Label-free fluorescence lifetime imaging for the assessment of cell viability in living tumor fragments.

Significance: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM). Aim: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death. Approach: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays. Results: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected. Conclusions: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.

Polydopamine Nanoparticles-Encapsulated Ferroptosis Inhibitor Ferstatin-1 Promotes GPX4 Expression by Down-Regulating NOX4 to Alleviate Myocardial Ischemia-Reperfusion Injury.

OBJECTIVE: Polydopamine nanoparticles (PDA NPs) are a promising topic in the fields of drug delivery, tissue engineering, bioimaging, etc. The present study aims to explore the impact of PDA NPs carrying ferroptosis inhibitor ferstatin-1 (Fer-1) on myocardial ischemia-reperfusion injury (MIRI). METHODS: After establishment of a rat model of MIRI and PDA NPs, the rats were divided into 4 groups: model group, sham operation group, Fer-1 group, and nano+Fer-1 group (n=8). To detect the effect of PDA NPs encapsulating Fer-1 on ferroptosis in MIRI rats, we further set up NOX4 overexpression group (pc-NOX4 group), NOX4 inhibitor group (Fulvene-5 group), nano+Fer-1+pc-NOX4 group, and nano+Fer-1+Fulvene-5 group (n=8). A CCK-8 assay was conducted to assess cell viability and staining to detect cardiomyocyte apoptosis and observe myocardial infraction. RESULTS: PDA NPs loaded with Fer-1 were successfully prepared with good safety and biocompatibility. Administration of PDA NPs carrying Fer-1 notably alleviated myocardial injury and hindered the process of ferroptosis in MIRI rats when inducing downregulation of NOX4 expression. Additionally, overexpression of GPX4 significantly attenuated myocardial injury in MIRI rats. While Fer-1 was shown to inhibit the expression of NOX4, the NOX4 inhibitor Fulvene-5 greatly elevated GPX4 and FTH1 expression in cardiomyocytes, and down-regulated the content of Fe2+, especially in the nanometer+Fer-1+Fulvene-5 group. CONCLUSION: With promising safety and biocompatibility, PDA NPs encapsulated Fer-1 decrease GPX4 and FTH1 expression by inhibiting the level of NOX4 in myocardial cells of MIRI rats, thereby suppressing ferroptosis of cardiomyocytes and alleviating myocardial injury.

NOX4 promotes tumor progression through the MAPK-MEK1/2-ERK1/2 axis in colorectal cancer.

BACKGROUND: Metabolic reprogramming plays a key role in cancer progression and clinical outcomes; however, the patterns and primary regulators of metabolic reprogramming in colorectal cancer (CRC) are not well understood. AIM: To explore the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in promoting progression of CRC. METHODS: We evaluated the expression and function of dysregulated and survival-related metabolic genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Consensus clustering was used to cluster CRC based on dysregulated metabolic genes. A prediction model was constructed based on survival-related metabolic genes. Sphere formation, migration, invasion, proliferation, apoptosis and clone formation was used to evaluate the biological function of NOX4 in CRC. mRNA sequencing was utilized to explore the alterations of gene expression NOX4 over-expression tumor cells. In vivo subcutaneous and lung metastasis mouse tumor model was used to explore the effect of NOX4 on tumor growth. RESULTS: We comprehensively analyzed 3341 metabolic genes in CRC and identified three clusters based on dysregulated metabolic genes. Among these genes, NOX4 was highly expressed in tumor tissues and correlated with worse survival. In vitro, NOX4 overexpression induced clone formation, migration, invasion, and stemness in CRC cells. Furthermore, RNA-sequencing analysis revealed that NOX4 overexpression activated the mitogen-activated protein kinase-MEK1/2-ERK1/2 signaling pathway. Trametinib, a MEK1/2 inhibitor, abolished the NOX4-mediated tumor progression. In vivo, NOX4 overexpression promoted subcutaneous tumor growth and lung metastasis, whereas trametinib treatment can reversed the metastasis. CONCLUSION: Our study comprehensively analyzed metabolic gene expression and highlighted the importance of NOX4 in promoting CRC metastasis, suggesting that trametinib could be a potential therapeutic drugs of CRC clinical therapy targeting NOX4.

