NADPH Oxidase Expression Research Articles

Hepatitis Delta Virus Antigens Trigger Oxidative Stress, Activate Antioxidant Nrf2/ARE Pathway, and Induce Unfolded Protein Response.

Hepatitis delta virus (HDV) is a viroid-like satellite that may co-infect individuals together with hepatitis B virus (HBV), as well as cause superinfection by infecting patients with chronic hepatitis B (CHB). Being a defective virus, HDV requires HBV structural proteins for virion production. Although the virus encodes just two forms of its single antigen, it enhances the progression of liver disease to cirrhosis in CHB patients and increases the incidence of hepatocellular carcinoma. HDV pathogenesis so far has been attributed to virus-induced humoral and cellular immune responses, while other factors have been neglected. Here, we evaluated the impact of the virus on the redox status of hepatocytes, as oxidative stress is believed to contribute to the pathogenesis of various viruses, including HBV and hepatitis C virus (HCV). We show that the overexpression of large HDV antigen (L-HDAg) or autonomous replication of the viral genome in cells leads to increased production of reactive oxygen species (ROS). It also leads to the upregulated expression of NADPH oxidases 1 and 4, cytochrome P450 2E1, and ER oxidoreductin 1α, which have previously been shown to mediate oxidative stress induced by HCV. Both HDV antigens also activated the Nrf2/ARE pathway, which controls the expression of a spectrum of antioxidant enzymes. Finally, HDV and its large antigen also induced endoplasmic reticulum (ER) stress and the concomitant unfolded protein response (UPR). In conclusion, HDV may enhance oxidative and ER stress induced by HBV, thus aggravating HBV-associated pathologies, including inflammation, liver fibrosis, and the development of cirrhosis and hepatocellular carcinoma.

Role of apigenin in high glucose-induced retinal microvascular endothelial cell dysfunction via regulating NOX4/p38 MAPK pathway in vitro.

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. 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. 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. Api might exert a beneficial role in HG-stimulated HRMECs through regulating NOX4/p38 MAPK pathway.

Carnosine Counteracts the Molecular Alterations Aβ Oligomers-Induced in Human Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) has been described as a progressive eye disease characterized by irreversible impairment of central vision, and unfortunately, an effective treatment is still not available. It is well-known that amyloid-beta (Aβ) peptide is one of the major culprits in causing neurodegeneration in Alzheimer's disease (AD). The extracellular accumulation of this peptide has also been found in drusen which lies under the retinal pigment epithelium (RPE) and represents one of the early signs of AMD pathology. Aβ aggregates, especially in the form of oligomers, are able to induce pro-oxidant (oxidative stress) and pro-inflammatory phenomena in RPE cells. ARPE-19 is a spontaneously arising human RPE cell line validated for drug discovery processes in AMD. In the present study, we employed ARPE-19 treated with Aβ oligomers, representing an in vitro model of AMD. We used a combination of methods, including ATPlite, quantitative real-time PCR, immunocytochemistry, as well as a fluorescent probe for reactive oxygen species to investigate the molecular alterations induced by Aβ oligomers. In particular, we found that Aβ exposure decreased the cell viability of ARPE-19 cells which was paralleled by increased inflammation (increased expression of pro-inflammatory mediators) and oxidative stress (increased expression of NADPH oxidase and ROS production) along with the destruction of ZO-1 tight junction protein. Once the damage was clarified, we investigated the therapeutic potential of carnosine, an endogenous dipeptide that is known to be reduced in AMD patients. Our findings demonstrate that carnosine was able to counteract most of the molecular alterations induced by the challenge of ARPE-19 with Aβ oligomers. These new findings obtained with ARPE-19 cells challenged with Aβ1-42 oligomers, along with the well-demonstrated multimodal mechanism of action of carnosine both in vitro and in vivo, able to prevent and/or counteract the dysfunctions elicited by Aβ oligomers, substantiate the neuroprotective potential of this dipeptide in the context of AMD pathology.

