NOX4-mediated astrocyte ferroptosis in Alzheimer's disease.

This study investigates NADPH oxidase 4 (NOX4) involvement in iron-mediated astrocyte cell death in Alzheimer's Disease (AD) using single-cell sequencing data and transcriptomes. We analyzed AD single-cell RNA sequencing data, identified astrocyte marker genes, and explored biological processes in astrocytes. We integrated AD-related chip data with ferroptosis-related genes, highlighting NOX4. We validated NOX4's role in ferroptosis and AD in vitro and in vivo. Astrocyte marker genes were enriched in AD, emphasizing their role. NOX4 emerged as a crucial player in astrocytic ferroptosis in AD. Silencing NOX4 mitigated ferroptosis, improved cognition, reduced Aß and p-Tau levels, and alleviated mitochondrial abnormalities. NOX4 promotes astrocytic ferroptosis, underscoring its significance in AD progression.

GC-MS analysis and pharmacological potentiality of Lasia spinosa (L.) Thwaites leaves and fruit extracts: an in vitro and in silico studies.

Lasia spinosa (L. spinosa) is widely used in Asian countries for treating various diseases and as a vegetable, yet its bioactive properties remain under-researched. It is traditionally utilized in Ayurveda and the AYUSH system of medicine for its medicinal properties, and commonly used to treat digestive disorders, respiratory issues, and inflammatory conditions. This study aims to identify the phytochemicals in L. spinosa leaves and fruit extracts and evaluate their biological activities. Phytochemicals in methanol extracts of L. spinosa fruits and leaves were identified by GC-MS analysis. Antioxidant and cytotoxic activities were assessed using the DPPH free radical and nitric oxide (NO) scavenging assay and brine shrimp lethality test. Antibacterial activity was evaluated against Shigella boydii, Shigella flexneri, Streptococcus iniae, and Streptococcus dysgalactiae, while antifungal properties were tested against Cercospora beticola and Rhizoctonia solani. Molecular docking was conducted to predict the effectiveness of L. spinosa phytochemicals against NADPH oxidase and the Shigella effector OspG. Nine compounds were detected from both extracts. The methanol leaves extract exhibited superior antioxidant activity compared to the fruit extract, with IC50 values of 111.81 ± 8.99 µg/ml and 174.81 ± 4.86 µg/ml, respectively, as determined by the DPPH scavenging assay. The nitric oxide (NO) scavenging assay also revealed higher potency in the leaves extract (IC50 = 138.59 ± 1.50 µg/ml) compared to the fruit extract (IC50 = 196.47 ± 1.72 µg/ml). Both extracts showed significant antimicrobial activity against all tested microorganisms. In silico studies indicated notable inhibitory activity of all phytochemicals against the target proteins, with Linoelaidic acid and 9-Octadecenamide, (Z)- exhibiting the highest activity against NADPH oxidase (PDB: 2cdu) and Shigella flexneri OspG effector kinase (PDB: 4bvu), respectively. These findings suggest that L. spinosa has potent antioxidant and antimicrobial activities. Compounds from this plant could serve as lead compounds for developing antioxidant and antibacterial agents. However, molecular studies should be addressed.

The role of reactive oxygen species in regulation of the plasma membrane H +-ATPase activity in Masson pine (Pinus massoniana Lamb.) roots responding to acid stress.