Disulfidptosis decoded: a journey through cell death mysteries, regulatory networks, disease paradigms and future directions.

Cell death is an important part of the life cycle, serving as a foundation for both the orderly development and the maintenance of physiological equilibrium within organisms. This process is fundamental, as it eliminates senescent, impaired, or aberrant cells while also promoting tissue regeneration and immunological responses. A novel paradigm of programmed cell death, known as disulfidptosis, has recently emerged in the scientific circle. Disulfidptosis is defined as the accumulation of cystine by cancer cells with high expression of the solute carrier family 7 member 11 (SLC7A11) during glucose starvation. This accumulation causes extensive disulfide linkages between F-actins, resulting in their contraction and subsequent detachment from the cellular membrane, triggering cellular death. The RAC1-WRC axis is involved in this phenomenon. Disulfidptosis sparked growing interest due to its potential applications in a variety of pathologies, particularly oncology, neurodegenerative disorders, and metabolic anomalies. Nonetheless, the complexities of its regulatory pathways remain elusive, and its precise molecular targets have yet to be definitively identified. This manuscript aims to meticulously dissect the historical evolution, molecular underpinnings, regulatory frameworks, and potential implications of disulfidptosis in various disease contexts, illuminating its promise as a groundbreaking therapeutic pathway and target.

NOX4 aggravates doxorubicin-induced cardiomyocyte pyroptosis by increasing reactive oxygen species content and activating the NLRP3 inflammasome.

Background: Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)-mediated reactive oxygen species (ROS) has been reported to induce cardiomyocyte apoptosis, but its effect on pyroptosis of cardiomyocytes has been rarely reported. This paper aimed to explore the effects of NOX4-mediated ROS production on doxorubicin (DOX)-induced myocardial injury and pyroptosis through nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome. Methods: HL-1 cells were treated with DOX or mice (30 mice were divided into five groups with six mice/group) underwent intraperitoneal injection with DOX (5 mg/kg, once a week, five times) to induce myocardial injury, followed by assessment of NOX4 and NLRP3 expression in cell supernatant and myocardial tissues. In cardiomyocyte HL-1 cells, cell proliferation was tested by MTT assay and the activity of ROS by probes. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) activity were evaluated by kits. The expression of pyroptosis proteins was assessed by western blotting. Subsequently, the expression of NOX4 or NLRP3 was altered to determine the effect of NOX4 or NLRP3 expression on cardiomyocyte injury and pyroptosis. The animal models were utilized to evaluate the changes in the cardiac function of mice using an echocardiographic system, with these parameters measured including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic diameter (LVEDD). Furthermore, the content of myocardial injury markers and the protein expression of pyroptosis proteins were determined to evaluate myocardial injury in the mice. Results: DOX treatment led to cardiomyocyte injury and pyroptosis, as evidenced by weakened LVEF, LVFS, and cell proliferation (P<0.05), elevated LVEDD, ROS, and MDA (P<0.05), increased expression of pyroptosis proteins (P<0.05), and decreased SOD and GSH (P<0.05). Additionally, NOX4 and NLRP3 were highly-expressed (P<0.05) in cell supernatant and myocardial tissues. In DOX-induced HL-1 cells, the overexpression of NOX4 intensified ROS levels to aggravate cardiomyocyte injury and pyroptosis, which was reversed by treatment of the ROS scavenger N-acetyl-cysteine. Furthermore, it was revealed that the combination of short hairpin RNA (sh)-NOX4 and overexpressed (oe)-NLRP3 reversed the cardioprotective effects of sh-NOX4 and increased myocardial tissue or cell injury and pyroptosis in vitro and in vivo. No mice died during the animal experiments, and only two were ruled out due to a weight loss greater than 20%. Conclusions: NOX4-mediated ROS production activated NLRP3 inflammasome, thereby aggravating DOX-induced myocardial injury in vitro and in vivo.

