NADPH Oxidase Research Articles

Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia-Reperfusion-Induced Kidney Injury.

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

Cross-Domain Text Mining of Pathophysiological Processes Associated with Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide. This study's goal was to identify the signaling drivers and pathways that modulate glomerular endothelial dysfunction in DKD via artificial intelligence-enabled literature-based discovery. Cross-domain text mining of 33+ million PubMed articles was performed with SemNet 2.0 to identify and rank multi-scalar and multi-factorial pathophysiological concepts related to DKD. A set of identified relevant genes and proteins that regulate different pathological events associated with DKD were analyzed and ranked using normalized mean HeteSim scores. High-ranking genes and proteins intersected three domains-DKD, the immune response, and glomerular endothelial cells. The top 10% of ranked concepts were mapped to the following biological functions: angiogenesis, apoptotic processes, cell adhesion, chemotaxis, growth factor signaling, vascular permeability, the nitric oxide response, oxidative stress, the cytokine response, macrophage signaling, NFκB factor activity, the TLR pathway, glucose metabolism, the inflammatory response, the ERK/MAPK signaling response, the JAK/STAT pathway, the T-cell-mediated response, the WNT/β-catenin pathway, the renin-angiotensin system, and NADPH oxidase activity. High-ranking genes and proteins were used to generate a protein-protein interaction network. The study results prioritized interactions or molecules involved in dysregulated signaling in DKD, which can be further assessed through biochemical network models or experiments.

Fennel seeds extract prevents fructose-induced cardiac dysfunction in a rat model of metabolic syndrome via targeting abdominal obesity, hyperuricemia and NF-κβ inflammatory pathway

BackgroundMetabolic syndrome (MetS) is commonly associated with increased risk of cardiac disease that affects a large number of world populations. ObjectiveThis research attempted to investigate the efficacy of fennel seeds extract (FSE) in preventing development of cardiac dysfunction in rats on fructose enriched diet for 3 months, as a model of MetS. Materials & methodsThirty adult Wistar male rats (160–170 g) were assigned into 5 groups including control, vehicle, FSE (200 mg/kg BW) and fructose (60%) fed rats with and without FSE. Following the last treatment, blood pressure, ECG and heart rate were measured. Next, blood and cardiac tissues were taken for biochemical and histological investigations. ResultsFeeding fructose exhibited characteristic features of MetS involving, hypertension, abnormal ECG, elevated heart rate, serum glucose, insulin, lipids and insulin resistance, accompanied by abdominal obesity, cardiac hypertrophy and hyperuricemia. Fructose fed rats also showed significant reduction in cardiac antioxidants (GSH, SOD, CAT) with elevation in oxidative stress indices (NADPH oxidase, O2.-, H2O2, MDA, PCO), NF-κβ, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), adhesion molecules (ICAM-1, VCAM-1) and serum cardiac biomarkers (AST, LDH, CK-MB, cTn-I). Histopathological changes evidenced by destruction of cardiac myofibrils, cytoplasmic vacuolization, and aggregation of inflammatory cells were also detected. Consumption of FSE showed high ability to alleviate fructose-induced hypertension, ECG abnormalities, cardiac hypertrophy, metabolic alterations, oxidative stress, inflammation and histological injury. ConclusionFindings could suggest FSE as a complementary supplement for preventing MetS and associated cardiac outcomes. However, well controlled clinical studies are still needed.

Redox Regulation of Phosphatase and Tensin Homolog by Bicarbonate and Hydrogen Peroxide: Implication of Peroxymonocarbonate in Cell Signaling.

Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) signaling pathway. Notably, its active site contains a cysteine residue that is susceptible to oxidation by hydrogen peroxide (H2O2). This oxidation inhibits the phosphatase function of PTEN, critically contributing to the activation of the PI3K/AKT pathway. Upon the stimulation of cell surface receptors, the activity of NADPH oxidase (NOX) generates a transient amount of H2O2, serving as a mediator in this pathway by oxidizing PTEN. The mechanism underlying this oxidation, occurring despite the presence of highly efficient and abundant cellular oxidant-protecting and reducing systems, continues to pose a perplexing conundrum. Here, we demonstrate that the presence of bicarbonate (HCO3-) promoted the rate of H2O2-mediated PTEN oxidation, probably through the formation of peroxymonocarbonate (HCO4-), and consequently potentiated the phosphorylation of AKT. Acetazolamide (ATZ), a carbonic anhydrase (CA) inhibitor, was shown to diminish the oxidation of PTEN. Thus, CA can also be considered as a modulator in this context. In essence, our findings consolidate the crucial role of HCO3- in the redox regulation of PTEN by H2O2, leading to the presumption that HCO4- is a signaling molecule during cellular physiological processes.

