miR-146a Decreases Inflammation and ROS Production in Aged Dermal Fibroblasts.

Aging is associated with a decline in the functionality of various cell types, including dermal fibroblasts, which play a crucial role in maintaining skin homeostasis and wound healing. Chronic inflammation and increased reactive oxygen species (ROS) production are hallmark features of aging, contributing to impaired wound healing. MicroRNA-146a (miR-146a) has been implicated as a critical regulator of inflammation and oxidative stress in different cell types, yet its role in aged dermal fibroblasts and its potential relevance to wound healing remains poorly understood. We hypothesize that miR-146a is differentially expressed in aged dermal fibroblasts and that overexpression of miR-146a will decrease aging-induced inflammatory responses and ROS production. Primary dermal fibroblasts were isolated from the skin of 17-week-old (young) and 88-week-old (aged) mice. Overexpression of miR-146a was achieved through miR-146a mimic transfection. ROS were detected using a reliable fluorogenic marker, 2,7-dichlorofluorescin diacetate. Real-time PCR was used to quantify relative gene expression. Our investigation revealed a significant reduction in miR-146a expression in aged dermal fibroblasts compared to their younger counterparts. Moreover, aged dermal fibroblasts exhibited heightened levels of inflammatory responses and increased ROS production. Importantly, the overexpression of miR-146a through miR-146a mimic transfection led to a substantial reduction in inflammatory responses through modulation of the NF-kB pathway in aged dermal fibroblasts. Additionally, the overexpression of miR-146a led to a substantial decrease in ROS production, achieved through the downregulation of NOX4 expression in aged dermal fibroblasts. These findings underscore the pivotal role of miR-146a in mitigating both inflammatory responses and ROS production in aged dermal fibroblasts, highlighting its potential as a therapeutic target for addressing age-related skin wound healing.

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.

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.

HOXD10 attenuates renal fibrosis by inhibiting NOX4-induced ferroptosis.

In chronic kidney disease (CKD), renal fibrosis is an unavoidable result of various manifestations. However, its pathogenesis is not yet fully understood. Here, we revealed the novel role of Homeobox D10 (HOXD10) in CKD-related fibrosis. HOXD10 expression was downregulated in CKD-related in vitro and in vivo fibrosis models. UUO model mice were administered adeno-associated virus (AAV) containing HOXD10, and HOXD10 overexpression plasmids were introduced into human proximal tubular epithelial cells induced by TGF-ß1. The levels of iron, reactive oxygen species (ROS), lipid ROS, the oxidized glutathione/total glutathione (GSSG/GSH) ratio, malonaldehyde (MDA), and superoxide dismutase (SOD) were determined using respective assay kits. Treatment with AAV-HOXD10 significantly attenuated fibrosis and renal dysfunction in UUO model mice by inhibiting NOX4 transcription, ferroptosis pathway activation, and oxidative stress. High levels of NOX4 transcription, ferroptosis pathway activation and profibrotic gene expression induced by TGF-ß1/erastin (a ferroptosis agonist) were abrogated by HOXD10 overexpression in HK-2 cells. Moreover, bisulfite sequencing PCR result determined that HOXD10 showed a hypermethylated level in TGF-ß1-treated HK-2 cells. The binding of HOXD10 to the NOX4 promoter was confirmed by chromatin immunoprecipitation (ChIP) analysis and dual-luciferase reporter assays. Targeting HOXD10 may represent an innovative therapeutic strategy for fibrosis treatment in CKD.

Increased PHLPP1 expression through ERK-4E-BP1 signaling axis drives nicotine induced oxidative stress related damage of cardiomyocytes.

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.

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.

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.

MYH7 R453C induced cardiac remodelling via activating TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS/NF-κB signalling pathways.

Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here, we generated transgenic MYH7 R453C and MYH6 R453C piglets and found both developed typical cardiac hypertrophy. Unexpectedly, we found serious fibrosis and cardiomyocyte loss in the ventricular of MYH7 R453C, not MYH6 R453C piglets, similar to HCM patients. Then, RNA-seq analysis and western blotting identified the activation of ERK1/2 and PI3K-Akt pathways in MYH7 R453C. Moreover, we observed an increased expression of fetal genes and an excess of reactive oxygen species (ROS) in MYH7 R453C piglet models, which was produced by Nox4 and subsequently induced inflammatory response. Additionally, the phosphorylation levels of Smad2/3, ERK1/2 and NF-kB p65 proteins were elevated in cardiomyocytes with the MYH7 R453C mutation. Furthermore, epigallocatechin gallate, a natural bioactive compound, could be used as a drug to reduce cell death by adjusting significant downregulation of the protein expression of Bax and upregulated Bcl-2 levels in the H9C2 models with MYH7 R453C mutation. In conclusion, our study illustrated that TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS pathways have synergistic effects on cardiac remodelling and inflammation in MYH7 R453C mutation.

Particulate Matter-Induced Neurotoxicity: Unveiling the Role of NOX4-Mediated ROS Production and Mitochondrial Dysfunction in Neuronal Apoptosis.

Urban air pollution, a significant environmental hazard, is linked to adverse health outcomes and increased mortality across various diseases. This study investigates the neurotoxic effects of particulate matter (PM), specifically PM2.5 and PM10, by examining their role in inducing oxidative stress and subsequent neuronal cell death. We highlight the novel finding that PM increases mitochondrial ROS production via stimulating NOX4 activity, not through its expression level in Neuro-2A cells. Additionally, PMs provoke ROS production via increasing the expression and activity of NOX2 in SH-SY5Y human neuroblastoma cells, implying differential regulation of NOX proteins. This increase in mitochondrial ROS triggers the opening of the mitochondrial permeability transition pore (mPTP), leading to apoptosis through key mediators, including caspase3, BAX, and Bcl2. Notably, the voltage-dependent anion-selective channel 1 (VDAC1) increases at 1 µg/mL of PM2.5, while PM10 triggers an increase from 10 µg/mL. At the same concentration (100 µg/mL), PM2.5 causes 1.4 times higher ROS production and 2.4 times higher NOX4 activity than PM10. The cytotoxic effects induced by PMs were alleviated by NOX inhibitors GKT137831 and Apocynin. In SH-SY5Y cells, both PM types increase ROS and NOX2 levels, leading to cell death, which Apocynin rescues. Variability in NADPH oxidase sources underscores the complexity of PM-induced neurotoxicity. Our findings highlight NOX4-driven ROS and mitochondrial dysfunction, suggesting a potential therapeutic approach for mitigating PM-induced neurotoxicity.

ADAM10 as a major activator of reactive oxygen species production and klotho shedding in podocytes under diabetic conditions.

Early stages of diabetes are characterized by elevations of insulin and glucose concentrations. Both factors stimulate reactive oxygen species (ROS) production, leading to impairments in podocyte function and disruption of the glomerular filtration barrier. Podocytes were recently shown to be an important source of αKlotho (αKL) expression. Low blood Klotho concentrations are also associated with an increase in albuminuria, especially in patients with diabetes. We investigated whether ADAM10, which is known to cleave αKL, is activated in glomeruli and podocytes under diabetic conditions and the potential mechanisms by which ADAM10 mediates ROS production and disturbances of the glomerular filtration barrier. In cultured human podocytes, high glucose increased ADAM10 expression, shedding, and activity, NADPH oxidase activity, ROS production, and albumin permeability. These effects of glucose were inhibited when cells were pretreated with an ADAM10 inhibitor or transfected with short-hairpin ADAM10 (shADAM10) or after the addition soluble Klotho. We also observed increases in ADAM10 activity, NOX4 expression, NADPH oxidase activity, and ROS production in αKL-depleted podocytes. This was accompanied by an increase in albumin permeability in shKL-expressing podocytes. The protein expression and activity of ADAM10 also increased in isolated glomeruli and urine samples from diabetic rats. Altogether, these results reveal a new mechanism by which hyperglycemia in diabetes increases albumin permeability through ADAM10 activation and an increase in oxidative stress via NOX4 enzyme activation. Moreover, αKlotho downregulates ADAM10 activity and supports redox balance, consequently protecting the slit diaphragm of podocyteσ under hyperglycemic conditions.

NADPH oxidase 4-derived hydrogen peroxide counterbalances testosterone-induced endothelial dysfunction and migration.

