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Background: The present study investigated the function of G protein-coupled receptor kinase 2 (GRK2) in acute liver injury (ALI) by cisplatin, and investigated the protective effect of pharmacological inhibition of GRK2. Methods: ALI models were generated in global adult hemizygous (ALI-Grk2±) mice and wild-type (WT) mice. Liver biochemistry parameters and histopathology were used to evaluate the severity of ALI and the protective effect of pharmacological inhibition of GRK2. GRK2-siRNA was used to knock down the expression of GRK2 in AML12 cells in vitro. Results: ALI model mice exhibited increased blood levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and abnormal liver pathology accompanied by imbalanced L-glutathione (GSH) levels. Cisplatin administration upregulated GKR2, p-GRK2 and NADPH oxidase 4 (NOX4) expression in the liver tissues of ALI model mice. Compared to WT mice injected with cisplatin, Grk2± mice that received cisplatin showed significant improvements in liver function and pathological performance, decreased NOX4 levels, reduced endoplasmic reticulum (ER) stress, and diminished liver cell apoptosis. In vitro, the transfection of AML12 cells with siRNA significantly reduced NOX4 expression and inhibited cisplatin-induced reactive oxygen species production, ER stress (increased levels of GRP94, GRP78, p-elF2α and CHOP) and apoptotic death. Moreover, pharmacological treatment with drugs that inhibit GRK2 (CP-25 or paroxetine) significantly ameliorated cisplatin-induced ALI by improving liver pathological manifestations, inhibiting oxidative stress and ER stress, and reducing liver cell apoptosis. Similar results were observed in vitro. Conclusions: GRK2 mediates the development of cisplatin-induced ALI by modulating NOX4 and ER stress. Pharmacological inhibition of GRK2 with CP-25 or paroxetine effectively alleviated ALI. GRK2 can be used as a potential target for the prevention and treatment of liver injury. [ABSTRACT FROM AUTHOR]

Oxidative stress is a primary contributor to cerebral ischemia/reperfusion (CI/R) injury, and the use of antioxidants represents a crucial therapeutic strategy for managing CI/R injury. This study aims to explore the antioxidant effects of benzyl ferulate on CI/R injury and elucidate its underlying mechanisms. In vivo models of CI/R injury and hypoxia/reoxygenation (H/R) injury in SH‐SY5Y cells were established, followed by treatment with benzyl ferulate. The extent of oxidative stress was assessed through evaluations of neurobiological function, cerebral infarct volume, reactive oxygen species (ROS), apoptosis levels, etc. Results indicated that benzyl ferulate significantly downregulated the expression of NADPH oxidase 2 (NOX) 2/NOX4 while upregulating miRNAs (652/532/92b) in CI/R rats or SH‐SY5Y cells. It also reduced total NOX enzyme activity, enhanced superoxide dismutase (SOD) activity, decreased ROS and malondialdehyde (MDA) production, and inhibited cleaved caspase‐3 and Bax expression—ultimately leading to reduced cell apoptosis. Benzyl ferulate effectively mitigates oxidative stress injuries of middle cerebral artery occlusion (MCAO) rats or SH‐SY5Y cells subjected to H/R, and its mechanism appears to involve modulation of the miRNAs (652/532/92b)/NOX2/4 axis. This study first proved that benzyl ferulate is a promising antioxidant candidate for treating CI/R injury. [ABSTRACT FROM AUTHOR]

Introduction: Pyroptosis is an inflammatory form of programmed cell death that plays a significant role in tumorigenesis. Cinobufotalin (CB), a bufadienolide extracted from toad venom, is associated with antitumor effects in various cancers, including liver cancer. However, the role of CB in pyroptosis and its underlying mechanisms have not been well characterized. Methods: MTT, Colony formation, EdU, Wound healing and Transwell migration and invasion assays were applied to determine the effects of CB on the proliferation, migration, and invasion ability of hepatocellular carcinoma (HCC) cells in vitro. The subcutaneous xenograft mouse model and pulmonary metastasis model were used to evaluate the effect of CB on HCC cells in vivo. PCR, western blot, immunohistochemistry, immunofluorescence, and ELISA were used to verify the expression of proliferation, migration, pyroptosis, and inflammation related molecules after CB treatment. Using si-RNA and inhibitors to interfere with NOX4 and HLRP3 expression to validate the key signaling pathways of pyroptosis induced by CB treatment. Results: In vivo experiments using nude mice with xenografted HCC cells and in vitro experiments with HCC cell lines demonstrated that CB treatment significantly inhibited the proliferation, migration, and invasiveness of HCC cells. CB treatment also showed dose-dependent activation of the NLRP3 inflammasome complex in HCC cells, leading to gasdermin D-induced pyroptosis. However, these effects were abrogated via the pretreatment of HCC cells with VX-765, a caspase-1 inhibitor. Additionally, CB increased the production of reactive oxygen species (ROS) and H2O2, along with upregulating NOX4 protein expression in HCC cells. Conversely, NOX4 silencing or pretreatment with VAS2870 (an NOX4 inhibitor) or NAC (an ROS scavenger) suppressed the activation of the NLRP3 inflammasome complex and pyroptosis in CB-treated HCC cells. Discussion: Our study demonstrated that CB suppressed the proliferation, migration, and invasiveness of HCC cells by inducing pyroptosis through the activation of the NOX4/NLRP3/GSDMD signaling pathway. Therefore, our results suggest that CB is a promising therapeutic agent for HCC. [ABSTRACT FROM AUTHOR]