Peroxidase gene TaPrx109-B1 enhances wheat tolerance to water deficit via modulating stomatal density.

Increasing the tolerance of crops to water deficit is crucial for the improvement of crop production in water-restricted regions. Here, a wheat peroxidase gene (TaPrx109-B1) belonging to the class III peroxidase gene family was identified and its function in water deficit tolerance was revealed. We demonstrated that overexpression of TaPrx109-B1 reduced leaf H2O2 level and stomatal density, increased leaf relative water content, water use efficiency, and tolerance to water deficit. The expression of TaEPF1 and TaEPF2, two key negative regulators of stomatal development, were significantly upregulated in TaPrx109-B1 overexpression lines. Furthermore, exogenous H2O2 downregulated the expression of TaEPF1 and TaEPF2 and increased stomatal density, while exogenous application of diphenyleneiodonium chloride, a potent NADPH oxidase inhibitor that repressed the synthesis of H2O2, upregulated the expression of TaEPF1 and TaEPF2, decreased stomatal density, and enhanced wheat tolerance to water deficit. These findings suggest that TaPrx109-B1 influences leaf stomatal density by modulation of H2O2 level and the expression of TaEPF1 and TaEPF2. The results of the field trial showed that overexpressing TaPrx109-B1 increased grain number per spike, which reduced the yield loss caused by water deficiency. Therefore, TaPrx109-B1 has great potential in breeding wheat varieties with improved water deficit tolerance.

NOX2 and NOX4 expression in monocytes and macrophages-extracellular vesicles in signalling and therapeutics.

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

Dexmedetomidine affects the NOX4/Nrf2 pathway to improve renal antioxidant capacity.

The study aimed to investigate the protective effects of dexmedetomidine (DEX) on renal injury caused by acute stress in rats and explore the protective pathways of DEX on rat kidneys in terms of oxidative stress. An acute restraint stress model was utilized, where rats were restrained for 3 hours after a 15-minute swim. Biochemical tests and histopathological sections were conducted to evaluate renal function, along with the measurement of oxidative stress and related pathway proteins. The open-field experiments validated the successful establishment of the acute stress model. Acute stress-induced renal injury led to increased NADPH oxidase 4 (NOX4) protein expression and decreased expression levels of nuclear transcription factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1). Following DEX treatment, there was a significant reduction in renal NOX4 expression. The DEX-treated group exhibited normalized renal biochemical results and less damage observed in pathological sections compared to the acute stress group. The findings suggest that DEX treatment during acute stress can impact the NOX4/Nrf2/HO-1/NQO1 signaling pathway and inhibit oxidative stress, thereby preventing acute stress-induced kidney injury. Additionally, DEX shows promise for clinical applications in stress syndromes.

Involvement of ROS and calcium ions in developing heat resistance and inducing antioxidant system of wheat seedlings under melatonin's effects.

The stress-protective effect of melatonin (N-acetyl-5-methoxytryptamine) on plant cells is mediated by key signaling mediators, in particular calcium ions and reactive oxygen species (ROS). However, the links between changes in calcium and redox homeostasis and the formation of adaptive responses of cultivated cereals (including wheat) to the action of high temperatures have not yet been studied. In the present study, we investigated the possible involvement of ROS and calcium ions as signaling mediators in developing heat resistance in wheat (Triticum aestivum L.) seedlings and activating their antioxidant system. Treatment of 3-day-old etiolated seedlings with melatonin solutions at concentrations 0.01-10µM increased their survival after exposure to 45°C for 10min. The most significant stress-protective effect was exerted by melatonin treatment at 1µM concentration. Under the influence of melatonin, a transient enhancement of superoxide anion radical (O2•-) generation and an increase in hydrogen peroxide content were observed in roots, with a maximum at 1h. Four hours after treatment with melatonin, the activity of catalase and guaiacol peroxidase increased in roots, while the activity of superoxide dismutase did not change significantly. After exposure to 45°C, the activity of catalase and guaiacol peroxidase was higher in the roots of melatonin-treated wheat seedlings, and the indices of ROS generation, content of the lipid peroxidation product malonic dialdehyde, and cell membrane damage were lower than in control seedlings. Melatonin-induced changes in root ROS generation and antioxidant enzyme activities were eliminated by pretreatment with the hydrogen peroxide scavenger dimethylthiourea (DMTU), NADPH oxidase inhibitor imidazole, and calcium antagonists (the extracellular calcium chelator EGTA and phospholipase C inhibitor neomycin). Treatment with DMTU, imidazole, EGTA, and neomycin also abolished the melatonin-induced increase in survival of wheat seedlings after heat stress. The role of calcium ions and ROS, generated with the participation of NADPH oxidase, as signaling mediators in the melatonin-induced antioxidant system and heat stress resistance of wheat seedlings have been demonstrated.