Resveratrol Improves Paclitaxel-Induced Cognitive Impairment in Mice by Activating SIRT1/PGC-1α Pathway to Regulate Neuronal State and Microglia Cell Polarization.

This study aimed to investigate the effect of resveratrol (Res) on paclitaxel (PTX)-induced cognitive impairment and elucidate the underlying molecular mechanisms. Morris Water Maze (MWM) test was used to evaluate the mice's spatial learning and memory abilities. Western blotting was applied to detect protein expression of receptor-interacting protein (RIP3), mixed lineage kinase domain-like protein (MLKL), silencing information regulator 2 related enzyme 1 (SIRT1), peroxisome proliferator activated receptor coactivator-1 (PGC-1α), NADPH oxidase 2 (NOX2), NOX4, postsynaptic density zone 95 (PSD95), arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS). Immunofluorescence of RIP3, MLKL, Arg-1, Iba-1 and iNOS was conducted to observe the apoptosis of hippocampal cells and the polarization of microglia. qRT-PCR was performed to detect BDNF mRNA expressions. DHE staining was used to assess the level of oxidative stress response. Golgi-Cox staining and dendritic spine counting were applied to visualize synaptic structural plasticity. Postsynaptic density was performed by transmission electron microscope. ELISA was used to detect the contents of tumour necrosis factor alpha (TNF-α), IL-1β, IL-4, and IL-10. PTX-induced cognitive impairment model was constructed after the application of PTX, represented as longer latency to platform and less platform crossing times over the whole period in PTX group. After Res treatment, the above indicators were reversed, indicating that cognitive function was improved. Moreover, Res reduced neuronal apoptosis and oxidative stress through SIRT1/PGC-1α pathway in mice, manifesting as down-regulated expression of RIP3, MLKL, NOX2 and NOX4. Meanwhile, Res increased the density of dendritic spines and the expression of PSD95 and BDNF, thereby ameliorating the PTX induced synaptic damage. Besides, M2 microglia was in the majority, eliciting the expression of anti-inflammatory cytokines IL-4 and IL-10 after Res treatment in PTX+Res group, while immunofluorescence images results demonstrated an decrease in the proportion of M2 microglia a following SIRT1 inhibitor EX-527. Res improves PTX-induced cognitive impairment in mice by activating SIRT1/PGC-1α pathways to regulate neuronal state and microglia cell polarization.

Study on the pharmacological mechanisms of sodium-glucose co-transporter 2 inhibitors in obesity-related atrial fibrillation based on network pharmacology and experimental verification.

Obesity is an independently risk factor of atrial fibrillation (AF). It is likely that the global burden of AF will escalate due to the current obesity epidemic. Weight loss can effectively reduce the risk of AF, while sodium-glucose co-transporter 2 inhibitors (SGLT2i) can reduce body weight, so SGLT2i are potentially effective for obesity-related AF. SGLT2i are a novel type of oral medication. This current study used network pharmacology to examine the potential mechanisms of SGLT2i in the treatment of obesity-related AF, and the therapeutic effects were assessed in vivo. Potential gene targets for SGLT2i in treating obesity-related AF were identified from public database. Cytoscape V3.7.1 was used to construct the "Drug-Target" and "Drug-Target-Disease" networks. The STRING database was applied to investigate the protein-protein interactions (PPIs). Additionally, the Bioconductor tools were used to analyze the Gene Ontology (GO) biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The efficacy of SGLT2i in treating obesity-related AF was investigated in vivo using a diet-induced obese C57BL/6J male mouse model. A number of indicators were assessed, including invasive electrophysiology, testing of blood samples, and expression detection of pathway targets. These experiments were used to verify the targets mined by network pharmacology. The results indicated that SGLT2i had 80 potential target genes during the treatment of obesity-related AF, and 10 hub genes were obtained by further screening. It was predicted that the treatment of obesity-related AF by SGLT2i involved the advanced glycation end product (AGE)-receptor for advanced glycation end product (RAGE) signaling pathway and other signaling pathways. In in vivo experiments, administration of SGLT2i (the SGLT2i + DIO group) resulted in a lower AF induction rate (P<0.05), decreased serum AGEs/soluble RAGE (sRAGE) ratio (P<0.01), and decreased expression of NADPH oxidase 2 (NOX2) (P<0.05) compared to untreated DIO mice (the DIO group). In this study, pharmacological network analysis and in vivo experiments demonstrated that SGLT2i acts on obesity-related AF by inhibiting the AGE-RAGE signaling pathway. These results offer fresh perspectives on the pharmacological actions of SGLT2i in treating obesity-related AF.