Periodontitis and infections with periodontal bacteria have been highlighted as risk factors for dementia. In recent years, attention has been drawn to the role of microglia cells in neurodegenerative diseases. However, there is limited knowledge of the influence of periodontal bacteria on microglia cells. The aim of the present study was to investigate the interactions between the periodontal bacteria Porphyromonas gingivalis and microglia cells and to unravel whether these interactions could contribute to the pathology of Alzheimer's disease. We found, through microarray analysis, that stimulation of microglia cells with P. gingivalis resulted in the upregulation of several Alzheimer's disease-associated genes, including NOX4. We also showed that P. gingivalis lipopolysaccharides (LPS) mediated reactive oxygen species (ROS) production and interleukin 6 (IL-6) and interleukin 8 (IL-8) induction via NOX4 in microglia. The viability of neurons was shown to be reduced by conditioned media from microglia cells stimulated with P. gingivalis LPS and the reduction was NOX4 dependent. The levels of total and phosphorylated tau in neurons were increased by conditioned media from microglia cells stimulated with P. gingivalis or LPS. This increase was NOX4- dependent. In summary, our findings provide us with a potential mechanistic explanation of how the periodontal pathogen P. gingivalis could trigger or exacerbate AD pathogenesis. [ABSTRACT FROM AUTHOR]

Introduction: Pyroptosis is an inflammatory form of programmed cell death that plays a significant role in tumorigenesis. Cinobufotalin (CB), a bufadienolide extracted from toad venom, is associated with antitumor effects in various cancers, including liver cancer. However, the role of CB in pyroptosis and its underlying mechanisms have not been well characterized. Methods: MTT, Colony formation, EdU, Wound healing and Transwell migration and invasion assays were applied to determine the effects of CB on the proliferation, migration, and invasion ability of hepatocellular carcinoma (HCC) cells in vitro. The subcutaneous xenograft mouse model and pulmonary metastasis model were used to evaluate the effect of CB on HCC cells in vivo. PCR, western blot, immunohistochemistry, immunofluorescence, and ELISA were used to verify the expression of proliferation, migration, pyroptosis, and inflammation related molecules after CB treatment. Using si-RNA and inhibitors to interfere with NOX4 and HLRP3 expression to validate the key signaling pathways of pyroptosis induced by CB treatment. Results: In vivo experiments using nude mice with xenografted HCC cells and in vitro experiments with HCC cell lines demonstrated that CB treatment significantly inhibited the proliferation, migration, and invasiveness of HCC cells. CB treatment also showed dose-dependent activation of the NLRP3 inflammasome complex in HCC cells, leading to gasdermin D-induced pyroptosis. However, these effects were abrogated via the pretreatment of HCC cells with VX-765, a caspase-1 inhibitor. Additionally, CB increased the production of reactive oxygen species (ROS) and H2O2, along with upregulating NOX4 protein expression in HCC cells. Conversely, NOX4 silencing or pretreatment with VAS2870 (an NOX4 inhibitor) or NAC (an ROS scavenger) suppressed the activation of the NLRP3 inflammasome complex and pyroptosis in CB-treated HCC cells. Discussion: Our study demonstrated that CB suppressed the proliferation, migration, and invasiveness of HCC cells by inducing pyroptosis through the activation of the NOX4/NLRP3/GSDMD signaling pathway. Therefore, our results suggest that CB is a promising therapeutic agent for HCC. [ABSTRACT FROM AUTHOR]

Background and purpose: Cannabidiol (CBD) is a phenolic terpene compound with anticancer, antioxidant, anti-inflammatory, antibacterial, neuroprotective, and anticonvulsant properties. Since the effects of CBD on sodium arsenite (As)-induced nephrotoxicity have not been fully determined, this study investigated the effect of CBD on As-induced nephrotoxicity by evaluating the NOX4 and NF-kB pathways in mice. Experimental approach: 48 male mice were divided into six groups (8 each) including group 1, receiving saline for 14 days; group 2, receiving CBD (10 mg/kg, intraperitoneally) from the 7th to the 14th day; group 3, receiving As (10 mg/kg) for 14 days by gavage; and treatment groups 4-6, receiving CBD (2.5, 5, and 10 mg/kg, i.p.) 1.5 h before As (10 mg/kg by gavage, for 14 days) from the 7th to the 14th day. Mice were anesthetized after overnight fasting on day 15, and the blood sample was collected from their hearts. The level of antioxidants and pro-inflammatory factors, the expression of ROS and TNF-α, NF-kB, NOX4, iNOS, cleaved PARP, and caspase-3 proteins were measured and histological studies were performed. Findings/Results: Exposure to As significantly increased kidney markers, oxidative stress, apoptosis, and inflammation in mice kidney tissue, and pretreatment with CBD reversed these changes. In addition, CBD significantly decreased the expression of NF-kB and NOX4, and the levels of pro-inflammatory factors and the expression of cleaved PARP and increased the level of antioxidants. Conclusion and implications: CBD ameliorated As-induced nephrotoxicity related to inhibiting oxidative stress, inflammation, and apoptosis, potentially through the NF-kB/Nox4 pathway.

Abstract Background Disruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell’s capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored. Methods An AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility. Results Our findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities. Conclusions This study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.

Rat H9c2 cardiomyocytes overexpressing the abscisic acid (ABA) hormone receptors LANCL1 and LANCL2 have an increased mitochondrial proton gradient, respiration, and vitality after hypoxia/reoxygenation. Our aim was to investigate the role of the ABA/LANCL1-2 system in ROS turnover in H9c2 cells. H9c2 cells were retrovirally infected to induce the overexpression or silencing of LANCL1 and LANCL2, without or with the concomitant silencing of the transcription factor ERRα. Enzymes involved in radical production or scavenging were studied by qRT-PCR and Western blot. The mitochondrial proton gradient and ROS were measured with specific fluorescent probes. ROS-generating enzymes decreased, ROS-scavenging enzymes increased, and mitochondrial ROS were reduced in LANCL1/2-overexpressing vs. control cells infected with the empty vector, while the opposite occurred in LANCL1/2-silenced cells. The knockdown of ERRα abrogated all beneficial effects on ROS turnover in LANCL1/2 overexpressing cells. Taken together, these results indicate that the ABA/LANCL1-2 system controls ROS turnover in H9c2 via ERRα. The ABA/LANCL system emerges as a promising target to improve cardiomyocyte mitochondrial function and resilience to oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Disruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell's capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored. Methods: An AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility. Results: Our findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities. Conclusions: This study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells. [ABSTRACT FROM AUTHOR]

