NADPH Oxidase 4 Overexpression Research Articles

Epigenetic mechanism of miR-26b-5p-enriched MSCs-EVs attenuates spinal cord injury

Mesenchymal stem cells (MSCs) and extracellular vesicles (EVs) are promising therapies for the treatment of spinal cord injury (SCI). This study sought to explore the epigenetic mechanism of miR-26b-5p-enriched MSCs-EVs in SCI. MSCs and MSCs-EVs were isolated and characterized. The SCI rat model was established, followed by Basso-Beattie-Bresnahan scoring and H&E staining. In vitro cell models were established in PC12 cells with lipopolysaccharide (LPS) treatment, followed by cell viability evaluation using CCK-8 assay. The levels of miR-26b-5p, lysine demethylase 6A (KDM6A), NADPH oxidase 4 (NOX4), reactive oxygen species (ROS), and inflammatory factors (TNF-α/IL-1β/IL-6) in tissues and cells were detected. The levels of cy3-lablled miR-26b-5p in tissues and cells were observed by confocal microscopy. The binding of miR-26b-5p to KDM6A 3′UTR and the enrichments of KDM6A and H3K27me3 at the NOX4 promoter were analyzed. MSCs-EVs attenuated motor dysfunction, inflammation, and oxidative stress in SCI rats. MSCs-EVs delivered miR-26b-5p into PC12 cells to reduce LPS-induced inflammation and ROS production and enhance cell viability. miR-26b-5p inhibited KDM6A, and KDM6A reduced H3K27me3 at the NOX4 promoter to promote NOX4. Overexpression of KDM6A or NOX4 reversed the alleviative role of MSCs-EVs in SCI or LPS-induced cell injury. Overall, MSCs-EVs delivered miR-26b-5p into cells to target the KDM6A/NOX4 axis and facilitate the recovery from SCI.

Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy.

Human corneal-endothelial cells (hCEnCs) are located on the inner layer of the cornea. Injury to CEnCs leads to permanent corneal edema, requiring corneal transplantation. NADPH oxidase 4 (NOX4) has been reported to be implicated in the pathogenesis of CEnCs diseases. Thus, we investigated the role of NOX4 in CEnCs in this study. In an animal study, siRNA for NOX4 (siNOX4) or plasmid for NOX4 (pNOX4) was introduced into the corneal endothelium of rats by electroporation, using a square-wave electroporator (ECM830, Havard apparatus) to decrease or increase the expression of NOX4, respectively, and the rat corneas were cryoinjured through contact with a metal rod of 3 mm diameter frozen in liquid nitrogen for 10 min. The immunofluorescence staining of NOX4 and 8-OHdG showed that the levels of NOX4 and 8-OHdG were decreased in the siNOX4 group compared to the siControl, and increased in the pNOX4 group compared to the pControl at one week after treatment. Without cryoinjury, corneal opacity was more severe, and the density of CEnCs was lower, in pNOX4-treated rats compared to pControl. After cryoinjury, the corneas were more transparent, and the CEnC density was higher, in siNOX4-treated rats. The hCEnCs were cultured and transfected with siNOX4 and pNOX4. The silencing of NOX4 in hCEnCs resulted in a normal cell shape, higher viability, and higher proliferation rate than those transfected with the siControl, while NOX4 overexpression had the opposite effect. NOX4 overexpression increased the number of senescent cells and intracellular oxidative stress levels. NOX4 overexpression increased ATF4 and ATF6 levels, and nuclear translocation of XBP-1, which is the endoplasmic reticulum (ER) stress marker, while the silencing of NOX4 had the opposite effect. Additionally, the mitochondrial membrane potential was hyperpolarized by the silencing of NOX4, and depolarized by NOX4 overexpression. The LC3II levels, a marker of autophagy, were decreased by the silencing of NOX4, and increased by NOX4 overexpression. In conclusion, NOX4 plays a pivotal role in the wound-healing and senescence of hCEnCs, by modulating oxidative stress, ER stress, and autophagy. The regulation of NOX4 may be a potential therapeutic strategy for regulating the homeostasis of CEnCs, and treating corneal-endothelial diseases.