TNF-α promotes expression of inflammatory factors by upregulating nicotinamide adenine dinucleotide phosphate oxidase-2 expression in human gingival fibroblasts.

Background/purpose: Periodontitis is a chronic infectious disease. The oxidative stress environment can cause or exacerbate the inflammation in periodontitis. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) may be the most important source of reactive oxygen species (ROS) in periodontal tissues. The pathological mechanism of periodontitis may be related to the increased ROS caused by enhanced NOX activity. The purpose was to investigate the effect of tumor necrosis factor (TNF-α) on inflammatory cytokines and ROS, and the role of NOX-2 in human gingival fibroblasts (HGFs). Materials and methods: HGFs were cultured and divided into the normal control group (NC group) and the inflammatory model group (TNF-α group) induced by 10 ng/ml TNF-α. Thereafter, NOX-2 siRNA was used to knock down NOX-2 gene expression. Quantitative real-time PCR was applied to detect IL-6, MCP-1, and NOX-2 mRNA levels. The levels of IL-6 and MCP-1 protein were examined by ELISA. The level of NOX-2 was evaluated by Western blot. ROS expression was measured by the fluorescence microplate. Results: The mRNA and protein expression levels of IL-6, MCP-1, and NOX-2 were significantly increased, and the expression of ROS was significantly elevated in response to 10 ng/ml TNF-α. Compared with the si-NC group, the mRNA and protein expression levels of IL-6 and MCP-1 were significantly down-regulated and ROS expression was significantly decreased in the si-NOX2 group stimulated by 10 ng/ml TNF-α. Conclusion: TNF-α promotes the expression of NOX-2 in human gingival fibroblasts and enhances the expression of inflammatory factors and ROS in human gingival fibroblasts through the upregulation of NOX-2 partly.

Nicotinamide adenine dinucleotide phosphate oxidase in pancreatic diseases: Mechanisms and future perspectives.

Pancreatitis and pancreatic cancer (PC) stand as the most worrisome ailments affecting the pancreas. Researchers have dedicated efforts to unraveling the mechanisms underlying these diseases, yet their true nature continues to elude their grasp. Within this realm, oxidative stress is often believed to play a causal and contributory role in the development of pancreatitis and PC. Excessive accumulation of reactive oxygen species (ROS) can cause oxidative stress, and the key enzyme responsible for inducing ROS production in cells is nicotinamide adenine dinucleotide phosphate hydrogen oxides (NOX). NOX contribute to pancreatic fibrosis and inflammation by generating ROS that injure acinar cells, activate pancreatic stellate cells, and mediate macrophage polarization. Excessive ROS production occurs during malignant transformation and pancreatic carcinogenesis, creating an oxidative microenvironment that can cause abnormal apoptosis, epithelial to mesenchymal transition and genomic instability. Therefore, understanding the role of NOX in pancreatic diseases contributes to a more in-depth exploration of the exact pathogenesis of these diseases. In this review, we aim to summarize the potential roles of NOX and its mechanism in pancreatic disorders, aiming to provide novel insights into understanding the mechanisms underlying these diseases.

Molecular mechanism of the NOS/NOX regulation of antibacterial activity in Eriocheir sinensis.

