ER-tethered RNA-binding protein controls NADPH oxidase translation for hydrogen peroxide homeostasis.

Hydrogen peroxide (H2O2) functions as a signaling molecule in diverse cellular processes. While cells have evolved the capability to detect and manage changes in H2O2 levels, the mechanisms regulating key H2O2-producing enzymes to maintain optimal levels, especially in pancreatic beta cells with notably weak antioxidative defense, remain unclear. We found that the protein EI24 responds to changes in H2O2 concentration and regulates the production of H2O2 by controlling the translation of NOX4, an enzyme that is constitutively active, achieved by recruiting an RNA-binding protein, RTRAF, to the 3'-UTR of Nox4. Depleting EI24 results in RTRAF relocating into the nucleus, releasing the brake on NOX4 translation. The excessive production of H2O2 by liberated NOX4 further suppresses the translation of the key transcription factor MafA, ultimately preventing its binding to the Ins2 gene promoter and subsequent transcription of insulin. Treatment with a specific NOX4 inhibitor or the antioxidant NAC reversed these effects and alleviated the diabetic symptoms in beta-cell specific Ei24-KO mice. This study revealed a new mechanism through which cells regulate oxidative stress at the translational level, involving an ER-tethered RNA-binding protein that controls the expression of the key H2O2-producing enzyme NOX4.

Myricetin Induces Ferroptosis and Inhibits Gastric Cancer Progression by Targeting NOX4.

Ferroptosis holds great potential as a therapeutic approach for gastric cancer (GC), a prevalent and deadly malignant tumor associated with high rates of incidence and mortality. Myricetin, well-known for its multifaceted biomedical attributes, particularly its anticancer properties, has yet to be thoroughly investigated regarding its involvement in ferroptosis. The aim of this research was to elucidate the impact of myricetin on ferroptosis in GC progression. The present study observed that myricetin could trigger ferroptosis in GC cells by enhancing malondialdehyde production and Fe2+ accumulation while suppressing glutathione levels. Mechanistically, myricetin directly interacted with NADPH oxidase 4 (NOX4), influencing its stability by inhibiting its ubiquitin degradation. Moreover, myricetin regulated the inhibition of ferroptosis induced by Helicobacter pylori cytotoxin-associated gene A (CagA) through the NOX4/NRF2/GPX4 pathway. In vivo experiments demonstrated that myricetin treatment significantly inhibited the growth of subcutaneous tumors in BALB/c nude mice. It was accompanied by increased NOX4 expression in tumor tissue and suppression of the NRF2/GPX4 antioxidant pathway. Therefore, this research underscores myricetin as a novel inducer of ferroptosis in GC cells through its interaction with NOX4. It is a promising candidate for GC treatment.

Gene Expression and Prognostic Value of NADPH Oxidase Enzymes in Breast Cancer.

NADPH oxidase enzymes (NOX) are involved in all stages of carcinogenesis, but their expression levels and prognostic value in breast cancer (BC) remain unclear. Thus, we aimed to assess the expression and prognostic value of NOX enzymes in BC samples using online databases. For this, mRNA expression from 290 normal breast tissue samples and 1904 BC samples obtained from studies on cBioPortal, Kaplan-Meier Plotter, and The Human Protein Atlas were analyzed. We found higher levels of NOX2, NOX4, and Dual oxidase 1 (DUOX1) in normal breast tissue. NOX1, NOX2, and NOX4 exhibited higher expression in BC, except for the basal subtype, where NOX4 expression was lower. DUOX1 mRNA levels were lower in all BC subtypes. NOX2, NOX4, and NOX5 mRNA levels increased with tumor progression stages, while NOX1 and DUOX1 expression decreased in more advanced stages. Moreover, patients with low expression of NOX1, NOX4, and DUOX1 had lower survival rates than those with high expression of these enzymes. In conclusion, our data suggest an overexpression of NOX enzymes in breast cancer, with certain isoforms showing a positive correlation with tumor progression.