Mutant p53 and NOX4 are modulators of a CCL5-driven pro-migratory secretome.

Previously, we showed wild-type (WT) and mutant (mt) forms of p53 differentially regulate ROS generation by NADPH oxidase-4 (NOX4). We found that WT-p53 suppresses TGF-β-induced NOX4, ROS production, and cell migration, whereas tumor-associated mt-p53 proteins enhance NOX4 expression and cell migration by TGF-β/SMAD3-dependent mechanisms. In this study, we investigated the role of mutant p53-induced NOX4 on the cancer cell secretome and the effects NOX4 signaling have on the tumor microenvironment (TME). We found conditioned media collected from H1299 lung epithelial cells stably expressing either mutant p53-R248Q or R273H promotes the migration and invasion of naïve H1299cells and chemotactic recruitment of THP-1 monocytes. These effects were diminished with conditioned media from cells co-transfected with dominant negative NOX4 (P437H). We utilized immunoblot-based cytokine array analysis to identify factors in mutant p53 H1299cell conditioned media that promote cell migration and invasion. We found CCL5 was significantly reduced in conditioned media from H1299cells co-expressing p53-R248Q and dominant negative NOX4. Moreover, neutralization of CCL5 reduced autocrine-mediated H1299cell mobility. Furthermore, CCL5 and TGF-beta from M2-polarized macrophages have a significant role in crosstalk and H1299cell migration and invasion. Collectively, our findings provide further insight into NOX4-based communication in the tumor microenvironment and its potential as a therapeutic target affecting metastatic disease progression.

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.

MAPK cascade and ROS metabolism are involved in GABA-induced disease resistance in red pitaya fruit

This study investigates the effects of γ-Aminobutyric acid (GABA) and mitogen-activated protein kinase (MAPK) inhibitor (PD98059) in disease resistance in red pitaya fruit. Results demonstrated that GABA treatment suppressed the growth of Gilbertella persicaria; increased H2O2 content and O2•- production rate; enhanced reduced glutathione and ascorbic acid contents, as well as increased the activities and transcripts of catalase (CAT), NADPH oxidase (NOX), peroxidise (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and up-regulated HcMYB111/24 expressions. Furthermore, GABA promoted the expressions of HcMAPKKK1-HcMAPKK2/4-HcMAPK1/7 cascades. However, PD98059 +GABA treatment decreased the disease resistance against G. persicaria and reduced H2O2 content; inhibited the activities and gene expressions of NOX, CAT, SOD, POD, GR and APX, and down-regulated the expressions of HcMAPK1/7. These findings suggest that GABA induces disease resistance by regulating ROS metabolism and MAPK cascades in red pitaya fruit.

The mechanism of oxidative stress in keloid fibroblasts and the experimental study of early application of angiotensin-converting enzyme inhibitor.