To elucidate the role of nitric oxide synthase (NOS), which produces the free radical nitric oxide (NO), and nicotinamide adenine dinucleotide phosphate oxidase (NOX), which produces the superoxide anion (O2-), in the innate immunity of Eriocheir sinensis, the full lengths of the NOS and NOX genes were cloned via rapid amplification of the cDNA ends and then expressed in the prokaryotic form to obtain the recombinant proteins, NOS-HIS and NOX-HIS. Through bacterial binding and stimulation experiments, the molecular mechanisms of NOS and NOX in the innate immunity of E. sinensis were explored. Based on the results, NOS and NOX were 5900 bp and 4504 bp long, respectively, and were evolutionarily conserved. Quantitative real-time PCR revealed that NOS and NOX were expressed in all studied tissues, and both were expressed in the highest amounts in hemocytes. NOS-HIS and NOX-HIS could bind to bacteria with different binding powers; their binding ability to gram-positive bacteria was higher than that of binding to gram-negative bacteria. After stimulation with Aeromonas hydrophila, NOS expression was significantly up-regulated at 3, 6, and 48 h, and NOX expression was significantly down-regulated at 3, 12, 24, and 48 h. After bacterial stimulation, the NOS enzyme activity in the serum of E. sinensis was also significantly up-regulated at 6 and 48 h, and the NOX enzyme activity was significantly down-regulated at 12 and 48 h, aligning with the gene expression trend. Moreover, the related free radical molecules, NO, O2-, and H2O2, tended to decrease after bacterial stimulation. Overall, the gene expression and enzyme activity of NOS and NOX had been changed respectively, and the contents of a series of free radical molecules (NO, O2- and H2O2) were induced in E. sinensis after bacterial stimulation, which then exert antibacterial immunity.

Empagliflozin prevents oxidative stress in human coronary artery endothelial cells via the NHE/PKC/NOX axis.

BACKGROUND: Empagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca2+), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation. METHODS: Human coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca2+ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na+/NCX/Ca2+ in the ROS inhibitory capacity of EMPA. RESULTS: Compared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-ß knockdown inhibits the NOX activation induced by Ca2+ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca2+ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch. CONCLUSION: EMPA reduced NOX activity via attenuation of the NHE/Na+/NCX/Ca2+/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.

Association Between Serum Levels of Nicotinamide-Adenine Dinucleotide Phosphate Oxidase 2 and the Development of Atrial Fibrillation After Isolated Coronary Artery Bypass Grafting: A Prospective Pilot Study.

INTRODUCTION: To investigate the levels of nicotinamide-adenine dinucleotide phosphate oxidase 2 (NOX2) in serum and pericardial drainage samples in the early stage after coronary artery bypass grafting (CABG) and determine whether NOX2 is predictive of postoperative atrial fibrillation (POAF). MATERIALS AND METHODS: This prospective pilot study involved 152 adults without history of atrial fibrillation who underwent first-time elective isolated CABG. Serum and pericardial fluid samples were simultaneously obtained from patients at baseline and 4, 12, and 24 h post operation. NOX2 levels were determined using enzyme-linked immunosorbent assays. The heart rhythm of patients was continuously monitored through a Holter monitor until discharge. Logistic regression and receiver-operating characteristic (ROC) curve analyses were performed, as appropriate. RESULTS: Fifty-one patients (33.6%) experienced in-hospital POAF. NOX2 concentration in serum and pericardial drainage samples was increased after surgery, reached its peak at 12 h, and gradually declined thereafter toward the baseline levels by 24 h. At 12 h, patients with POAF had higher levels of serum NOX2 than those without (3.96 ± 0.35 vs. 3.70 ± 0.75 µg/mL, respectively, p = 0.004). There were no discernible differences in pericardial NOX2 between the 2 groups. Multivariate analysis revealed that serum NOX2 at 12 h post operation was the strongest independent predictor of POAF (odds ratio: 2.179, 95% confidence interval: 1.084-4.377). The area under the ROC curve of the POAF predictive model was 0.732 (95% confidence interval: 0.654-0.801). CONCLUSION: Serum NOX2 may be useful in the identification of POAF. Larger studies are warranted to substantiate these findings.