Reactive oxygen species augment contractile responses of saphenous artery in 10-15-day-old but not adult rats: Substantial role of NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases (NOX, a key source of ROS in vascular cells) are involved in the regulation of vascular tone, but this has been explored mainly for adult organisms. Importantly, the mechanisms of vascular tone regulation differ significantly in early postnatal ontogenesis and adulthood, while the vasomotor role of ROS in immature systemic arteries is poorly understood. We tested the hypothesis that the functional contribution of NADPH oxidase-derived ROS to the regulation of peripheral arterial tone is higher in the early postnatal period than in adulthood. We studied saphenous arteries from 10- to 15-day-old ("young") and 3- to 4-month-old ("adult") male rats using lucigenin-enhanced chemiluminescence, quantitative PCR, Western blotting, and isometric myography. We demonstrated that both basal and NADPH-stimulated superoxide anion radical (O2•-) production was significantly higher in the arteries from young in comparison to adult rats. Importantly, pan-inhibitor of NADPH oxidase VAS2870 (10 µM) reduced NADPH-induced O2•- production in arteries of young rats. Saphenous arteries of both young and adult rats demonstrated high levels of Nox2 and Nox4 mRNAs, while Nox1 and Nox3 mRNAs were not detected. The protein contents of NOX2 and NOX4 were significantly higher in arterial tissue of young compared to adult animals. Moreover, VAS2870 (10 µM) had no effect on methoxamine-induced contractile responses of adult arteries but decreased them significantly in young arteries; such effect of VAS2870 persisted after removal of the endothelium. Finally, NOX2 inhibitor GSK2795039 (10 µM), but not NOX1/4 inhibitor GKT137831 (10 µM) weakened methoxamine-induced contractile responses of arteries from young rats. Thus, ROS produced by NOX2 have a pronounced contractile influence in saphenous artery smooth muscle cells of young, but not adult rats, which is associated with the increased vascular content of NOX2 protein at this age.

CircSTRBP contributes to H2O2-induced lens epithelium cell dysfunction through increasing NOX4 mRNA stability by recruiting IGF2BP1.

Previous studies have shown that the development of age-related cataract (ARC) is involved in lens epithelium dysfunction, which is associated with abnormally expressed circular RNAs (circRNAs). The current work aims to probe the role of circSTRBP (hsa_circ_0088,427) in hydrogen peroxide (H2O2)-induced lens epitheliums. Lens epithelium tissues were harvested from ARC or normal subjects (n = 23). CircSTRBP, spermatid perinuclear RNA binding protein (STRBP), and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation, cycle progression, and apoptosis were assessed using 5-ethynyl-2'-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and flow cytometry assays. Caspase 3 activity, reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidases (GSH-PX) levels were detected using corresponding kits. NOX4 protein level was determined using Western blot. The interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and circSTRBP or NOX4 was assessed through RNA immunoprecipitation (RIP). CircSTRBP and NOX4 abundances were increased in lens epithelium samples from ARC patients and H2O2-treated SRA01/04 cells. CircSTRBP knockdown might abolish H2O2-triggered SRA01/04 cell proliferation repression and apoptosis and oxidative stress promotion. In mechanism, circSTRBP is bound with IGF2BP1 and improves the stability and expression of NOX4 mRNA in SRA01/04 cells. CircSTRBP facilitated H2O2-induced SRA01/04 cell apoptosis and oxidative stress through by enhancing NOX4 mRNA stability via recruiting IGF2BP1, providing novel insights for ARC progression and treatment.

Rare coding variants in NOX4 link high ROS levels to psoriatic arthritis mutilans.

Psoriatic arthritis mutilans (PAM) is the rarest and most severe form of psoriatic arthritis, characterized by erosions of the small joints and osteolysis leading to joint disruption. Despite its severity, the underlying mechanisms are unknown, and no susceptibility genes have hitherto been identified. We aimed to investigate the genetic basis of PAM by performing massive parallel sequencing in sixty-one patients from the PAM Nordic cohort. We found rare variants in the NADPH oxidase 4 (NOX4) in four patients. In silico predictions show that the identified variants are potentially damaging. NOXs are the only enzymes producing reactive oxygen species (ROS). NOX4 is specifically involved in the differentiation of osteoclasts, the cells implicated in bone resorption. Functional follow-up studies using cell culture, zebrafish models, and measurement of ROS in patients uncovered that these NOX4 variants increase ROS levels both in vitro and in vivo. We propose NOX4 as the first candidate susceptibility gene for PAM. Our study links high levels of ROS caused by NOX4 variants to the development of PAM, offering a potential therapeutic target.

Cannabidiol regulates the activation of hepatic stellate cells by modulating the NOX4 and NF-κB pathways.

