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| Immunofluorescence staining of NOX4 around the haematoma firmly colocalised in astrocytes, neurons, microglia and vascular endothelial cells. (A) Representative immunofluorescent images illustrated immunoreactivity of the injured basal ganglia region to NOX4 (green), GFAP (red), NeuN (red), Iba-1 (red), and CD34 (red). Nuclei are stained with DAPI (blue); the brain tissues were collected and used for immunofluorescence analysis on the third day after the rat ICH model (Continued)
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Intracerebral hemorrhage (ICH) is a common and severe neurological disorder that can effectively induce oxidative stress responses. NADPH oxidase 4 (NOX4) is a member of the NOX family of oxidases. It is expressed in the brain normally and involved in cell signal transduction and the removal of harmful substances. In some pathological conditions, i...
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Context 1
... is mainly expressed in neurons, astrocytes, vascular endothelial cells and microglia following ICH Immunohistochemistry and confocal microscopy imaging demonstrated that the elevated immunoreactivity of NOX4 was predominantly located in the neurons, astrocytes, vascular endothelial cells, and microglia in the injured basal ganglia. This observation is supported by a line-scan analysis of the channel intensity of NOX4/NEUN, NOX4/GFAP, NOX4/CD34, NOX4/Iba-1 and DAPI, which showed strongly overlapping NOX4 (green) and NEUN/GFAP/CD34/Iba-1 (red) intensity peaks (Figures 2A-E). ...
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... Knockout of NOX4 also reduces the production of reactive oxygen species in mitochondria, alleviates mitochondrial damage, prevents the accumulation of secondary reactive oxygen species, reduces neuronal pyroptosis, and helps alleviate secondary brain injury after intracerebral hemorrhage in rats [34]. ROS, as metabolic byproducts, when in excess, can instigate oxidative stress and inflammation [35]. Their role in activating NLRP3 inflammasomes is well-established, with ROS inhibition shown to ameliorate brain damage via NLRP3 downregulation [36]. ...
Background
Intracerebral hemorrhage (ICH) is a critical neurological condition with few treatment options, where secondary immune responses and specific cell death forms, like pyroptosis, worsen brain damage. Pyroptosis involves gasdermin-mediated membrane pores, increasing inflammation and neural harm, with the NLRP3/Caspase-1/GSDMD pathway being central to this process. Peroxiredoxin II (Prx II), recognized for its mitochondrial protection and reactive oxygen species (ROS) scavenging abilities, appears as a promising neuronal pyroptosis modulator. However, its exact role and action mechanisms need clearer definition. This research aims to explore Prx II impact on neuronal pyroptosis and elucidate its mechanisms, especially regarding endoplasmic reticulum (ER) stress and oxidative stress-induced neuronal damage modulation.
Methods and results
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Conclusions
Our study not only underlines Prx II importance in neuroprotection but also opens new therapeutic intervention avenues in intracerebral hemorrhage, stressing the complex interplay between redox regulation, ER stress, and mitochondrial dynamics in neuroinflammation and cell death management.
... Sections were incubated with antibodies cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) (1:200 dilution) overnight at 4 °C. Secondary antibodies (1:200 dilution) were added and incubated for 1 h at room temperature (Xie et al. 2020). The sections were stained with 3, 3-diaminobenzidine (DAB) chromogenic solution. ...
Objective and design
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Materials and methods
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Results
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... Activation of NOX4 (heterodimerized with p22phox), which does not require classical cytosolic subunits or their homologs, predominantly produces H 2 O 2 (Lyle et al., 2009). NOX4 is widely expressed in various tissues, including the brain (Xie et al., 2020), and some brain regions have been investigated in relation to NOX4 elevation and neurodegenerative diseases. It has further been established that NOX4 expression is upregulated in the hippocampus of animal models and patients with both AD (Zhu et al., 2020;Tao et al., 2021;Luengo et al., 2022) and PD (Choi et al., 2019;Boonpraman et al., 2023), as well as in the cortex of individuals with AD (Bruce-Keller et al., 2011;Fragoso-Morales et al., 2021;Park et al., 2021). ...