Impact of amino acid substitutions in hepatitis C virus core region on the severe oxidative stress.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, leading to liver steatosis, fibrosis, and hepatocellular carcinoma (HCC). Despite the accumulation of clinical data showing the impact of amino acid substitutions at positions 70 (R70Q/H) and/or 91 (L91M) in the HCV core protein in progressive liver diseases, including HCC, the underlying mechanisms have not been elucidated. We analyzed 72 liver biopsy specimens from patients with chronic HCV genotype 1b (HCV-1b) infection prior to antiviral treatment. Levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor erythroid 2-related factor 2 (NRF2) in the nucleus were quantified using liver tissue immunohistochemistry. The effects of amino acid substitutions in the HCV core region on hepatocellular oxidative stress were investigated using wild-type or double-mutant (R70Q/H+L91M) HCV-1b core transfection and stable expression in human hepatoma HuH-7 cells. Overall, 24, 19, 11, and 18 patients had the wild-type, R70Q/H, L91M, and R70Q/H+L91M genotypes, respectively, in the HCV core. A significantly higher accumulation of hepatocellular 8-OHdG and a lower NRF2/8-OHdG ratio were observed in patients with R70Q/H+L91M than in those with the wild-type disease. Increased levels of intracellular superoxide and hydrogen peroxide in the cytoplasm and mitochondria, mRNA expression of enzymes generating oxidative stress, and nuclear expression of nicotinamide adenine dinucleotide phosphate oxidase 4 were augmented in cells treated with R70Q+L91M. HCV core proteins harboring either or both substitutions of R70Q/H or L91M enhanced hepatocellular oxidative stress in vivo and in vitro. These amino acid substitutions may affect HCC development by enhancing hepatic oxidative stress in patients with chronic HCV-1b infection.

Aspirin protects human trophoblast HTR-8/SVneo cells from H2O2-Induced oxidative stress via NADPH/ROS pathway.

INTRODUCTION: Pre-eclampsia (PE) is a pregnancy complication that can lead to maternal, fetal, and neonatal deaths in clinical practice. Accumulation of trophoblastic reactive oxygen species (ROS), which could result in oxidative stress and cell apoptosis, is considered to play an important role in PE pathology. It has been reported that aspirin has a positive effect on PE treatment in high-risk pregnant women. METHODS: In vitro, extravillous trophoblast cell line (HTR-8/SVneo) were treated with hydrogen peroxide (H2O2, 150 µM) after the presence of aspirin (90 and 120 µM) with or without GKT137831 (a Nox4 inhibitor, 20 µM). A series of experiments including CCK-8 assays, flow cytometry, biochemical testing, and Western Blotting etc. verified the protective effects and potential mechanisms of aspirin against oxidative stress-induced damage in PE. RESULTS: Our results demonstrated that H2O2 induces oxidative stress and apoptosis in HTR8/SVneo cells. However, aspirin pretreatment rescue cell viability and reduce LDH activity of HTR-8/SVneo cells. Aspirin can suppress the ROS overproduction and MDA level while increase SOD content and CAT activity. In addition, aspirin pretreatment significantly alleviated cell apoptosis and suppressed the expression of Nox4 and its subunits (p22phox and p47phox) at protein and mRNA levels. The above results were more obvious after the combination of aspirin with GKT137831. DISCUSSION: This study demonstrated that aspirin protects human trophoblasts against H2O2-induced oxidative stress and cell apoptosis via suppressing NADPH/ROS pathway. These findings provide novel insights for the application of aspirin as a protective and curative agent against PE.

[Intervention effect of apocynin on silicosis induced by silica in rats].