NOX4 promotes tumor progression through the MAPK-MEK1/2-ERK1/2 axis in colorectal cancer.

Metabolic reprogramming plays a key role in cancer progression and clinical outcomes; however, the patterns and primary regulators of metabolic reprogramming in colorectal cancer (CRC) are not well understood. To explore the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in promoting progression of CRC. We evaluated the expression and function of dysregulated and survival-related metabolic genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Consensus clustering was used to cluster CRC based on dysregulated metabolic genes. A prediction model was constructed based on survival-related metabolic genes. Sphere formation, migration, invasion, proliferation, apoptosis and clone formation was used to evaluate the biological function of NOX4 in CRC. mRNA sequencing was utilized to explore the alterations of gene expression NOX4 over-expression tumor cells. In vivo subcutaneous and lung metastasis mouse tumor model was used to explore the effect of NOX4 on tumor growth. We comprehensively analyzed 3341 metabolic genes in CRC and identified three clusters based on dysregulated metabolic genes. Among these genes, NOX4 was highly expressed in tumor tissues and correlated with worse survival. In vitro, NOX4 overexpression induced clone formation, migration, invasion, and stemness in CRC cells. Furthermore, RNA-sequencing analysis revealed that NOX4 overexpression activated the mitogen-activated protein kinase-MEK1/2-ERK1/2 signaling pathway. Trametinib, a MEK1/2 inhibitor, abolished the NOX4-mediated tumor progression. In vivo, NOX4 overexpression promoted subcutaneous tumor growth and lung metastasis, whereas trametinib treatment can reversed the metastasis. Our study comprehensively analyzed metabolic gene expression and highlighted the importance of NOX4 in promoting CRC metastasis, suggesting that trametinib could be a potential therapeutic drugs of CRC clinical therapy targeting NOX4.

Saikosaponin a promotes neutrophil extracellular trap formation and bactericidal activity

This study aimed to investigate the effects of SSa, one of the major triterpenoid saponins extracted from Radix bupleuri, on neutrophil extracellular trap (NET) formation and the mechanism associated with this process. Using Sytox green and immunofluorescence assays, we found SSa rapidly induced NET formation, which depended on NADPH oxidase (NOX)-independent ROS production and autophagy. Pharmacologic inhibitor studies indicated that ERK and PI3K/AKT signalling were also required for SSa-induced NET formation, whereas protein arginine deiminase 4 (PAD4) was not required. Furthermore, we found that SSa promoted neutrophil bactericidal activity mainly through NET formation. Based on flow cytometry and the Cell Counting Kit-8 (CCK-8) assays, the results demonstrated that SSa-induced NET formation occurred without neutrophil death. Taken together, these findings indicated that SSa could be a potential natural product to boost innate immune defense against pathogen attack via NET formation.

