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Tetrahydrobiopterin (BH4) deficiency is caused by genetic abnormalities that impair its biosynthesis and recycling, which trigger neurochemical, metabolic, and redox imbalances. Low BH4 levels are also associated with hypoxia, reperfusion reoxygenation, endothelial dysfunction, and other conditions that are not genetically determined. The exact cause of changes in BH4 in nongenetic disorders is not entirely understood, but a role for oxidant species has been implicated. The oxidation of BH4 generates several products, including 7,8-dihydrobiopterin (BH2), the accumulation of which is predicted in cells with low dihydrofolate reductase activity. The relative efficiency of oxidant species at causing variations in BH4/BH2 levels in cells furnished with several antioxidant enzymes has not yet been systematically analyzed. This study examined the quantitative changes of BH4/BH2 in cells challenged with several oxidants. We showed that BH2 is not a major product of treatments with hydrogen peroxide or RSL3, as indicated by the moderate effect of dihydrofolate reductase-inhibitor methotrexate on the accumulation of BH2. However, we found a net loss in BH4/BH2, suggesting that products other than BH2 were generated. These reactions were further examined in NOX4-expressing HEK cells producing hydrogen peroxide. These cells showed slightly decreased BH4/BH2 ratios compared with HEK wild-type cells, and, again, methotrexate treatment moderately increased BH2 levels. In contrast, peroxynitrite-producing RAW 264.7 cells showed dramatically decreased BH4 levels without BH2 accumulation. Following the activation of peroxynitrite production with PMA in lipopolysaccharide-treated cells, we also found a significant time-dependent decline in GTPCH-I protein levels. We conclude that hydrogen peroxide is the least effective oxidant species at decreasing intracellular BH4 levels, while peroxynitrite is highly effective by targeting GTPCH-I and BH4 directly. Moreover, we conclude that BH4/BH2 levels are not a determinant of RSL3 cytotoxicity.

Diabetic nephropathy (DN), a leading cause of end-stage renal disease, remains a formidable challenge in diabetes management due to the complex nature of its pathogenesis, particularly the epithelial-mesenchymal transition (EMT) process. Our innovative study leverages network pharmacology to explore the therapeutic potentials of Myricetin, a natural flavonoid, focusing on its effects against NOX4, a critical mediator in DN progression. This investigation marks a pioneering approach by integrating network pharmacology to predict and elucidate the inhibitory relationship between Myricetin and NOX4. Utilizing a high-fat diet/streptozotocin (HFD/STZ) induced DN mouse model, we delved into the effects of Myricetin on renal EMT processes. Through network pharmacology analyses coupled with molecular docking studies, we identified and confirmed Myricetin's binding efficacy to NOX4. Extensive in vitro and in vivo experiments further established Myricetin's significant impact on mitigating EMT by modulating the NOX4-NF-κB-Snail signaling pathway. Results from our research demonstrated notable improvements in renal function and reductions in tissue fibrosis among treated HFD/STZ mice. By curtailing NOX4 expression, Myricetin effectively reduced reactive oxygen species (ROS) production, thereby inhibiting NF-κB activation and subsequent Snail expression, crucial steps in the EMT pathway. Supported by both theoretical predictions and empirical validations, this study unveils the mechanism underlying Myricetin's modulation of EMT in DN through disrupting the NOX4-NF-κB-Snail axis. These findings not only contribute a new therapeutic avenue for DN treatment but also underscore the utility of network pharmacology in advancing drug discovery processes.

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-β/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-β/NOX4 signaling pathway.

Inflammatory macrophages within the synovium play a pivotal role in the progression of arthritis inflammation. Effective drug therapy targeting inflammatory macrophages has long been a goal for clinicians and researchers. The standard approach for treating osteoarthritis (OA) involves systemic treatment and local injection. However, the high incidence of side effects associated with long-term drug administration increases the risk of complications in patients. Additionally, the rapid clearance of the joint cavity poses a biological barrier to the therapeutic effect. NADPH oxidase 4 (NOX4) is an enzyme protein regulating the cellular redox state by generating reactive oxygen species (ROS) within the cell. In this study, we designed and fabricated a hydrogel microsphere consisting of methyl methacrylate (MMA) and polyvinyl acetate (PVA) as the outer layer structure. We then loaded GLX351322 (GLX), a novel selective NOX4 inhibitor, into hydrogel microspheres through self-assembly with the compound polyethylene glycol ketone mercaptan (mPEG-TK) containing a disulfide bond, forming nanoparticles (mPEG-TK-GLX), thus creating a two-layer drug-loaded microspheres capsule with ROS-responsive and slow-releasing capabilities. Our results demonstrate that mPEG-TK-GLX@PVA-MMA effectively suppressed TBHP-induced inflammation, ROS production, and ferroptosis, indicating a promising curative strategy for OA and other inflammatory diseases in the future.