Objective: To investigate the intervention effect and its mechanism of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) on silicosis induced by silica (SiO(2)) in rats. Methods: In October 2021, 24 SPF SD male rats were divided into control group, silicosis model group and apocynin intervention group according to random number table method, with 8 rats in each group. SiO(2) was exposed by one-time intratracheal instillation. The rats in the apocynin intervention group were intraperitoneally injected with apocynin 50 mg/kg, 3 times a week, on the second day after treatment. The rats were sacrificed 28 days later, and lung coefficients were calculated after lung tissues were weighed. Hematoxylin-eosin staining and Masson staining were used to observe the lung histopathological changes in each group, respectively. The levels of NOX, reactive oxygen species (ROS), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in lung tissue were detected. The expressions of interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The level of hydroxyproline (HYP) was detected by alkaline hydrolysate. The expressions of transforming growth factor beta 1 (TGF-ß1), E-cadherin (E-cad) and α-smooth muscle actin (α-SMA) in lung tissue were detected by Western blotting. Results: Compared with the control group, the body weight of silicosis model group was decreased, the lung tissue showed obvious inflammatory infiltration and fibrosis, and the levels of lung coefficient, IL-1ß, IL-6, TNF-α and TGF-ß1 were significantly increased (P<0.05). Compared with the silicosis model group, the lung tissue injury in the apocynin intervention group was significantly improved, the lung coefficient, NOX, ROS, MDA, IL-1ß, IL-6, TNF-α and TGF-ß1 levels were decreased, and the activity of GSH-Px was increased (P<0.05). Compared with the silicosis model group, the expressions of HYP and α-SMA were decreased and the level of E-cad was increased in the apocynin intervention group (P<0.05) . Conclusion: Apocynin may alleviate SiO(2)-induced fibrosis in silicosis rats by reducing oxidative stress, the release of inflammatory factors and inhibiting the process of epithelial-mesenchymal transition.

Inhibitory Effect of Quercetin on Oxidative Endogen Enzymes: A Focus on Putative Binding Modes.

Oxidative stress is defined as an imbalance between the production of free radicals and reactive oxygen species (ROS) and the ability of the body to neutralize them by anti-oxidant defense systems. Cells can produce ROS during physiological processes, but excessive ROS can lead to non-specific and irreversible damage to biological molecules, such as DNA, lipids, and proteins. Mitochondria mainly produce endogenous ROS during both physiological and pathological conditions. Enzymes like nicotinamide adenine dinucleotide phosphate oxidase (NOX), xanthine oxidase (XO), lipoxygenase (LOX), myeloperoxidase (MPO), and monoamine oxidase (MAO) contribute to this process. The body has enzymatic and non-enzymatic defense systems to neutralize ROS. The intake of bioactive phenols, like quercetin (Que), can protect against pro-oxidative damage by quenching ROS through a non-enzymatic system. In this study, we evaluate the ability of Que to target endogenous oxidant enzymes involved in ROS production and explore the mechanisms of action underlying its anti-oxidant properties. Que can act as a free radical scavenger by donating electrons through the negative charges in its phenolic and ketone groups. Additionally, it can effectively inhibit the activity of several endogenous oxidative enzymes by binding them with high affinity and specificity. Que had the best molecular docking results with XO, followed by MAO-A, 5-LOX, NOX, and MPO. Que's binding to these enzymes was confirmed by subsequent molecular dynamics, revealing different stability phases depending on the enzyme bound. The 500 ns simulation showed a net evolution of binding for NOX and MPO. These findings suggest that Que has potential as a natural therapy for diseases related to oxidative stress.

Stachydrine Hydrochloride Regulates the NOX2-ROS-Signaling Axis in Pressure-Overload-Induced Heart Failure.

