P47phox Research Articles

NCF1-dependent production of ROS protects against lupus by regulating plasmacytoid dendritic cell development and functions.

Low capacity to produce reactive oxygen species (ROS) due to mutations in neutrophil cytosolic factor 1 (NCF1/p47phox), a component of NADPH oxidase 2 (NOX2) complex, is strongly associated with systemic lupus erythematosus in both humans and mouse models. Here, we aim to identify the key immune cell type(s) and cellular mechanisms driving lupus pathogenesis under the condition of NCF1-dependent ROS deficiency. Using a set of cell-specific Cre-deleter, the human NCF1-339 variant knock-in, and transgenic mouse strains, we show that low ROS production in plasmacytoid dendritic cells (pDCs) exacerbates both pristane-induced lupus and a newly established Yaa-related spontaneous model by promoting pDC accumulation in multiple organs during lupus development, accompanied by elevated IFNα levels and expression of IFN-stimulated genes. Mechanistic studies reveal that ROS deficiency enhances pDC generation through the AKT/mTOR pathway and CCR2-mediated migration to tissues, which together with hyperactivation of the redox-sensitive STING/IFNα/JAK1/STAT1 cascade further augments type I IFN responses. More importantly, by suppressing these pathways, restoration of NOX2-derived ROS specifically in pDCs protects against lupus. These discoveries explain the causative effect of dysfunctional NCF1 in lupus and demonstrate the protective role of pDC-derived ROS in disease development driven by NCF1-dependent ROS deficiency.

A novel lysosome-related gene signature coupled with gleason score for prognosis prediction in prostate cancer.

Background: Prostate cancer (PCa) is highly heterogeneous, which makes it difficult to precisely distinguish the clinical stages and histological grades of tumor lesions, thereby leading to large amounts of under- and over-treatment. Thus, we expect the development of novel prediction approaches for the prevention of inadequate therapies. The emerging evidence demonstrates the pivotal role of lysosome-related mechanisms in the prognosis of PCa. In this study, we aimed to identify a lysosome-related prognostic predictor in PCa for future therapies. Methods: The PCa samples involved in this study were gathered from The Cancer Genome Atlas database (TCGA) (n = 552) and cBioPortal database (n = 82). During screening, we categorized PCa patients into two immune groups based on median ssGSEA scores. Then, the Gleason score and lysosome-related genes were included and screened out by using a univariate Cox regression analysis and the least absolute shrinkage and selection operation (LASSO) analysis. Following further analysis, the probability of progression free interval (PFI) was modeled by using unadjusted Kaplan-Meier estimation curves and a multivariable Cox regression analysis. A receiver operating characteristic (ROC) curve, nomogram and calibration curve were used to examine the predictive value of this model in discriminating progression events from non-events. The model was trained and repeatedly validated by creating a training set (n = 400), an internal validation set (n = 100) and an external validation (n = 82) from the cohort. Results: Following grouping by ssGSEA score, the Gleason score and two LRGs-neutrophil cytosolic factor 1 (NCF1) and gamma-interferon-inducible lysosomal thiol reductase (IFI30)-were screened out to differentiate patients with or without progression (1-year AUC = 0.787; 3-year AUC = 0.798; 5-year AUC = 0.772; 10-year AUC = 0.832). Patients with a higher risk showed poorer outcomes (p < 0.0001) and a higher cumulative hazard (p < 0.0001). Besides this, our risk model combined LRGs with the Gleason score and presented a more accurate prediction of PCa prognosis than the Gleason score alone. In three validation sets, our model still achieved high prediction rates. Conclusion: In conclusion, this novel lysosome-related gene signature, coupled with the Gleason score, works well in PCa for prognosis prediction.

Identification of m6A regulator-mediated RNA methylation modification patterns and key immune-related genes involved in atrial fibrillation.

The role of m6A in the regulation of the immune microenvironment in atrial fibrillation (AF) remains unclear. This study systematically evaluated the RNA modification patterns mediated by differential m6A regulators in 62 AF samples, identified the pattern of immune cell infiltration in AF and identified several immune-related genes associated with AF. A total of six key differential m6A regulators between healthy subjects and AF patients were identified by the random forest classifier. Three distinct RNA modification patterns (m6A cluster-A, -B and -C) among AF samples were identified based on the expression of 6 key m6A regulators. Differential infiltrating immune cells and HALLMARKS signaling pathways between normal and AF samples as well as among samples with three distinct m6A modification patterns were identified. A total of 16 overlapping key genes were identified by weighted gene coexpression network analysis (WGCNA) combined with two machine learning methods. The expression levels of the NCF2 and HCST genes were different between controls and AF patient samples as well as among samples with the distinct m6A modification patterns. RT-qPCR also proved that the expression of NCF2 and HCST was significantly increased in AF patients compared with control participants. These results suggested that m6A modification plays a key role in the complexity and diversity of the immune microenvironment of AF. Immunotyping of patients with AF will help to develop more accurate immunotherapy strategies for those with a significant immune response. The NCF2 and HCST genes may be novel biomarkers for the accurate diagnosis and immunotherapy of AF.