Influence of intestinal microbiota on autophagy in colorectal cancer.

e15505 Background: Gut microbiota favors butyrate-forming bacteria and maintains homeostatic balance which is revoked during the development of colorectal cancer (CRC)-associated dysbiosis. Butyrate is a short chain fatty acid that acts as a lysine deacetylase inhibitor and is known to activate the AMPK pathway as well as inhibiting inflammation and ROS production. The role of AMPK activation in diabetes-associated complications including colorectal cancer is poorly understood. This project examines the role of dysbiosis and butyrate on CRC onset and disease progression. Methods: C57BL/6- Apctm1Tyj/J ( APC) mice that spontaneously develop CRC were used and the study included four groups of mice (n = 3 for each): (1) Control C57BL/6, (2) APC, (3) APC treated with probiotics rich in butyrate forming bacteria and (4) APC treated with butyrate. Probiotics and butyrate were administered by oral gavage and intraperitoneally, respectively. Feces were collected at early (when CRC is developed at 8 weeks of age) and late (at 16 weeks of age, day of sacrifice) timepoints, and microbial composition was identified by 16S rRNA sequencing. Mice colons following sacrifice were also harvested for molecular, anatomical, and histological analyses. Results: The butyrate kinase gene was assessed in fecal samples. Butyrate forming bacteria were significantly reduced in APC mice but elevated in APC receiving probiotics when compared to control mice. Anatomically, polyp number was reduced in groups receiving probiotics and butyrate when compared to APC mice without treatment (p < 0.01 for each). Histologically, butyrate and probiotics ameliorated the histological changes observed in in APC mice as assessed by PAS staining. Furthermore, we observed a decrease in goblet cells, and increased number of crypts on distal colon histology. The expression of NADPH Oxidase 4 (NOX4) and TIGAR, a P53 target protein expression were significantly increased in APC mice and restored following the administration of probiotics or butyrate. ROS production assessed by HPLC showed similar pattern across our 4 groups. The expression of the autophagy markers Beclin-1, ATG-12 and LC3-β were restored to control levels after probiotics and butyrate administration. 16S rRNA sequencing of fecal samples revealed significant differences in microbial composition between groups, most significantly at 16 weeks with Lachnospiraceae (ASV 175), Muribaculaceae (ASV 53) and Muribaculaceae (ASV 584) were decreased in APC when compared to control mice ( < -7 Log2 fold change each). Conclusions: Collectively, our data suggests colorectal cancer is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria. This disruption leads to over-expression of NOX4, and ROS production associated with alteration in autophagy, thus worsening gastrointestinal complications and CRC. Restoring butyrate forming bacteria might serve as an effective adjuvant to therapy in CRC.

Luteolin suppresses inflammation and oxidative stress in chronic obstructive pulmonary disease through inhibition of the NOX4‐mediated NF‐κB signaling pathway

Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation that predominantly affects the lung and peripheral airways. Previous investigation has underlined the efficacy of luteolin in the treatment of inflammation-related symptoms. Accordingly, our study concentrates on unveiling the effect of luteolin on COPD. Mice or A549 cells were treated with cigarette smoke (CS) to establish COPD models in vivo and in vitro. Then, the serum and bronchoalveolar lavage fluidof mice were harvested. The lung tissues of mice were subjected to hematoxylin-eosin staining to observe the degree of damage. The inflammation and oxidative stress factors level were calculated via enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. The expressions of nuclear factor-kappa B (NF-κB) pathway-related factors were detected by Western blot. In in vivo experiments, CS treatment reduced the weight of mice and promoted lung tissue damage, while luteolin attenuated the effect of CS on the mice. Moreover, luteolin inhibited the inflammation factors level, oxidative stress, and NADPH oxidase 4 (NOX4)-mediated NF-κB signaling pathway in CS-induced COPD mice. Similar results were obtained in in vitro experiments that luteolin alleviated CS-induced inflammation, oxidative stress, and NOX4-mediated NF-κB signaling pathway activation in CS-treated A549 cells. Besides, NOX4 overexpression offset the impacts of luteolin on the CS-induced A549 cells. Luteolin alleviates inflammation and oxidative stress in COPD via NOX4-mediated NF-κB signaling pathway, which provides a theoretical basis for the treatment of COPD with luteolin.

Perfluorooctane sulfonate-induced apoptosis in kidney cells by triggering the NOX4/ROS/JNK axis and antagonism of cannabidiol.