Diseases like Alzheimer’s and Parkinson’s diseases are defined by inflammation and the damage neurons undergo due to oxidative stress. A primary reactive oxygen species contributor in the central nervous system, NADPH oxidase 4, is viewed as a potential therapeutic touchstone and indicative marker for these ailments. This in-depth review brings to light distinct features of NADPH oxidase 4, responsible for generating superoxide and hydrogen peroxide, emphasizing its pivotal role in activating glial cells, inciting inflammation, and disturbing neuronal functions. Significantly, malfunctioning astrocytes, forming the majority in the central nervous system, play a part in advancing neurodegenerative diseases, due to their reactive oxygen species and inflammatory factor secretion. Our study reveals that aiming at NADPH oxidase 4 within astrocytes could be a viable treatment pathway to reduce oxidative damage and halt neurodegenerative processes. Adjusting NADPH oxidase 4 activity might influence the neuroinflammatory cytokine levels, including myeloperoxidase and osteopontin, offering better prospects for conditions like Alzheimer’s disease and Parkinson’s disease. This review sheds light on the role of NADPH oxidase 4 in neural degeneration, emphasizing its drug target potential, and paving the path for novel treatment approaches to combat these severe conditions.
... In infectious disorders, it plays a vital role in defending the host against invading microorganisms (Heyworth et al., 2003). However, in some pathological conditions, NOX4 mediates inflammation and cellular aging through ROS production (Babior, 1999;Wu et al., 2006;Xie et al., 2020). Recent research suggests that NOX4 has a neuroprotective role by regulating ROS and calcium levels, preventing hyperexcitability and neuronal death (Gola et In Silico Evaluation of Nutri-Pharmacological Potentials of Phytochemicals Fatoki et al. al., 2023). ...
Sorghum (Sorghum bicolor L. Moench) ranks as the fifth most widely grown cereal globally, and its grain is gluten-free, containing about 26 phytochemicals which are mostly phenolics. The objective of this study was to predict pharmacokinetics of sorghum grain phytochemicals and some molecular targets that may impact good health in human. The methods used were in silico pharmacokinetic prediction, target prediction, target gene network analysis, docking and molecular dynamics simulation. The results showed that ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, and vanillic acid were blood-brain barrier permeant, while p-coumaric acid and gallic acid have high gastrointestinal absorption. The results indicated that p-coumaric acid has 100% probability of target on aldose reductase and estrogen receptor, while gallic acid had 100% probability of target on alpha-(1,3)-fucosyltransferase 7. The docking analyses revealed that p-coumaric acid bind to aldose reductase with an affinity of -7.759 kcal.mol-1 while gallic acid bind to alpha-(1,3)-fucosyltransferase 7 with binding affinity of -5.512 kcal.mol-1. Overall, binding energy ΔGbind (Total) at 0 ns was slightly higher than that of 100 ns for p-coumaric acid - aldose reductase complex (-56.631 to -53.546 kcal.mol-1). This study provides valuable insights into the potential pharmacological actions of phytochemicals in sorghum grains.
... Anti-202-3p or NC-Anti were transfected into mice brains as described previously 48 . Briefly, the mice were deeply anesthetized and the plasmid premixed with transfection reagent was microinjected into the left lateral ventricle at a rate of 0.2 µL/min. ...