Lutein Induces Reactive Oxygen Species-Mediated Apoptosis in Gastric Cancer AGS Cells via NADPH Oxidase Activation.

Disruption of apoptosis leads to cancer cell progression; thus, anticancer agents target apoptosis of cancer cells. Reactive oxygen species (ROS) induce apoptosis by activating caspases and caspase-dependent DNase, leading to DNA fragmentation. ROS increase the expression of apoptotic protein Bax, which is mediated by activation of nuclear factor-κB (NF--κB). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of endogenous ROS, and its activation is involved in apoptosis. Lutein, an oxygenated carotenoid and known antioxidant, is abundant in leafy dark green vegetables, such as spinach and kale, and in yellow-colored foods, such as corn and egg yolk. High amounts of lutein increase ROS levels and exhibit anticancer activity. However, its anticancer mechanism remains unclear. This study aimed to determine whether lutein activates NADPH oxidase to produce ROS and induce apoptosis in gastric cancer AGS cells. Lutein increased ROS levels and promoted the activation of NADPH oxidase by increasing the translocation of NADPH oxidase subunit p47 phox to the cell membrane. It increased NF-κB activation and apoptotic indices, such as Bax, caspase-3 cleavage, and DNA fragmentation, and decreased Bcl-2, cell viability, and colony formation in AGS cells. The specific NADPH oxidase inhibitor ML171, and the known antioxidant N-acetyl cysteine reversed lutein-induced cell death, DNA fragmentation, and NF-κB DNA-binding activity in AGS cells. These results suggest that lutein-induced ROS production is dependent on NADPH oxidase, which mediates NF-κB activation and apoptosis in gastric cancer AGS cells. Therefore, lutein supplementation may be beneficial for increasing ROS-mediated apoptosis in gastric cancer cells.

Role of NCF2 as a potential prognostic factor and immune infiltration indicator in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related deaths globally. The tumor microenvironment (TME) plays a crucial role in the prognosis and treatment of HCC. Hence, it is important to exploit new biomarkers for survival surveillance and TME estimation of HCC. HCC samples data was collected from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database, and clinical samples were collected from our center. The TME of HCC were explored with ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data), ssGSEA (single sample Gene Sets Enrichment Analysis) and CIBERSORT algorithm. Differentially expressed genes were analyzed with functional enrichment analysis. Immunohistochemistry was implemented to validate the results. Based on TCGA database, we found that Neutrophil Cytosolic Factor 2 (NCF2) was significantly associated with the prognosis of HCC patients, involved in immune-related biological processes of HCC and closely associated with some types of immunocompetent cells. The survival analysis based on NCF2 expression assessed by immunohistochemistry also confirmed that NCF2-positive group had a shorter relapse free survival (RFS) and overall survival (OS) than NCF2-negative group. Multivariate Cox regression revealed NCF2 expression level and lymphovascular space invasion (LVSI) were independent risk factors for HCC patients. Receiver operating characteristic curves showed that the combination of NCF2 and LVSI had higher predictive efficacy on the 1-year RFS rate and 5-year OS rate than each of them alone. Besides, the expression level of NCF2 was positively associated with M0 and M2 macrophages infiltration. Furthermore, NCF2 expression was positively correlated with CSF1, IL4, IL10, CD206, CD163, CSF1R and TGFβ1. We proposed that higher NCF2 expression predicted an adverse prognosis and more M2 macrophages infiltration in HCC patients.

A missense variant in NCF1 is associated with susceptibility to unexplained recurrent spontaneous abortion.