Periodontitis and infections with periodontal bacteria have been highlighted as risk factors for dementia. In recent years, attention has been drawn to the role of microglia cells in neurodegenerative diseases. However, there is limited knowledge of the influence of periodontal bacteria on microglia cells. The aim of the present study was to investigate the interactions between the periodontal bacteria Porphyromonas gingivalis and microglia cells and to unravel whether these interactions could contribute to the pathology of Alzheimer’s disease. We found, through microarray analysis, that stimulation of microglia cells with P. gingivalis resulted in the upregulation of several Alzheimer’s disease-associated genes, including NOX4. We also showed that P. gingivalis lipopolysaccharides (LPS) mediated reactive oxygen species (ROS) production and interleukin 6 (IL-6) and interleukin 8 (IL-8) induction via NOX4 in microglia. The viability of neurons was shown to be reduced by conditioned media from microglia cells stimulated with P. gingivalis LPS and the reduction was NOX4 dependent. The levels of total and phosphorylated tau in neurons were increased by conditioned media from microglia cells stimulated with P. gingivalis or LPS. This increase was NOX4-dependent. In summary, our findings provide us with a potential mechanistic explanation of how the periodontal pathogen P. gingivalis could trigger or exacerbate AD pathogenesis.

Spinal cord injury (SCI) results in severe atrophy of skeletal muscle in paralyzed regions, and a decrease in the force generated by muscle per unit of cross-sectional area. Oxidation of skeletal muscle ryanodine 1 receptors (RyR1) reduces contractile force as a result of reduced binding of calstabin 1 to RyR1. One cause of RyR1 oxidation is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4). We have previously shown that, in rats, RyR1 was oxidized and bound less to calstabin 1 at 56 days after SCI by spinal cord transection. Here, we used a conditional knockout (KO) mouse model of Nox4 in skeletal muscle to investigate the role of Nox4 in reduced muscle specific force after SCI. Peak twitch force of extensor digitorum longus muscles in control mice after SCI was reduced by 42% compared with sham-operated controls, but was increased by ?43% in SCI Nox4 conditional KO mice compared with SCI controls, although it remained less than that for sham-operated controls. Unlike what was previously observed in rats after SCI, the expression of Nox4 was not increased in gastrocnemius muscle, and binding of calstabin 1 to RyR1 was not reduced in this muscle. The results suggest that Nox4 is directly involved in reduction in muscle twitch force after SCI, although further studies are needed to understand the mechanistic basis for this linkage.

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.

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

Hinokitiol (HKT) is one of the essential oil components found in the heartwood of Cupressaceae plants, and has been reported to have various bioactive effects, including anti-inflammatory effects. However, the improving effect of HKT on periodontitis, which is characterized by periodontal tissue inflammation and alveolar bone loss, has not been clearly revealed. Therefore, we investigated the periodontitis-alleviating effect of HKT and the related molecular mechanisms in human periodontal ligament cells. According to the study results, HKT downregulated SIRT1 and NOX4, which were increased by Porphyromonas gingivalis Lipopolysaccharide (PG-LPS) stimulation and were found to regulate pro-inflammatory mediators and oxidative stress through SIRT1/NOX4 signals. Additionally, by increasing the expression of osteogenic makers such as alkaline phosphatase, osteogenic induction of human periodontal ligament (HPDL) cells, which had been reduced by PG-LPS, was restored. Furthermore, we confirmed that NOX4 expression was regulated through regulation of SIRT1 expression with HKT. The in vitro effect of HKT on improving periodontitis was proven using the periodontal inflammation model, which induces periodontal inflammation using ligature, a representative in vivo model. According to in vivo results, HKT alleviated periodontal inflammation and restored damaged alveolar bone in a concentration-dependent manner in the periodontal inflammation model. Through this experiment, the positive effects of HKT on relieving periodontal tissue inflammation and recovering damaged alveolar bone, which are important treatment strategies for periodontitis, were confirmed. Therefore, these results suggest that HKT has potential in the treatment of periodontitis. [ABSTRACT FROM AUTHOR]