Ischemic stroke (IS) is associated with changes in gene expression patterns in the ischemic penumbra and extensive neurovascular inflammation. However, the key molecules related to the inflammatory response in the acute phase of IS remain unclear. To address this knowledge gap, conducted a study using Gene Set Enrichment Analysis (GSEA) on two gene expression profiles, GSE58720 and GSE202659, downloaded from the GEO database. We screened differentially expressed genes (DEGs) using GEO2R and analyzed 170 differentially expressed intersection genes for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis. We also used Metascape, DAVID, STRING, Cytoscape, and TargetScan to identify candidate miRNAs and genes. The targeted genes and miRNA molecule were clarified using the mice middle cerebral artery occlusion-reperfusion (MCAO/R) model. Our findings revealed that 170 genes were correlated with cytokine production and inflammatory cell activation, as determined by GO and KEGG analyses. Cluster analysis identified 11 hub genes highly associated with neuroinflammation: Ccl7, Tnf, Ccl4, Timp1, Ccl3, Ccr1, Sele, Ccr2, Tlr4, Ptgs2, and Il6. TargetScan results suggested that Ptgs2, Tlr4, and Ccr2 might be regulated by miR-202-3p. In the MCAO/R model, the level of miR-202-3p decreased, while the levels of Ptgs2, Tlr4, and Ccr2 increased compared to the sham group. Knockdown of miR-202-3p exacerbated ischemic reperfusion injury (IRI) through neuroinflammation both in vivo and in vitro. Our study also demonstrated that mRNA and protein levels of Ptgs2, Tlr4, and Ccr2 increased in the MCAO/R model with miR-202-3p knockdown. These findings suggest that differentially expressed genes, including Ptgs2, Tlr4, and Ccr2 may play crucial roles in the neuroinflammation of IS, and their expression may be negatively regulated by miR-202-3p. Our study provides new insights into the regulation of neuroinflammation in IS.
... Several studies have shown that NOX4 is crucial for pathophysiological changes after ICH. When NOX4 expression was down-regulated after ICH, oxidative stress, apoptosis, BBB defects, and brain swelling were significantly reduced (Xie et al., 2020). ...
Intracerebral hemorrhage is a life-threatening condition with a high fatality rate and severe sequelae. However, there is currently no treatment available for intracerebral hemorrhage, unlike for other stroke subtypes. Recent studies have indicated that mitochondrial dysfunction and mitophagy likely relate to the pathophysiology of intracerebral hemorrhage. Mitophagy, or selective autophagy of mitochondria, is an essential pathway to preserve mitochondrial homeostasis by clearing up damaged mitochondria. Mitophagy markedly contributes to the reduction of secondary brain injury caused by mitochondrial dysfunction after intracerebral hemorrhage. This review provides an overview of the mitochondrial dysfunction that occurs after intracerebral hemorrhage and the underlying mechanisms regarding how mitophagy regulates it, and discusses the new direction of therapeutic strategies targeting mitophagy for intracerebral hemorrhage, aiming to determine the close connection between mitophagy and intracerebral hemorrhage and identify new therapies to modulate mitophagy after intracerebral hemorrhage. In conclusion, although only a small number of drugs modulating mitophagy in intracerebral hemorrhage have been found thus far, most of which are in the preclinical stage and require further investigation, mitophagy is still a very valid and promising therapeutic target for intracerebral hemorrhage in the long run.
... ROS production also contributes significantly to ischemic injury in the brain (Abramov et al., 2007), resulting in necrosis and apoptotic cell death, eventually leading to brain injury. In addition, accumulated ROS in focal ischemia damages the blood-brain barrier (Xie et al., 2020) and further induces secondary brain tissue injury via the inflammatory response (Chen & Li, 2020). ...
Background: The transfer of mitochondria from healthy mesenchymal stem cells (MSCs) to injured MSCs has been shown to have potential therapeutic benefits for neural cell post-ischemic stroke. Specifically, functional mitochondria can perform their normal functions after being internalized by stressed cells, leading to host cell survival. However, while this approach shows promise, there is still a lack of understanding regarding which neural cells can internalize functional mitochondria and the regulatory mechanisms involved. To address this gap, we investigated the ability of different neural cells to internalize exogenous functional mitochondria extracted from MSCs.