Unexplained recurrent spontaneous abortion (URSA) is a major concern in reproductive medicine. Neutrophil cytosolic factor 1 (NCF1) polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases. We investigated the association of the missense single nucleotide polymorphism (SNP) rs201802880 (NCF1-339) in NCF1 with URSA and explored its function. We performed NCF1-339 SNP genotyping of samples from 152 Chinese patients with URSA and 72 healthy controls using nested PCR and TaqMan assays. ROS production and RELA (NF-κB subunit) expression in the blood of participants with different NCF1-339 genotypes were determined. The frequencies of the wild-type (GG) and mutant (GA) genotypes remarkably differed between the URSA and control groups. The mutant genotype was associated with an increased risk of recurrent abortion. Furthermore, ROS levels in the URSA group with the GG genotype were significantly higher than those in the group with the GA genotype (p < 0.05). RELA expression in URSA patients with the GA genotype was considerably higher than that in control individuals with the GG genotype. These findings indicate that mutations in NCF1 may increase the risk of URSA via the NADP/ROS/NF-κB signaling pathway, which has implications for the diagnosis and treatment of URSA.

ROS Regulate NCF2, Key Metabolic Enzymes and MDA Levels to Affect the Growth of Fusarium solani

Fusarium solani is the most significant pathogen that causes root rot in wolfberry, which has led to serious economic losses in terms of production. As an important enzyme in organisms, NADPH oxidase produces ROS. However, the mechanism of ROS mediated by NADPH oxidase in the growth of F. solani has not been studied. In this study, F. solani colonies were treated with 40 µmol/L DPI and 0.0012% H2O2. The growth rate in terms of colonies, number of spores, key gene expression levels, activity of key enzymes and the content of key products of ROS metabolic pathways were determined. The results showed that the growth rate of colonies treated by DPI decreased by 19.43%, the number of macroconidia increased by 231.03%, the IOD/area values of O2− and H2O2 decreased by 34.88% and 16.97%, respectively, the expression levels in terms of NCF2, SOD1, CTA1 and PXMP4 significantly decreased and the activities of SOD, CAT and POD decreased significantly, while the MDA content increased significantly. Additionally, in the case of the colonies treated with exogenous H2O2, the MDA content decreased significantly while the other indicators increased. Taken together, the NCF2 gene is involved in regulating the activity of NADPH oxidase and regulates the products of O2− and ROS metabolism enzyme genes and their activities to affect colony growth in the F. solani growth process.

Glial cell reactivity and oxidative stress prevention in Alzheimer’s disease mice model by an optimized NMDA receptor antagonist

In Alzheimer's disease pathology, several neuronal processes are dysregulated by excitotoxicity including neuroinflammation and oxidative stress (OS). New therapeutic agents capable of modulating such processes are needed to foster neuroprotection. Here, the effect of an optimised NMDA receptor antagonist, UB-ALT-EV and memantine, as a gold standard, have been evaluated in 5XFAD mice. Following treatment with UB-ALT-EV, nor memantine, changes in the calcineurin (CaN)/NFAT pathway were detected. UB-ALT-EV increased neurotropic factors (Bdnf, Vgf and Ngf) gene expression. Treatments reduced astrocytic and microglial reactivity as revealed by glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) quantification. Interestingly, only UB-ALT-EV was able to reduce gene expression of Trem2, a marker of microglial activation and NF-κB. Pro-inflammatory cytokines Il-1β, Ifn-γ, Ccl2 and Ccl3 were down-regulated in UB-ALT-EV-treated mice but not in memantine-treated mice. Interestingly, the anti-inflammatory markers of the M2-migroglial phenotype, chitinase-like 3 (Ym1) and Arginase-1(Arg1), were up-regulated after treatment with UB-ALT-EV. Since iNOS gene expression decreased after UB-ALT-EV treatment, a qPCR array containing 84 OS-related genes was performed. We found changes in Il-19, Il-22, Gpx6, Ncf1, Aox1 and Vim gene expression after UB-ALT-EV. Hence, our results reveal a robust effect on neuroinflammation and OS processes after UB-ALT-EV treatment, surpassing the memantine effect in 5XFAD.

Detection of NCF-2 Gene Expression in Egyptian Children with Chronic Granulomatous Disease

Detection of NCF-2 Gene Expression in Egyptian Children with Chronic Granulomatous Disease

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

Impact of single-nucleotide variants and nutritional status on population pharmacokinetics of Doxorubicin, and its effect on cardiotoxicity in children with leukemia.