Perfluorooctane sulfonate (PFOS) is one of the persistent organic pollutants (POPs), which can cause severe nephrotoxicity in mammals. Cannabinol (CBD), a nonpsychoactive cannabinoid obtained from the cannabis plant, has attracted attention in recent years for its excellent antioxidant properties. NADPH oxidase 4 (NOX4) has an important effect in supporting normal renal physiological function. The potential mechanisms of PFOS nephrotoxicity and whether CBD can prevent renal damage caused by PFOS remain unclear. This work aimed to study the mechanisms of PFOS-induced kidney damage and the protective role of CBD against PFOS-induced kidney damage. We demonstrated that PFOS led to renal insufficiency and structural damage in mice, induced overexpression of NOX4 and the onset of oxidative stress, and activated apoptosis of the mitochondrial pathway via the JNK signaling pathway. However, treatment with CBD reversed these changes. For further investigation of the potential mechanism of PFOS-induced renal cell apoptosis, the expression of NOX4 was inhibited in vitro experiments using Apocynin, an effective NOX4 inhibitor. The outcomes showed that PFOS-induced ROS production and JNK signaling pathway activation and apoptosis in human embryonic kidney (HEK293) cells were significantly reduced after inhibition of NOX4. This suggests that PFOS-induced NOX4 overexpression serves as an upstream event for JNK pathway activation. In conclusion, the findings suggest that PFOS induces apoptosis in renal cells via the NOX4/ROS/JNK pathway. Meanwhile, CBD alleviated PFOS-induced renal apoptosis through the inhibition of NOX4/ROS/JNK axis activation.

Reduction of NADPH oxidase 4 in adipocytes contributes to the anti-obesity effect of dihydroartemisinin

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

NADPH oxidase 4 regulate the glycolytic metabolic reprogramming of microglial cells to promote M1 polarization.

This work aimed to investigate the role and mechanism of NADPH oxidase 4 (NOX4) in the polarization of microglial cells.Microglial cells were transfected with the NOX4 overexpression plasmid (pGL3-NOX4), and later treated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce its M1 polarization. Later, the F4/80 + CD86 + cell proportion was detected by flow cytometry (FCM), the inflammatory factor expression levels were analyzed through enzyme-linked immunosorbent assay (ELISA), while ionized calcium binding adapter molecule 1(IBA-1) and PKM2 expression were measured by immunofluorescence (IF) staining. In addition, dichlorodihydrofluorescein diacetate probe was utilized to detect the reactive oxygen species(ROS) levels, glucose uptake, and glycolysis, as well as lactic acid level. The expression of glycolytic enzymes PKM2, HK2, and citrate (Si)-synthas(CS) was detected by Western-blot (WB) assay. Moreover, the polarization level of microglial cells was detected after ROS expression was suppressed by the ROS inhibitor N-acetylcysteine (NAC). In mouse experiments, LPS was applied in inducing central neuroinflammation in NOX4 knockdown mouse model (KO) and wild-type mice (WT). Thereafter, the inflammatory factor levels and lactic acid level in mouse tissues were detected; IBA-1 and CD86 expression in mice was measured by IF staining; and the expression of glycolytic enzymes PKM2, HK2, and CS in the central nervous system (CNS) was also detected.After NOX4 overexpression in microglial cells, the M1 polarization level was upregulated, the F4/80 + CD86 + cell proportion increased, and inflammatory factors were upregulated. At the same time, the expression of glycolytic enzymes PKM2, HK2, and CS was upregulated. NAC pretreatment suppressed the effects of NOX4, reduced the F4/80 + CD86 + cell proportion, and suppressed the expression of PKM2, HK2, and CS. In the mouse model, the expression levels of CD86 in KO group decreased, and the inflammatory factors were also downregulated.NOX4 promotes glycolysis of microglial cells via ROS, thus accelerating M1 polarization and inflammatory factor expression. In this regard, NOX4 is promising as a new target for the treatment of neuroinflammation.

Regulation of FOXO3 on neuronal ferroptosis after intracerebral hemorrhage via modulating NOX4 transcription.