Objective To investigate the protective effects of an angiotensin-converting enzyme inhibitor after inducing oxidative stress on keloid fibroblasts. Methods Primary keloid fibroblasts were isolated and cultured by enzyme digestion combined with the tissue adhesion method in vitro, and the third to fifth generations of cells were selected for the experiment. For 24 hours, keloid fibroblasts were treated with different concentrations of hydrogen peroxide. Different concentrations of angiotensin-converting enzyme inhibitor were added to the keloid fibroblast culture medium, and then the cells were treated with hydrogen peroxide for 24 hours. Results With the increase of hydrogen peroxide concentration, the growth of keloid fibroblasts was inhibited and the levels of malondialdehyde, superoxide dismutase, and reactive oxygen species increased gradually, accompanied by an increase in the expression of nicotinamide adenine dinucleotide phosphate oxidase and collagen I mRNA. The expression of nicotinamide adenine dinucleotide phosphate oxidase-mRNA in keloid fibroblasts and the formation of reactive oxygen species in keloid fibroblasts were induced by different concentrations of angiotensin II, and the most significant effect was at 10-5 mmol/mL. The effects of diphenyleneiodonium chloride (NOX inhibitor), N-acetylcysteine (reactive oxygen species inhibitor) and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) RNA treatment on angiotensin II-induced nicotinamide adenine dinucleotide phosphate oxidase and collagen I increased significantly. Hydrogen peroxide and angiotensin II alone or combined can induce NADPH oxidase and reactive oxygen species expression in keloid fibroblasts. When the angiotensin-converting enzyme inhibitor was added, the expression of NADPH oxidase and reactive oxygen species in keloid induced by hydrogen peroxide and angiotensin II could be inhibited. Conclusion Oxidative stress can lead to increased expression of reactive oxygen species, NADPH oxidase and collagen I in keloid fibroblasts, suggesting oxidative stress mediates the migration of human keloid fibroblasts and extracellular matrix synthesis.

PRDX6-mediated pulmonary artery endothelial cell ferroptosis contributes to monocrotaline-induced pulmonary hypertension

Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder whose underlying pathogenesis is unknown. Our previous study showed that pulmonary endothelial cell (PAEC) ferroptosis is involved in the progression of PH by releasing High-mobility group box 1 (HMGB1) and activating Toll-like receptor 4/NOD-like receptor family pyrin domain containing 3 (TLR4/NLRP3) inflammasome signalling. The precise mechanisms that regulate ferroptosis in PH are unclear. This study aimed to investigate the effect of peroxiredoxin 6 (PRDX6) on PAEC ferroptosis in PH. A rat model of PH was established with monocrotaline (MCT), and the distribution and expression of PRDX6 in the pulmonary artery were examined. Lentiviral vectors carrying PRDX6 (LV-PRDX6) were transfected into PAECs and injected into MCT-induced PH rats. Cell viability, MDA levels, reactive oxygen species (ROS) levels, labile iron pool (LIP) levels and mitochondrial morphology were examined. Ferroptosis-related proteins (NADPH oxidase-4 (NOX4), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1(FTH1)), TLR4, NLRP3 inflammasome markers, HMGB1 and inflammatory cytokines were examined. Pulmonary vascular remodelling and right ventricular structure and function were measured. PRDX6 was expressed in PAECs and was significantly decreased in PH. PRDX6 overexpression significantly inhibited ferroptosis in PAECs under PH conditions in vitro and in vivo, as indicated by increased cell viability, decreased MDA, ROS and LIP levels, inhibited mitochondrial damage, upregulated GPX4 and FTH1 expression, and downregulated NOX4 expression. PRDX6 overexpression attenuated pulmonary vascular remodelling and changes in right ventricle structure and function in MCT-induced PH rats. Moreover, PRDX6 overexpression prevented HMGB1 release by PAECs and decreased TLR4 and NLRP3 inflammasome expression and inflammatory cytokine release in macrophages, while RSL3, a specific activator of ferroptosis, reversed these effects. Taken together, these findings indicate that PRDX6 regulates PAEC ferroptosis through the release of HMGB1 and activation of the TLR4/NLRP3 inflammasome signalling pathway, providing novel therapeutic targets for the treatment of PH.

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.