Doxorubicin is an important antineoplastic agent with wide interindividual variability in response to treatment and in its cardiotoxic effects. To determine the effect of genotypic status of three single-nucleotide variants in ABCC1, NCF4, and CBR3 genes and nutritional status assessed by body mass index, on the population pharmacokinetics of Doxorubicin and its cardiotoxic effects in pediatric patients with leukemia. Seventy pediatric patients treated with Doxorubicin were studied, in which 189 biological samples were obtained to determine Doxorubicin concentrations (1 to 3 samples per patient) at different times, for 20 h. Low body mass index and age ≤ 7 years were associated with decreased clearance of Doxorubicin, and female gender was associated with increased clearance of Doxorubicin. Low BMI and low height were associated with a decrease and increase, respectively, in the intercompartmental clearance (Q) of Doxorubicin. TT homozygosity of the single-nucleotide variant rs3743527 of the ABCC1 gene was associated with an increase in clearance and decreased area under the curve, AA homozygosity of the single-nucleotide variant rs1883112 of the NCF4 gene was associated with a decrease in the volume of distribution in the peripheral compartment (V2), and GG homozygosity of CBR3 rs1056892 with increasing area under the curve. Some covariates studied are directly related to the increase or decrease of the pharmacokinetic parameters of Doxorubicin. Decreased clearance, V2, and increased area under the curve were associated with systolic dysfunction, and decreased Q and V2 were associated with diastolic dysfunction. These results may contribute to the effective and safe use of Doxorubicin in pediatric patients with leukemia.

The NCF1 variant p.R90H aggravates autoimmunity by facilitating the activation of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are a professional type I IFN producer that play critical roles in the pathogenesis of autoimmune diseases. However, both genetic regulation of the function of pDCs and their relationships with autoimmunity are largely undetermined. Here, we investigated the causality of the neutrophil cytosolic factor 1 (NCF1) missense variant, which is one of the most significant associated risk variants for lupus, and found that the substitution of arginine (R) for histidine (H) at position 90 in the NCF1 protein (NCF1 p.R90H) led to excessive activation of pDCs. A mechanism study demonstrated that p.R90H reduced the affinity of NCF1 for phospholipids, thereby impairing endosomal localization of NCF1. As NCF1 is a subunit of the NADPH oxidase 2 (NOX2) complex, this impairment led to an acidified endosomal pH and facilitated downstream TLR signaling. Consistently, the homozygous knockin mice manifested aggravated lupus progression in a pDC-dependent lupus model. More important, pharmaceutical intervention revealed that hydroxychloroquine (HCQ) could antagonize the detrimental function of NCF1 p.R90H in the lupus model and systemic lupus erythematosus samples, supporting the idea that NCF1 p.R90H could be identified as a genetic biomarker for HCQ application. Therefore, our study provides insights into the genetic control of pDC function and a paradigm for applying genetic variants to improve targeted therapy for autoimmune diseases.

Multifocal Trichosporon asahii Infection in a Patient With Chronic Granulomatous Disease

A 20-year-old female with chronic granulomatous disease (CGD) presented with a 10-day history of left hip pain and intermittent fever to an outpatient clinic in the Midwestern United States. She reported constant, moderate to severe pain over the posterior aspect of the left hip with distal radiation that improved with movement. She denied any joint swelling, decrease in range of motion, discoloration, rash, or edema. A review of systems was significant for a nonproductive cough without dyspnea and a 3-kg weight loss during the last 4 months. The patient’s CGD resulted from biallelic pathogenic variants in the neutrophil cytosolic factor 1 (NCF1) gene, an autosomal-recessive form of CGD. She was originally from the United Arab Emirates (UAE) and had arrived in the United States 30 days before presentation for allogeneic hematopoietic stem cell transplantation (HSCT) evaluation. Previous history was notable for Streptococcus intermedius liver abscess treated 4 years before presentation. She denied any significant animal exposure or known exposure to individuals with active tuberculosis infection. She had poor adherence to her previously prescribed itraconazole and trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis regimen.

Distinct histopathological phenotypes of severe alcoholic hepatitis suggest different mechanisms driving liver injury and failure.

Intrahepatic neutrophil infiltration has been implicated in severe alcoholic hepatitis (SAH) pathogenesis; however, the mechanism underlying neutrophil-induced injury in SAH remains obscure. This translational study aims to describe the patterns of intrahepatic neutrophil infiltration and its involvement in SAH pathogenesis. Immunohistochemistry analyses of explanted livers identified two SAH phenotypes despite a similar clinical presentation, one with high intrahepatic neutrophils (Neuhi), but low levels of CD8+ T cells, and vice versa. RNA-Seq analyses demonstrated that neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with hepatic inflammation and disease progression. To study specifically the mechanisms related to Neuhi in AH patients and liver injury, we used the mouse model of chronic-plus-binge ethanol feeding and found that myeloid-specific deletion of the Ncf1 gene abolished ethanol-induced hepatic inflammation and steatosis. RNA-Seq analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic hepatitis (AH) by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key antiinflammatory and antifibrotic microRNA). In conclusion, two distinct histopathological phenotypes based on liver immune phenotyping are observed in SAH patients, suggesting a separate mechanism driving liver injury and/or failure in these patients.