Intracerebral hemorrhage (ICH) is known to trigger neuronal ferroptosis while forkhead box O3 (FOXO3) is implicated in ICH. This study aimed to determine the specific effect of FOXO3 on neuronal ferroptosis after ICH. The ICH mouse model was established through the injection of bacterial collagenase type IV and the cell model was established in Hemin-induced HT-22 cells. Subsequently, neurological functions, brain water content, and histopathological changes in mice were assessed. HT-22 cell activity was examined via cell counting kit-8 (CCK-8) method, and the levels of FOXO3, NADPH oxidase 4 (NOX4), and glutathione peroxidase 4 (GPX4) in brain tissues and HT-22 cells were measured. Fe2+ concentration and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) in the tissues and cells were examined. The binding relationship between FOXO3 and the NOX4 promoter region was determined via chromatin-immunoprecipitation (Ch-IP). Rescue experiments were designed to probe the role of NOX4 in the regulation of FOXO3 on neuronal ferroptosis. FOXO3 was highly-expressed in ICH models while silencing FOXO3 alleviated brain damage, edema, and inflammatory infiltration in ICH mice. Meanwhile, silencing FOXO3 enhanced cell activity, diminished ROS and MDA activities and Fe2+ concentration, and elevated GSH and GPX4 levels in the tissues or cells. FOXO3 could bind to the NOX4 promoter and upregulate NOX4 transcription. NOX4 overexpression partially neutralized the repressive role of silencing FOXO3 in neuronal ferroptosis. Silencing FOXO3 attenuated ICH-induced neuronal ferroptosis via down-regulating NOX4 transcription levels, thus ameliorating post-ICH brain damage.

Role of the OTUB1/IRF7/NOX4 axis in oxidative stress injury and inflammatory responses in mice with Parkinson's disease.

Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders and is associated with oxidative stress injury (OSI) and inflammatory responses. This study sought to investigate the mechanism of ovarian tumour domain-containing ubiquitin aldehyde binding 1 (OTUB1) in OSI and inflammatory responses in PD, providing a theoretical foundation for PD treatment. The PD mouse model was established by an intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, followed by behavioural tests, observation of brain pathological changes, and quantification of inflammatory (TNF-α, IL-1β, and IL-10) and OS (ROS, SOD, and MDA) factors. Next, the expression levels of OTUB1, interferon regulatory factor 7 (IRF7), and NADPH oxidase 4 (NOX4) levels were determined by real-time quantitative polymerase chain reaction and western blot assay, the binding of OTUB1 to IRF7 was analysed by co-immunoprecipitation, and the ubiquitination level of IRF7 and the enrichment and binding of IRF7 and the NOX4 promoter were measured by chromatin immunoprecipitation and dual-luciferase assays. Afterwards, rescue experiments were performed with IRF7 or NOX4 overexpression in OTUB1 knockout PD mice. OTUB1 was upregulated in brain tissues of PD mice. Inhibition of OTUB1 alleviated PD progression, OSI, and inflammatory responses. OTUB1 stabilized IRF7 through deubiquitination, and IRF7 bound to the NOX4 promoter to promote NOX4 expression. IRF7 or NOX4 overexpression reversed the effects of silencing OTUB1 on OSI and inflammatory responses in PD mice. OTUB1-mediated deubiquitination stabilized IRF7 and upregulated NOX4 expression, thereby promoting OSI and inflammatory responses in PD mice.

Long Noncoding RNA-5657 Inhibits the Proliferation of Pancreatic Cells and Insulin Secretion in Gestational Diabetes by Inhibiting the NADPH Oxidase 4 (NOX4)

Multiple comorbidities are observed during pregnancy and there are many challenges for pregnant women with a previous history of diabetes. Pancreatic β-cell dysfunction is associated with gestational diabetes mellitus (GDM). This study intends to explore LncRNA-5657’s effect on proapoptotic cell proliferation and insulin secretion in gestational diabetes. Real-time quantitative fluorescence PCR measured LncRNA-5657 expression in GDM patient placental tissues along with analysis of cell activity, proliferation and insulin secretion by MTT assay, BrdU staining and ELISA. The relation between LncRNA-5657 and NOX4 was assessed by dual luciferase reporter assay. LncRNA-5657 was overexpressed in placental tissue of GDM patients compared to normal pregnant women. LncRNA-5657 overexpression promoted cell proliferation and insulin secretion, which was reversed by LncRNA-5657 downregulation. In addition, LncRNA-5657 negatively regulated NOX4 expression. Overexpression of NOX4 abolished the effect of LncRNA-5657 mimics on pancreatic β-cells, and knockdown of LncRNA-5657 promoted insulin secretion by upregulating NOX4 expression. In conclusion, LncRNA-5657 inhibits insulin secretion and β-cell proliferation by targeting NOX4, indicating that LncRNA-5657 might be a novel target for the treatment of GDM.