To understand the role of reactive oxygen species (ROS) in regulation of the plasma membrane (PM) H+-ATPase in acid-stressed Masson pine roots, different acidity (pH 6.6 as the control, pH 5.6, and pH 4.6) of simulated acid rain (SAR) added with and without external chemicals [H2O2, enzyme inhibitors, and ROS scavenger] was prepared. After 30 days of SAR exposure, the plant morphological phenotype attributes, levels of cellular ROS and lipid peroxidation, enzymatic activities of antioxidants, PM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and PM H+-ATPase activity in pine seedlings were measured. Compared to the control, the growth of pine seedlings exposed to SAR in the presence or absence of H2O2 was well-maintained, but the application of Na3VO4, 1,3-Dimethyl-2-thiourea, N, N-dimethylthiourea (DMTU), and diphenyleneiodonium chloride (DPI) caused a substantial growth inhibition. In addition, SAR exposure, SAR with H2O2 treatment, and SAR with Na3VO4 treatment increased the cellular H2O2 content, O2·- content, and malondialdehyde (MDA) content, while the use of DMTU and DPI lead to relatively low levels. Similarly, the enzymatic activities of antioxidants, PM NADPH oxidase, and PM H+-ATPase in acid stressed pine seedlings elevated with the increasing acidity. A significant stimulation of these enzymatic activities obtained from SAR with H2O2 treatment was observed, whereas which decreased obviously with the addition of Na3VO4, DMTU, and DPI (P < 0.05). Moreover, a positive correlation was found between plant morphological attributes and the PM H+-ATPase activity (P < 0.05). Besides, the PM H+-ATPase activity positively correlated with the cellular ROS contents and the enzymatic activities of antioxidants and PM NADPH oxidase (P < 0.05). Therefore, the PM H+-ATPase is instrumental in the growth of pine seedlings resisting to acid stress by enhancing its activity. The process involves the signaling transduction of cellular ROS and coordination with PM NADPH oxidase.

Tamarixetin ameliorates cerebral ischemia-reperfusion injury via suppressing nicotinamide adenine dinucleotide phosphate oxidase 2/nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3 inflammasome activation.

Tamarixetin, a natural dietary flavone, exhibits remarkable potential for the treatment of ischemic stroke. The present article aimed to explore the impact of tamarixetin on ischemic stroke and elucidate the underlying mechanisms. Effects of tamarixetin on ischemic stroke were evaluated in rats using the middle cerebral artery occlusion and reperfusion (MCAO/R) model, by assessing the neurological deficit scores, brain water content, brain infraction, and neuronal damage. The levels of proinflammatory cytokines, NLRP3 inflammasome activation, reactive oxygen species (ROS) production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were measured in MCAO/R rats and lipopolysaccharide-stimulated cells. Tamarixetin administration improved the neurological dysfunction and neuronal loss in MCAO/R rats. In addition, tamarixetin reduced microglial hyperactivation and proinflammatory cytokines expression in vivo and in vitro. Tamarixetin attenuated NF-κB p65 phosphorylation and promoter activity, reduced NLRP3 expression and caspase-1 cleavage, and downregulated IL-1ß and IL-18 secretions to suppress NLRP3 inflammasome activation. The levels of superoxide anion, hydrogen peroxide, and ROS were also suppressed by tamarixetin. The downregulation of NADP+ and NADPH levels, and gp91phox expression indicated the ameliorative effects of tamarixetin on NADPH oxidase activation. In the gp91phox knockdown cells treated with lipopolysaccharide, the effects of tamarixetin on NADPH oxidase activation, ROS generation, and NLRP3 inflammasome activation were diminished. Moreover, tamarixetin protects neurons against microglial hyperactivation in vitro. Our findings support the potential of tamarixetin as a therapeutic agent for ischemic stroke, and its mechanism of action involves the inhibition of NADPH oxidase-NLRP3 inflammasome signaling.

Mitochondrial dysfunction and metabolic reprogramming induce macrophage pro-inflammatory phenotype switch and atherosclerosis progression in aging.

Introduction: Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages. Methods: We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet. Results: Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1ß secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis. Discussion: Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.

Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Potentiation of the axis involving pentose phosphate pathway/NADPH oxidase/reactive oxygen species drives higher IL-10 production in monocytes of Sub-Saharan Africans.

Cellular metabolism is a key determinant of immune cell function. Here we found that CD14+ monocytes from Sub-Saharan Africans produce higher levels of IL-10 following TLR-4 stimulation and are bioenergetically distinct from monocytes from Europeans. Through metabolomic profiling, we identified the higher IL-10 production to be driven by increased baseline production of NADPH oxidase-dependent reactive oxygen species, supported by enhanced pentose phosphate pathway activity. Together, these data indicate that NADPH oxidase-derived ROS is a metabolic checkpoint in monocytes that governs their inflammatory profile and uncovers a metabolic basis for immunological differences across geographically distinct populations.

Orthologs of NOX5 and EC-SOD/SOD3: dNox and dSod3 Impact Egg Hardening Process and Egg Laying in Reproductive Function of Drosophila melanogaster.

The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.

Ca2+-mediated reactive oxygen species signaling regulates cell repair after mechanical wounding in the red alga Griffithsia monilis.