TRPM2 regulates autophagy to participate in hepatitis B virus replication.

Hepatitis B virus (HBV) affects over 300 million people across the world and is further associated with the self-digesting process of autophagy. Accordingly, the current study set out to explore the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in HBV replication. Firstly, Huh-7 cells were transfected with the pHBV1.3 plasmid to detect the expression patterns of TRPM2 and neutrophil cytosolic factor 1 (p47 phox), followed by evaluating the role of TRPM2 in autophagy and HBV replication and exploring the interaction between TRPM2 and p47 phox. Collaborative experiments were further designed to explore the role of p47 phox and autophagy in TRPM2 regulation of HBV replication, in addition to animal experimentation to validate the role of TRPM2/p47 phox axis in vivo. It was found that TRPM2 up-regulation was associated with HBV replication. On the other hand, silencing of TRPM2 inhibited HBV replication and autophagy in vitro and in vivo, as evidenced by reduced HBV DNA load, HBV mRNA, HBeAg and HBsAg, and diminished autophagic spot number, LC3 II/I ratio, Beclin-1 expressions and increased p62 expressions. Mechanistic experimentation illustrated that TRPM2 interacted with p47 phox and positively regulated p47 phox, such that p47 phox up-regulation or use of Rapamycin (autophagy activator) weakened the inhibitory role of silencing TRPM2. Collectively, our findings indicated that HBV infection promotes TRPM2 expression, and TRPM2 interacts with p47 phox to induce autophagy and facilitate HVB replication.

Characterization of chronic relapsing antibody mediated arthritis in mice with a mutation in Ncf1 causing reduced oxidative burst

Rheumatoid arthritis (RA) is a chronic autoimmune disorder affecting joints with a hallmark of autoantibody production. Mannan-enhanced collagen type II (COL2) antibody induced arthritis (mCAIA) in neutrophil cytosolic factor 1(Ncf1) mutation mouse is a chronic disease model imitating RA in mice. In this study, we characterize the chronic phase of mCAIA in Ncf1 mutated (BQ.Ncf1m1j/m1j) mice. Arthritis was induced by an intravenous injection of anti-COL2 monoclonal antibodies on day 0 followed by intra-peritoneal injections of mannan (from Saccharomyces cerevisiae) on days 3 and 65 in BQ.Ncf1m1j/m1j and BQ mice. Bone erosion was analysed by computed tomography (CT) and blood cell phenotypes by flow cytometry. Cytokines and anti-COL2 antibodies were analyzed with multiplex bead-based assays. The arthritis in the Ncf1m1j/m1j mice developed with a chronic and relapsing disease course, which was followed for 200 days and bone erosions of articular joints were evaluated. An increased number of circulating CD11b+ Ly6G+ neutrophils were observed during the chronic phase, together with a higher level of G-CSF (granulocyte colony-stimulating factor) and TNF-α. In conclusion, the chronic relapsing arthritis of mCAIA in the Ncf1m1j/m1j mice develop bone erosions associated with a sustained neutrophil type of inflammatory responses.

Enteric glia regulates the expression of neutrophil inflammatory factors during acute inflammation

Abstract Inflammation is an event in functional gastrointestinal disorders that has detrimental effects in the enteric nervous system. Recent advances in our understanding of the immune and enteric nervous system indicate that enteric glial cells modulate immune cells in the gastrointestinal tract. Previous single-nuclei multiomic analysis has shown that genes that encode for and influence neutrophil function, Neutrophil cytosolic factor 4 and C-X-C motif chemokine ligand 5, are changed in enteric glia during peak colitis. We hypothesize that enteric glia regulate the expression of neutrophil modulating factors during acute inflammation. We tested this using the acute dinitrobenzene sulfonic acid colitis model. Animal body weight and gross macroscopic damage were recorded as indications of overall inflammation and tissue was harvested for immunohistochemical analysis. Enteric glial cells were labeled with antibodies against Neutrophil cytosolic factor 4 and C-X-C motif chemokine ligand 5. Results suggest no significant difference in labeling for immunological markers in enteric glial cells of dinitrobenzene sulfonic acid treated mice. Results also show labeling in some neurons that may or may not be specific. Further experimentation is needed to elucidate the mechanisms of enteric neurons and glia in gut inflammation. These studies will contribute to further understand how immune responses are regulated in the gut and impact the general knowledge of gut disease processes. This work was supported by the National Institute of Health grants R01DK103723 and R01DK120862 to BDG and DPR was supported by the National Institute of Health Bridge to the Ph.D. in Neuroscience grant 2R25NS090989.