Hyperoside exerts osteoprotective effect on dexamethasone-induced osteoblasts by targeting NADPH Oxidase 4 (NOX4) to inhibit the reactive oxygen species (ROS) accumulation and activate c-Jun N-terminal kinase (JNK) pathway

ABSTRACT Hyperoside (Hyp) is a flavonoid active compound deriving from Chinese herbal medicines. Increasing studies have implicated that Hyp may serve as a predominant promoting factor in osteoblast differentiation. This paper investigates whether Hyp could relieve glucocorticoid-induced osteonecrosis of the femoral head (GONFH) via promoting osteoblast survival and differentiation as well as to uncover its potential mechanism. GONFH cell model was induced by treating MC3T3-E1 cells with dexamethasone (DEX). The viability, apoptosis, and osteogenic differentiation of DEX-induced cells with the presence or absence of Hyp were assessed by CCK-8, Tunel, ALP assay, and ARS staining, respectively. The NADPH Oxidase 4 (NOX4) overexpression was performed by transfection with overexpression vector. Besides, western blot was used to determine the levels of apoptosis-, osteogenic differentiation-, and c-Jun N-terminal kinase (JNK) signaling-related proteins. It was noticed that Hyp caused no significant effects on the viability of MC3T3-E1 cells without any treatment but significantly enhanced the viability of DEX-induced cells. Besides, Hyp inhibited the apoptosis in DEX-induced cells but enhanced ALP activity and calcium nodule formation. Additionally, Hyp declined NOX4 expression in DEX-induced cells. However, NOX4 overexpression partially reversed the impacts of Hyp on DEX-exposed MC3T3-E1 cells. Finally, Hyp suppressed the activation of ROS/JNK pathway in DEX-induced cells, which was then counteracted by NOX4 overexpression. In conclusion, Hyp could promote the survival and differentiation of DEX-induced osteoblasts by targeting NOX4 to inhibit the ROS/JNK pathway. These results provide evidence for the application of Hyp in treating GONFH.

NOX4 regulates TGFβ-induced proliferation and self-renewal in glioblastoma stem cells.

Glioblastoma (GBM) is the most aggressive and common glioma subtype, with a median survival of 15 months after diagnosis. Current treatments have limited therapeutic efficacy; thus, more effective approaches are needed. The glioblastoma tumoural mass is characterised by a small cellular subpopulation – glioblastoma stem cells (GSCs) – that has been held responsible for glioblastoma initiation, cell invasion, proliferation, relapse and resistance to chemo‐ and radiotherapy. Targeted therapies against GSCs are crucial, as is understanding the molecular mechanisms that govern the GSCs. Transforming growth factor β (TGFβ) signalling and reactive oxygen species (ROS) production are known to govern and regulate cancer stem cell biology. Among the differentially expressed genes regulated by TGFβ in a transcriptomic analysis of two different patient‐derived GSCs, we found NADPH oxidase 4 (NOX4) as one of the top upregulated genes. Interestingly, when patient tissues were analysed, NOX4 expression was found to be higher in GSCs versus differentiated cells. A functional analysis of the role of NOX4 downstream of TGFβ in several patient‐derived GSCs showed that TGFβ does indeed induce NOX4 expression and increases ROS production in a NOX4‐dependent manner. NOX4 downstream of TGFβ regulates GSC proliferation, and NOX4 expression is necessary for TGFβ‐induced expression of stem cell markers and of the transcription factor nuclear factor erythroid 2‐related factor 2 (NRF2), which in turn controls the cell’s antioxidant and metabolic responses. Interestingly, overexpression of NOX4 recapitulates the effects induced by TGFβ in GSCs: enhanced proliferation, stemness and NRF2 expression. In conclusion, this work functionally establishes NOX4 as a key mediator of GSC biology.

NOX4 promotes mucosal barrier injury in inflammatory bowel disease by mediating macrophages M1 polarization through ROS