Mechanical damage to a cell can be fatal, and the cell must reseal its membrane and restore homeostasis to survive. Plant cell repair involves additional steps such as rebuilding vacuoles, rearranging chloroplasts, and remodeling the cell wall. When we pierced a Griffithsia monilis cell with a glass needle, a large amount of intracellular contents was released, but the cell membrane resealed in less than a second. The turgor of the vacuole was quickly restored, and the punctured cell returned to its original shape within an hour. Organelles such as chloroplasts and nuclei migrated to the wound site for 12 h and then dispersed throughout the cell after the wound was covered by a new cell wall. Using fluorescent probes, high levels of reactive oxygen species (ROS) and calcium were detected at the wound site from 3 h after wounding, which disappeared when cell repair was complete. Wounding in a solution containing ROS scavengers inhibited cellular repair, and inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity or blocking calcium influx reversibly inhibited cell repair. Oryzalin reversibly inhibited both chloroplast movement and ROS production during cell repair. Our results show that cell repair in G. monilis is regulated by calcium-mediated ROS signaling and that microtubules serve as mechanical effectors.

NADPH Alters DUOX1 Calcium Responsiveness.

Hydrogen peroxide is a key element in redox signaling and in setting cellular redox tone. DUOX1 and DUOX2, that directly synthesize hydrogen peroxide, are the most abundant NADPH oxidase transcripts in most epithelia. DUOX1 and DUOX2 hydrogen peroxide synthesis is regulated by intracellular calcium transients and thus cells can respond to signals and initiate responses by increasing cellular hydrogen peroxide synthesis. Nevertheless, many details of their enzymatic regulation are still unexplored. DUOX1 and DUOXA1 were expressed in HEK293T cells and activity was studied in homogenates and membrane fractions. When DUOX1 homogenates or membranes were pre-incubated in NADPH and started with addition of Ca2+, to mimic intracellular activation, progress curves were distinctly different from those pre-incubated in Ca2+ and started with NADPH. The Ca2+ EC50 for DUOX1's initial rate when pre-incubated in Ca2+, was three orders of magnitude lower (EC50 âˆ¼ 10-6 M) than with preincubation in NADPH (EC50 âˆ¼ 10-3 M). In addition, activity was several fold lower with Ca2+ start. Identical results were obtained using homogenates and membrane fractions. The data suggested that DUOX1 Ca2+ binding in expected physiological signaling conditions only slowly leads to maximal hydrogen peroxide synthesis and that full hydrogen peroxide synthesis activity in vivo only can occur when encountering extremely high concentration Ca2+ signals. Thus, a complex interplay of intracellular NADPH and Ca2+ concentrations regulate DUOX1 over a wide extent and may limit DUOX1 activity to a restricted range and spatial distribution.

Deubiquitinating Enzyme USP19 Regulates Ferroptosis and Mitochondrial Damage in SH-SY5Y Cells by Targeting the NOX4 Protein.

Background: Ferroptosis is extremely relevant to the progression of neurodegenerative pathologies such as Alzheimer's disease (AD). Ubiquitin-specific proteases (USP) can affect the NADPH oxidase family. Objective: Our study aimed to elucidate the potential role and molecular basis of a certain USP19 in reducing ferroptosis and mitochondrial injury in AD cells by targeting NOX4 stability. Methods: The deubiquitinase USP family gene USP19, which affects the stability of NOX4 protein, was first screened. The cell model of AD was constructed after interfering with SH-SY5Y cells by Aß1-40, and then SH-SY5Y cells were infected with lentiviral vectors to knock down USP19 and overexpress NOX4, respectively. Finally, the groups were tested for cell viability, changes in cellular mitochondrial membrane potential, lipid reactive oxygen species, intracellular iron metabolism, and NOX4, Mf1, Mf2, and Drp1 protein expression. Results: 5 µmol/L Aß1-40 intervened in SH-SY5Y cells for 24 h to construct a cell model of AD. Knockdown of USP19 decreased the expression of NOX4 protein, promoted the expression of mitochondrial fusion proteins Mnf1 and Mnf2, and inhibited the expression of the splitting protein Drp1. Furthermore, USP19 knockdown decreased mitochondrial membrane potential, SOD, MDA, intracellular iron content and increased GSH/GSSG ratio in SH-SY5Y cells. Our study revealed that NOX4 protein interacts with USP19 and knockdown of USP19 enhanced ubiquitination to maintain NOX4 protein stability. Conclusions: USP19 attenuates mitochondrial damage in SH-SY5Y cells by targeting NOX4 protein with Aß1-40.