TrPLD1 and TrPLD2 modulate reactive oxygen species production and pathogenicity in Trichothecium roseum infected apple fruit

Phospholipase D (PLD) plays an important role during pathogen-host interaction, which not only hydrolyzes the phosphodiester bond in the phospholipid structure of the host cell membrane, resulting in the cell membrane structure destruction of the host, but also produces phosphatidic acid that can interact with NADPH oxidase (NOX) on the cell plasma membrane, resulting in reactive oxygen species (ROS) accumulation and improving the pathogenicity of the pathogen. However, the ROS production of filamentous fungi by PLD modulation and its pathogenicity on postharvest fruit and vegetables have not been documented. In this study, we constructed ΔTrPLD1 and ΔTrPLD2 mutants and complement strains of Trichothecium roseum by homologous recombination, and analyzed ROS production and its pathogenicity on apple fruit. The results showed that the deletion of TrPLD1 and TrPLD2 increased the sensitivity to oxidative stress and reduced ROS production of T. roseum mycelia. During ΔTrPLD1 mutant of T. roseum infecting apple fruit, the gene expression and activity of NOX decreased, which lead to the reduction of superoxide anion production rate. The gene expressions and activities of superoxide dismutase and peroxidase were up-regulated, the expression and activity of catalase were down-regulated, and the activation of ascorbic acid-glutathione cycle, ultimately enhanced the production of H2O2 in apple tissue, H2O2 acted as signal molecule to trigger disease resistance of fruit, thereby reducing the pathogenicity on apple fruit. The ΔTrPLD2 mutant had no significant effect on ROS production during pathogen infecting apple fruit. In summary, ΔTrPLD1 and ΔTrPLD2 mutants were highly sensitive to oxidative stress, and TrPLD1 played more important role in T. roseum infected apple fruit by regulating ROS production.

Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis

BackgroundIncreased acid sphingomyelinase (ASMase) activity is associated with insulin resistance and cardiac dysfunction. However, the effects of ASMase on diabetic cardiomyopathy (DCM) and the molecular mechanism(s) underlying remain to be elucidated. We here investigated whether ASMase caused DCM through NADPH oxidase 4-mediated apoptosis.Methods and resultsWe used pharmacological and genetic approaches coupled with study of murine and cell line samples to reveal the mechanisms initiated by ASMase in diabetic hearts. The protein expression and activity of ASMase were upregulated, meanwhile ceramide accumulation was increased in the myocardium of HFD mice. Inhibition of ASMase with imipramine (20 mg Kg−1 d−1) or siRNA reduced cardiomyocyte apoptosis, fibrosis, and mitigated cardiac hypertrophy and cardiac dysfunction in HFD mice. The similar effects were observed in cardiomyocytes treated with high glucose (HG, 30 mmol L−1) + palmitic acid (PA, 100 μmol L−1) or C16 ceramide (CER, 20 μmol L−1). Interestingly, the cardioprotective effect of ASMase inhibition was not accompanied by reduced ceramide accumulation, indicating a ceramide-independent manner. The mechanism may involve activated NADPH oxidase 4 (NOX4), increased ROS generation and triggered apoptosis. Suppression of NOX4 with apocynin prevented HG + PA and CER incubation induced Nppb and Myh7 pro-hypertrophic gene expression, ROS production and apoptosis in H9c2 cells. Furthermore, cardiomyocyte-specific ASMase knockout (ASMaseMyh6KO) restored HFD-induced cardiac dysfunction, remodeling, and apoptosis, whereas NOX4 protein expression was downregulated.ConclusionsThese results demonstrated that HFD-mediated activation of cardiomyocyte ASMase could increase NOX4 expression, which may stimulate oxidative stress, apoptosis, and then cause metabolic cardiomyopathy.

NADPH oxidase 4 deficiency attenuates experimental osteoarthritis in mice

ObjectiveLow-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In...

Pharmacological reversal of post-transcriptional alterations implicated in alcohol-induced alveolar macrophage dysfunction.