NADPH oxidase 4 (NOX4) plays an important role in transporting electrons in the mitochondrial respiratory chain, which is also one major source of ROS. This study investigates the mechanism by which NOX4 promotes the M1 polarization of intestinal macrophages in inflammatory bowel disease (IBD) through ROS.Dextran sulfate sodium (DSS) was used to induce the inflammatory bowel disease (IBD) in wild-type (C57BL/6N, WT) and NOX4 knockout (C57BL/6N-NOX4em1cyagen, KO) mice. Body weights of mice were dynamically monitored and the disease active index (DAI) scores were assessed. H&E staining was performed to examine pathological changes, and immunohistochemical (IHC) staining was conducted to measure the expressions of TJ proteins (ZO-1, Occludin) and CD11c. Tissue ROS labeling was accomplished with ROS probe. More ucosal permeability was assessed by FITC-D. Tissue inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA), while the expressions of TJ proteins (ZO-1, Occludin) were measured through Western Blotting. After NOX4 inhibitor pretreatment of intestinal macrophages in vitro, polarization was induced by lipopolysaccharide (LPS) and IFN-γ, followed by determination of polarization degree. The polarized intestinal macrophages were co-cultured with Caco-2 cells, and their effect on the monolayer cell permeability was evaluated.DSS can induce the intestinal inflammation and mucosal barrier injury in mice. Besides, it can enhance the FITC-D permeability, reduce the TJ protein levels, and promote the CD11c and ROS expressions. In KO mice, intestinal inflammation was alleviated and barrier permeability was reduced. Moreover, the TJ protein levels were higher than those of WT mice, while the CD11c and ROS were down-regulated. In WT mice, the intestinal inflammation and barrier permeability could also be reduced after treatment with NOX4 inhibitor. Overexpression of NOX4 in intestinal macrophages could promote the macrophage M1 polarization while improving the barrier integrity of Caco-2 monolayer cells.NOX4 is capable of promoting M1 polarization of intestinal macrophages through ROS, thereby further aggravating the intestinal inflammation and mucosal barrier injury in IBD. NOX4 has potential as a novel therapeutic target for IBD.

NOX4-derived ROS-induced overexpression of FOXM1 regulates aerobic glycolysis in glioblastoma

BackgroundIncreased expression of the transcription factor Forkhead box M1 (FOXM1) has been reported to play an important role in the progression and development of multiple tumors, but the molecular mechanisms that regulate FOXM1 expression remain unknown, and the role of FOXM1 in aerobic glycolysis is still not clear.MethodsThe expression of FOXM1 and NADPH oxidase 4 (NOX4) in normal brain tissues and glioma was detected in data from the TCGA database and in our specimens. The effect of NOX4 on the expression of FOXM1 was determined by Western blot, qPCR, reactive oxygen species (ROS) production assays, and luciferase assays. The functions of NOX4 and FOXM1 in aerobic glycolysis in glioblastoma cells were determined by a series of experiments, such as Western blot, extracellular acidification rate (ECAR), lactate production, and intracellular ATP level assays. A xenograft mouse model was established to test our findings in vivo.ResultsThe expression of FOXM1 and NOX4 was increased in glioma specimens compared with normal brain tissues and correlated with poor clinical outcomes. Aberrant mitochondrial reactive oxygen species (ROS) generation of NOX4 induced FOXM1 expression. Mechanistic studies demonstrated that NOX4-derived MitoROS exert their regulatory role on FOXM1 by mediating hypoxia-inducible factor 1α (HIF-1α) stabilization. Further research showed that NOX4-derived MitoROS-induced HIF-1α directly activates the transcription of FOXM1 and results in increased FOXM1 expression. Overexpression of NOX4 or FOXM1 promoted aerobic glycolysis, whereas knockdown of NOX4 or FOXM1 significantly suppressed aerobic glycolysis, in glioblastoma cells. NOX4-induced aerobic glycolysis was dependent on elevated FOXM1 expression, as FOXM1 knockdown abolished NOX4-induced aerobic glycolysis in glioblastoma cells both in vitro and in vivo.ConclusionIncreased expression of FOXM1 induced by NOX4-derived MitoROS plays a pivotal role in aerobic glycolysis, and our findings suggest that inhibition of NOX4-FOXM1 signaling may present a potential therapeutic target for glioblastoma treatment.

Poncirin ameliorates oxygen glucose deprivation/reperfusion injury in cortical neurons via inhibiting NOX4-mediated NLRP3 inflammasome activation

Poncirin, a natural flavonoid present abundantly in citrus fruits, possesses anti-oxidant and anti-inflammatory activities that contribute to neuroprotection, but its roles and mechanisms in neuronal injury is still poorly understood. In this study, an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in primary cortical neurons to induce neuronal injury in vitro. Poncirin effectively attenuated OGD/R-induced neuronal damage by enhancing cell viability, restraining lactate dehydrogenase release, and reducing apoptosis of neurons. Poncirin restrained mitochondrial dysfunction and oxidative stress by increasing mitochondrial membrane potential, declining reactive oxygen species production, lessening malondialdehyde generation, and increasing the activities of antioxidant enzymes in OGD/R-treated neurons. Poncirin also repressed inflammatory responses by reducing the secretion of pro-inflammatory factors, and inhibiting NLRP3 inflammasome activation. Importantly, poncirin administration notably abolished OGD/R-induced upregulation of NADPH oxidase 4 (NOX4), and overexpression of NOX4 neutralized poncirin-mediated neuroprotection. In conclusion, poncirin protects cortical neurons from OGD/R injury via inhibiting NOX4/ROS/NLRP3 axis.

NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases

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

A Positive Feed Forward Loop between Wnt/β-Catenin and NOX4 Promotes Silicon Dioxide-Induced Epithelial-Mesenchymal Transition of Lung Epithelial Cells.

Silicosis is a chronic fibrotic lung disease caused by the accumulation of silica dust in the distal lung. Canonical Wnt signaling and NADPH oxidase 4 (NOX4) have been demonstrated to play a crucial role in the pathogenesis of pulmonary fibrosis including silicosis. However, the underlying mechanisms of crosstalk between these two signalings are not fully understood. In the present study, we aimed to explore the interaction of Wnt/β-catenin and NOX4 of human epithelial cells in response to an exposure of silica dust. Results demonstrated an elevated expression of key components of Wnt/β-catenin signaling and NOX4 in the lungs of silicon dioxide- (SiO2-) induced silicosis mice. Furthermore, the activated Wnt/β-catenin and NOX4 signaling are accompanied by an inhibition of cell proliferation, an increase of ROS production and cell apoptosis, and an upregulation of profibrogenic factors in BEAS-2B human lung epithelial cells exposed to SiO2. A mechanistic study further demonstrated that the Wnt3a-mediated activation of canonical Wnt signaling could augment the SiO2-induced NOX4 expression and reactive oxygen species (ROS) production but reduced glutathione (GSH), while Wnt inhibitor DKK1 exhibited an opposite effect to Wnt3a. Vice versa, an overexpression of NOX4 further activated SiO2-induced Wnt/β-catenin signaling and NFE2-related factor 2 (Nrf2) antioxidant response along with a reduction of GSH, whereas the shRNA-mediated knockdown of NOX4 showed an opposite effect to NOX4 overexpression. These results imply a positive feed forward loop between Wnt/β-catenin and NOX4 signaling that may promote epithelial-mesenchymal transition (EMT) of lung epithelial cells in response to an exposure of silica dust, which may thus provide an insight into the profibrogenic role of Wnt/β-catenin and NOX4 crosstalk in lung epithelial cell injury and pathogenesis of silicosis.

Abstract 3571: Oxidative stress and the ovarian surface epithelium: TGF-β1 stimulation of IOSE cell lines reveals novel upregulation of NADPH oxidase 4, a membrane bound hydrogen peroxide producing enzyme

Abstract Normal ovarian surface epithelium (OSE) has been widely studied as the origin of ovarian cancer. Repetitive ovulation has been implicated in cancer initiation based on damage and inflammation incurred to the OSE upon continuous cycles of tear and repair. Transforming growth factor-beta (TGF-β) plays a role in this normal ovulatory cycle, having been shown to inhibit human OSE proliferation and induce apoptosis, which may prevent the over-proliferation of cells. NADPH oxidase 4 (NOX4), a membrane-associated hydrogen peroxide-producing enzyme, has previously been shown to be sensitive to TGF-β1, with increased expression stimulated in smooth muscle cells and fibroblasts. Here we describe for the first time TGF-β1 induced upregulation of NOX4 in immortalized OSE (IOSE) cells (IOSE121, IOSE144, IOSE364, IOSE386 and IOSE397). This time-dependent increased expression (>5 fold at 2 ng/ml TGF-β1, 24 h) was simulated by direct overexpression of NOX4 in IOSE397 cells and has demonstrated that increased NOX4, in the absence of further growth factor related effects, affords induction of p18 and p21. In addition, using immunohistochemical evaluation of human ovarian cancers in tissue microarrays (n=17 patients), we observed significantly increased expression of NOX4, suggesting that NOX4 upregulation may contribute to the malignant transformation of ovarian cells. Citation Format: Jennifer L. Meitzler, Becky A. Diebold, Smitha Antony, Yongzhong Wu, Agnes Juhasz, Iris Dahan, Guojian Jiang, Jiamo Lu, Stephen Wang, Mariam M. Konaté, Krishnendu Roy, James H. Doroshow. Oxidative stress and the ovarian surface epithelium: TGF-β1 stimulation of IOSE cell lines reveals novel upregulation of NADPH oxidase 4, a membrane bound hydrogen peroxide producing enzyme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3571.