High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

Hederagenin improves renal fibrosis in diabetic nephropathy by regulating Smad3/NOX4/SLC7A11 signaling-mediated tubular cell ferroptosis.

Diabetic nephropathy (DN) is a common complication of diabetes, characterized by renal fibrosis and poor patient prognosis. Hederagenin (HDG) has shown promising improvement in chronic kidney disease (CKD) kidney fibrosis, but its mechanism in DN-induced kidney fibrosis remains unclear. In this study, a model of diabetic nephropathy (DN) in mice was induced by intraperitoneal injection of streptozocin (50 mg/kg), while in vitro, high glucose (25 mM) was used to induce HK2 cell damage, simulating tubular injury in DN kidneys. The improvement of HDG treatment intervention was evaluated by observing changes in renal function, pathological structural damage, and the expression of fibrosis-related proteins in renal tubular cells. The results demonstrate that HDG intervention alleviates renal dysfunction and pathological damage in DN mice, accompanied by reduced expression of fibrotic markers α-smooth muscle actin (α-SMA), fibronectin (FN) and Collagen-I. Mechanistically, this study found that HDG can inhibit ferroptosis and fibrosis induced by the ferroptosis inducer Erastin (1 µM) in renal tubular cells. Phosphorylation of Smad3 promotes ferroptosis in renal tubular cells. After using its specific inhibitor SIS3 (4 µM), the expression of downstream target protein NADPH oxidase 4 (NOX4) significantly decreases, while the level of glutathione peroxidase 4 (GPX4) is notably restored, mitigating ferroptosis. Smad3 overexpression attenuates the therapeutic effect of HDG on tubular cell fibrosis induced by high glucose. These results demonstrate HDG inhibits Smad3 phosphorylation, thereby reducing the expression of NOX4 and enhancing the expression of GPX4, ultimately attenuating ferroptosis induced renal fibrosis. These findings suggest that HDG offer therapeutic potential for DN renal fibrosis by targeting Smad3-mediated ferroptosis in renal tubular cells.

Continuous TNF-α exposure in mammary epithelial cells promotes cancer phenotype acquisition via EGFR/TNFR2 activation.

Tumor necrosis factor alpha (TNF-α), an abundant inflammatory cytokine in the tumor microenvironment (TME), is linked to breast cancer growth and metastasis. In this study, we established MCF10A cell lines incubated with TNF-α to investigate the effects of continuous TNF-α exposure on the phenotypic change of normal mammary epithelial cells. The established MCF10A-LE cell line, through long-term exposure to TNF-α, displayed cancer-like features, including increased proliferation, migration, and sustained survival signaling even in the absence of TNF-α stimulation. Unlike the short-term exposed cell line MCF10A-SE, MCF10A-LE exhibited elevated levels of epidermal growth factor receptor (EGFR) and subsequent TNF receptor 2 (TNFR2), and silencing of EGFR or TNFR2 suppressed the cancer-like phenotype of MCF10A-LE. Notably, we demonstrated that the elevated levels of NAD(P)H oxidase 4 (NOX4) and the resulting increase in reactive oxygen species (ROS) were associated with EGFR/TNFR2 elevation in MCF10A-LE. Furthermore, mammosphere-forming capacity and the expression of cancer stem cell (CSC) markers increased in MCF10A-LE. Silencing of EGFR reversed these effects, indicating the acquisition of CSC-like properties via EGFR signaling. In conclusion, our results reveal that continuous TNF-α exposure activates the EGFR/TNFR2 signaling pathway via the NOX4/ROS axis, promoting neoplastic changes in mammary epithelial cells within the inflammatory TME.

Autophagy caused by oxidative stress promotes TGF-ß1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells.