Alcohol use disorders (AUD) cause alveolar macrophage (AM) immune dysfunction and increase risk of lung infections. Excessive alcohol use causes AM oxidative stress, which impairs AM phagocytosis and pathogen clearance from the alveolar space. Alcohol induces expression of NADPH oxidases (Noxes), primary sources of oxidative stress in AM. In contrast, alcohol decreases AM peroxisome proliferator-activated receptor gamma (PPARγ), a critical regulator of AM immune function. To explore the underlying molecular mechanisms for these effects of alcohol, we hypothesized that ethanol promotes CCAAT/enhancer-binding protein beta (C/EBPβ)-mediated suppression of Nox-related microRNAs (miRs), in turn enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. We also hypothesized that PPARγ activation with pioglitazone (PIO) would reverse alcohol-induced C/EBPβ expression and attenuate AM oxidative stress and phagocytic dysfunction. Cells from the mouse AM cell line (MH-S) were exposed to ethanol invitro or primary AM were isolated from mice fed ethanol invivo. Ethanol enhanced C/EBPβ expression, decreased Nox 1-related miR-1264 and Nox 2-related miR-107 levels, and increased Nox1, Nox2, and Nox 4 expression in MH-S cells invitro and mouse AM invivo. These alcohol-induced AM derangements were abrogated by loss of C/EBPβ, overexpression of miRs-1264 or -107, or PIO treatment. These findings identify C/EBPβ and Nox-related miRs as novel therapeutic targets for PPARγ ligands, which could provide a translatable strategy to mitigate susceptibility to lung infections in people with a history of AUD. These studies further clarify the molecular underpinnings for a previous clinical trial using short-term PIO treatment to improve AM immunity in AUD individuals.

Endothelial cell TRPA1 activity exacerbates cerebral hemorrhage during severe hypertension.

Introduction: Hypoxia-induced dilation of cerebral arteries orchestrated by Ca2+-permeable transient receptor potential ankyrin 1 (TRPA1) cation channels on endothelial cells is neuroprotective during ischemic stroke, but it is unknown if the channel has a similar impact during hemorrhagic stroke. TRPA1 channels are endogenously activated by lipid peroxide metabolites generated by reactive oxygen species (ROS). Uncontrolled hypertension, a primary risk factor for the development of hemorrhagic stroke, is associated with increased ROS production and oxidative stress. Therefore, we hypothesized that TRPA1 channel activity is increased during hemorrhagic stroke. Methods: Severe, chronic hypertension was induced in control (Trpa1 fl/fl) and endothelial cell-specific TRPA1 knockout (Trpa1-ecKO) mice using a combination of chronic angiotensin II administration, a high-salt diet, and the addition of a nitric oxide synthase inhibitor to drinking water. Blood pressure was measured in awake, freely-moving mice using surgically placed radiotelemetry transmitters. TRPA1-dependent cerebral artery dilation was evaluated with pressure myography, and expression of TRPA1 and NADPH oxidase (NOX) isoforms in arteries from both groups was determined using PCR and Western blotting techniques. In addition, ROS generation capacity was evaluated using a lucigenin assay. Histology was performed to examine intracerebral hemorrhage lesion size and location. Results: All animals became hypertensive, and a majority developed intracerebral hemorrhages or died of unknown causes. Baseline blood pressure and responses to the hypertensive stimulus did not differ between groups. Expression of TRPA1 in cerebral arteries from control mice was not altered after 28 days of treatment, but expression of three NOX isoforms and the capacity for ROS generation was increased in hypertensive animals. NOX-dependent activation of TRPA1 channels dilated cerebral arteries from hypertensive animals to a greater extent compared with controls. The number of intracerebral hemorrhage lesions in hypertensive animals did not differ between control and Trpa1-ecKO animals but were significantly smaller in Trpa1-ecKO mice. Morbidity and mortality did not differ between groups. Discussion: We conclude that endothelial cell TRPA1 channel activity increases cerebral blood flow during hypertension resulting in increased extravasation of blood during intracerebral hemorrhage events; however, this effect does not impact overall survival. Our data suggest that blocking TRPA1 channels may not be helpful for treating hypertension-associated hemorrhagic stroke in a clinical setting.