566-P: Sulodexide Suppresses NLRP3 Inflammasome Activation via NOX4/ROS Signaling in the Retinal Microvasculature

Aims: Sulodexide (SDX) protects endothelial cells via restoring endothelial glycocalyx, but the effect of SDX on endothelial inflammation remains unknown. We showed that SDX potently inhibited the activation of NLRP3 inflammasome in diabetic retinas, but the underlying mechanism was not clear. As reactive oxygen species (ROS) activates NLRP3 inflammasome, and NADPH oxidase 4 (NOX4) is a key generator of ROS in endothelial cells, this study thus investigated if NOX4/ROS signaling mediates the effect of SDX on NLRP3 inflammasome, hoping to provide novel insights into SDX in treating diabetic retinopathy (DR). Methods: Diabetes was induced in mice by high fat diet and streptozotocin, followed by i.p. injection of SDX (10mg/kg) every other day for 3 months. Human retinal microvascular endothelial cells were exposed to high glucose, accompanied with overexpression or knocking down of NOX4 and a serial concentration of SDX. The retinal levels of NOX4, NLRP3 inflammasome, inflammatory factors, junction components, vascular leakage and ROS levels were determined. Results: SDX treatment dramatically suppressed the hyperglycemia-induced overexpression of NOX4 and activation of NLRP3 inflammasome in retinas compared to controls. Moreover, SDX preserved the levels of ZO-1, VE-cadherin and ameliorated the retinal vascular permeability. In addition, SDX significantly ameliorated high glucose-induced NOX4 up-regulation, ROS production and NLRP3 inflammasome activation, and restored the reduction of ZO-1 and VE-cadherin in endothelial cells. Conversely, overexpression of NOX4 exacerbated endothelial inflammation through over production of ROS and subsequent activation of NLRP3 inflammasome, which was rescued by SDX treatment. Conclusion: Our study suggests that SDX protects the retinal microvasculature by inhibiting NOX4/ROS-induced NLRP3 inflammasome activation, thus provides novel evidence of utilizing SDX for the treatment of DR. Disclosure T. Li: None. X. He: None. R. Peng: None. X. Fu: None. G. Shi: None. Y. Chen: None. Funding Research and Development Plans in Key Areas of Guangdong Province (2019B020227003); National Natural Science Foundation of China (81770826); National Key Research and Development Program of China (2017YFA0105803)

Tumoral NOX4 recruits M2 tumor-associated macrophages via ROS/PI3K signaling-dependent various cytokine production to promote NSCLC growth

M2-type tumor-associated macrophages (TAMs) infiltration contributes to cancer malignant progression. However, the mechanisms for controlling recruitment and M2 polarization of macrophages by cancer cells are largely unclear. NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and mediates cancer progression. NOXs are in close relation with cancer-related inflammation, nevertheless, whether tumoral NOXs influence microenvironmental macrophages remains undentified. This study found that there was a close association between NOX4 expression and macrophage chemotaxis in patients with NSCLC analyzed using TCGA RNA-sequencing data. NOX4 in NSCLC cells (A549 and Calu-1 cell lines) efficiently enhanced murine peritoneal macrophage migration and induces M2 polarization. Immunohistochemical analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206. The mechanical study revealed that tumoral NOX4-induced reactive oxygen species (ROS) stimulated various cytokine production, including CCL7, IL8, CSF-1 and VEGF-C, via PI3K/Akt signaling-dependent manner. Blockade of the function of these cytokines reversed NOX4 effect on macrophages. Specifically, the results showed that tumoral NOX4-educated M2 macrophages exhibited elevated JNK activity, expressed and released HB-EGF, thus facilitating NSCLC proliferation in vitro. Pretreatment of macrophages with JNK inhibitor blocked tumoral NOX4-induced HB-EGF production in M2 macrophages. Finally, in a xenograft mouse model, overexpression of NOX4 in A549 cells enhanced the tumor growth. Elimination of ROS by NAC or inhibition of NOX4 activity by GKT137831 suppressed tumor growth accompanied by reduction in macrophage infiltration and the percentage of M2 macrophages. In conclusion, our study indicates that tumoral NOX4 recruits M2 TAMs via ROS/PI3K signaling-dependent various cytokine production, thus contributing NSCLC cell growth.