Cannabidiol (CBD) is an extract of natural cannabinoids that has therapeutic implications for a variety of ailments, such as neurological diseases, cardiomyopathy, and diabetes, due to its strong anti-inflammatory and oxidative stress properties. Our purpose was to reveal the possible underlying mechanisms and effect of CBD on the glucose oxidase (GO)-induced activation of HSC-T6 and LX-2 cells. The results showed that CBD effectively inhibited the proliferation and activation of HSC-T6 and LX-2 cells, and reduced the production of profibrotic factors to different degrees. CBD disrupted the NOX4 signalling pathway in activated HSC-T6 and LX-2 cells, reduced ROS and MDA levels, and increased SOD and GSH levels, thereby stabilizing the oxidative imbalance. CBD significantly inhibited the phosphorylation and degradation of NF-κB and IκBα, and decreased the release of TNF-α, IL-1ß and IL-6. Moreover, CBD and an NF-κB-specific inhibitor (CAPE) effectively inhibited the expression of α-SMA, COL I, TNF-α and IL-1ß to promote collagen metabolism and inhibit the inflammatory response. Overall, CBD inhibited HSCs activation through a and the mechanism involving the inhibition of NOX4 and NF-κB-dependent ROS regulation, thereby reducing inflammation and ameliorating oxidative imbalances.

Development and validation of a new diagnostic prediction model of ENHO and NOX4 for early diagnosis of systemic sclerosis.

Objective: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis. The challenge of early diagnosis, along with the lack of effective treatments for fibrosis, contribute to poor therapeutic outcomes and high mortality of SSc. Therefore, there is an urgent need to identify suitable biomarkers for early diagnosis of SSc. Methods: Three skin gene expression datasets of SSc patients and healthy controls were downloaded from Gene Expression Omnibus (GEO) database (GSE130955, GSE58095, and GSE181549). GSE130955 (48 early diffuse cutaneous SSc and 33 controls) were utilized to screen differentially expressed genes (DEGs) between SSc and normal skin samples. Least absolute shrinkage and selection operator (LASSO) regression and support vector machine recursive feature elimination (SVM-RFE) were performed to identify diagnostic genes and construct a diagnostic prediction model. The results were further validated in GSE58095 (61 SSc and 36 controls) and GSE181549 (113 SSc and 44 controls) datasets. Receiver operating characteristic (ROC) curves were applied for assessing the level of diagnostic ability. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to verify the diagnostic genes in skin tissues of out cohort (10 SSc and 5 controls). Immune infiltration analysis were performed using CIBERSORT algorithm. Results: A total of 200 DEGs were identified between SSc and normal skin samples. Functional enrichment analysis revealed that these DEGs may be involved in the pathogenesis of SSc, such as extracellular matrix remodeling, cell-cell interactions, and metabolism. Subsequently, two critical genes (ENHO and NOX4) were identified by LASSO and SVM-RFE. ENHO was found down-regulated while NOX4 was up-regulated in skin of SSc patients and their expression levels were validated by above three datasets and our cohort. Notably, these differential expressions were more pronounced in patients with diffuse cutaneous SSc than in those with limited cutaneous SSc. Next, we developed a novel diagnostic model for SSc using ENHO and NOX4, which demonstrated strong predictive power in above three cohorts and in our own cohort. Furthermore, immune infiltration analysis revealed dysregulated levels of various immune cell subtypes within early SSc skin specimens, and a negative correlation was observed between the levels of ENHO and Macrophages M1 and M2, while a positive correlation was observed between the levels of NOX4 and Macrophages M1 and M2. Conclusion: This study identified ENHO and NOX4 as novel biomarkers that can be serve as a diagnostic prediction model for early detection of SSc and play a potential role in the pathogenesis of the disease.

Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis.