Circ_005077 accelerates myocardial lipotoxicity induced by high-fat diet via CyPA/p47PHOX mediated ferroptosis

The long-term high-fat diet (HFD) can cause myocardial lipotoxicity, which is characterized pathologically by myocardial hypertrophy, fibrosis, and remodeling and clinically by cardiac dysfunction and heart failure in patients with obesity and diabetes. Circular RNAs (circRNAs), a novel class of noncoding RNA characterized by a ring formation through covalent bonds, play a critical role in various cardiovascular diseases. However, few studies have been conducted to investigate the role and mechanism of circRNA in myocardial lipotoxicity. Here, we found that circ_005077, formed by exon 2–4 of Crmp1, was significantly upregulated in the myocardium of an HFD-fed rat. Furthermore, we identified circ_005077 as a novel ferroptosis-related regulator that plays a role in palmitic acid (PA) and HFD-induced myocardial lipotoxicity in vitro and in vivo. Mechanically, circ_005077 interacted with Cyclophilin A (CyPA) and inhibited its degradation via the ubiquitination proteasome system (UBS), thus promoting the interaction between CyPA and p47phox to enhance the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for ROS generation, subsequently inducing ferroptosis. Therefore, our results provide new insights into the mechanisms of myocardial lipotoxicity, potentially leading to the identification of a novel therapeutic target for the treatment of myocardial lipotoxicity in the future.

Teneligliptin mitigates diabetic cardiomyopathy by inhibiting activation of the NLRP3 inflammasome

BACKGROUND Diabetic cardiomyopathy (DCM), which is a complication of diabetes, poses a great threat to public health. Recent studies have confirmed the role of NLRP3 (NOD-like receptor protein 3) activation in DCM development through the inflammatory response. Teneligliptin is an oral hypoglycemic dipeptidyl peptidase-IV inhibitor used to treat diabetes. Teneligliptin has recently been reported to have anti-inflammatory and protective effects on myocardial cells. AIM To examine the therapeutic effects of teneligliptin on DCM in diabetic mice. METHODS Streptozotocin was administered to induce diabetes in mice, followed by treatment with 30 mg/kg teneligliptin. RESULTS Marked increases in cardiomyocyte area and cardiac hypertrophy indicator heart weight/tibia length reductions in fractional shortening, ejection fraction, and heart rate; increases in creatine kinase-MB (CK-MB), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels; and upregulated NADPH oxidase 4 were observed in diabetic mice, all of which were significantly reversed by teneligliptin. Moreover, NLRP3 inflammasome activation and increased release of interleukin-1β in diabetic mice were inhibited by teneligliptin. Primary mouse cardiomyocytes were treated with high glucose (30 mmol/L) with or without teneligliptin (2.5 or 5 µM) for 24 h. NLRP3 inflammasome activation. Increases in CK-MB, AST, and LDH levels in glucose-stimulated cardiomyocytes were markedly inhibited by teneligliptin, and AMP (p-adenosine 5‘-monophosphate)-p-AMPK (activated protein kinase) levels were increased. Furthermore, the beneficial effects of teneligliptin on hyperglycaemia-induced cardiomyocytes were abolished by the AMPK signaling inhibitor compound C. CONCLUSION Overall, teneligliptin mitigated DCM by mitigating activation of the NLRP3 inflammasome.

Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Oxidative Stress: A Culprit in the Progression of Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the principal culprit behind chronic kidney disease (CKD), ultimately developing end-stage renal disease (ESRD) and necessitating costly dialysis or kidney transplantation. The limited therapeutic efficiency among individuals with DKD is a result of our finite understanding of its pathogenesis. DKD is the result of complex interactions between various factors. Oxidative stress is a fundamental factor that can establish a link between hyperglycemia and the vascular complications frequently encountered in diabetes, particularly DKD. It is crucial to recognize the essential and integral role of oxidative stress in the development of diabetic vascular complications, particularly DKD. Hyperglycemia is the primary culprit that can trigger an upsurge in the production of reactive oxygen species (ROS), ultimately sparking oxidative stress. The main endogenous sources of ROS include mitochondrial ROS production, NADPH oxidases (Nox), uncoupled endothelial nitric oxide synthase (eNOS), xanthine oxidase (XO), cytochrome P450 (CYP450), and lipoxygenase. Under persistent high glucose levels, immune cells, the complement system, advanced glycation end products (AGEs), protein kinase C (PKC), polyol pathway, and the hexosamine pathway are activated. Consequently, the oxidant-antioxidant balance within the body is disrupted, which triggers a series of reactions in various downstream pathways, including phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), transforming growth factor beta/p38-mitogen-activated protein kinase (TGF-β/p38-MAPK), nuclear factor kappa B (NF-κB), adenosine monophosphate-activated protein kinase (AMPK), and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. The disease might persist even if strict glucose control is achieved, which can be attributed to epigenetic modifications. The treatment of DKD remains an unresolved issue. Therefore, reducing ROS is an intriguing therapeutic target. The clinical trials have shown that bardoxolone methyl, a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, blood glucose-lowering drugs, such as sodium-glucose cotransporter 2 inhibitors, and glucagon-like peptide-1 receptor agonists can effectively slow down the progression of DKD by reducing oxidative stress. Other antioxidants, including vitamins, lipoic acid, Nox inhibitors, epigenetic regulators, and complement inhibitors, present a promising therapeutic option for the treatment of DKD. In this review, we conduct a thorough assessment of both preclinical studies and current findings from clinical studies that focus on targeted interventions aimed at manipulating these pathways. We aim to provide a comprehensive overview of the current state of research in this area and identify key areas for future exploration.