Polymorphic Variants of the Neutrophil Cytosolic Factor 2 Gene: Associations with Susceptibility to Type 2 Diabetes Mellitus and Cardiovascular Autonomic Neuropathy

Polymorphic Variants of the Neutrophil Cytosolic Factor 2 Gene: Associations with Susceptibility to Type 2 Diabetes Mellitus and Cardiovascular Autonomic Neuropathy

Euphorbia supina Extracts Block NADPH Oxidase-Mediated, Ceramide-Induced Apoptosis Initiated by Diesel Particulate Matter.

Air pollutants contribute to the development of diseases such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary cancer, cardiovascular problems, and some skin diseases. We recently found that a major air pollutant, diesel particulate matter (DPM), induces apoptosis in human keratinocytes by increasing a proapoptotic lipid mediator, ceramide. DPM activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which stimulates sphingomyelinase, leading to an increased conversion of sphingomyelin to ceramide. Interestingly, we characterized that although NOX is a reactive oxygen species (ROS) generator, the activation of sphingomyelinases by NOX is an ROS-independent mechanism. A Korean weed, prostrate spurge Euphorbia supina Rafin (ESR), has been used for centuries as a folk medicine to treat bronchitis, hepatitis, hemorrhage, and skin inflammation. Flavonoids, terpenes and tannins are enriched in ESR, and although ESR has proven antioxidative activity, its biological activities are largely unknown. Here, we investigate whether and how ESR protects keratinocytes against DPM-mediated apoptosis. We found that ESR-extracts (ESR-Ex) protect keratinocytes from DPM-induced apoptosis by inhibiting NOX activation in keratinocytes in response to DPM. We also demonstrated that ESR-Ex suppresses NOX activation via a blockage of the aryl hydrocarbon receptor (AhR) activation-mediated transcription of neutrophil cytosolic factor 1 (NCF1)/p47phox, a subunit of NOX. Our study reveals previously uncharacterized biological activity of ESR-Ex; i.e., its inhibition of Ahr and NOX activation. Thus, because the inhibition of NOX has already been developed to treat NOX-mediated diseases, including various types of cardiovascular diseases and cancers, initiated by air pollutants and because AhR activation contributes to the development of chronic inflammatory diseases, our study provides further advantages for the medical use of ESR.

Choline and citicoline ameliorate oxidative stress in acute kidney injury in rats.

The purpose of this study is to investigate the effects of cholinergic anti-inflammatory pathway (CAP)-activating drugs, choline and citicoline (Cytidinediphosphate-choline, CDP-choline), on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) parameters and the contribution of NADPH Oxidase4 (NOX4) p22phox. Endotoxemia induces a systemic inflammatory response characterized by the production of pro-inflammatory mediators and reactive oxygen species (ROS), which eventually develops acute kidney injury (AKI). NADPH Oxidase4 (NOX4) p22phox pathway contributes to the development of endotoxemia-induced AKI. Inflammatory response can be controlled by CAP. Expressions levels of KIM-1, TNF-α, NOX4, p22phox and NFκB in the kidney tissues of rats were analyzed via RT-PCR in experimental groups; 1. Control, 2. LPS (10 mg/kg) + saline, 3. LPS + CDP-choline (375 mg/kg) and 4. LPS + choline (90 mg/kg). Choline and ROS levels in kidney tissues were also measured by a spectrofluorometric assay. LPS-induced elevations of ROS levels were decreased by CDP-choline or choline administration (p < 0.001). LPS-elevated KIM-1, TNFα, NOX4, p22 phox, and NFκB expressions were significantly decreased by choline or CDP-choline treatments (p < 0.001). Decreased ROS production in kidney tissues in treatment groups suggests that choline or CDP-choline may have therapeutic potential in endotoxemia-associated AKI via downregulating NOX4 and p22phox expressions (Tab. 1, Fig. 5, Ref. 45). Text in PDF www.elis.sk Keywords: endotoxemia, choline, cytidine diphosphate choline, acute kidney injury, reactive oxygen species.