Nrf2 regulates the expression of NOX1 in TNF-α-induced A549 cells

Acute lung injury causes severe inflammation and oxidative stress in lung tissues. In this study, we analyzed the potential regulatory role of nuclear factor erythroid-2-related factor 2 (Nrf2) on NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced inflammation and oxidative stress in human type II alveolar epithelial cells. In this study, A549 cells were transfected with Nrf2 siRNA and overexpression vectors for 6 h before being induced by TNF-α for 24 h. TNF-α upregulated the expression of NOX1 and Nrf2 in A549 cells. Furthermore, overexpression of Nrf2 could reduce TNF-α-induced NF-κB mRNA and protein expression after transfection with the Nrf2 siRNA vector, and the levels of IL-6, IL-8, ROS, and malondialdehyde (MDA) in TNF-α-induced A549 cells increased, while the level of total antioxidation capability (T-AOC) decreased. On the other hand, the overexpression of Nrf2 decreased the levels of IL-6, IL-8, ROS, and MDA, while increasing T-AOC. The mRNA and protein levels of NOX1 were dramatically increased by TNF-α, while those changes were notably suppressed by Nrf2 overexpression. Further studies demonstrated that Nrf2 suppressed NOX1 transcription by binding to the -1199 to -1189 bp (ATTACACAGCA) region of the NOX1 promoter in TNF-α-stimulated A549 cells. Our study suggests that Nrf2 may bind to and regulate NOX1 expression to antagonize TNF-α-induced inflammatory reaction and oxidative stress in A549 cells.

Dexamethasone Induces Senescence-Associated Changes in Trabecular Meshwork Cells by Increasing ROS Levels Via the TGFβ/Smad3-NOX4 Axis

Glaucoma is a serious complication of glucocorticoid (GC) therapy arising through elevations in intraocular pressure (IOP). Dexamethasone (DEX) is reported to contribute to elevated IOP through different effects on the trabecular meshwork but whether DEX contributes to glaucoma development through the induction of cellular senescence is still unclear. We explored the actions of DEX on transformed human trabecular meshwork cells (HTMCs) using RNA-seq and conducted bioinformatic analyses to determine the affected pathways. Among the 4,103 differentially expressed genes identified in transformed HTMCs treated with 400 nM DEX (2,036 upregulated and 2,067 downregulated genes, respectively), bioinformatic analyses revealed significant enrichment and potential interplay between the transforming growth factor beta (TGFβ)41; signaling and cellular senescence pathways. DEX treatment induced senescence changes in primary and transformed HTMCs as indicated by increases in SA-β-gal positivity, interleukin (IL)-6 secretion, and senescence-associated heterochromatin foci (SAHF) along with selective accumulation of senescence marker p15 and elevations in reactive oxygen species (ROS) levels. Notably, the DEX-induced senescence changes were rescued by treatment with the TGFβ/Smad3 pathway inhibitor SIS3. Furthermore, we show that DEX increases cellular ROS levels via upregulation of NADPH oxidase 4 (NOX4) through activation of Smad3, and that SIS3 decreases ROS levels by downregulating NOX4. Instructively, inhibiting NOX4 with GLX351322 and scavenging ROS with NAC were both effective in preventing DEX-induced senescence changes. Similarly, we found in the mouse model that DEX-ac upregulated p15 and NOX4 expression in the trabecular meshwork, with cotreatment with GLX351322 alleviating elevations in IOP. We establish that DEX induces senescence changes in HTMCs by increasing ROS levels via the TGFβ/Smad3/NOX4 axis, increasing IOP and contributing to glaucoma development.

Association of the Toll-like receptor 4 and NOX4 gene and protein levels in asthmatic patients with metabolic syndrome: A case-control study.