NOX4 and its association with myeloperoxidase and osteopontin in regulating endochondral ossification.

IMPORTANCE: Endochondral ossification plays an important role in skeletal development. Recent studies have suggested a link between increased intracellular reactive oxygen species (ROS) and skeletal disorders. Moreover, previous studies have revealed that increasing the levels of myeloperoxidase (MPO) and osteopontin (OPN) while inhibiting NADPH oxidase 4 (NOX4) can enhance bone growth. This investigation provides further evidence by showing a direct link between NOX4 and MPO, OPN in bone function. OBJECTIVE: This study investigates NOX4, an enzyme producing hydrogen peroxide, in endochondral ossification and bone remodeling. NOX4's role in osteoblast formation and osteogenic signaling pathways is explored. METHODS: Using NOX4-deficient (NOX4-/-) and ovariectomized (OVX) mice, we identify NOX4's potential mediators in bone maturation. RESULTS: NOX4-/- mice displayed significant differences in bone mass and structure. Compared to the normal Control and OVX groups. Hematoxylin and eosin staining showed NOX4-/- mice had the highest trabecular bone volume, while OVX had the lowest. Proteomic analysis revealed significantly elevated MPO and OPN levels in bone marrow-derived cells in NOX4-/- mice. Immunohistochemistry confirmed increased MPO, OPN, and collagen II (COLII) near the epiphyseal plate. Collagen and chondrogenesis analysis supported enhanced bone development in NOX4-/- mice. CONCLUSIONS AND RELEVANCE: Our results emphasize NOX4's significance in bone morphology, mesenchymal stem cell proteomics, immunohistochemistry, collagen levels, and chondrogenesis. NOX4 deficiency enhances bone development and endochondral ossification, potentially through increased MPO, OPN, and COLII expression. These findings suggest therapeutic implications for skeletal disorders.

Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury.

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Enhanced effect of limb remote ischemic postconditioning combined with paeoniflorin on alleviating cerebral ischemic injury via neutrophil NADPH pathway.

BACKGROUND: Limb remote ischemic postconditioning (LRIP) and paeoniflorin (PF) both can ameliorate cerebral ischemia reperfusion (I/R) injury. At present, whether LRIP combined with PF can achieve better therapeutic effect is unknown. PURPOSE: This study explored the alleviating effect and mechanism of LRIP in combination with PF on cerebral I/R injury in rats. METHODS: Middle cerebral artery occlusion (MCAO) surgery was performed on rats except Sham group. Then PF (2.5 mg/kg, 5 mg/kg, 10 mg/kg) was administrated by intraperitoneal injection 10 min before the start of reperfusion. LRIP was operated on the left femoral artery at 0 h of reperfusion. Behavioral testing was used to assess neurological impairment, while TTC staining was used to examine infarct volume. Protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox in neutrophils from rat peripheral blood were tested by Western blot. Rat bone marrow neutrophils were extracted and incubated for 24 h with serum from rats after LRIP combined with PF. p38 MAPK inhibitor group was administrated SB203580 while the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor group was administrated Apocynin. Neutrophils were stimulated by fMLP (10 µM). Reactive oxygen species (ROS) production and protein expression of MyD88, TRAF6, p38 MAPK, and p47phox (ser 304 and ser 345) were detected. RESULTS: LRIP combined with PF (5 mg/kg) reduced cerebral infarct volume, ameliorated neurological deficit score (NDS), decreased fMLP-stimulated ROS release and downregulated the protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox (ser 304 and ser 345) in neutrophils. CONCLUSION: The protective effect of LRIP combined with PF on cerebral I/R injury was better than either alone. Taken together, we provided solid evidence to demonstrate that the combination of LRIP and PF had potential to alleviate cerebral I/R injury, which was regulated by MyD88-TRAF6-p38 MAPK pathway and neutrophil NADPH oxidase pathway.

Reynosin protects neuronal cells from microglial neuroinflammation by suppressing NLRP3 inflammasome activation mediated by NADPH oxidase.

