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.

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.

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.

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.

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.

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.

The phosphokinase activity of IRE1ɑ prevents the oxidative stress injury through miR-25/Nox4 pathway after ICH.

BACKGROUND: Endoplasmic reticulum (ER) stress and oxidative stress are the major pathologies encountered after intracerebral hemorrhage (ICH). Inositol-requiring enzyme-1 alpha (IRE1α) is the most evolutionarily conserved ER stress sensor, which plays a role in monitoring and responding to the accumulation of unfolded/misfolded proteins in the ER lumen. Recent studies have shown that ER stress is profoundly related to oxidative stress in physiological or pathological conditions. The purpose of this study was to investigate the role of IRE1α in oxidative stress and the potential mechanism. METHODS: A mouse model of ICH was established by autologous blood injection. The IRE1α phosphokinase inhibitor KIRA6 was administrated intranasally at 1 h after ICH, antagomiR-25 and agomiR-25 were injected intraventricularly at 24 h before ICH. Western blot analysis, RT-qPCR, immunofluorescence staining, hematoma volume, neurobehavioral tests, dihydroethidium (DHE) staining, H2O2 content, brain water content, body weight, Hematoxylin and Eosin (HE) staining, Nissl staining, Morris Water Maze (MWM) and Elevated Plus Maze (EPM) were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), miR-25-3p, and Nox4 were increased in the ICH model. Administration of KIRA6 downregulated miR-25-3p expression, upregulated Nox4 expression, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, reduced body weight, aggravated spatial learning and memory deficits, and increased anxiety levels. Then antagomiR-25 further upregulated the expression of Nox4, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, whereas agomiR-25 reversed the effects promoted by KIRA6. CONCLUSION: The IRE1α phosphokinase activity is involved in the oxidative stress response through miR-25/Nox4 pathway in the mouse ICH brain.

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.

Mitochondria- and NOX4-dependent antioxidant defense mitigates progression to nonalcoholic steatohepatitis in obesity.

Nonalcoholic fatty liver disease (NAFLD) is prevalent in the majority of individuals with obesity, but in a subset of these individuals, it progresses to nonalcoholic steatohepatitis (0NASH) and fibrosis. The mechanisms that prevent NASH and fibrosis in the majority of patients with NAFLD remain unclear. Here, we report that NAD(P)H oxidase 4 (NOX4) and nuclear factor erythroid 2-related factor 2 (NFE2L2) were elevated in hepatocytes early in disease progression to prevent NASH and fibrosis. Mitochondria-derived ROS activated NFE2L2 to induce the expression of NOX4, which in turn generated H2O2 to exacerbate the NFE2L2 antioxidant defense response. The deletion or inhibition of NOX4 in hepatocytes decreased ROS and attenuated antioxidant defense to promote mitochondrial oxidative stress, damage proteins and lipids, diminish insulin signaling, and promote cell death upon oxidant challenge. Hepatocyte NOX4 deletion in high-fat diet-fed obese mice, which otherwise develop steatosis, but not NASH, resulted in hepatic oxidative damage, inflammation, and T cell recruitment to drive NASH and fibrosis, whereas NOX4 overexpression tempered the development of NASH and fibrosis in mice fed a NASH-promoting diet. Thus, mitochondria- and NOX4-derived ROS function in concert to drive a NFE2L2 antioxidant defense response to attenuate oxidative liver damage and progression to NASH and fibrosis in obesity.

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells.

BACKGROUND: The increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study. METHODS: The predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified. RESULTS: There were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR = 1.93, 95% CI 1.49-2.49, P < 0.001) and a higher percentage of lymph node metastases (OR = 3.22, 95% CI 2.18-4.29, P < 0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P < 0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P < 0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with "CRISPR HDR" or "CRISPR excision." Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment. CONCLUSIONS: The NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.

Downregulation of NOX4 improves airway remodeling and inflammation by the TGF-ß1-Smad2/3 pathway in asthma.

Asthma is a respiratory inflammatory disease, and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is involved in the progression of respiratory diseases. However, the role of NOX4 in asthma remains unclear. In the present study, we aimed to explore the effects of NOX4 on airway remodeling and inflammation. NOX4 expression was measured using immunocytochemistry (IHC), western blot, and real-time PCR (qPCR). Lung tissues were stained using the H&E assay. ELISA was used to examine the levels of airway remodeling-related indicators, and qPCR was used to detect airway inflammatory factors. The results indicated that NOX4 is highly expressed in lung tissues, bronchoalveolar lavage fluid (BALF), and serum of OVA-treated mice. Inhibition of NOX4 alleviated OVA-induced airway remodeling and inflammation. Similarly, TGF-ß1 was also upregulated in BALF and serum OVA-induced mice. Inhibition of TGF-ß1 signaling also improved airway remodeling and inflammation induced by OVA. Moreover, the downregulation of NOX4 inactivated the TGF-ß1-Smad2/3 pathway, and TGF-ß1 decreased Smad2/3 expression. Moreover, inhibition of the TGF-ß1 was enhanced, while TGF-ß1 reversed the effects on airway remodeling and inflammation induced by NOX4 inhibition. Taken together, the downregulation of NOX4 improves airway remodeling and inflammation via inactivation of the TGF-ß1-Smad2/3 pathway in asthma mice, suggesting that NOX4 may be a therapeutic target for asthma.

miR-100a-5p-enriched exosomes derived from mesenchymal stem cells enhance the anti-oxidant effect in a Parkinson's disease model via regulation of Nox4/ROS/Nrf2 signaling.

BACKGROUND: The pathogenesis of Parkinson's disease (PD) has not been fully elucidated, and there are no effective disease-modifying drugs for the treatment of PD. Mesenchymal stem cells have been used to treat several diseases, but are not readily available. METHODS: Here, we used phenotypically uniform trophoblast stage-derived mesenchymal stem cells (T-MSCs) from embryonic stem cells, which are capable of stable production, and their exosomes (T-MSCs-Exo) to explore the molecular mechanisms involved in dopaminergic (DA) neuron protection in PD models using experimental assays (e.g., western blotting, immunofluorescence and immunohistochemistry staining). RESULTS: We assessed the levels of DA neuron injury and oxidative stress in MPTP-induced PD mice and MPP+-induced MN9D cells after treating them with T-MSCs or T-MSCs-Exo. Furthermore, T-MSCs-Exo miRNA sequencing analysis revealed that miR-100-5p-enriched T-MSCs-Exo directly targeted the 3' UTR of NOX4, which could protect against the loss of DA neurons, maintain nigro-striatal system function, ameliorate motor deficits, and reduce oxidative stress via the Nox4-ROS-Nrf2 axis in PD models. CONCLUSIONS: The study suggests that miR-100-5p-enriched T-MSCs-Exo may be a promising biological agent for the treatment of PD. Schematic summary of the mechanism underlying the neuroprotective actions of T-MSCs-Exo in PD. T-MSCs Exo may inhibit the expression level of the target gene NOX4 by delivering miR-100-5p, thereby reducing ROS production and alleviating oxidative stress via the Nox4-ROS-Nrf2 axis, thus improving DA neuron damage in PD.