The NADPH oxidase NOX4 that releases H2O2 can mediate vasoprotective mechanisms under pathophysiological conditions in conductive arteries. However, the role of NOX4 in resistance arteries and in perivascular adipose tissue is not well understood. We hypothesized that NOX4 is of functional importance in resistance arteries and perivascular adipose tissue under dyslipidemia conditions. We detected elevated NOX4 expression in murine and human vessels under dyslipidemia. Diminishing Nox4 under these conditions led to endothelial dysfunction in resistance arteries. The mesenteric arteries of Nox4−/−/Ldlr−/− mice revealed decreased eNos mRNA expression. Inhibition of eNOS in those vessels did not affect vascular function, while in Ldlr−/− mice endothelial function was significantly altered. Anticontractile properties of perivascular adipose tissue at resistance arteries were diminished in Nox4−/−/Ldlr−/− compared with Ldlr−/− mice. In addition, the presence of perivascular adipose tissue further worsened endothelial dysfunction in mesenteric arteries under dyslipidemia conditions. Perivascular adipose tissue from mesenteric arteries revealed a higher expression of markers of white adipocytes compared to markers of beige/brown adipocytes. Among those white adipocyte markers, leptin was significantly less expressed in perivascular adipose tissue from Nox4−/−/Ldlr−/− mice compared with Ldlr−/− mice. Furthermore, in human perivascular adipose tissue with a profound pattern of white adipocyte marker genes, we detected a correlation of NOX4 and LEP expression. In addition, incubating arterial vessels with leptin induced nitrite release, indicating increased eNOS activity. In humans, a higher expression of leptin in perivascular adipose tissue correlated with eNOS expression in the corresponding left internal mammary artery. In conclusion, vascular function of resistance arteries was dependent on Nox4-derived H2O2, especially under dyslipidemia conditions. Perivascular adipose tissue of the mesenteric arteries with white adipose tissue characteristics further aggravated endothelial function through reduced leptin-eNOS signaling. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are a type of cytoplasmic vesicles secreted by a variety of cells. EVs originating from cells have been known to participate in cell communication, antigen presentation, immune cell activation, tolerance induction, etc. These EVs can also carry the active form of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Hydrogen (NADPH) oxidase, which is very essential for the production of reactive oxygen species (ROS) and that can then modulate processes such as cell regeneration. The aim of this study is to characterize the EVs isolated from U-937 and THP-1 cells, identify the NADPH oxidase (NOX) isoforms, and to determine whether EVs can modulate NOX4 and NOX2 in monocytes and macrophages. In our study, isolated EVs of U-937 were characterized using dynamic light scattering (DLS) spectroscopy and immunoblotting. The results showed that the exogenous addition of differentiation agents (either phorbol 12-myristate 13-acetate (PMA) or ascorbic acid) or the supplementation of EVs used in the study did not cause any stress leading to alterations in cell proliferation and viability. In cells cocultured with EVs for 72 h, strong suppression of NOX4 and NOX2 is evident when monocytes transform into macrophagic cells. We also observed lower levels of oxidative stress measured using immunoblotting and electron paramagnetic resonance spectroscopy under the EVs co-cultured condition, which also indicates that EVs might contribute significantly by acting as an antioxidant source, which agrees with previous studies that hypothesized the role of EVs in therapeutics. Therefore, our results provide evidence for NOX regulation by EVs in addition to its role as an antioxidant cargo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? NADPH oxidases (NOXs) are induced following ischemia-reperfusion (IR), which aggravates acute kidney injury NOX4 inhibition improves kidney and mitochondria function in IR, and dampens renal injury What is the implication of the main finding? These findings can facilitate development of new preventive strategies of IR-induced AKI, which is a global medical problem. Background: Acute kidney injury (AKI) is a sudden episode of kidney failure which is frequently observed at intensive care units and related to high morbidity/mortality. Although AKI can have many different causes, ischemia–reperfusion (IR) injury is the main cause of AKI. Mechanistically, NADPH oxidases (NOXs) are involved in the pathophysiology contributing to oxidative stress following IR. Previous reports have indicated that knockout of NOX4 may offer protection in cardiac and brain IR, but there is currently less knowledge about how this could be exploited therapeutically and whether this could have significant protection in IR-induced AKI. Aim: To investigate the hypothesis that a novel and specific NOX4 inhibitor (GLX7013114) may have therapeutic potential on kidney and mitochondrial function in a mouse model of IR-induced AKI. Methods: Kidneys of male C57BL/6J mice were clamped for 20 min, and the NOX4 inhibitor (GLX7013114) was administered via osmotic minipump during reperfusion. Following 3 days of reperfusion, kidney function (i.e., glomerular filtration rate, GFR) was calculated from FITC-inulin clearance and mitochondrial function was assessed by high-resolution respirometry. Renal histopathological evaluations (i.e., hematoxylin–eosin) and TUNEL staining were performed for apoptotic evaluation. Results: NOX4 inhibition during reperfusion significantly improved kidney function, as evidenced by a better-maintained GFR (p < 0.05) and lower levels of blood urea nitrogen (p < 0.05) compared to untreated IR animals. Moreover, IR caused significant tubular injuries that were attenuated by simultaneous NOX4 inhibition (p < 0.01). In addition, the level of renal apoptosis was significantly reduced in IR animals with NOX4 inhibition (p < 0.05). These favorable effects of the NOX4 inhibitor were accompanied by enhanced Nrf2 Ser40 phosphorylation and conserved mitochondrial function, as evidenced by the better-preserved activity of all mitochondrial complexes. Conclusion: Specific NOX4 inhibition, at the time of reperfusion, significantly preserves mitochondrial and kidney function. These novel findings may have clinical implications for future treatments aimed at preventing AKI and related adverse events, especially in high-risk hospitalized patients. [ABSTRACT FROM AUTHOR]

Chronic hyperglycemia induces intrarenal oxidative stress due to the excessive production of reactive oxygen species (ROS), leading to a cascade of events that contribute to the development and progression of diabetic kidney disease (DKD). NOX5, a pro-oxidant NADPH oxidase isoform, has been identified as a significant contributor to renal ROS in humans. Elevated levels of renal ROS contribute to endothelial cell dysfunction and associated inflammation, causing increased endothelial permeability, which can disrupt the renal ecosystem, leading to progressive albuminuria and renal fibrosis in DKD. This study specifically examines the contribution of endothelial cell-specific human NOX5 expression in renal pathology in a transgenic mouse model of DKD. This study additionally compares NOX5 with the previously characterized NADPH oxidase, NOX4, in terms of their relative roles in DKD. Regardless of NOX4 pathway, this study found that endothelial cell-specific expression of NOX5 exacerbates renal injury, albuminuria and fibrosis. This is attributed to the activation of the endothelial mesenchymal transition (EMT) pathway via enhanced ROS formation and the modulation of redox-sensitive factors. These findings underscore the potential therapeutic significance of NOX5 inhibition in human DKD. The study proposes that inhibiting NOX5 could be a promising approach for mitigating the progression of DKD and strengthens the case for the development of NOX5-specific inhibitors as a potential therapeutic intervention. [ABSTRACT FROM AUTHOR]

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.

Glioblastomas are known for their poor clinical prognosis, with recurrent tumors often exhibiting greater invasiveness and faster growth rates compared to primary tumors. To understand the intratumoral changes driving this phenomenon, we employed single-cell sequencing to analyze the differences between two pairs of primary and recurrent glioblastomas. Our findings revealed an upregulation of ferroptosis in endothelial cells within recurrent tumors, identified by the significant overexpression of the NOX4 gene. Further analysis indicated that knocking down NOX4 in endothelial cells reduced the activity of the ferroptosis pathway. Utilizing conditioned media from endothelial cells with lower ferroptosis activity, we observed a decrease in the growth rate of glioblastoma cells. These results highlighted the complex role of ferroptosis within tumors and suggested that targeting ferroptosis in the treatment of glioblastomas requires careful consideration of its effects on endothelial cells, as it may otherwise produce counterproductive outcomes.