Cisplatin is a chemotherapeutant widely used in treating solid tumors, with the common side effect of acute kidney injury (AKI). Developing effective useful agent for preventing or treating cisplatin-induced AKI is of great importance. In this study, we investigate the protective effect of vaccarin, a chemical entity of flavonoid glycoside, against cisplatin-induced AKI. Cisplatin-treated C57BL/6J mice and human kidney-2 (HK-2) cells were used as the model of cisplatin-induced AKI. The levels of blood urea nitrogen (BUN) and creatine (Cr) levels and periodic acid-Schiff staining (PAS) scores decreased when vaccarin was administrated. Vaccarin had no impact on renal platinum accumulation, which was detected by the ICP-MS 6 h after cisplatin injection. Moreover, vaccarin can significantly alleviate the product of reactive oxygen species and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) in cisplatin-induced AKI, both in vivo and in vitro. In addition, vaccarin decreased the receptor-interacting protein kinase 1 (RIPK1) related programmed necrosis (necroptosis), cell apoptosis (shown by the protein levels of cleaved-caspase3 and flow cytometry) and inflammation (shown by the decreased levels of NLRP3, p-P65 and the mRNA of several inflammatory factors). NOX4 inhibitor GLX351322 (GLX) and NOX4 kowndown by siRNA have equivalent protective effect of vaccarin in vitro. When vaccarin was administered together with GLX or NOX4 siRNA, this protective effect of vaccarin did not further increase, as indicating by the index of oxidative stress, cell viability, necroptosis and apoptosis. In conclusion, vaccarin can alleviate cisplatin-induced AKI via inhibiting NOX4.

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.

Diabetic vascular complications can affect both microvascular and macrovascular. Diabetic microvascular complications, such as diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and diabetic cardiomyopathy, are believed to be caused by oxidative stress. The Nox family of NADPH oxidases is a significant source of reactive oxygen species and plays a crucial role in regulating redox signaling, particularly in response to high glucose and diabetes mellitus. This review aims to provide an overview of the current knowledge about the role of Nox4 and its regulatory mechanisms in diabetic microangiopathies. Especially, the latest novel advances in the upregulation of Nox4 that aggravate various cell types within diabetic kidney disease will be highlighted. Interestingly, this review also presents the mechanisms by which Nox4 regulates diabetic microangiopathy from novel perspectives such as epigenetics. Besides, we emphasize Nox4 as a therapeutic target for treating microvascular complications of diabetes and summarize drugs, inhibitors, and dietary components targeting Nox4 as important therapeutic measures in preventing and treating diabetic microangiopathy. Additionally, this review also sums up the evidence related to Nox4 and diabetic macroangiopathy.

Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-β) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix (ECM) and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-β-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-β in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-β treatment, Nox4 did not influence skin myofibroblast differentiation although a putative NOX4 inhibitor GKT137831 and a flavoprotein inhibitor diphenylene iodonium mitigated this mechanism. Transcriptomic analysis revealed upregulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3 in Nox4-deficient fibroblasts, which had however no impact on fibroblast bioenergetics. Altogether, we provide extensive evidence that NOX4 is dispensable for wound healing and skin fibroblast to myofibroblast differentiation, and suggest that another H2O2-generating flavoprotein drives this mechanism.

Artemisinin derivatives have been found to have anti-obesity effects recently, but the mechanism is still controversial. Herein, long-term DHA treatment in obese mice significantly reduced the body weight and improved glucose metabolism. However, short-term DHA treatment did not affect glucose metabolism in obese mice, suggesting that the improved glucose metabolism in mice with DHA treatment could be secondary to body weight reduction. Consistent with previous reports, we observed that DHA inhibited the differentiation of adipocytes. Mechanistically, DHA significantly reduced the expression of NADPH oxidase 4 (NOX4) in white adipose tissue (WAT) of mice and differentiated adipocytes, and using NOX4 siRNA or the NOX4 inhibitor GKT137831 significantly attenuated adipocyte differentiation. Over-expression of NOX4 partially reversed the inhibition effect of DHA on adipogenic differentiation of preadipocytes. In addition, targeted proteomics analysis showed that DHA improved the abnormality of metabolic pathways. In conclusion, DHA significantly reduced fat mass and improved glucose metabolism in obese mice, possibly by inhibiting NOX4 expression to suppress adipocyte differentiation and lipid accumulation in adipocytes.

Hesperidin possesses myriads of pharmacological benefits, including anti-inflammatory and antioxidant properties. Herein, we speculated that the described pharmacological benefits of hesperidin might be due to its potentiating action on SIRT1; thereby, inhibition of NOX4. We developed diabetic neuropathy in Sprague-Dawley rats by feeding them a high-fat diet (HFD) for 12 weeks. We checked the effect of hesperidin on the level of oxidative stress, inflammatory markers, NOX4, and SIRT1 by biochemical analysis, histopathology, immunoblotting, immunocytochemistry, and real-time qPCR in HFD-fed rats and Palmitate encountered rat glial C6 cells. Hesperidin administration improved mechanical, thermal allodynia, and glucose homeostasis. There was a decrease in oxidative stress and inflammation and an enhanced level of antioxidant enzymes. Besides, the expression of NOX4 was down-regulated, while SIRT1 was upregulated. Interestingly, hesperidin treatment protected them from oxidative and inflammatory damage by upregulating SIRT1 and inhibiting NOX4 expression.