Tumor-associated macrophages (TAMs) are often associated with chemoresistance and resultant poor clinical outcome in solid tumors. Here, we demonstrated that TAMs-released chemokine-C-C motif chemokine 22 (CCL22) in esophageal squamous cell carcinoma (ESCC) stroma was tightly correlated with the chemoresistance of ESCC patients. TAMs-secreted CCL22 was able to block the growth inhibitory and apoptosis-promoting effects of cisplatin on ESCC cells. Mechanistically, CCL22 stimulated intratumoral diacylglycerol kinase α (DGKα) to produce phosphatidic acid (PA), which suppressed the activity of NADPH oxidase 4 (NOX4) and then blocked the overproduction of intratumoral reactive species oxygen (ROS) induced by cisplatin. CCL22 activated DGKα/nuclear factor-κB (NF-κB) axis to upregulate the level of several members of ATP binding cassette (ABC) transporter superfamily, including ABC sub-family G member 4 (ABCG4), ABC sub-family A member 3 (ABCA3), and ABC sub-family A member 5 (ABCA5), to lower the intratumoral concentration of cisplatin. Consequently, these processes induced the cisplatin resistance in ESCC cells. In xenografted models, targeting DGKα with 5'-cholesterol-conjugated small-interfering (si) RNA enhanced the chemosensitivity of cisplatin in ESCC treatment, especially in the context of TAMs. Our data establish the correlation between the TAMs-induced intratumoral metabolic product/ROS axis and chemotherapy efficacy in ESCC treatment and reveal relevant molecular mechanisms.

DRD4 alleviates acute kidney injury by suppressing ISG15/NOX4 axis-associated oxidative stress.

Acute kidney injury (AKI) is a life-threatening health condition associated with increasing morbidity and mortality. Despite extensive research on the mechanisms underlying AKI, effective clinical tools for prediction and treatment remain scarce. Oxidative stress and mitochondrial damage play a critical role in AKI and dopamine D4 receptor (DRD4) has been confirmed to be associated with oxidative stress. In this study, we hypothesized that DRD4 could attenuate AKI through its antioxidative and antiapoptotic effects. In vivo, DRD4 was remarkably decreased in the kidneys of mice subjected to ischemia/reperfusion injury (IRI) or cisplatin treatment. Notably, DRD4 significantly attenuated nephrotoxicity by suppressing oxidative stress and enhancing mitochondrial bioenergetics through the downregulation of reactive oxygen species (ROS) generation and NADPH oxidase 4 (NOX4) expression. In vitro, DRD4 demonstrated the ability to ameliorate oxidative stress-induced apoptosis in HK-2 cells subjected to hypoxia/reoxygenation- or cisplatin treatment. Transcriptome sequencing revealed that, mechanistically, DRD4 reduced the expression of its downstream target, interferon-stimulated gene 15 (ISG15), suppressing NOX4 ISGylation, enhancing the ubiquitination of NOX4, leading to its degradation, and ultimately counteracting oxidative stress-induced AKI. Altogether, these findings underscore the significance of DRD4 in AKI and elucidate DRD4 as a potential protectant against IRI or cisplatin-induced nephrotoxicity.

Intermittent short-duration reoxygenation relieves high-altitude pulmonary hypertension via NOX4/H2O2/PPAR-γ axis.

High-altitude pulmonary hypertension (HAPH) is a severe and progressive disease that can lead to right heart failure. Intermittent short-duration reoxygenation at high altitude is effective in alleviating HAPH; however, the underlying mechanisms are unclear. In the present study, a simulated 5,000-m hypoxia rat model and hypoxic cultured pulmonary artery smooth muscle cells (PASMCs) were used to evaluate the effect and mechanisms of intermittent short-duration reoxygenation. The results showed that intermittent 3-h/per day reoxygenation (I3) effectively attenuated chronic hypoxia-induced pulmonary hypertension and reduced the content of H2O2 and the expression of NADPH oxidase 4 (NOX4) in lung tissues. In combination with I3, while the NOX inhibitor apocynin did not further alleviate HAPH, the mitochondrial antioxidant MitoQ did. Furthermore, in PASMCs, I3 attenuated hypoxia-induced PASMCs proliferation and reversed the activated HIF-1α/NOX4/PPAR-γ axis under hypoxia. Targeting this axis offset the protective effect of I3 on hypoxia-induced PASMCs proliferation. The present study is novel in revealing a new mechanism for preventing HAPH and provides insights into the optimization of intermittent short-duration reoxygenation.

C/EBPα aggravates renal fibrosis in CKD through the NOX4-ROS-apoptosis pathway in tubular epithelial cells.