Epithelial-to-mesenchymal transition (EMT) is one of the main causes of peritoneal fibrosis. However, the pathophysiological mechanisms of EMT, specifically its relationship with autophagy, are still unknown. This study aimed to evaluate the role of autophagy in transforming growth factor-beta 1 (TGF-ß1)-induced EMT in human peritoneal mesothelial cells (HPMCs). Primary cultured HPMCs were treated with TGF-ß1 (2 and 5 ng/mL) and changes in autophagy markers and the relationship between autophagy and EMT were evaluated. We also identified changes in EMT- and autophagy-related signaling pathways after autophagy and NADPH oxidase 4 (NOX4) inhibition. TGF-ß1 increased the generation of NOX4 and reactive oxygen species (ROS) in HPMCs, resulting in mitochondrial damage. Treatment with GKT137831 (20 µM), a NOX1/4 inhibitor, reduced ROS in the mitochondria of HPMC cells and reduced TGF-ß1-induced mitochondrial damage. Additionally, the indirect inhibition of autophagy by GKT137831 (20 µM) downregulated TGF-ß1-induced EMT, whereas direct inhibition of autophagy using 3-methyladenine (3-MA) (2 mM) or autophagy-related gene 5 (ATG5) gene silencing decreased the TGF-ß1-induced EMT in HPMCs. The suppressor of mothers against decapentaplegic 2/3 (Smad2/3), autophagy-related phosphoinositide 3-kinase (PI3K) class III, and protein kinase B (Akt) pathways, and mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular signal-regulated kinase (ERK) and P38, were involved in TGF-ß1-induced EMT. Autophagy and NOX4 inhibition suppressed the activation of these signaling pathways. Direct inhibition of autophagy and its indirect inhibition through the reduction of mitochondrial damage by upstream NOX4 inhibition reduced EMT in HPMCs. These results suggest that autophagy could serve as a therapeutic target for the prevention of peritoneal fibrosis in patients undergoing peritoneal dialysis.

A novel Senescence-Based prognostic model unveils tumor interactions and drug resistance in colorectal cancer.

BACKGROUND: In China, CRC incidence is escalating. The main hurdles are heterogeneity and drug resistance. This research delves into cellular senescence in CRC, aiming to devise a prognostic model and pinpoint mechanisms impacting drug resistance. METHODS: Mendelian randomization (MR) analysis confirmed the association between CRC and cellular aging. The Cancer Genome Atlas (TCGA)-CRC data served as the training set, with GSE38832 and GSE39582 as validation sets. Various bioinformatics methods were employed to construct and validate a risk model. CRC cells with NADPH Oxidase 4 (NOX4) knockout were generated using CRISPR-Cas9 technology. Protein blotting and colony formation assays elucidated the role of NOX4 in CRC cell aging and drug resistance. RESULTS: A prognostic model, derived from dataset analysis, uncovered a link between high-risk groups and cancer progression. Notable differences in the tumor microenvironment were observed between risk groups. Finally, NOX4 was found to be linked with aging and drug resistance in CRC. CONCLUSION: This research presents a novel senescence-based CRC prognosis model. It identifies NOX4's role in CRC drug resistance, suggesting it is a potential treatment target.

Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.

Emotional Stress Induces Adaptive Response in Rat Lymphocytes to Subsequent Ionizing Radiation Exposure.

We studied the molecular mechanisms of cross-adaptation to ionizing radiation (1 Gy) of lymphocytes isolated from rats subjected to emotional stress. The effects of chronic (CES; various types of stress exposure) and acute (AES; forced swimming) emotional stress in rats on indicators of oxidative stress, cell death, and levels of NRF2 and NOX4 proteins involved in the development of the adaptive response were analyzed in isolated lymphocytes. It was found that stress induced an adaptive response in rat lymphocytes and triggered processes similar to the adaptive response induced by low doses of ionizing radiation: an increase in the level of oxidized DNA and cell death, as well as an increase in the content of NOX4 and NRF2 proteins. In animals subjected to emotional stress, suppressed DNA oxidation in response to irradiation, reduced levels of protective factor NRF2, as well as lymphocyte death were observed.

Comprehensive bioinformatics analysis of NOX4 as a biomarker for pan-cancer prognosis and immune infiltration.