Background: Oxidative stress and the resultant hyperpermeability play a vital role in the pathogenesis of diabetic retinopathy (DR). Poldip2 has been implicated in H2O2 production, but the effects of capsaicin on poldip2 have not been reported. Methods: Diabetic Sprague-Dawley (SD) rats induced with STZ were treated with capsaicin or AAV9-poldip2-shRNA, and human retinal microvascular endothelial cells (HRMECs) were treated with capsaicin or poldip2 siRNA. Results: Current data indicated that the expression of PPARγ, poldip2, Nox4, VCAM-1, HIF-1α, and VEGF increased in rat retinas with DR and in HRMECs treated with high glucose. The production of ROS or H2O2 in the tissues, serum, and cells increased, and the paracellular permeability of cultured HRMECs with high glucose significantly increased. In addition, overt hyperpermeability of retinal microvessels and increased retinal neovascularization in diabetic rats were observed. However, capsaicin treatment inhibited these increases and suppressed the expression of PPARγ by enhancing its phosphorylation and ubiquitination in the retinas of DR rats. Poldip2 knockdown in HRMECs or its silencing in the retina of DR rats concomitantly led to reduced levels of Nox4, VCAM-1, HIF-1α, VEGF, ROS, and H2O2, and the paracellular permeability of HRMECs or the hyperpermeability of retinal microvessels in diabetic rat retinas decreased. Similarly, after PPARγ knockdown in HRMECs, poldip2, Nox4, HIF-1α, VEGF, ROS, and H2O2 decreased, and the monolayer paracellular permeability was reduced accordingly. Conclusion: Capsaicin may ameliorate diabetic retinopathy by activating TRPV1 and suppressing the PPARγ-poldip2-Nox4 pathway.

Breast cancer arises as a result of multiple interactions between environmental and genetic factors. Conventionally, breast cancer is treated based on histopathological and clinical features. DNA technologies like the human genome microarray are now partially integrated into clinical practice and are used for developing new “personalized medicines” and “pharmacogenetics” for improving the efficiency and safety of cancer medications. We investigated the effects of four established therapies—for ER+ ductal breast cancer—on the differential gene expression. The therapies included single agent tamoxifen, two-agent docetaxel and capecitabine, or combined three-agents CAF (cyclophosphamide, doxorubicin, and fluorouracil) and CMF (cyclophosphamide, methotrexate, and fluorouracil). Genevestigator 8.1.0 was used to compare five datasets from patients with infiltrating ductal carcinoma, untreated or treated with selected drugs, to those from the healthy control. We identified 74 differentially expressed genes involved in three pathways, i.e., apoptosis (extrinsic and intrinsic), oxidative signaling, and PI3K/Akt signaling. The treatments affected the expression of apoptotic genes (TNFRSF10B [TRAIL], FAS, CASP3/6/7/8, PMAIP1 [NOXA], BNIP3L, BNIP3, BCL2A1, and BCL2), the oxidative stress-related genes (NOX4, XDH, MAOA, GSR, GPX3, and SOD3), and the PI3K/Akt pathway gene (ERBB2 [HER2]). Breast cancer treatments are complex with varying drug responses and efficacy among patients. This necessitates identifying novel biomarkers for predicting the drug response, using available data and new technologies. GSR, NOX4, CASP3, and ERBB2 are potential biomarkers for predicting the treatment response in primary ER+ ductal breast carcinoma.

A role for ferroptosis in articular cartilage destruction associated with rheumatoid arthritis (RA) has not been identified. We previously reported transient receptor potential melastatin 7 (TRPM7) expression was correlated with RA cartilage destruction. Herein, we further characterized a role for TRPM7 in chondrocyte ferroptosis. The expression of TRPM7 was found to be elevated in articular chondrocytes derived from adjuvant arthritis (AA) rats, human RA patients, and cultured chondrocytes treated with the ferroptosis inducer, erastin. TRPM7 knockdown or pharmacological inhibition protected primary rat articular chondrocytes and human chondrocytes (C28/I2 cells) from ferroptosis. Moreover, TRPM7 channel activity was demonstrated to contribute to chondrocyte ferroptosis by elevation of intracellular Ca2+. Mechanistically, the PKCα-NOX4 axis was found to respond to stimulation with erastin, which resulted in TRPM7-mediated chondrocyte ferroptosis. Meanwhile, PKCα was shown to directly bind to NOX4, which could be reduced by TRPM7 channel inhibition. Adeno-associated virus 9-mediated TRPM7 silencing or TRPM7 blockade with 2-APB alleviated articular cartilage destruction in AA rats and inhibited chondrocyte ferroptosis. Collectively, both genetic and pharmacological inhibitions of TRPM7 attenuated articular cartilage damage and chondrocyte ferroptosis via the PKCα-NOX4 axis, suggesting that TRPM7-mediated chondrocyte ferroptosis is a promising target for the prevention and treatment of RA.

