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Activated microglia and macrophages exert dual beneficial and detrimental roles after central nervous system injury, which are thought to be due to their polarization along a continuum from a classical pro-inflammatory M1-like state to an alternative anti-inflammatory M2-like state. The goal of the present study was to analyze the temporal dynamics of microglia/macrophage polarization within the lesion micro-environment following traumatic brain injury (TBI) using a moderate-level controlled cortical impact (CCI) model in mice. We performed a detailed phenotypic analysis of M1- and M2-like polarized microglia/macrophages, as well as nicotinamide adenine dinucleotide phosphate oxidase (NOX2) expression, through 7 days post-injury using real-time polymerase chain reaction (qPCR), flow cytometry and image analyses. We demonstrated that microglia/macrophages express both M1- and M2-like phenotypic markers early after TBI, but the transient up-regulation of the M2-like phenotype was replaced by a predominant M1- or mixed transitional (Mtran) phenotype that expressed high levels of NOX2 at 7 days post-injury. The shift towards the M1-like and Mtran phenotype was associated with increased cortical and hippocampal neurodegeneration. In a follow up study, we administered a selective NOX2 inhibitor, gp91ds-tat, to CCI mice starting at 24 h post-injury to investigate the relationship between NOX2 and M1-like/Mtran phenotypes. Delayed gp91ds-tat treatment altered M1-/M2-like balance in favor of the anti-inflammatory M2-like phenotype, and significantly reduced oxidative damage in neurons at 7 days post-injury. Therefore, our data suggest that despite M1-like and M2-like polarized microglia/macrophages being activated after TBI, the early M2-like response becomes dysfunctional over time, resulting in development of pathological M1-like and Mtran phenotypes driven by increased NOX2 activity.

BackgroundIntracerebral hemorrhage (ICH) is a severe condition associated with high mortality and disability rates. Oxidative stress plays a critical role in the development of secondary brain injury (SBI) following ICH. Previous research has demonstrated that Annao Pingchong decoction (ANPCD) treatment for ICH has antioxidant effects, but the exact mechanism is not yet fully understood.ObjectiveThis study aimed to investigate the neuroprotective effects of ANPCD on oxidative stress and neuronal apoptosis after ICH by targeting the receptor for advanced glycation end products (RAGE)-NADPH oxidase (NOX) 2/4 signaling axis.MethodsThe research involved the creation of rat ICH models, the mNSS assay to assess neurological function, Nissl staining to evaluate neuronal damage, and biochemical assays to measure oxidative and antioxidant levels. The expression of RAGE-NOX2/4 axis proteins was analyzed using western blotting and immunofluorescence, while neuronal apoptosis was assessed with TUNEL staining. Furthermore, after performing quality control of drug-containing serum using UPLC-MS/MS, we employed an in vitro model of heme-induced injury in rat cortical neurons to investigate the neuroprotective mechanisms of ANPCD utilizing RAGE inhibitors.ResultsThe findings indicated that ANPCD improved neurological deficits, reduced neuronal damage, decreased ROS and MDA levels, and increased the activities enzymatic activities of SOD, CAT, GSH and GPX. Additionally, it suppressed the RAGE-NOX2/4 signaling axis and neuronal apoptosis.ConclusionANPCD exhibits neuroprotective effects by inhibiting the RAGE-NOX2/4 signaling axis, thereby alleviating neuronal oxidative stress and apoptosis following ICH.

Background: Proprotein convertase subtilisin kexin type 9 inhibitors (PCSK9i) lower LDL-cholesterol and slow atherosclerosis preventing cardiovascular events. While it is known that circulating PCSK9 enhances platelet activation (PA) and that PCSK9i reduce it, the underlying mechanism is not still clarified. Methods: In a multicenter before–after study in 80 heterozygous familial hypercholesterolemia (HeFH) patients on treatment with maximum tolerated statin dose ± ezetimibe, PA, soluble-NOX2-derived peptide (sNOX2-dp), and oxidized-LDL (ox-LDL) were measured before and after six months of PCSK9i treatment. In vitro study investigates the effects of plasma from HeFH patients before and after PCK9i on PA in washed platelets (wPLTs) from healthy subjects. Results: Compared to baseline, PCSK9i reduced the serum levels of LDL-c, ox-LDL, Thromboxane (Tx) B2, sNOX2-dp, and PCSK9 (p < 0.001). The decrease of TxB2 correlates with that of ox-LDL, while ox-LDL reduction correlated with PCSK9 and sNOX2-dp delta. In vitro study demonstrated that wPLTs resuspended in plasma from HeFH after PCSK9i treatment induced lower PA and sNOX2-dp release than those obtained using plasma before PCSK9i treatment. This reduction was vanished by adding ox-LDL. ox-LDL-induced PA was blunted by CD36, LOX1, and NOX2 inhibition. Conclusions: PCSK9i treatment reduces PA modulating NOX2 activity and in turn ox-LDL formation in HeFH patients.