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.

Ummul Khair Imaduddin,1 Afiat Berbudi,2,&ast; Enny Rohmawaty3,&ast; 1Graduate School of Master Program in Anti Aging and Aesthetic Medicine, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia; 2Parasitology Division, Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia; 3Pharmacology & Therapy Division, Departement of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia&ast;These authors contributed equally to this workCorrespondence: Enny Rohmawaty, Pharmacology & Therapy Division, Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang, KM.21, Hegarmanah, Kec. Jatinangor, Kabupaten Sumedang, Bandung, West Java, 45363, Indonesia, Email e.rohmawaty@unpad.ac.idPurpose: To explore the potential therapeutic effects of Physalis angulata L. (Ciplukan) extract on lung fibrosis resolution in a Bleomycin-induced mouse model, researchers conducted a comprehensive study. The study focused on key genes associated with fibrosis progression, including Nox4, Mmp8, Klf4, and FAS, and assessed their mRNA expression levels following the administration of Ciplukan extract.Methods: A Bleomycin-induced mice model was divided into seven groups to investigate the effects of ciplukan extract on fibrosis-related gene expressions. Mice were induced with subcutaneously injected Bleomycin to generate lung fibrosis and given different doses of the Ciplukan extract for four weeks. Lung fibrosis mRNA expression was analyzed by semi-quantitative PCR for Nox4, Klf4, Mmp8, and FAS.Results: The administration of ciplukan extract resulted in a significant decrease in mRNA expression of Nox4 with p-value=0.000, Mmp8 with p-value =0.002, and Klf4 with p-value =0.007, indicating potential antifibrotic effects. However, FAS expression remained unchanged (p-value=0.127).Conclusion: Ciplukan extract exhibited promising effects on fibrosis-related gene expressions, particularly Nox4, Mmp8, and Klf4. This study suggests that the extract has the potential to intervene in fibrosis progression, offering a potential avenue for therapeutic strategies.Keywords: ciplukan, FAS, Klf4, lung fibrosis, Mmp8, Nox4, pulmonary fibrosis, Physalis angulata L

Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

Simple Summary: NOX4, PDE11A and GHR have previously been screened as candidate genes for litter size in sheep. Therefore, it is necessary to validate these genes in the sheep population and identify loci that are associated with litter size. In this study, the results of association analysis showed that c.1057-4C > T in NOX4 and c.1983C > T in PDE11A were significantly associated with litter size in Small Tail Han sheep, and their effects were independent of each other. In summary, this study provided two new genetic markers for improving litter size in sheep. In previous studies, NOX4, PDE11A and GHR genes have been screened as important candidate genes for litter size in sheep by using the GWAS method; however, neither their effects on litter size nor the loci associated with litter size have been identified. In this study, three candidate loci (c.1057-4C > T in NOX4, c.1983C > T in PDE11A and c.1618C > T in GHR) were first screened based on our previous resequencing data of 10 sheep breeds. After the three loci were genotyped using Sequenom MassARRAY technology, we carried out population genetics analysis on the three loci and performed association analysis between the polymorphism of the three loci and the litter size of sheep. The results of population genetics analysis suggested that c.1057-4C > T in NOX4 and c.1983C > T in PDE11A may be subject to natural or artificial selection. The results of association analysis indicated that litter size was significantly associated with c.1057-4C > T in NOX4 and c.1983C > T in PDE11A (p < 0.05) in Small Tail Han sheep, and there was no significant interaction effect between the two loci on the litter size. In summary, c.1057-4C > T in NOX4 and c.1983 C > T in PDE11A can be considered candidate molecular markers for improving litter size in sheep. [ABSTRACT FROM AUTHOR]

[Display omitted] • Androgen-dependent benign prostatic hyperplasia (BPH) model exerts excessive ROS, which was revealed by the involvement of the AR and NOX4 networks. • In BPH in vitro and in vivo model, the NADPH oxidase inhibitor Apocynin (Apo) suppresses inflammation and proliferation to suppress BPH through the AR/NOX4/TGF-β signaling. • Apo exerts anti-proliferative, anti-inflammatory, and anti-oxidant effects, highlighting the potential of Apo as a therapeutic agent for treatment of BPH. 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. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro- oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adultmice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function. Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2,wetestedthe hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult -KD mice. Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KDmice. Targeting cardiac expression in KDmice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase. Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function. [ABSTRACT FROM AUTHOR]

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

The hydrogen peroxide (H2O2)‐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, H2O2 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 H2O2 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 H2O2 production. [ABSTRACT FROM AUTHOR]

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

Extracellular vesicles (EVs) are a type of cytoplasmic vesicles secreted by a variety of cells. EVs originating from cells have been known to participate in cell communication, antigen presentation, immune cell activation, tolerance induction, etc. These EVs can also carry the active form of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Hydrogen (NADPH) oxidase, which is very essential for the production of reactive oxygen species (ROS) and that can then modulate processes such as cell regeneration. The aim of this study is to characterize the EVs isolated from U-937 and THP-1 cells, identify the NADPH oxidase (NOX) isoforms, and to determine whether EVs can modulate NOX4 and NOX2 in monocytes and macrophages. In our study, isolated EVs of U-937 were characterized using dynamic light scattering (DLS) spectroscopy and immunoblotting. The results showed that the exogenous addition of differentiation agents (either phorbol 12-myristate 13-acetate (PMA) or ascorbic acid) or the supplementation of EVs used in the study did not cause any stress leading to alterations in cell proliferation and viability. In cells co-cultured with EVs for 72 h, strong suppression of NOX4 and NOX2 is evident when monocytes transform into macrophagic cells. We also observed lower levels of oxidative stress measured using immunoblotting and electron paramagnetic resonance spectroscopy under the EVs co-cultured condition, which also indicates that EVs might contribute significantly by acting as an antioxidant source, which agrees with previous studies that hypothesized the role of EVs in therapeutics. Therefore, our results provide evidence for NOX regulation by EVs in addition to its role as an antioxidant cargo.