Rationale: Nox4 is a constitutively active NADPH oxidase that constantly produces low levels of H2O2. Thereby, Nox4 contributes to cell homeostasis and long-term processes, such as differentiation. The high expression of Nox4 seen in endothelial cells contrasts with the low abundance of Nox4 in stem cells, which are accordingly characterized by low levels of H2O2. We hypothesize that Nox4 is a major contributor to endothelial differentiation, is induced during the process of differentiation, and facilitates homeostasis of the resulting endothelial cells. Objective: To determine the role of No×4 in differentiation of murine inducible pluripotent stem cells (miPSC) into endothelial cells (ECs). Methods and results: miPSC, generated from mouse embryonic wildtype (WT) and Nox4−/− fibroblasts, were differentiated into endothelial cells (miPSC-EC) by stimulation with BMP4 and VEGF. During this process, Nox4 expression increased and knockout of Nox4 prolonged the abundance of pluripotency markers, while expression of endothelial markers was delayed in differentiating Nox4-depleted iPSCs. Eventually, angiogenic capacity of iPSC-ECs is reduced in Nox4 deficient cells, indicating that an absence of Nox4 diminishes stability of the reached phenotype. As an underlying mechanism, we identified JmjD3 as a redox target of Nox4. iPSC-ECs lacking Nox4 display a lower nuclear abundance of the histone demethylase JmjD3, resulting in an increased triple methylation of histone 3 (H3K27me3), which serves as a repressive mark for several genes involved in differentiation. Conclusions: Nox4 promotes differentiation of miPSCs into ECs by oxidation of JmjD3 and subsequent demethylation of H3K27me3, which forced endothelial differentiation and stability.

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.

Diclofenac (DF), a non-steroidal anti-inflammatory drug, is commonly used to relieve pain and inflammation. High doses of DF might induce acute kidney injury (AKI), particularly in elderly, a known vulnerable population., Aim: We aimed to assess the protective role of melatonin (Mel) on DF-induced AKI in aged rats and to highlight the underpinning mechanisms include, oxidative stress and inflammation focusing on microRNA-34a (miR-34a), nuclear factor erythroid-2-related factor-2/hemeoxygenase-1 (Nrf2/HO-1) and NLR family-pyrin domain containing-3 (NLRP3) inflammasome pathways, and to elucidate the possibility of epithelial sodium channel (ENaC) involvement., Materials and Methods: Thirty old male Wistar rats were allocated randomly into 3 groups: Control, DF and Mel-DF groups., Key Findings: Melatonin provided nephroprotective effects against DF-induced AKI via attenuating the expression of renal miR-34a and subsequently promoting the signaling of Nrf2/HO-1 with elevation of the antioxidant defense capacity and suppressing NLRP3 inflammasomes. Melatonin alleviated DF-induced hypernatremia via decreasing the ENaC expression. Renal histopathological examination revealed significant reduction in vascular congestion, mononuclear infiltration, glomerulo-tubular damage, fibrosis and TNF-α optical density., Significance: It can be assumed that melatonin is a promising safe therapeutic agent in controlling DF-induced AKI in elderly., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hashimoto's thyroiditis (HT) is a prevalent autoimmune thyroid disease, necessitating further research to identify effective treatment strategies. Two key pathophysiological factors of HT are inflammation and oxidative stress. Petunidin (PET) is an anthocyanin with anti-inflammatory and antioxidant properties. This study aimed to investigate the effect and mechanism of PET on HT. C57BL/6N mice were injected with thyroglobulin emulsified with adjuvant to establish the HT animal model. Our results showed that PET administration decreased the concentrations of TPOAb, TgAb, T3, T4, IgG, IgA and IgM in HT mice, accompanied by significant alterations in follicle shape and increased lymphocyte infiltrations. Additionally, the apoptosis rate, ROS level, MDA content, CD4+ level, IFN-γ and IL-17A levels, as well as the concentrations of IFN-γ and IL-17, were elevated in HT mice and reduced by PET treatment. Furthermore, HT patients exhibited higher levels of NOX4 and PKM2, which were positively correlated with TPOAb, IFN-γ, and IL-17 concentrations. In HT mice, PET therapy decreased the expression of PKM2 and NOX4 proteins. In summary, PET can improve thyroid dysfunction by suppressing apoptosis, oxidative stress and Th1/Th17 differentiation through regulation of the NOX4/PKM2 axis in HT mice, suggesting its promising potential for HT intervention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

Objective: The NADPH oxidase Nox4 is an important source of H2O2. Nox4-derived H2O2 limits vascular inflammation and promotes smooth muscle differentiation. On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery injury model. Methods and results: Genetic deletion of Nox4 by a tamoxifen-activated Cre-Lox-system did not impact on neointima formation in the carotid artery wire injury model. To understand this unexpected finding, time-resolved single-cell RNA-sequencing (scRNAseq) from injured carotid arteries of control mice and massive-analysis-of-cDNA-ends (MACE)-RNAseq from the neointima harvested by laser capture microdissection of control and Nox4 knockout mice was performed. This revealed that resting smooth muscle cells (SMCs) and fibroblasts exhibit high Nox4 expression, but that the proliferating de-differentiated SMCs, which give rise to the neointima, have low Nox4 expression. In line with this, the first weeks after injury, gene expression was unchanged between the carotid artery neointimas of control and Nox4 knockout mice. Conclusion: Upon vascular injury, Nox4 expression is transiently lost in the cells which comprise the neointima. NADPH oxidase 4 therefore does not interfere with restenosis development after wire-induced vascular injury.

Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondrial dysfunction is linked to oxidative stress and reactive oxygen species (ROS) in neurotoxicity during AD. Impaired mitochondrial metabolism has been associated with mitochondrial dysfunction in brain damage of AD. While the role of NADPH oxidase 4 (NOX4), a major source of ROS, has been identified in brain damage, the mechanism by which NOX4 regulates ferroptosis of astrocytes in AD remains unclear. Here, we show that the protein levels of NOX4 were significantly elevated in impaired astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. The levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), a marker of oxidative stress-induced lipid peroxidation, were significantly also elevated in impaired astrocytes of patients with AD and mouse AD. We demonstrate that the over-expression of NOX4 significantly increases the impairment of mitochondrial metabolism by inhibition of mitochondrial respiration and ATP production via the reduction of five protein complexes in the mitochondrial ETC in human astrocytes. Moreover, the elevation of NOX4 induces oxidative stress by mitochondrial ROS (mtROS) production, mitochondrial fragmentation, and inhibition of cellular antioxidant process in human astrocytes. Furthermore, the elevation of NOX4 increased ferroptosis-dependent cytotoxicity by the activation of oxidative stress-induced lipid peroxidation in human astrocytes. These results suggest that NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in AD.

Chalcone is a polyphenolic compound found abundantly in natural plant components. They have been acclaimed as potential antitumor compounds in multiple tumor cells. However, not much attention has been paid to elucidate its antitumor mechanism of action. Here, chalcone was demonstrated to trigger endoplasmic reticulum (ER) stress-induced apoptosis through sulfonation of IRE1α by ER-localized NADPH oxidase 4 (NOX4). IRE1α-sulfonation at a cysteine residue was shown to induce “regulated IRE1α-dependent decay” (RIDD) of mRNA rather than specific splicing of XBP1. The IRE1α sulfonation-induced RIDD degraded miR-23b, enhancing the expression of NOX4. The expression of NOX4 was also upregulated in breast, and prostate cancer tissue. In chalcone-administered mice in vivo, tumor growth was regressed by the consistent mechanisms “NOX4-IRE1α sulfonation-RIDD”. Similarly, NOX4 activation and IRE1α sulfonation were also highly increased under severe ER stress conditions. Together, these findings suggest chalcone as a lead anticancer compound where it acts through NOX4-IRE1α-RIDD-miR-23b axis providing a promising vision of chalcone derivatives’ anticancer mechanism.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-β) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4−/−) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4−/− and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4−/− mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-β pathway. In fact, levels of active TGF-β1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-β1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.

Objective: The immediate signals that couple exercise to metabolic adaptations are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) produces reactive oxygen species (ROS) and plays a significant role in metabolic and vascular adaptation during stress conditions. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism. Methods: Mice were subjected to acute exercise to assess their immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and the time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and their subsequent substrate oxidation was measured. Results: We identified key exercise-responsive metabolic genes that depend on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for the expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Global Nox4 deletion resulted in decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Furthermore, Nox4-deficient mice demonstrated impaired adaptation to chronic exercise as measured by the time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Importantly, mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise. Conclusions: We report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in the Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting inter-tissue cross-talk between the endothelium and skeletal muscle in response to exercise.

Background: Cissus quadrangularis L. is commonly known as hadjod and traditionally used as a medicinal plant for the treatment of osteoporosis, dyspepsia, tumors, chronic ulcers, and hepatic diseases. Existing studies have proclaimed the proinflammatory property of NOX4 through instigating ROS and oxidative stress, which is considered to be one of the instrumental causes of steatohepatitis. Purpose: To investigate the protective effect of ethanolic extract of C. quadrangularis (EECQ) on the steatohepatitis associated with type 2 diabetes (DM) through inhibition of NOX4. Methods: An experimental DM-induced steatohepatitis was developed by feeding a high-fat diet for 12 weeks in Sprague Dawley rats. Treatment of EECQ was given at a dose of 200 mg/kg (po), for six weeks. Glucose and insulin tolerance test was conducted. Hematoxylin and eosin and picrosirius red staining of liver sections was done. Steatosis and lobular inflammation were scored, and hepatocyte ballooning was assessed to confirm the NASH. qPCR, Immunofluorescence, and Immunoblotting determined the mRNA and protein expression of NOX4. Results: NASH group rats manifested features of DM, marked by aggravated glucose and insulin tolerance tests. Moreover, the DM rats exhibited the presence of steatohepatitis, characterized by elevated serum lipid content, aminotransferases, and presence of steatosis, lobular inflammation, and ballooning in the liver section. Nevertheless, EECQ treatment prevented all of these abnormalities. Besides, the level of AGEs and the expression of its receptor, RAGE was found to be increased in DM rats. Parallelly, the expression of NOX4 and the accompanying level of ROS, oxidative stress markers, and inflammatory cytokines were determined to be surged in palmitic acid exposed HepG2 cells and DM rats. However, EECQ treatment exemplified safeguards from the soared level of the aforementioned markers. Conclusion: Collectively, the above findings propounded the protective effect of EECQ against steatohepatitis. Nonetheless, further studies on humans necessitate to scrutinize and reinforce its therapeutic potential so that it may be employed as a remedy for DM-induced steatohepatitis.