Understanding the contributing of influence inflammatory biomarkers in asthmatic patients with metabolic syndrome is more important. Whereby, the present study considering the important association of NADPH oxidase4 (NOX4) and Toll- like receptor4 (TLR4) in the respiratory inflammatory responses in asthmatic patients with metabolic syndrome (AS-MetS) and asthmatic (AS) patients. In this case-control study, 30 AS and 34 AS-MetS patients were enrolled. The Peripheral blood mononuclear cells (PBMCs) mRNA and protein levels of TLR4 and NOX4 were measured by qRT-PCR and western blot, respectively. Then their correlation was evaluated. The significant down-regulation of mRNA and protein PBMCs expression levels of TLR4 were observed in the AS-MetS group in comparison to AS one (P=0.03), but the NOX4 expression was non-significant. Additionally, the significant correlation was exhibited between mRNA expression levels of NOX4 and TLR4 in both AS-MetS (r= 0.440, P=0.009) and AS groups (r=0.909, P=0.0001). The association between TLR4 mRNA level and triglyceride in AS-MetS group (r=0.454, P=0.008,) and also white blood cells (WBC) in AS group (r= -0.507, P=0.006,) were significant. The metabolic syndrome can significantly influence the expressions of TLR4 in AS-MetS. This study indicated that TLR4 and NOX4 altogether may provide valuable molecular knowledge of their relation with metabolic syndrome criteria for finding major pathways in different phenotype of asthma.

Oleic acid-induced metastasis of KRAS/p53-mutant colorectal cancer relies on concurrent KRAS activation and IL-8 expression bypassing EGFR activation.

Background: Multigene mutations in colorectal cancer (CRC), including KRAS, BRAF, and p53, afford high metastatic ability and resistance to EGFR-targeting therapy. Understanding the molecular mechanisms regulating anti-EGFR-resistant CRC metastasis can improve CRC therapy. This study aimed to investigate the effects of IL-8 and the activation of KRAS on reactive oxygen species (ROS) production and metastasis of hyperlipidemia-associated CRC harboring mutations of KRAS and p53. Methods: The cytokine array analysis determined the up-expression of secreted factors, including IL-8. The clinical relevance of the relationship between IL-8 and angiopoietin-like 4 (ANGPTL4) was examined in CRC patients from National Cheng Kung University Hospital and TCGA dataset. Expressions of IL-8, ANGPTL4, NADPH oxidase 4 (NOX4), and epithelial-mesenchymal transition (EMT) markers in free fatty acids (FFAs)-treated KRAS/p53 mutant CRC cells were determined. The hyperlipidemia-triggered metastatic ability of CRC cells under treatments of antioxidants, statin, and cetuximab or knockdown of IL-8, KRAS, and EGFR was evaluated in vitro and in vivo. In addition, the effects of antioxidants and depletion of IL-8 and KRAS on the correlation between ROS production and hyperlipidemia-promoted CRC metastasis were also clarified. Results: In this study, we found that free fatty acids promoted KRAS/p53-mutant but not single-mutant or non-mutant CRC cell metastasis. IL-8, the most abundant secreted factor in KRAS/p53-mutant cells, was correlated with the upregulation of NOX4 expression and ROS production under oleic acid (OA)-treated conditions. In addition, the metastasis of KRAS/p53-mutant CRC relies on the ANGPTL4/IL-8/NOX4 axis and the activation of KRAS. The antioxidants and inactivation of KRAS also inhibited OA-induced EMT and metastasis. Although KRAS mediated EGF- and OA-promoted CRC cell invasion, the inhibition of EGFR did not affect OA-induced ANGPTL4/IL-8/NOX4 axis and CRC metastasis. The high-fat diet mice fed with vitamin E and statin or in IL-8-depleted cells significantly inhibited tumor extravasation and metastatic lung growth of CRC. Conclusion: The antioxidants, statins, and targeting IL-8 may provide better outcomes for treating metastatic CRC that harbors multigene mutations and anti-EGFR resistance.