Methods: Functional mitochondria (F-Mito) isolated from umbilical cord derived-MSCs (UCMSCs) were labeled with lentivirus of HBLV-mito-dsred-Null-PURO vector. The ability of stressed cells to internalize F-Mito was analyzed using a mouse (C57BL/6 J) middle cerebral artery occlusion (MCAO) model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. The cell viability was measured by CCK-8 kit. Time-course of intracellular ROS levels in stressed cells were analyzed by DCFH-DA staining after OGD/R and F-Mito treatment. MitoSOX, Mitotracker and WGA labeling were used to assess the relationship between ROS levels and the uptake of F-Mito at the single-cell level. Pharmacological modulation of ROS was performed using acetylcysteine (ROS inhibitor).
Results: Our findings demonstrate that neurons and endothelial cells are more effective at internalizing mitochondria than astrocytes, both in vitro and in vivo, using an ischemia-reperfusion model. Additionally, internalized F-Mito decreases host cell reactive oxygen species (ROS) levels and rescues survival. Importantly, we found that the ROS response in stressed cells after ischemia is a crucial determinant in positively mediating the internalization of F-Mito by host cells, and inhibiting the generation of ROS chemicals in host cells may decrease the internalization of F-Mito. These results offer insight into how exogenous mitochondria rescue neural cells via ROS response in an ischemic stroke model. Overall, our study provides solid evidence for the translational application of MSC-derived mitochondria as a promising treatment for ischemic stroke.
... Therefore, ICH represents a serious issue to human health. Oxidative damage, which is caused by an imbalance between the production of reactive oxygen species (ROS) and efficient elimination of ROS by recovery enzymes, plays a pivotal role in the poor prognosis of ICH (Xie et al., 2020). Heme oxygenase-1 (HO-1), a limiting enzyme for heme catabolism and iron production, is induced by several stress factors such as oxidative stress and hypoxia (Chau, 2015;Chen et al., 2019b). ...
Recent studies have indicated that suppressing oxidative stress and ferroptosis can considerably improve the prognosis of intracerebral hemorrhage (ICH). Withaferin A (WFA), a natural compound, exhibits a positive effect on a number of neurological diseases. However, the effects of WFA on oxidative stress and ferroptosis-mediated signaling pathways to ICH remain unknown. In this study, we investigated the neuroprotective effects and underlying mechanism for WFA in the regulation of ICH-induced oxidative stress and ferroptosis. We established a mouse model of ICH by injection of autologous tail artery blood into the caudate nucleus and an in vitro cell model of hemin-induced ICH. WFA was injected intracerebroventricularly at 0.1, 1 or 5 µg/kg once daily for 7 days, starting immediately after ICH operation. WFA markedly reduced brain tissue injury and iron deposition and improved neurological function in a dose-dependent manner 7 days after cerebral hemorrhage. Through in vitro experiments, cell viability test showed that WFA protected SH-SY5Y neuronal cells against hemin-induced cell injury. Enzyme-linked immunosorbent assays in vitro and in vivo showed that WFA markedly decreased the level of malondialdehyde, an oxidative stress marker, and increased the activities of anti-oxidative stress markers superoxide dismutase and glutathione peroxidase after ICH. Western blot assay, quantitative polymerase chain reaction and immunofluorescence results demonstrated that WFA activated the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling axis, promoted translocation of Nrf2 from the cytoplasm to nucleus, and increased HO-1 expression. Silencing Nrf2 with siRNA completely reversed HO-1 expression, oxidative stress and protective effects of WFA. Furthermore, WFA reduced hemin-induced ferroptosis. However, after treatment with an HO-1 inhibitor, the neuroprotective effects of WFA against hemin-induced ferroptosis were weakened. MTT test results showed that WFA combined with ferrostatin-1 reduced hemin-induced SH-SY5Y neuronal cell injury. Our findings reveal that WFA treatment alleviated ICH injury-induced ferroptosis and oxidative stress through activating the Nrf2/HO-1 pathway, which may highlight a potential role of WFA for the treatment of ICH.