BACKGROUND: Chronic kidney disease (CKD) is a prevalent renal disorder with various risk factors. Emerging evidence indicates that the transcriptional factor CCAAT/enhancer binding protein alpha (C/EBPα) may be associated with renal fibrosis. However, the precise role of C/EBPα in CKD progression remains unexplored. METHODS: We investigated the involvement of C/EBPα in CKD using two distinct mouse models induced by folic acid (FA) and unilateral ureteral obstruction (UUO). Additionally, we used RNA sequencing and KEGG analysis to identify potential downstream pathways governed by C/EBPα. FINDINGS: Cebpa knockout significantly shielded mice from renal fibrosis and reduced reactive oxygen species (ROS) levels in both the FA and UUO models. Primary tubular epithelial cells (PTECs) lacking Cebpa exhibited reduced apoptosis and ROS accumulation following treatment with TGF-ß. RNA sequencing analysis suggested that apoptosis is among the primary pathways regulated by C/EBPα, and identified NADPH oxidoreductase 4 (NOX4) as a key protein upregulated upon C/EBPα induction (ICCB280). Treatment with l-Theanine, a potential NOX4 inhibitor, mitigated renal fibrosis and inflammation in both the FA and UUO mouse models. INTERPRETATION: Our study unveils a role for C/EBPα in suppressing renal fibrosis, mitigating ROS accumulation, and reducing cell apoptosis. Furthermore, we investigate whether these protective effects are mediated by C/EBPα's regulation of NOX4 expression. These findings present a promising therapeutic target for modulating ROS and apoptosis in renal tubular cells, potentially offering an approach to treating CKD and other fibrotic diseases.

Targeting benign prostate hyperplasia treatments: AR/TGF-ß/NOX4 inhibition by apocynin suppresses inflammation and proliferation.

INTRODUCTION: Apocynin (Apo), an NADPH oxidase (NOX) inhibitor, has been widely used to treat various inflammatory diseases. However, the therapeutic effects of Apo on benign prostatic hyperplasia (BPH), a multifactorial disease associated with chronic inflammation and hormone imbalance, remain unknown. OBJECTIVES: The link between androgen signaling, reactive oxygen species (ROS), and prostate cell proliferation may contribute to the pathogenesis of BPH; therefore, the aim of this study was to identify the specific signaling pathway involved and to demonstrate whether the anti-oxidant Apo plays a role in the prevention and treatment of BPH. METHODS: Ingenuity pathway analysis and si-RNA transfection were conducted to demonstrate the androgen receptor (AR) and NOX4 linkage in BPH. Pathological markers of BPH were measured by H&E staining, immunoblotting, ELISA, qRT-PCR, and immunofluorescence to examine the effect of Apo. Rats stimulated with testosterone and BPH-1 cells were used as BPH models. RESULTS: AR and NOX4 network-mediated oxidative stress was upregulated in the BPH model. Next, we examined the effects of Apo on oxidative stress and chronic prostatic inflammation in BPH mouse models. In a testosterone-induced BPH rat model, Apo alleviated pathological prostate enlargement and suppressed androgen/AR signaling. Apo suppressed the upregulation of proinflammatory markers and promoted the expression of anti-oxidant factors. Furthermore, Apo regulated the TGF-ß/Glut9/activin pathway and macrophage programming. In BPH-1 cells, Apo suppressed AR-mediated proliferation and upregulation of TGFB and NOX4 expression by alleviating oxidative stress. Apo activated anti-oxidant and anti-inflammatory systems and regulated macrophage polarization in BPH-1 cells. AR knockdown partially abolished the beneficial effects of Apo in prostate cells, indicating AR-dependent effects of Apo. CONCLUSION: In contrast with existing BPH therapies, Apo may provide a new application for prostatic disease treatment, especially for BPH, by targeting the AR/TGF-ß/NOX4 signaling pathway.

Ang-(1-7)/Mas axis ameliorates bleomycin-induced pulmonary fibrosis in mice via restoration of Nox4-Nrf2 redox homeostasis.

Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease characterized by diffuse alveolar inflammation, fibroblast differentiation, and the excessive deposition of extracellular matrix. During the progression of PF, redox imbalance caused by excessive reactive oxygen species (ROS) production can result in further destruction of lung tissue. At present, data on the role of NADPH oxidase-4 (Nox4)-nuclear factor erythroid 2-related factor 2 (Nrf2) redox imbalance in PF are limited. The angiotensin (1-7) [Ang-(1-7)]/Mas axis is a protective axis in the renin-angiotensin system (RAS) that exerts antifibrotic effects. Therefore, this study aimed to investigate the role of the Ang-(1-7)/Mas axis in PF and to explore its mechanism in depth. The results revealed that the Ang-(1-7)/Mas axis inhibited TGF-ß1-induced lung fibroblast differentiation, inflammation and fibrosis in bleomycin (BLM)-treated lung tissue. A mechanistic study suggested that the Ang-(1-7)/Mas axis may restore Nox4-Nrf2 redox homeostasis by upregulating the level of p62, reducing oxidative stress and the inflammatory response and thus delaying the progression of lung fibrosis. This study provides a theoretical basis for exploring the mechanisms of PF and therapeutic targets for PF.