Our previous studies revealed the protection of stachydrine hydrochloride (STA) against cardiopathological remodeling. One of the underlying mechanisms involves the calcium/calmodulin-dependent protein kinase Ⅱ (CaMKII). However, the way STA influences CaMKII needs to be further investigated. The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-coupled reactive oxygen species (ROS) overproduction putatively induces the oxidative activation of CaMKII, resulting in the occurrence of pathological cardiac remodeling and dysfunction in experimental models of mice. Thus, in this study, we assessed the role of the NOX2-ROS signal axis in STA cardioprotection. The transverse aortic constriction (TAC)-induced heart failure model of mice, the phenylephrine-induced hypertrophic model of neonatal rat primary cardiomyocytes, and the H2O2-induced oxidative stress models of adult mouse primary cardiomyocytes and H9c2 cells were employed. The echocardiography and histological staining were applied to assess the cardiac effect of STA (6 mg/kg/d or 12 mg/kg/d), which was given by gavage. NOX2, ROS, and excitation-contraction (EC) coupling were detected by Western blotting, immunofluorescence, and calcium transient-contraction synchronous recordings. ROS and ROS-dependent cardiac fibrosis were alleviated in STA-treated TAC mice, demonstrating improved left ventricular ejection fraction and hypertrophy. In the heart failure model of mice and the hypertrophic model of cardiomyocytes, STA depressed NOX2 protein expression and activation, as shown by inhibited translocation of its phosphorylation, p67phox and p47phox, from the cytoplasm to the cell membrane. Furthermore, in cardiomyocytes under oxidative stress, STA suppressed NOX2-related cytosolic Ca2+ overload, enhanced cell contractility, and decreased Ca2+-dependent regulatory protein expression, including CaMKⅡ and Ryanodine receptor calcium release channels. Cardioprotection of STA against pressure overload-induced pathological cardiac remodeling correlates with the NOX2-coupled ROS signaling cascade.

Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia.

BACKGROUND: The urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. METHODS: The effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model. Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. RESULTS: We initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1ß, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. CONCLUSION: Crebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

Novel mutations in CYBB Gene Cause X-linked chronic Granulomatous Disease in Pakistani patients.

BACKGROUND: Chronic Granulomatous Disease (CGD) is a primary immunodeficiency that causes susceptibility to recurrent fungal and bacterial infections. The CYBB gene encodes gp91phox component of the Phagocytic Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and specifically, X-linked CGD is caused by mutations in the CYBB gene, located on the X chromosome. The aim of the study was to characterize functional and genetic mutations in X-linked CGD. METHODS: Functional analysis was conducted on the whole blood of seventeen male individuals who were suspected to have X-linked chronic granulomatous disease (CGD). Flow cytometry was employed to assess the capacity of NADPH oxidase, measuring both H2O2 production and gp91phox protein expression in neutrophils. Additionally, DNA Sanger sequencing was performed for genetic analysis. The pathogenicity of novel mutations was assessed by pathogenicity prediction tools. RESULT: Among the seventeen patients evaluated, five patients (P1, P2, P3, P4, and P5) displayed impaired H2O2 production by their neutrophils upon stimulation with Phorbol myristate acetate (PMA), accompanied by abnormal gp91phox expression. DNA sequencing of the CYBB gene identified specific mutations in each patient. In P1 and P2 (previously reported cases), a hemizygous missense mutation, c.925G > A/p.E309K was identified. In P3 and P4 (novel cases), hemizygous nonsense mutations, c.216T > A/p.C72X were found. Lastly, in P5 (also a novel case), a hemizygous missense mutation, c.732T > G/p.C244W was detected. These mutations reside in exons 9,3 and 7 of the CYBB gene, respectively. CONCLUSIONS: The current study contributes to the understanding of the clinical and genetic spectrum associated with X-linked chronic granulomatous disease (CGD). It highlights the significance of early diagnosis in CGD and emphasizes the importance of lifelong prophylaxis to prevent severe infections.

ZC3H13 reduced DUOX1-mediated ferroptosis in laryngeal squamous cell carcinoma cells through m6A-dependent modification.