Oxidative stress is a primary contributor to cerebral ischemia/reperfusion (CI/R) injury, and the use of antioxidants represents a crucial therapeutic strategy for managing CI/R injury. This study aims to explore the antioxidant effects of benzyl ferulate on CI/R injury and elucidate its underlying mechanisms. In vivo models of CI/R injury and hypoxia/reoxygenation (H/R) injury in SH‐SY5Y cells were established, followed by treatment with benzyl ferulate. The extent of oxidative stress was assessed through evaluations of neurobiological function, cerebral infarct volume, reactive oxygen species (ROS), apoptosis levels, etc. Results indicated that benzyl ferulate significantly downregulated the expression of NADPH oxidase 2 (NOX) 2/NOX4 while upregulating miRNAs (652/532/92b) in CI/R rats or SH‐SY5Y cells. It also reduced total NOX enzyme activity, enhanced superoxide dismutase (SOD) activity, decreased ROS and malondialdehyde (MDA) production, and inhibited cleaved caspase‐3 and Bax expression—ultimately leading to reduced cell apoptosis. Benzyl ferulate effectively mitigates oxidative stress injuries of middle cerebral artery occlusion (MCAO) rats or SH‐SY5Y cells subjected to H/R, and its mechanism appears to involve modulation of the miRNAs (652/532/92b)/NOX2/4 axis. This study first proved that benzyl ferulate is a promising antioxidant candidate for treating CI/R injury.

NADPH oxidase enzymes (NOXs) are a family of enzymes generating superoxide, which form reactive oxygen species. NOX2 activity is a causative agent for the progression of many diseases: neurodegenerative, cardiovascular, immune dysregulations, and even hereditary diseases and cancer. Administering antioxidants helps in inhibiting NOX2 activity; however, the development of selective inhibitors may provide greater improvement in the therapy of diseases. Here, an optimized synthesis of two most promising NOX2 inhibitors based on the 3-(indolin-6-yl)-4-(N-pyrazole-sulfonamide)-1H-pyrrolo [2,3-b]pyridine structure, namely, GSK2795039 and NCATS-SM7270, and an isomeric derivative of the same class, IMBIOC-1, is reported. The new modified procedures simplify the isolation, reduce byproduct formation, and improve the yields in 0.1–1 g scale preparations. Molecular modeling of the structures of NOX2 complexes with inhibitors validated their binding at the same site as NADPH, with IMBIOC-1 forming the largest number of intermolecular interactions with the NOX2 active site. Testing the effects of the compounds on amyloid beta-induced oxidative stress and toxicity in HMC3 microglial cells showed that all three inhibitors completely prevented the pathological amyloid-beta effect. At the same time, NCATS-SM7270 and IMBIOC-1 provided a stronger protective effect on microglial cell survival than GSK2795039, which allowed us to assert the potential of those compounds as neuroprotective agents.

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N‐acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2‐siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2‐associated oxidative stress in an AMPK‐dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.