Obesity is regarded as an abnormal expansion and excessive accumulation of fat mass in white adipose tissue. The involvement of oxidative stress in the development of obesity is still unclear. Although mainly present in peroxisomes, catalase scavenges intracellular H2O2 at toxic levels. Therefore, we used catalase-knockout (CKO) mice to elucidate the involvement of excessive H2O2 in the development of obesity. CKO mice with C57BL/6J background gained more weight with higher body fat mass with age than age-matched wild-type (WT) mice fed with either chow or high-fat diets. This phenomenon was attenuated by concomitant treatment with the antioxidants, melatonin or N-acetyl cysteine. Moreover, CKO mouse embryonic fibroblasts (MEFs) appeared to differentiate to adipocytes more easily than WT MEFs, showing increased H2O2 concentrations. Using 3T3-L1-derived adipocytes transfected with catalase-small interfering RNA, we confirmed that a more prominent lipogenesis occurred in catalase-deficient cells than in WT cells. Catalase-deficient adipocytes presented increased nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression but decreased adenosine monophosphate-activated protein kinase (AMPK) expression. Treatment with a NOX4 inhibitor or AMPK activator rescued the propensity for obesity of CKO mice. These findings suggest that excessive H2O2 and related oxidative stress increase body fat mass via both adipogenesis and lipogenesis. Manipulating NOX4 and AMPK in white adipocytes may be a therapeutic tool against obesity augmented by oxidative stress.

Dysregulated redox signaling and oxidative injury are associated with inflammatory processes and fibrosis. H2O2 generation by NOX4 has been suggested as a key driver in the development of fibrosis and a small molecule drug is under evaluation in clinical trials for idiopathic pulmonary fibrosis and primary biliary cholangitis. Fibrosis is a common complication in Crohn's disease (CD) leading to stricture formation in 35–40% of patients, who require surgical interventions in the absence of therapeutic options. Here we assess NOX4 expression in CD patients with inflammatory or stricturing disease and examine whether loss of NOX4 is beneficial in acute and fibrotic intestinal disease. NOX4 was upregulated in inflamed mucosal tissue of CD and ulcerative colitis (UC) patients, in CD ileal strictures, and in mice with intestinal inflammation. Nox4 deficiency in mice promoted pathogen colonization and exacerbated tissue injury in acute bacterial and chemical colitis. In contrast, in two chronic injury models aberrant tissue remodeling and fibrosis-related gene expression did not differ substantially between Nox4−/− mice and wildtype mice, suggesting that Nox4 is dispensable in TGF-β1-driven intestinal fibrogenesis. While animal models do not recapitulate all the hallmarks of CD fibrosis, the tissue-protective role of Nox4 warrants a cautious approach to pharmacological inhibitors.

Summary: Cytosolic proteins are required for regulation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) isozymes. Here we show that Src homology 3 (SH3) domain-containing YSC84-like 1 (SH3YL1), as a Nox4 cytosolic regulator, mediates lipopolysaccharide (LPS)-induced H2O2 generation, leading to acute kidney injury. The SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins (SYLF) region and SH3 domain of SH3YL1 contribute to formation of a complex with Nox4-p22phox. Interaction of p22phox with SH3YL1 is triggered by LPS, and the complex induces H2O2 generation and pro-inflammatory cytokine expression in mouse tubular epithelial cells. After LPS injection, SH3YL1 knockout mice show lower levels of acute kidney injury biomarkers, decreased secretion of pro-inflammatory cytokines, decreased infiltration of macrophages, and reduced tubular damage compared with wild-type (WT) mice. The results strongly suggest that SH3YL1 is involved in renal failure in LPS-induced acute kidney injury (AKI) mice. We demonstrate that formation of a ternary complex of p22phox-SH3YL1-Nox4, leading to H2O2 generation, induces severe renal failure in the LPS-induced AKI model.

NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces H2O2 which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived H2O2 oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. H2O2 disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with H2O2, induction of Nox4 expression by treatment of the cells with TGFβ and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not.Taken together, Nox4 oxidizes HDAC4, increases its phosphorylation, and eventually ensures proper tube formation by endothelial cells.