Abstract 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

Abstract 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.

Rat H9c2 cardiomyocytes overexpressing the abscisic acid (ABA) hormone receptors LANCL1 and LANCL2 have an increased mitochondrial proton gradient, respiration, and vitality after hypoxia/reoxygenation. Our aim was to investigate the role of the ABA/LANCL1-2 system in ROS turnover in H9c2 cells. H9c2 cells were retrovirally infected to induce the overexpression or silencing of LANCL1 and LANCL2, without or with the concomitant silencing of the transcription factor ERRα. Enzymes involved in radical production or scavenging were studied by qRT-PCR and Western blot. The mitochondrial proton gradient and ROS were measured with specific fluorescent probes. ROS-generating enzymes decreased, ROS-scavenging enzymes increased, and mitochondrial ROS were reduced in LANCL1/2-overexpressing vs. control cells infected with the empty vector, while the opposite occurred in LANCL1/2-silenced cells. The knockdown of ERRα abrogated all beneficial effects on ROS turnover in LANCL1/2 overexpressing cells. Taken together, these results indicate that the ABA/LANCL1-2 system controls ROS turnover in H9c2 via ERRα. The ABA/LANCL system emerges as a promising target to improve cardiomyocyte mitochondrial function and resilience to oxidative stress.

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro-oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adult mice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function.Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2, we tested the hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult Angptl2-KD mice.Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac Nox4 expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac Nox4 expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KD mice. Targeting cardiac Nox4 expression in KD mice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase.Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function.

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.

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. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN), a severe diabetes complication, causes kidney morphological and structural changes due to extracellular matrix accumulation. This accumulation is caused mainly by oxidative stress. Semi-essential amino acid derivative taurine has powerful antioxidant and antifibrotic effects. The aim of this study was to investigate the renoprotective effects of taurine through its possible roles in oxidative stress, extracellular matrix proteins, and the signaling pathways associated with the accumulation of extracellular matrix proteins in DN rats. 29 Wistar albino rats were randomly separated into control, taurine, diabetes, and diabetes + taurine groups. Diabetes animals were injected 45 mg/kg streptozosine. Taurine is given by adding to drinking water as 1% (w/v). Urine, serum, and kidney tissue were collected from rats for biochemical and histological analysis after 12 weeks. According to the studies, taurine significantly reduces the levels of malondialdehyde (MDA), total oxidant status (TOS), and protein expression of NADPH oxidase 4 (NOX4) that increase in diabetic kidney tissue. Also, decreased superoxide dismutase (SOD) activity levels significantly increased with taurine in diabetic rats. Moreover, increased mRNA and protein levels of fibronectin decreased with taurine. The matrix metalloproteinase (MMP)-2 and MMP-9 activities and their mRNA levels increased significantly, and this increase was significantly summed with taurine. There was a decrease in mRNA expression of Extracellular matrix metalloproteinase inducer (EMMPRIN). Taurine significantly increased this decrease. Diabetes increased mRNA expressions of transforming growth factor (TGF)-β and Smad2/3. Taurine significantly reduced this induction. TGF-β protein expression, p38, and Smad2/3 activations were also inhibited, but taurine was suppressed significantly. All these findings indicate that taurine may be an effective practical strategy to prevent renal diabetic injury. [ABSTRACT FROM AUTHOR]

Currently, the treatment for sepsis-induced acute lung injury mainly involves mechanical ventilation with limited use of drugs, highlighting the urgent need for new therapeutic options. As a pivotal aspect of acute lung injury, the pathologic activation and apoptosis of endothelial cells related to oxidative stress play a crucial role in disease progression, with NOX4 and Nrf2 being important targets in regulating ROS production and clearance. Echinacoside, extracted from the traditional Chinese herbal plant Cistanche deserticola, possesses diverse biological activities. However, its role in sepsis-induced acute lung injury remains unexplored. Moreover, although some studies have demonstrated the regulation of NOX4 expression by SIRT1, the specific mechanisms are yet to be elucidated. Therefore, this study aimed to investigate the effects of echinacoside on sepsis-induced acute lung injury and oxidative stress in mice and to explore the intricate regulatory mechanism of SIRT1 on NOX4. We found that echinacoside inhibited sepsis-induced acute lung injury and oxidative stress while preserving endothelial function. In vitro experiments demonstrated that echinacoside activated SIRT1 and promoted its expression. The activated SIRT1 was competitively bound to p22 phox, inhibiting the activation of NOX4 and facilitating the ubiquitination and degradation of NOX4. Additionally, SIRT1 deacetylated Nrf2, promoting the downstream expression of antioxidant enzymes, thus enhancing the NOX4-Nrf2 axis and mitigating oxidative stress-induced endothelial cell pathologic activation and mitochondrial pathway apoptosis. The SIRT1-mediated anti-inflammatory and antioxidant effects of echinacoside were validated in vivo. Consequently, the SIRT1-regulated NOX4-Nrf2 axis may represent a crucial target for echinacoside in the treatment of sepsis-induced acute lung injury. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and its prevalence increases with age. The irregular and rapid contraction of the atria can lead to ineffective blood pumping, local blood stasis, blood clots, ischemic stroke, and heart failure. NADPH oxidases (NOX) and mitochondria are the main sources of reactive oxygen species in the heart, and dysregulated activation of NOX and mitochondrial dysfunction are associated with AF pathogenesis. NOX- and mitochondria-derived oxidative stress contribute to the onset of paroxysmal AF by inducing electrophysiological changes in atrial myocytes and structural remodeling in the atria. Because high atrial activity causes cardiac myocytes to expend extremely high energy to maintain excitation-contraction coupling during persistent AF, mitochondria, the primary energy source, undergo metabolic stress, affecting their morphology, Ca2+ handling, and ATP generation. In this review, we discuss the role of oxidative stress in activating AF-triggered activities, regulating intracellular Ca2+ handling, and functional and anatomical reentry mechanisms, all of which are associated with AF initiation, perpetuation, and progression. Changes in the extracellular matrix, inflammation, ion channel expression and function, myofibril structure, and mitochondrial function occur during the early transitional stages of AF, opening a window of opportunity to target NOX and mitochondria-derived oxidative stress using isoform-specific NOX inhibitors and mitochondrial ROS scavengers, as well as drugs that improve mitochondrial dynamics and metabolism to treat persistent AF and its transition to permanent AF. [ABSTRACT FROM AUTHOR]