... Kuroda et al. reported that NOX4 is the main source of oxidative stress in heart failure [5]. It was also reported that inhibition of NOX4/ROS could suppress neuronal and blood-brain barrier injury in intracerebral hemorrhage [18]. Moreover, hypertrophic stimuli triggered the expression of NOX4 in cardiac myocytes, which can promote apoptosis and mitochondrial dysfunction [19]. ...
... In the paraventricular nucleus, inhibition of NOX4 improves MI-induced cardiac dysfunction by suppressing the apoptosis of periinfarct and sympathoexcitation [20]. In addition, Xie et al. reported that NOX4 inhibition can improve the blood-brain barrier damage and attenuate oxidative stress caused by intracerebral hemorrhage [18]. NOX4 is closely related to oxidative stress in cardiovascular and cerebrovascular diseases, however, the specific quantitative relationship between NOX4 and oxidative stress in MI model is still unknown. ...
... Noticeably, NOX4 expression positively correlated with the oxidative stress factors (H 2 O 2 and MDA), and negatively correlated with the antioxidants (GPx and SOD). Interestingly, similar pattern was observed in brain injury, Casas et al. reported that the expression level of NOX4 was elevated after brain injury, and Xie et al. found that both NOX4 and oxidative stress levels were increased in the brain with intracerebral hemorrhage [18,33]. Ours and other groups' studies suggest that NOX4 plays an important role in the oxidative stress imbalance that happens in the injury tissues. ...
Oxidative stress closely related to the progression and severity of myocardial infarction (MI). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is one of the major enzymes that generate reactive oxygen species (ROS) in cardiovascular system. Here, we aim to elucidate the pathological role of NOX4 in MI. MI mouse model was created by the coronary artery ligation. NOX4 was specifically knocked down in heart through intramyocardial injection of siRNA. NOX4 expression and oxidative stress indicators were determined at different time points using qRT-PCR, Western blot, and ELISA, and then analyzed by Pearson’s correlation. Cardiac function was evaluated by using echocardiographic technique. NOX4 was upregulated in myocardial tissues of MI mice, which positively correlated with the elevation of oxidative stress indicators. Knockdown of NOX4 in heart significantly reduced the production of ROS and the level of oxidative stress in left ventricle tissues, which was accompanied by significant improvement of cardiac function in MI mice. Selective knockdown of NOX4 in heart attenuates MI-induced oxidative stress and improves cardiac function, suggesting inhibition of NOX4/ROS axis in heart using siRNA is a potential therapeutic treatment for MI-induced cardiac dysfunction.
... Additionally, NOX4 −/− mice did not show changes in the expression levels of NOX2 within the brain (data not shown), an observation known from other tissues before [44,45]. More interestingly, NOX4 expression is elevated during hypoxia and brain ischemia model [46], after intracerebral hemorrhage [47] and traumatic brain injury [43], all stress conditions associated with ROS excess and detrimental neuronal damage. Thereby we questioned if the elevated NOX4 levels are involved in this neuronal damage or if this is a compensatory mechanism since NOX4 might induce an antioxidative response. ...
Hyperexcitability is associated with neuronal dysfunction, cellular death, and consequently neurodegeneration. Redox disbalance can contribute to hyperexcitation and increased reactive oxygen species (ROS) levels are observed in various neurological diseases. NOX4 is an NADPH oxidase known to produce ROS and might have a regulating function during oxidative stress. We, therefore, aimed to determine the role of NOX4 on neuronal firing, hyperexcitability, and hyperexcitability-induced changes in neural network function. Using a multidimensional approach of an in vivo model of hyperexcitability, proteomic analysis, and cellular function analysis of ROS, mitochondrial integrity, and calcium levels, we demonstrate that NOX4 is neuroprotective by regulating ROS and calcium homeostasis and thereby preventing hyperexcitability and consequently neuronal death. These results implicate NOX4 as a potential redox regulator that is beneficial in hyperexcitability and thereby might have an important role in neurodegeneration.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00018-023-04758-z.
