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Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.

Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein–protein interactions but also metabolite-dependent interactions. Based on this protein–metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4. Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood–brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein–metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.

Ferroptotic cell death is characterized by iron-dependent lipid peroxidation that is initiated by ferrous iron and H 2 O 2 via Fenton reaction, in which the role of activating transcription factor 3 (ATF3) remains elusive. Brucine is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica , which has shown potent antitumor activity against various tumors, including glioma. In this study, we showed that brucine inhibited glioma cell growth in vitro and in vivo, which was paralleled by nuclear translocation of ATF3, lipid peroxidation, and increases of iron and H 2 O 2 . Furthermore, brucine-induced lipid peroxidation was inhibited or exacerbated when intracellular iron was chelated by deferoxamine (500 μM) or improved by ferric ammonium citrate (500 μM). Suppression of lipid peroxidation with lipophilic antioxidants ferrostatin-1 (50 μM) or liproxstatin-1 (30 μM) rescued brucine-induced glioma cell death. Moreover, knockdown of ATF3 prevented brucine-induced accumulation of iron and H 2 O 2 and glioma cell death. We revealed that brucine induced ATF3 upregulation and translocation into nuclei via activation of ER stress. ATF3 promoted brucine-induced H 2 O 2 accumulation via upregulating NOX4 and SOD1 to generate H 2 O 2 on one hand, and downregulating catalase and xCT to prevent H 2 O 2 degradation on the other hand. H 2 O 2 then contributed to brucine-triggered iron increase and transferrin receptor upregulation, as well as lipid peroxidation. This was further verified by treating glioma cells with exogenous H 2 O 2 alone. Moreover, H 2 O 2 reversely exacerbated brucine-induced ER stress. Taken together, ATF3 contributes to brucine-induced glioma cell ferroptosis via increasing H 2 O 2 and iron.

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. Graphical Abstract 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.

Ferroptosis is a regulated cell death nexus linking metabolism, redox biology and diseases including cancer. The aim of the present study was to identify a ferroptosis‐related gene prognostic signature for stomach adenocarcinoma (STAD) by systematic analysis of transcriptional profiles from The Cancer Genome Atlas (TCGA), GEO and a clinical cohort from our centre. We developed a predictive model based on three ferroptosis‐related genes (CHAC1, NOX4 and HIF1A), gene expression data and corresponding clinical outcomes were obtained from the TCGA database, and the reliability of this model was verified with GSE15459 and 51 queues in our centre. ROC curve showed better predictive ability using the risk score. Immune cell enrichment analysis demonstrated that the types of immune cells and their expression levels in the high‐risk group were significantly different from those in the low‐risk group. The experimental results confirmed that NOX4 was upregulated and CHAC1 was downregulated in the STAD tissues compared with the normal stomach mucosal tissues (p < 0.05). In sum, the ferroptosis‐related gene signature can accurately predict the outcomes of patients with STAD, providing valuable insights for personalized treatment. As the signature also has relevance to the immune characteristics, it may help improve the efficacy of personalized immunotherapy.

Background The recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear. Methods HepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives. Results Here,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. n -acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress. Conclusion These data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.

Panax notoginseng , a traditional Chinese medicine, exerts beneficial effect on diabetic kidney disease (DKD), but its mechanism is not well clarified. In this study we investigated the effects of ginsenoside Rb1 (Rb1), the main active ingredients of Panax notoginseng , in alleviating podocyte injury in diabetic nephropathy and the underlying mechanisms. In cultured mouse podocyte cells, Rb1 (10 μM) significantly inhibited high glucose-induced cell apoptosis and mitochondrial injury. Furthermore, Rb1 treatment reversed high glucose-induced increases in Cyto c , Caspase 9 and mitochondrial regulatory protein NOX4, but did not affect the upregulated expression of aldose reductase (AR). Molecular docking analysis revealed that Rb1 could combine with AR and inhibited its activity. We compared the effects of Rb1 with eparestat, a known aldose reductase inhibitor, in high glucose-treated podocytes, and found that both alleviated high glucose-induced cell apoptosis and mitochondrial damage, and Rb1 was more effective in inhibiting apoptosis. In AR-overexpressing podocytes, Rb1 (10 μM) inhibited AR-mediated ROS overproduction and protected against high glucose-induced mitochondrial injury. In streptozotocin-induced DKD mice, administration of Rb1 (40 mg·kg −1 ·d −1 , ig, for 7 weeks) significantly mitigated diabetic-induced glomerular injuries, such as glomerular hypertrophy and mesangial matrix expansion, and reduced the expression of apoptotic proteins. Collectively, Rb1 combines with AR to alleviate high glucose-induced podocyte apoptosis and mitochondrial damage, and effectively mitigates the progression of diabetic kidney disease.