Recent studies showed increased expression of complements in various neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. However, the mechanism regulating the expression of complements and their roles in the pathogenesis of neurodegeneration are unclear. We hypothesized that acute neuroinflammation increases the expression and activation of brain complements, which, in turn, participate in chronic neuroinflammation and progressive neurodegeneration. We initially focused on the complement component C3, because C3 can activate microglia by binding to C3 receptors and attaching to damaged neurons destined to be phagocytosed by microglia. We found that complement C3 is upregulated in lipopolysaccharide (LPS)-stimulated neuron/glial cultures. Mechanistic studies revealed that microglia-released proinflammatory factors initiated the enhanced expression of C3 in astroglia during acute neuroinflammation. On the other hand, the sustained C3 expression during chronic neuroinflammation requires releasing damage-associated molecule patterns (DAMPs) from damaged/degenerating brain cells. Our results suggested that DAMPs might act on microglial integrin receptor Mac1 to trigger the activation of NADPH oxidase (NOX2). Activated microglial NOX2 increases the production of extracellular reactive oxygen species (ROS), elevating the levels of intracellular ROS of astroglia and sustaining the astroglial C3 expression. This was supported by the findings showing reduced C3 expression and attenuated neurodegeneration in LPS-treated neuron/glial cultures prepared from mice deficient in Mac1 or NOX2. LPS-induced neurodegeneration and oxidative stress are significantly reduced in C3 KO neuron/glial cultures and mouse brains. Together, this study provides the first evidence demonstrating the role of C3 in regulating chronic neuroinflammation and in driving progressive neurodegeneration.

Cisplatin is a commonly used chemotherapeutic agent in cancer treatment. However, its high nephrotoxicity limits its therapeutic application and efficacy. Cisplatin induces nephrotoxicity mainly through oxidative stress and inflammation. Reactive oxygen species (ROS) in the kidneys mainly arise from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 2 (NOX2), which is highly upregulated during ischemia-reperfusion injury and diabetes mellitus. However, its role in cisplatin-induced acute kidney injury (AKI) remains unknown. A 8-10-week-old NOX2 gene-knockout and wild-type mice were injected with 25 mg/kg cisplatin intraperitoneally for experiments. We investigated the role of NOX2 in cisplatin-induced AKI and found that NOX2-mediated ROS production is a key inflammatory mediator of proximal tubular cell injury in cisplatin-induced AKI. NOX2 gene-knockout alleviated cisplatin-induced renal function decline, tubular injury score, kidney injury molecule-1(Kim-1) expression, and interleukin (IL)-6 and IL-1α levels with a reduction of ROS production. Moreover, in cisplatin-induced AKI, intercellular adhesion molecule 1 (ICAM-1) and the chemoattractant CXC ligand 1 (CXCL1) were highly expressed in association with neutrophil infiltration, which were all attenuated by deletion of NOX2. These data indicate that NOX2 aggravates cisplatin nephrotoxicity by promoting ROS-mediated tissue injury and neutrophil infiltration. Thus, appropriate targeting of NOX2/ROS pathway may minimize the risk of cisplatin-induced kidney injury in patients receiving cancer therapy.

Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here, we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2, and M1 and M4 helices of p22 are involved in the heterodimer formation. Dehydrogenase (DH) domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain, leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the DH in a productive docked conformation.

Neuroinflammation and oxidative stress are key drivers of various ocular diseases. Experimental hypoxia, modeled using cobalt chloride (CoCl2), induces hypoxia-inducible factor 1-alpha (HIF-1α) stabilization, mitochondrial dysfunction, and excessive reactive oxygen species (ROS) production, primarily via the NADPH oxidase 2 (Nox2)–voltage-gated proton channel Hv1 axis. Although Botulinum neurotoxin type A (BoNT/A) is classically recognized for SNAP-25 cleavage, recent studies suggest broader anti-inflammatory and neuroprotective effects. We evaluated BoNT/A in R28 retinal precursor cells and ex vivo retinal explants subjected to CoCl2-induced hypoxic stress. BoNT/A pretreatment attenuated CoCl2-induced upregulation of HIF-1α, Hv1, Nox2, NOD-like receptor protein 3 (NLRP3), COX2, and nuclear factor kappa B (NF-κB), while enhancing protective mediators including suppressor of cytokine signaling 3 (SOCS3), Growth Associated Protein 43 (Gap43), and Syntaxin12. Brn3a expression and retinal architecture were preserved, apoptotic cell death reduced, and glial activation suppressed. Moreover, BoNT/A decreased mitochondrial ROS accumulation, restored voltage-dependent anion channel 1 (VDAC1) distribution, and partially stabilized intracellular pH. These findings indicate that BoNT/A mitigates oxidative stress and inflammation in hypoxia-driven retinal injury, at least in part, via modulation of the Nox2–Hv1–ROS axis, and support its potential as a therapeutic candidate for ocular disorders associated with hypoxia and neuroinflammation.