Laryngeal squamous cell carcinoma (LSCC) is the second most common head and neck cancer. To identify the link between ferroptosis and LSCC, we targeted the dual oxidase 1 (DUOX1) gene. This study aimed to reveal the intrinsic mechanism by which the DUOX1-zinc-finger CCCH domain-containing protein 13 (ZC3H13) ferroptosis axis affected the LSCC process. GEPIA was used to investigate the expression of DUOX1 in LSCC, and the expression levels of DUOX1 and ZC3H13 were manipulated by overexpression and RNA interference. MTT assay was used to detect cell proliferation. Chromatin immunoprecipitation (CHIP) detected the binding of DUOX1 and ZC3H13, and ROS assessment and intracellular Fe2+ content determination were performed to examine the ferroptosis. MeRIP was used to analyze the m6A methylation of DUOX1. Ferroptosis-related proteins were detected by qRT-PCR. DUOX1 was found to be poorly expressed in LSCC cells, low DUOX1 level promoted LSCC cell proliferation, and low ZC3H13 level decreased LSCC cell proliferation. Besides, there was an interaction between DUOX1 and ZC3H13. DUOX1 could inhibit the expression levels of ferroptosis-related genes GPX4 and F1H1 in LSCC cells DUOX1 inhibited the expression levels of ROS and ferroptosis-related genes GPX4 and F1H1 and increased intracellular iron content in LSCC cells, but ZC3H13 reversed this phenomenon by inhibiting DUOX1 gene through m6A methylation modification. ZC3H13 reduced DUOX1-mediated ferroptosis in LSCC cells through m6A-dependent modification. The regulatory pathway of DUOX1 and ferroptosis are potential targets for designing diagnostic and combination therapeutic strategies for LSCC patients.

In what area of biology has a "new" type of cell death been discovered?

Cell death is a fundamental physiological process that occurs in all organisms and is crucial to each organism's evolution, ability to maintain a stable internal environment, and the development of multiple organ systems. Disulfidptosis is a new mode of cell death that is triggered when cells with high expression of solute carrier family 7 member 11 (SLC7A11) are exposed to glucose starvation to initiate the process of cell death. The disulfidptosis mechanism is a programmed cell death mode that triggers cell death through reduction-oxidation (REDOX) reactions and disulfur bond formation. In disulfidptosis, disulfur bonds play a crucial role and cause the protein in the cell to undergo conformational changes, eventually leading to cell death. This mode of cell death has unique characteristics and regulatory mechanisms in comparison with other modes of cell death. In recent years, an increasing number of studies have shown that the disulfidptosis mechanism plays a key role in the occurrence and development of a variety of diseases. For example, cancer, cardiovascular diseases, neurodegenerative diseases, and liver diseases are all closely related to cell disulfidptosis mechanisms. Therefore, it is of paramount clinical significance to conduct in-depth research regarding this mechanism. This review summarizes the research progress on the disulfidptosis mechanism, including its discovery history, regulatory mechanism, related proteins, and signaling pathways. Potential applications of the disulfidptosis mechanism in disease therapy and future research directions are also discussed. This mechanism represents another subversive discovery after ferroptosis, and provides both a fresh perspective and an innovative strategy for the treatment of cancer, as well as inspiration for the treatment of other diseases.

Crystal Structures of 6-Phosphogluconate Dehydrogenase from Corynebacterium glutamicum.

Corynebacterium glutamicum (C. glutamicum) has been considered a very important and meaningful industrial microorganism for the production of amino acids worldwide. To produce amino acids, cells require nicotinamide adenine dinucleotide phosphate (NADPH), which is a biological reducing agent. The pentose phosphate pathway (PPP) can supply NADPH in cells via the 6-phosphogluconate dehydrogenase (6PGD) enzyme, which is an oxidoreductase that converts 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P), to produce NADPH. In this study, we identified the crystal structure of 6PGD_apo and 6PGD_NADP from C. glutamicum ATCC 13032 (Cg6PGD) and reported our biological research based on this structure. We identified the substrate binding site and co-factor binding site of Cg6PGD, which are crucial for understanding this enzyme. Based on the findings of our research, Cg6PGD is expected to be used as a NADPH resource in the food industry and as a drug target in the pharmaceutical industry.