NADPH oxidase 4-mediating oxidative stress contributes to endometriosis.

NADPH oxidase 4 (NOX4) plays an important role in the regulation of oxidative stress, which is associated with endometriosis. This study aims to investigate the effects of NOX4 in endometriosis and its molecular mechanisms. Clinical specimens were collected, and human endometrial stromal cells (HESCs) were isolated. The knockdown of NOX4 cell lines was established on the HESCs and induced by peritoneal fluid. The levels of NOX4 were determined by using immunohistochemistry (IHC) staining, western blotting, and qPCR, respectively. The levels of oxidative stress markers were determined by using western blotting and ELISAs, respectively. The correlation of NOX4 and oxidative stress markers was analyzed by the Pearson correlation coefficient. The levels of NOX4 were dramatically elevated in the ectopic endometrium. Besides, oxidative stress biomarkers were also dysregulated in the ectopic endometrium as compared to the normal endometrium. Pearson's correlation coefficient analysis revealed a relationship between NOX4 and oxidative stress biomarkers in the ectopic endometrium. NOX4 modulated the expressions of oxidative stress markers in endometrial stromal cells stimulated by the peritoneal fluid from endometriosis. The effects of NOX4 on endometriosis are in part by its regulatory effects against oxidative stress.

Hypoxia stabilizes the H2 O2 -producing oxidase Nox4 in cardiomyocytes via suppressing autophagy-related lysosomal degradation.

The hydrogen peroxide (H2 O2 )-producing NADPH oxidase Nox4, forming a heterodimer with p22phox , is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H2 O2 production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4-catalyzed H2 O2 production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22phox . Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3-methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy-related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4-catalyzed H2 O2 production.

Advanced glycated end-products inhibit dilation through constitutive endothelial RAGE and Nox1/4 in rat isolated skeletal muscle arteries.

OBJECTIVE: This study investigated the actions of advanced glycated end-products (AGE), their receptors (RAGE), and NAD(P)H oxidase (Nox) subtypes 1, 2, and 4 on mechanisms of endothelium-dependent dilation of the rat cremaster muscle artery (CMA). METHODS: Immunofluorescence studies were used to examine expression of RAGE in rat arteries. ROS accumulation was measured using luminescence and fluorescence assays. Functional studies were performed using pressure myography. RESULTS: High levels of RAGE expression were shown in the endothelial cells of the CMA, compared with low endothelial expression in middle cerebral and mesenteric arteries and the aorta. Exogenous AGE (in vitro glycated bovine serum albumin) stimulated H2O2 accumulation in CMA, which was prevented by the RAGE antagonist FPS-ZM1, the NAD(P)H oxidase (Nox) inhibitor apocynin and inhibited by the Nox1/4 inhibitor setanaxib, but not the Nox2 inhibitor GSK2795039. In functional studies, AGE inhibited vasodilation of CMA stimulated by acetylcholine, sodium nitroprusside, and the BKCa activator NS1619, but not adenosine-induced dilation. FPS-ZM1, apocynin, and setanaxib prevented the inhibitory effects of AGE on responses to acetylcholine and NS-1619. CONCLUSION: These observations suggest RAGE are constitutively expressed in the endothelium of the rat CMA and may be activated by AGE to stimulate Nox1/4 and ROS formation with resulting inhibition of NO and BKCa-mediated endothelium-dependent dilation.

Neutrophil membrane-engineered Panax ginseng root-derived exosomes loaded miRNA 182-5p targets NOX4/Drp-1/NLRP3 signal pathway to alleviate acute lung injury in sepsis: experimental studies.