Neuroinflammation, mediated by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) inflammasome, is a significant contributor to the pathogenesis of neurodegenerative diseases (NDDs). Reynosin, a natural sesquiterpene lactone (SL), exhibits a broad spectrum of pharmacological effects, suggesting its potential therapeutic value. However, the effects and mechanism of reynosin on neuroinflammation remain elusive. The current study explores the effects and mechanisms of reynosin on neuroinflammation using mice and BV-2 microglial cells treated with lipopolysaccharide (LPS). Our findings reveal that reynosin effectively reduces microglial inflammation in vitro, as demonstrated by decreased CD11b expression and lowered interleukin-1 beta (IL-1ß) and interleukin-18 (IL-18) mRNA and protein levels. Correspondingly, in vivo, results showed a reduction in the number of Iba-1 positive cells and alleviation of morphological alterations, alongside decreased expressions of IL-1ß and IL-18. Further analysis indicates that reynosin inhibits NLRP3 inflammasome activation, evidenced by reduced transcription of NLRP3 and caspase-1, diminished NLRP3 protein expression, inhibited apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and decreased caspase-1 self-cleavage. Additionally, reynosin curtailed the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, demonstrated by reduced NADP+ and NADPH levels, downregulation of gp91phox mRNA, protein expression, suppression of p47phox expression and translocation to the membrane. Moreover, reynosin exhibited a neuroprotective effect against microglial inflammation in vivo and in vitro. These collective findings underscore reynosin's capacity to mitigate microglial inflammation by inhibiting the NLRP3 inflammasome, thus highlighting its potential as a therapeutic agent for managing neuroinflammation.

Inhibitory Action of 1,3,5-Trihydroxybenzene on UVB-Induced NADPH Oxidase 4 through AMPK and JNK Signaling Pathways.

Specific sensitivity of the skin to ultraviolet B (UVB) rays is one of the mechanisms responsible for widespread skin damage. This study tested whether 1,3,5-trihydroxybenzene (THB), a compound abundant in marine products, might inhibit UVB radiation-induced NADPH oxidase 4 (NOX4) in both human HaCaT keratinocytes and mouse dorsal skin and explore its cytoprotective mechanism. The mechanism of action was determined using western blotting, immunocytochemistry, NADP+/NADPH assay, reactive oxygen species (ROS) detection, and cell viability assay. THB attenuated UVB-induced NOX4 expression both in vitro and in vivo, and suppressed UVB-induced ROS generation via NADP+ production, resulting in increased cell viability with decreased apoptosis. THB also reduced the expression of UVB-induced phosphorylated AMP-activated protein kinase (AMPK) and phosphorylated c-Jun N-terminal kinase (JNK). THB suppressed UVB-induced NOX4 expression and ROS generation by inhibiting AMPK and JNK signaling pathways, thereby inhibiting cellular damage. These results showed that THB could be developed as a UV protectant.

The peptidyl-prolyl isomerase Pin1 controls GM-CSF-induced priming of NADPH oxidase in human neutrophils and priming at inflammatory sites.

The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation.

Ca2+ binding shifts dimeric dual oxidase's truncated EF-hand domain to monomer.

Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca2+ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca2+ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca2+ binding (Ka = 107 M-1) while EF1 shows a weaker binding (Ka = 105 M-1), resulting in the burial of its negatively charged residues. The Ca2+ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca2+-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca2+ binding is vital for its Duox's domain-domain interaction for electron transfer.

Development of new peptidomimetic NADPH oxidase inhibitors with antithrombotic properties.

The ability of synthetic peptides inhibitors of NOX1 to effectively block the production of ROS by the enzyme was studied with different methodologies. Specifically, taking advantage of our understanding of the active epitope of the regulatory NOX1 subunit NOXA1 as a potent inhibitor of NOX1-derived O2•- formation, a panel of peptidomimetic derivatives of this peptide were designed and synthesized with the aim of improving their activity and increasing their stability in plasma. The results highlighted that improved efficacy and potency was found for both the peptide-peptoid hybrid GS2, whereas stapled peptide AC5 and its precursor showed higher stability despite lower biological potency. This study showed that minimal structural modifications of NOXA1 peptides are required to improve both their potency and stability to finally achieve best candidates as new potential anti-thrombotic drugs.