The ability for skeletal muscle to perform optimally can be affected by the regulation of Ca2+ within the triadic junctional space at rest. Reactive oxygen species impact muscle performance due to changes in oxidative stress, damage and redox regulation of signaling cascades. The interplay between ROS and Ca2+ signaling at the triad of skeletal muscle is therefore important to understand as it can impact the performance of healthy and diseased muscle. Here, we aimed to examine how changes in Ca2+ and redox signaling within the junctional space micro-domain of the mouse skeletal muscle fibre alters the homeostasis of these complexes. The dystrophic mdx mouse model displays increased RyR1 Ca2+ leak and increased NAD(P)H Oxidase 2 ROS. These alterations make the mdx mouse an ideal model for understanding how ROS and Ca2+ handling impact each other. We hypothesised that elevated t-tubular Nox2 ROS increases RyR1 Ca2+ leak contributing to an increase in cytoplasmic Ca2+, which could then initiate protein degradation and impaired cellular functions such as autophagy and ER stress. We found that inhibiting Nox2 ROS did not decrease RyR1 Ca2+ leak observed in dystrophin-deficient skeletal muscle. Intriguingly, another NAD(P)H isoform, Nox4, is upregulated in mice unable to produce Nox2 ROS and when inhibited reduced RyR1 Ca2+ leak. Our findings support a model in which Nox4 ROS induces RyR1 Ca2+ leak and the increased junctional space [Ca2+] exacerbates Nox2 ROS; with the cumulative effect of disruption of downstream cellular processes that would ultimately contribute to reduced muscle or cellular performance.

Increased pulmonary vascular permeability due to endothelial cell (EC) barrier dysfunction is a major pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI), which is a devastating critical illness with high incidence and excessive mortality. Activation of NADPH oxidase (NOX) induces EC dysfunction via production of reactive oxygen species (ROS). However, the role(s) of NOX isoform(s), and their downstream signaling events, in the development of ARDS/ALI have remained unclear. Cecal Ligation Puncture (CLP) was used to induce preclinical septic ALI in wild-type mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA in vivo. The survival rate of the CLP group at 24 h (26.6%, control siRNA treated) was substantially improved by NOX4 knockdown (52.9%). Mice lacking NOX2 or p47phox, however, had worse outcomes after CLP (survival rates at 0% and 8.3% respectively), whereas NOX1-silenced mice had similar survival rate (30%). NOX4 knockdown attenuated lung ROS production in septic mice, whereas NOX1 knockdown, NOX2 knockout, or p47phox knockout in mice had no effects. In addition, NOX4 knockdown attenuated redox-sensitive activation of the CaMKII/ERK1/2/MLCK pathway, and restored expression of EC tight junction proteins ZO-1 and Occludin to maintain EC barrier integrity. Correspondingly, NOX4 knockdown in cultured human lung microvascular ECs also reduced LPS-induced ROS production, CaMKII/ERK1/2/MLCK activation and EC barrier dysfunction. Scavenging superoxide in vitro and in vivo with TEMPO, or inhibiting CaMKII activation with KN93, had similar effects as NOX4 knockdown in preserving EC barrier dysfunction. In summary, we have identified a novel, selective and causal role of NOX4 (versus other NOX isoforms) in inducing lung EC barrier dysfunction and injury/mortality in a preclinical CLP-induced septic model, which involves redox-sensitive activation of CaMKII/ERK1/2/MLCK pathway. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.

Recurrence and metastasis remain the major cause of cancer mortality. Even for early-stage lung cancer, adjuvant chemotherapy yields merely slight increase to patient survival. EF-hand domain-containing protein D2 (EFHD2) has recently been implicated in recurrence of patients with stage I lung adenocarcinoma. In this study, we investigated the correlation between EFHD2 and chemoresistance in non-small cell lung cancer (NSCLC). High expression of EFHD2 was significantly associated with poor overall survival of NSCLC patients with chemotherapy in in silica analysis. Ectopic EFHD2 overexpression increased cisplatin resistance, whereas EFHD2 knockdown improved chemoresponse. Mechanistically, EFHD2 induced the production of NADPH oxidase 4 (NOX4) and in turn the increase of intracellular reactive oxygen species (ROS), consequently activating membrane expression of the ATP-binding cassette subfamily C member 1 (ABCC1) for drug efflux. Non-steroidal anti-inflammatory drug (NSAID) ibuprofen suppressed EFHD2 expression by leading to the proteasomal and lysosomal degradation of EFHD2 through a cyclooxygenase (COX)-independent mechanism. Combining ibuprofen with cisplatin enhanced antitumor responsiveness in a murine xenograft model in comparison with the individual treatment. In conclusion, we demonstrate that EFHD2 promotes chemoresistance through the NOX4-ROS-ABCC1 axis and therefore developing EFHD2-targeting strategies may offer a new avenue to improve adjuvant chemotherapy of lung cancer.

NADPH oxidase 4 (NOX4) is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients), esophagus (12/18 patients), bladder (10/19 patients), ovary (6/17 patients), and prostate (7/19 patients), as well as malignant melanoma (7/15 patients) when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

Cancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion.