Neuregulin‐1 (NRG‐1) is reported to be cardioprotective through the extracellular‐regulated protein kinase (ERK) 1/2 pathway in myocardial ischaemia‐reperfusion injury (MIRI). NOX4‐induced ROS activated NLRP3 inflammasome and exacerbates MIRI. This study aims to investigate whether NRG‐1 can suppress NOX4 by ERK1/2 and consequently inhibit the NLRP3/caspase‐1 signal in MIRI. The myocardial infarct size (IS) was measured by TTC‐Evans blue staining. Immunohistochemical staining, real‐time quantitative PCR (RT‐qPCR) and Western blotting were used for detection of the factors, such as NOX4, ERK1/2, NLRP3, caspase‐1 and IL‐1β .The IS in the NRG‐1 (3 μg/kg, intravenous) group was lower than that in the IR group. Immunohistochemical analysis revealed NRG‐1 decreased 4HNE and NOX4. The RT‐qPCR and Western blot analyses revealed that NRG‐1 mitigated the IR‐induced up‐regulation of NOX4 and ROS production. Compared with the IR group, the NRG‐1 group exhibited a higher level of P‐ERK1/2 and a lower level of NLRP3. In the Langendorff model, PD98059 inhibited ERK1/2 and up‐regulated the expression of NOX4, NLRP3, caspase‐1 and IL‐1β, which exacerbated oxidative stress and inflammation. In conclusion, NRG‐1 can reduce ROS production by inhibiting NOX4 through ERK1/2 and inhibit the NLRP3/caspase‐1 pathway to attenuate myocardial oxidative damage and inflammation in MIRI.

Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

High ambient temperatures are a critical challenge in the poultry industry which is a key producer of the animal-based food. To evaluate heat stress levels, various parameters have been used, including growth rates, blood metabolites, and hormones. The most recent advances have explored expression profiling of genes that may play vital roles under stress. A high ambient temperature adversely affects nutrient uptake and is known to modulate the expression of genes encoding for sodium-dependent glucose transporters, glucose transporters, excitatory amino acid transporters, and fatty acid-binding proteins which are responsible for the absorption of macronutrients in the intestine. Various defensive activities are stimulated to protect the cell of different tissues from the heat-generated stress, including expression of early stress response genes coding for heat shock protein (HSP), c-FOS like protein, brain-derived neurotrophic factor (BDNF), and neuronal nitric oxide synthase (nNOS); antioxidant enzyme genes such as superoxide dismutase (SOD), catalase (CAT), and nicotinamide adenine dinucleotide phosphate oxidase (NOX4); and immune-related genes such as cytokines and toll-like receptors (TLRs). The potential role of HSPs in protecting the cell from stress and their presence in several tissues make them suitable markers to be evaluated under heat stress. BDNF and c-FOS genes expressed in the hypothalamus help cells to adapt to an adverse environment. Heat causes damage to the cell by generating reactive oxygen species (ROS). The NOX4 gene is the inducer of ROS under heat stress, which is in turns controlled by antioxidant enzymes such as SOD and CAT. TLRs are responsible for protecting against pathogenic attacks arising from enhanced membrane permeability, and cytokines help in controlling the pathogen and maintaining homeostasis. Thus, the evaluation of nutrient transporters and defense mechanisms using the latest molecular biology tools has made it possible to shed light on the complex cellular mechanism of heat-stressed chickens. As the impacts of heat stress on the above-mentioned aspects are beyond the extent to which the reduced growth performance could be explained, heat stress has more specific effects on the regulation of these genes than previously thought. Graphical abstract Effect of heat exposure on the nutrient transporters, antioxidants, and immune inflammation in chickens. Most of the nutrient transporters were suppressed under heat stress. Increase in the production of reactive oxygen species resulted in enhanced production of antioxidant enzymes. Expression of various proinflammatory cytokines and toll-like receptors were enhanced due to heat stress in chicken.