BACKGROUND: NADPH oxidase 4 (NOX4) has been proven to be associated with the prognosis of tumors in multiple cancers and can serve as a potential immunotherapy target to provide new treatment options for various tumors. In this study, our aim is to conduct an in-depth investigation of NOX4 across a range of cancer types to determine the relationship between NOX4 and tumors. METHODS: Utilizing large-scale transcriptomic and clinical data from public databases, a systematic examination of NOX4 expression patterns was performed in pan-cancer cohorts. Survival analysis, methylation analysis, and correlation studies were employed to assess the diagnostic and prognostic significance of NOX4 in diverse cancer types. Additionally, an exploration of the relationship between NOX4 expression and immune infiltration across various tumors was conducted. RESULTS: The analyses unveiled a consistent upregulation of NOX4 expression in multiple cancer types relative to normal tissues, indicating its potential as a universal cancer biomarker. Elevated NOX4 expression significantly correlated with poor overall survival in several cancers. Furthermore, the study demonstrated a robust correlation between NOX4 expression and immune cell infiltration, signifying its involvement in the modulation of the tumor microenvironment. CONCLUSIONS: This study imparts valuable insights into the potential applications of NOX4 in cancer research, highlighting its significance as a multifaceted biomarker with diagnostic, prognostic, and immunomodulatory implications across diverse malignancies.

Binge alcohol induces NRF2-related antioxidant response in the skeletal muscle of female mice.

BACKGROUND: Chronic alcohol enhances oxidative stress, but the temporal response of antioxidant genes in skeletal muscle following a binge drinking episode remains unknown. METHODS: Experiment 1: C57BL/6Hsd female mice received an IP injection of saline (CON; n = 39) or ethanol (ETOH; n = 39) (5 g/kg). Gastrocnemius muscles were collected from baseline (untreated; n = 3), CON (n = 3), and ETOH (n = 3) mice every 4 h for 48 h. Experiment 2: Gastrocnemius muscles were collected from control-fed (CON-FED; n = 17), control-fasted (CON-FAST; n = 18), or alcohol-fed (ETOH-FED; n = 18) mice every 4hrs for 20hrs after saline or ethanol (5 g/kg). RESULTS: EtOH enhanced Superoxide dismutase 1 (Sod1) and NADPH Oxidase 4 (Nox4) from 24 to 48hr after the binge, while Sod2 and Nox2 were suppressed. Nuclear factor erythroid-derived 2-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) increased 12hrs after intoxication. Cytochrome P450 oxidoreductase (Por), Heme oxygenase 1 (Ho1), Peroxiredoxin 6 (Prdx6), Glutamate-cysteine ligase catalytic subunit (Gclc), Glutamate-cysteine ligase modifier subunit (Gclm), and Glutathione-disulfide reductase (Gsr) were increased by ETOH starting 12-16hrs post-binge. Fasting had similar effects on Nrf2 compared to alcohol, but downstream targets of NRF2, including Por, Ho1, Gclc, and Gclm, were differentially altered with fasting and EtOH. CONCLUSION: These data suggest that acute alcohol intoxication induced markers of oxidative stress and antioxidant signaling through the NRF2 pathway and that there were effects of alcohol independent of a possible decrease in food intake caused by binge intoxication.

NOX4 regulates gastric cancer cell invasion and proliferation by increasing ferroptosis sensitivity through regulating ROS.

OBJECTIVE: We assessed NOX4 expression in gastric cancer (GC), its prognostic significance, and underlying mechanisms, focusing on promoting ferroptosis through increased ROS production. METHODS: We evaluated NOX4 expression in GC tissues via immunohistochemistry and analyzed correlations with clinicopathological characteristics using TCGA and clinical data. Impacts of manipulating NOX4 levels on GC cell invasiveness, proliferation, and sensitivity to ferroptosis inducers were investigated. RESULTS: Significantly higher NOX4 expression in GC tissues versus normal adjacent tissues correlated with decreased overall survival and increased tumor aggressiveness. NOX4 was an independent predictor of poor prognosis. Functionally, NOX4 manipulation influenced ROS levels, with overexpression enhancing production. Inhibition of NOX4 or application of antioxidants reduced cancer cell invasion and proliferation. Importantly, NOX4-overexpressing cells showed increased sensitivity to ferroptosis inducers, indicating synergistic effects between NOX4 and ferroptosis in suppressing GC progression. CONCLUSION: Our findings highlight NOX4's potential as a therapeutic target in GC, where modulation can enhance efficacy of ferroptosis-inducing treatments, offering a promising strategy for combating this malignancy.