Our previous studies indicated that the benzene metabolite hydroquinone (HQ) evokes the ROS/p38 MAPK/protein phosphatase 2A/tristetraprolin axis, leading to increased TNF-α expression in human acute myeloid leukemia cell lines U937 and HL-60. In this study, we aimed to identify the upstream pathway involved in ROS-mediated TNF-α expression. HQ treatment increased SIDT2 expression, which subsequently decreased miR-25 and SIRT3 expression in U937 cells. Notably, miR-25 downregulation promoted SIDT2 expression in HQ-treated U937 cells. SIDT2 induced lysosomal degradation of SIRT3 mRNA, but inhibited miR-25 expression through a lysosome-independent pathway. MiR-25 inhibition reduced NOX4 mRNA turnover, resulting in increased NOX4 protein levels. NOX4 induces mitochondrial ROS production and HuR downregulation. Restoration of HuR expression increased SIRT3 expression, suggesting that NOX4-mediated HuR downregulation promotes SIDT2-mediated degradation of SIRT3 mRNA. Inhibition of NOX4 or SIRT3 overexpression abolished HQ-induced ROS production, thereby abolishing TNF-α upregulation. Overall, these results indicate that SIDT2 regulates the miR-25/NOX4/HuR axis and SIRT3 mRNA destabilization, leading to ROS-mediated TNF-α upregulation in HQ-treated U937 cells. HQ-induced increase in TNF-α expression in HL-60 cells was also mediated through a similar pathway. [ABSTRACT FROM AUTHOR]

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.

AIM: To investigate the retinoprotective role of Apigenin (Api) against high glucose (HG)-induced human retinal microvascular endothelial cells (HRMECs), and to explore its regulatory mechanism. METHODS: HRMECs were stimulated by HG for 48h to establish the in vitro cell model. Different concentrations of Api (2.5, 5, and 10 μmol/L) were applied for treatment. Cell counting kit-8 (CCK-8), Transwell, and tube formation assays were performed to examine the effects of Api on the viability, migration, and angiogenesis in HG-induced HRMECs. Vascular permeability was evaluated by Evans blue dye. The inflammatory cytokines and oxidative stress-related factors were measured using their commercial kits. Protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and p38 mitogen-activated protein kinase (MAPK) was measured by Western blot. RESULTS: Api prevented HG-induced HRMECs viability, migration, angiogenesis, and vascular permeability in a concentration-dependent manner. Meanwhile, Api also concentration-dependently inhibited inflammation and oxidative stress in HRMECs exposed to HG. In addition, HG caused an elevated expression of NOX4, which was retarded by Api treatment. HG stimulation facilitated the activation of p38 MAPK signaling in HRMECs, and Api could weaken this activation partly via downregulating NOX4 expression. Furthermore, overexpression of NOX4 or activation of p38 MAPK signaling greatly weakened the protective role of Api against HG-stimulated HRMECs. CONCLUSION: Api might exert a beneficial role in HG-stimulated HRMECs through regulating NOX4/p38 MAPK pathway.

The NADPH oxidase NOX4 that releases H2O2 can mediate vasoprotective mechanisms under pathophysiological conditions in conductive arteries. However, the role of NOX4 in resistance arteries and in perivascular adipose tissue is not well understood. We hypothesized that NOX4 is of functional importance in resistance arteries and perivascular adipose tissue under dyslipidemia conditions. We detected elevated NOX4 expression in murine and human vessels under dyslipidemia. Diminishing Nox4 under these conditions led to endothelial dysfunction in resistance arteries. The mesenteric arteries of Nox4−/−/Ldlr−/− mice revealed decreased eNos mRNA expression. Inhibition of eNOS in those vessels did not affect vascular function, while in Ldlr−/− mice endothelial function was significantly altered. Anticontractile properties of perivascular adipose tissue at resistance arteries were diminished in Nox4−/−/Ldlr−/− compared with Ldlr−/− mice. In addition, the presence of perivascular adipose tissue further worsened endothelial dysfunction in mesenteric arteries under dyslipidemia conditions. Perivascular adipose tissue from mesenteric arteries revealed a higher expression of markers of white adipocytes compared to markers of beige/brown adipocytes. Among those white adipocyte markers, leptin was significantly less expressed in perivascular adipose tissue from Nox4−/−/Ldlr−/− mice compared with Ldlr−/− mice. Furthermore, in human perivascular adipose tissue with a profound pattern of white adipocyte marker genes, we detected a correlation of NOX4 and LEP expression. In addition, incubating arterial vessels with leptin induced nitrite release, indicating increased eNOS activity. In humans, a higher expression of leptin in perivascular adipose tissue correlated with eNOS expression in the corresponding left internal mammary artery. In conclusion, vascular function of resistance arteries was dependent on Nox4-derived H2O2, especially under dyslipidemia conditions. Perivascular adipose tissue of the mesenteric arteries with white adipose tissue characteristics further aggravated endothelial function through reduced leptin-eNOS signaling.