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-β. 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tanshinone IIA (TSA), an active component isolated from Danshen, possess high medicinal values against atherosclerosis by reducing vascular oxidative stress, inhibiting platelet aggregation, and protecting the endothelium from damage. The periodontal pathogen Porphyromonas gingivalis (P. gingivalis) has been proven to accelerate the development of atherosclerosis. We aim to determine the effects of TSA on P. gingivalis-induced atherosclerosis in ApoE-knockout (ApoE-/-) mice. After feeding with a high-lipid diet and infected with P. gingivalis three times per week for four weeks, TSA-treated (60 mg/kg/d) mice greatly inhibited atherosclerotic lesions both morphologically and biochemically and exhibited significantly reduction ROS, 8-OHdG, and ox-LDL levels in serum compared with P. gingivalis-infected mice. Additionally, TSA-treated mice were observed a marked reduction of ROS, 8-OHdG and ox-LDL in the serum, mRNA levels of COX-2, LOX-1, NOX2 and NOX4 in the aorta, as well as the levels of NOX2, NOX4, and NF-κB. These results suggest that TSA attenuates oxidative stress by decreasing NOX2 and NOX4 and downregulating NF-κB signaling pathway, which might be contributed to the amelioration of atherosclerosis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aims: Dietary supplementation with ursolic acid (UA) prevents monocyte dysfunction in diabetic mice and protects mice against atherosclerosis and loss of renal function. The goal of this study was to determine the molecular mechanism by which UA prevents monocyte dysfunction induced by metabolic stress. Methods and results: Metabolic stress sensitizes or “primes” human THP-1 monocytes and murine peritoneal macrophages to the chemoattractant MCP-1, converting these cells into a hyper-chemotactic phenotype. UA protected THP-1 monocytes and peritoneal macrophages against metabolic priming and prevented their hyper-reactivity to MCP-1. UA blocked the metabolic stress-induced increase in global protein-S-glutathionylation, a measure of cellular thiol oxidative stress, and normalized actin-S-glutathionylation. UA also restored MAPK phosphatase-1 (MKP1) protein expression and phosphatase activity, decreased by metabolic priming, and normalized p38 MAPK activation. Neither metabolic stress nor UA supplementation altered mRNA or protein levels of glutaredoxin-1, the principal enzyme responsible for the reduction of mixed disulfides between glutathione and protein thiols in these cells. However, the induction of Nox4 by metabolic stress, required for metabolic priming, was inhibited by UA in both THP-1 monocytes and peritoneal macrophages. Conclusion: UA protects THP-1 monocytes against dysfunction by suppressing metabolic stress-induced Nox4 expression, thereby preventing the Nox4-dependent dysregulation of redox-sensitive processes, including actin turnover and MAPK-signaling, two key processes that control monocyte migration and adhesion. This study provides a novel mechanism for the anti-inflammatory and athero- and renoprotective properties of UA and suggests that dysfunctional blood monocytes may be primary targets of UA and related compounds.

Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-β has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-β would have undesired systemic side effects due to the multiple physiological functions of TGF-β. Further characterization of the downstream signaling pathway(s) involved in TGF-β-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-β-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-β/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders.

Aim: The current work aims to demonstrate the potential defensive function of venlafaxine (VLF) in cardiotoxicity and nephrotoxicity caused by cisplatin (CP), that could be by modulating extracellular signal-regulated kinase (ERK)1/2 and nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase NOX4 pathways., Main Methods: Five groups of rats were used, as follow: three control groups (control, carboxymethyl cellulose, and VLF), CP group got CP once (7 mg/kg, intraperitoneally, i.p.), and (CP+ VLF) group got CP once then after 1 h they got VLF {50 mg/kg daily, orally for 14 days}. At the end of the study; electrocardiogram (ECG) was recorded for anaesthized rats then blood samples and tissues were taken for biochemical and histopathological investigations. Caspase 3, a marker of cellular damage and apoptosis was detected by immunohistochemistry., Key Findings: CP treatment significantly impaired cardiac functions as evidenced by changes in rats' ECG. Cardiac enzymes, renal markers and inflammatory markers were increased with decreased activities of the total antioxidant capacity, superoxide dismutase and glutathione peroxidase. Also, ERK1/2 and NOX4 were upregulated with histopathological and immunohistochemical alterations of heart and kidney. While, VLF markedly alleviated CP-induced functional cardiac abnormalities and improved ECG pattern. It reduced both cardiac and renal biomarkers, oxidative stress, proinflammatory cytokine with ERK1/2 and NOX4 downregulation, improved the histopathological and immunohistochemical changes induced by cisplatin in heart and kidney., Significance: VLF treatment impedes cardiotoxicity and nephrotoxicity caused by CP. This beneficial effect was mediated through reduction of oxidative stress, inflammation, and apoptosis by targeting the ERK1/2 and NOX4., Competing Interests: Declaration of competing interest There are no conflicts of interest to report for any of the authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)