BACKGROUND: The purpose of this study was to prepare neutrophil membrane-engineered Panax ginseng root-derived exosomes (N-exo) and investigate the effects of N-exo microRNA (miRNA) 182-5p (N-exo-miRNA 182-5p) on acute lung injury (ALI) in sepsis. METHODS: Panax ginseng root-derived exosomes were separated by differential centrifugation. Neutrophil membrane engineering was performed on exo to obtain N-exo. miRNA182-5p was transmitted into N-exo by electroporation technology to obtain N-exo-miRNA 182-5p. LPS was used to establish an in-vivo and in-vitro model of ALI of sepsis to evaluate the anti-inflammatory effect of N-exo-miRNA 182-5p. RESULTS: The results of transmission electron microscope showed that exo was a double-layer membrane structure like a saucer. Nanoparticle size analysis showed that the average particle size of exo was 129.7 nm. Further, compared with exo, the level of miRNA182-5p was significantly increased in N-exo. The experimental results showed that N-exo-miRNA 182-5p significantly improved ALI via target regulation of NOX4/Drp-1/NLRP3 signal pathway in vivo and in vitro . CONCLUSION: In conclusion, this study prepared a novel engineered exosome (N-exo and N-exo-miRNA 182-5p significantly improved ALI in sepsis via target regulation of NOX4/Drp-1/NLRP3 signal pathway, providing new ideas and methods for treatment of ALI in sepsis.

NOX4 alleviates breast cancer cell aggressiveness by co-ordinating mitochondrial turnover through PGC1α/Drp1 axis.

Triple Negative Breast Cancer (TNBC) is a highly aggressive form of breast cancer, with few treatment options. This study investigates the complex molecular mechanism by which NADPH oxidase 4 (NOX4), a major ROS producer in mitochondria, affects the aggressiveness of luminal and triple-negative breast cancer cells (TNBCs). We found that NOX4 expression was differentially regulated in luminal and TNBC cells, with a positive correlation to their epithelial characteristics. Time dependent analysis revealed that TNBCs exhibits higher steady-state ROS levels than luminal cells, but NOX4 silencing increased ROS levels in luminal breast cancer cells and enhanced their ability to migrate and invade. In contrast, NOX4 over expression in TNBCs had the opposite effect. The mouse tail-vein experiment showed that the group injected with NOX4 silenced luminal cells had a higher number of lung metastases compared to the control group. Mechanistically, NOX4 enhanced PGC1α dependent mitochondrial biogenesis and attenuated Drp1-mediated mitochondrial fission in luminal breast cancer cells, leading to an increased mitochondrial mass and elongated mitochondrial morphology. Interestingly, NOX4 silencing increased mitochondrial ROS (mtROS) levels without affecting mitochondrial (Δψm) and cellular integrity. Inhibition of Drp1-dependent fission with Mdivi1 reversed the effect of NOX4-dependent mitochondrial biogenesis, dynamics, and migration of breast cancer cells. Our findings suggest that NOX4 expression diminishes from luminal to a triple negative state, accompanied by elevated ROS levels, which may modulate mitochondrial turnover to attain an aggressive phenotype. The study provides potential insights for targeted therapies for TNBCs.

NADPH oxidase 4 in mouse ß cells participates in inflammation on chronic nutrient overload.

OBJECTIVE: By exposing mice carrying a deletion of NADPH oxidase isoform 4, NOX4, specifically in pancreatic ß cells (ßNOX4-/-) to nutrient excess stimulated by a high-fat diet (HFD), this study aimed to elucidate the role of ß-cell redox status in the development of meta-inflammation within the diabetic phenotype. METHODS: The authors performed basic phenotyping of ßNOX4-/- mice on HFD involving insulin and glycemic analyses, histochemistry of adipocytes, indirect calorimetry, and cytokine analyses. To characterize local inflammation, the study used caspase-1 activity assay, interleukin-1ß immunochemistry, and real-time polymerase chain reaction during coculturing of ß cells with macrophages. RESULTS: The phenotype of ßNOX4-/- mice on HFD was not associated with hyperinsulinemia and hyperglycemia but showed accumulation of excessive lipids in epididymal fat and ß cells. Surprisingly, mice showed significantly reduced systemic inflammation. Decreased interleukin-1ß protein levels and downregulated NLRP3-inflammasome activity were observed on chronic glucose overload in ßNOX4-/- isolated islets and NOX4-silenced INS1-E cells resulting in attenuated proinflammatory polarization of macrophages/monocytes in vitro and in situ and reduced local islet inflammation. CONCLUSIONS: Experimental evidence suggests that NOX4 pro-oxidant activity in ß cells is involved in NLRP3-inflammasome activation during chronic nutrient overload and participates in local inflammatory signaling and perhaps toward peripheral tissues, contributing to a diabetic inflammatory phenotype.