Hinokitiol (HKT) is one of the essential oil components found in the heartwood of Cupressaceae plants, and has been reported to have various bioactive effects, including anti-inflammatory effects. However, the improving effect of HKT on periodontitis, which is characterized by periodontal tissue inflammation and alveolar bone loss, has not been clearly revealed. Therefore, we investigated the periodontitis-alleviating effect of HKT and the related molecular mechanisms in human periodontal ligament cells. According to the study results, HKT downregulated SIRT1 and NOX4, which were increased by Porphyromonas gingivalis Lipopolysaccharide (PG-LPS) stimulation and were found to regulate pro-inflammatory mediators and oxidative stress through SIRT1/NOX4 signals. Additionally, by increasing the expression of osteogenic makers such as alkaline phosphatase, osteogenic induction of human periodontal ligament (HPDL) cells, which had been reduced by PG-LPS, was restored. Furthermore, we confirmed that NOX4 expression was regulated through regulation of SIRT1 expression with HKT. The in vitro effect of HKT on improving periodontitis was proven using the periodontal inflammation model, which induces periodontal inflammation using ligature, a representative in vivo model. According to in vivo results, HKT alleviated periodontal inflammation and restored damaged alveolar bone in a concentration-dependent manner in the periodontal inflammation model. Through this experiment, the positive effects of HKT on relieving periodontal tissue inflammation and recovering damaged alveolar bone, which are important treatment strategies for periodontitis, were confirmed. Therefore, these results suggest that HKT has potential in the treatment of periodontitis.

Background: Acute kidney injury (AKI) is a sudden episode of kidney failure which is frequently observed at intensive care units and related to high morbidity/mortality. Although AKI can have many different causes, ischemia–reperfusion (IR) injury is the main cause of AKI. Mechanistically, NADPH oxidases (NOXs) are involved in the pathophysiology contributing to oxidative stress following IR. Previous reports have indicated that knockout of NOX4 may offer protection in cardiac and brain IR, but there is currently less knowledge about how this could be exploited therapeutically and whether this could have significant protection in IR-induced AKI. Aim: To investigate the hypothesis that a novel and specific NOX4 inhibitor (GLX7013114) may have therapeutic potential on kidney and mitochondrial function in a mouse model of IR-induced AKI. Methods: Kidneys of male C57BL/6J mice were clamped for 20 min, and the NOX4 inhibitor (GLX7013114) was administered via osmotic minipump during reperfusion. Following 3 days of reperfusion, kidney function (i.e., glomerular filtration rate, GFR) was calculated from FITC-inulin clearance and mitochondrial function was assessed by high-resolution respirometry. Renal histopathological evaluations (i.e., hematoxylin–eosin) and TUNEL staining were performed for apoptotic evaluation. Results: NOX4 inhibition during reperfusion significantly improved kidney function, as evidenced by a better-maintained GFR (p < 0.05) and lower levels of blood urea nitrogen (p < 0.05) compared to untreated IR animals. Moreover, IR caused significant tubular injuries that were attenuated by simultaneous NOX4 inhibition (p < 0.01). In addition, the level of renal apoptosis was significantly reduced in IR animals with NOX4 inhibition (p < 0.05). These favorable effects of the NOX4 inhibitor were accompanied by enhanced Nrf2 Ser40 phosphorylation and conserved mitochondrial function, as evidenced by the better-preserved activity of all mitochondrial complexes. Conclusion: Specific NOX4 inhibition, at the time of reperfusion, significantly preserves mitochondrial and kidney function. These novel findings may have clinical implications for future treatments aimed at preventing AKI and related adverse events, especially in high-risk hospitalized patients.

AbstractDiabetic kidney disease (DKD) is a severe complication of type 2 diabetes mellitus (T2DM). However, the pathogenesis of DKD remains unclear, and effective treatment strategies are still lacking. Ginsenoside Rg1 (Rg1) has been reported to improve DKD, but the mechanism is unclear. NADPH oxidase 4 (NOX4) is an essential reactive oxygen species (ROS) source in the kidney. The mitogen-activated protein kinase (MAPK) signaling may exacerbate renal fibrosis. Therefore, we hypothesized that Rg1 might alleviate renal injury and fibrosis by inhibiting NOX4 and MAPK signaling in T2DM-induced DKD. We found that Rg1 significantly improves lipid deposition, fibrosis, and ROS production and reduces NOX4, p22phox, p47phox, p-ERK, p-JNK, and p-P38 MAPK expressions in the T2DM mice kidneys. We also found that the high-fat diet treatment in mice and the palmitate (PA) and PA + HG (high glucose) exposure in human mesangial cells could significantly induce lipid deposition, ROS production, fibrosis, and the activation of NOX4–MAPK signaling. The results suggest that high lipid and glucose may play a significant role in DKD progression, while Rg1 may attenuate renal fibrosis by inhibiting NOX4–MAPK signaling.

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 role of inflammation and immunity in the pathomechanism of neurodegenerative diseases has become increasingly relevant within the past few years. In this context, the NOD-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in the activation of inflammatory responses by promoting the maturation and secretion of pro-inflammatory cytokines such as interleukin-1β and interleukin-18. We hypothesized that the interplay between nuclear factor erythroid 2-related factor 2 (Nrf2) and NADPH oxidase 4 (NOX4) may play a critical role in the activation of the NLRP3 inflammasome and subsequent inflammatory responses. After priming mixed glial cultures with lipopolysaccharide (LPS), cells were stimulated with ATP, showing a significant reduction of IL1-β release in NOX4 and Nrf2 KO mice. Importantly, NOX4 inhibition using GKT136901 also reduced IL-1β release, as in NOX4 KO mixed glial cultures. Moreover, we measured NOX4 and NLRP3 expression in wild-type mixed glial cultures following LPS treatment, observing that both increased after TLR4 activation, while 24 h treatment with tert-butylhydroquinone, a potent Nrf2 inducer, significantly reduced NLRP3 expression. LPS administration resulted in significant cognitive impairment compared to the control group. Indeed, LPS also modified the expression of NLRP3 and NOX4 in mouse hippocampus. However, mice treated with GKT136901 after LPS impairment showed a significantly improved discrimination index and recovered the expression of inflammatory genes to normal levels compared with wild-type animals. Hence, we here validate NOX4 as a key player in NLRP3 inflammasome activation, suggesting NOX4 pharmacological inhibition as a potent therapeutic approach in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]