbate-induced intracellular ROS formation and cytotoxicity in human glioblastoma cells by pre-treatment of the tumor cells with ferric iron, as well as caspase-3 independence of cell death induced by high-dose ascorbate. Instead, the cell death mechanism caused by high-dose ascorbate in glioblastoma cells shows evidence of ferroptosis. The results of the present work provide insights into the efficacy and mode of action of pharmacological ascorbate for the therapy of glioblastoma, as well as indications for possible approaches to increase the effectiveness of ascorbate treatment.

Decursin ameliorates neurotoxicity induced by glutamate through restraining ferroptosis by up-regulating FTH1 in SH-SY5Y neuroblastoma cells

Alzheimer’s disease (AD) is the most common form of neurodegeneration which currently has no effective treatment. Ferroptosis is a new style of programmed cell death and is widely implicated in the pathogenesis and progression of AD. Decursin has been shown widely neuroprotective effects but poorly understood about the underlying mechanisms between decursin and ferroptosis in AD. Here, the protective effect of decursin and the underlying mechanism under glutamate treatment in SH-SY5Y cells was investigated. SH-SY5Y cells were cultured with glutamate in the presence or absence of decursin. The safe concentrations of decursin on SH-SY5Y cells were measured via CCK-8. Furthermore, LDH content, antioxidant enzyme activities including GPx, CAT and SOD, MDA contents, GSH levels, ROS formation, MMP, mitochondria ultrastructure morphology change, and intracellular Fe2+ levels were measured to investigate the influence of decursin and Fer-1 on ferroptosis in glutamate-treated SH-SY5Y cells. Moreover, the expressions of ferroptosis-related proteins were determined by Western blot. As a result, glutamate-induced cell survival was markedly elevated and morphological change was improved by decursin administrated in SH-SY5Y cells. Furthermore, decursin could reversed the decreased antioxidant enzyme activities, GSH levels, GPX4n and FTH1 expression, as well as the increased iron levels, LDH, MDA, ROS formation, and MMP, which showed similar effects to Fer-1, the specific ferroptosis inhibitor. Therefore, the inhibitory effect of decursin on ferroptosis probably was partially governed by FTH1 expression to regulate the cellular iron homeostasis. Additionally, decursin facilitated the translocation of Nrf2 from the cytoplasm to the nucleus. Taken together, our data for the first time suggest that decursin could ameliorate neurotoxicity induced by glutamate by attenuating ferroptosis via alleviating cellular iron levels by up-regulating FTH1 expression which is attributing to its promotion of Nrf2 translocation into the nucleus in SH-SY5Y neuroblastoma cells. Hence, decursin might be a novel and promising therapeutic option for AD. In addition, our study also provided some new clues to potential target for the intervention and therapy of AD.

Enhancing Zea mays L. seedling growth with He[sbnd]Ne laser-irradiated Alcaligenes sp. E1 to mitigate salinity stress

Plant growth-promoting rhizobacteria (PGPR) are a beneficial strategy to improve agriculture and mitigate abiotic stress. To increase the efficacy of this approach, the utilization of HeNe laser radiation (L) and methylene blue (MB) as photosensitizer is commonly suggested. This study evaluated the role of the IAA-producing rhizobacterial strain Alcaligenes sp. E1 on maize growth under salt stress (150 mM NaCl) with the application of laser and methylene blue. The results showed that treatment of Alcaligenes sp. E1 with red laser and methylene blue exhibited a maximum increase in shoot length (44 %), root length (49 %), fresh weight (34 %), dry weight (45.6 %), chlorophyll a (51 %), and carotenoids (71 %) over the stressed control. In addition, it demonstrated more activity to antioxidant activities (SOD, CAT, GSH, and TAC) and limited oxidative damage resulting from ROS formation. In conclusion, Alcaligenes sp. has a good ability to stimulate maize growth when applied with laser radiation and photosensitizer under normal and saline conditions. Thus, we propose that red laser and photosensitizer will have promising applications in crop biostimulation and sustainable agriculture.

Sirt6 overexpression relieves ferroptosis and delays the progression of diabetic nephropathy via Nrf2/GPX4 pathway

Objective Sirt6, reactive oxygen species and ferroptosis may participate in the pathogenesis of Diabetic Nephropathy (DN). Exploring the relationship between Sirt6, oxidative stress, and ferroptosis provides new scientific ideas to DN. Methods Human podocytes were stimulated with 30 mM glucose and 5.5 mM glucose. The mice of db/db group were randomly divided into two groups:12 weeks and 16 weeks. Collect mouse blood and urine specimens and renal cortices for investigations. HE, Masson, PAS and immunohistochemical staining were used to observe pathological changes. Western blot, RT-qPCR and immunofluorescence staining were used to evaluate expression of relevant molecules. CCK8 method was introduced to observe cell viability. The changes of podocyte mitochondrial membrane potential and mitochondrial morphology in each group were determined by JC-1 staining and Mito-Tracker. Results The expression level of Sirt6, Nrf2, SLC7A11, HO1, SOD2 and GPX4 were reduced, while ACSL4 was increased in DN. Blood glucose, BUN, Scr, TG, T-CHO and 24h urine protein were upregulated, while ALB was reduced in diabetic group. The treatment of Ferrostatin-1 significantly improved these changes, which proved ferroptosis was involved in the development of DN. Overexpression of Sirt6 might ameliorate the oxidation irritable reaction and ferroptosis. Sirt6 plasmid transfection increased mitochondrial membrane potential and protected morphology and structure of mitochondria. The application of Sirt6 siRNA could aggravated the damage manifestations. Conclusion High glucose stimulation could decrease the antioxidant capacity and increase formation of ROS and lipid peroxidation. Sirt6 might alleviate HG-induced mitochondrial dysfunction, podocyte injury and ferroptosis through regulating Nrf2/GPX4 pathway.

Delta-9-Tetrahydrocannabinol Blocks Bone Marrow-Derived Macrophage Differentiation through Elimination of Reactive Oxygen Species.

Macrophages are vital components of the immune system and serve as the first line of defense against pathogens. Macrophage colony-stimulating factor (M-CSF) induces macrophage differentiation from bone marrow-derived cells (BMDCs). Δ9-tetrahydrocannabiol (THC), a phytocannabinoid from the Cannabis plant, has profound anti-inflammatory properties with significant effects on myeloid cells. To investigate the effect of THC on macrophage differentiation, we cultured BMDCs with M-CSF in the presence of THC. Interestingly, THC markedly blocked the differentiation of BMDCs into CD45 + CD11b + F4/80+ macrophages. The effect of THC was independent of cannabinoid receptors CB1, and CB2, as well as other potential receptors such as GPR18, GPR55, and Adenosine 2A Receptor. RNA-seq analysis revealed that the THC-treated BMDCs displayed a significant increase in the expression of NRF2-ARE-related genes. KEGG pathway analysis revealed that the expression profiles of THC-treated cells correlated with ferroptosis and glutathione metabolism pathways. Fluorescence-based labile iron assays showed that the THC-treated BMDCs had significantly increased iron levels. Finally, THC-exposed BMDCs showed decreased levels of intracellular ROS. THC has the unique molecular property to block the Fenton Reaction, thus preventing the increase in intracellular ROS that is normally induced by high iron levels. Together, these studies demonstrated that THC blocks M-CSF-induced macrophage differentiation by inhibiting ROS production through both the induction of NRF2-ARE-related gene expression and the prevention of ROS formation via the Fenton Reaction.