NOX-derived ROS modulates the antibacterial immune responses in coelomocytes of Apostichopus japonicus

As an important generator of reactive oxygen species (ROS) in eukaryotic cells, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is essential to antimicrobial host defense. AjNOX, a member of the NOX family, was identified in Apostichopus japonicus during the current investigation. It encodes a polypeptide with 571 amino acids, and it has an open reading frame of 1716 bp. The inferred amino acid sequence of AjNOX includes a transmembrane domain, a ferric reductase domain, a flavin adenine dinucleotide-binding-8 domain, and a NADPH-binding-6 domain. AjNOX shares 53.03%–42.39% sequence homology with other NOX proteins found in vertebrates and invertebrates in accordance with multiple sequence alignment. AjNOX was found in close proximity to the sea urchin SpNOX and the vertebrate NOX1–3 on the phylogenetic tree. The AjNOX gene was expressed in all of the test tissues including the body wall, muscle, intestine, respiratory tree, and coelomocytes. After being challenged with Vibrio splendidus, sea cucumber coelomocytes showed a remarkable increase in the protein and mRNA levels of AjNOX. ROS was produced less remarkably in coelomocytes as a result of AjNOX RNA interference. When the expression of AjNOX was suppressed by siRNA or inhibitor, the ability of coelomocytes to remove bacteria was drastically decreased. Reducing AjNOX-derived ROS decreased the production of inflammatory cytokines and coelomocyte apoptosis. All of these findings indicate that AjNOX may be crucial to the antibacterial immune response against the invasive pathogen V. splendidus in sea cucumbers A. japonicus.

NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis

The endogenous metabolite of estradiol, estradiol 17β-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.

In ovo feeding of α-ketoglutaric acid improves hepatic antioxidant-gene expression, plasma antioxidant activities and decreases body temperature without affecting broiler body weight under cyclic heat stress

The broiler industry is adversely affected by the rise in global temperature. This study investigated the effects of in ovo feeding of α-ketoglutaric acid (AKG) on growth performance, organ weight, plasma metabolite, plasma oxidative stress, rectal temperature (RT), and hepatic mRNA expression of antioxidant-related genes in Arbor Acres broilers subjected to cyclic heat stress (HS). Three hundred fifty fertile eggs during incubation were divided into 5 groups according to AKG concentrations and temperature conditions. After dissolving AKG in distilled water at 0, 0.5, 1.0, and 1.5, 0% AKG was in ovo administered to 2 of the 5 groups whereas the remaining 3 groups received 0.5, 1.0, and 1.5%, respectively. From d 29 to 34 of age, 4 groups of birds received heat stress (HS) at 31°C ± 1°C for 6 h per day while the other group was kept at room temperature (21°C ± 1°C; NT). So, the 5 treatment groups were: 1) 0AKG-NT, where chicks hatched from eggs receiving 0% AKG were reared under thermoneutral conditions. 2) 0AKG-HS, where chicks hatched from eggs receiving 0% AKG were reared under cyclic HS conditions. 3) 0.5AKG-HS, where chicks hatched from eggs receiving 0.5% AKG were reared under cyclic HS conditions. 4) 1.0AKG-HS, where chicks hatched from eggs receiving 1.0% AKG were reared under cyclic HS conditions. 5) 1.5AKG-HS, where chicks hatched from eggs receiving 1.5% AKG were reared under cyclic HS conditions. HS significantly reduced body weight change (ΔBW %) and average daily gain (ADG) without affecting average daily feed intake (ADFI). Feed conversion ratio (FCR) was significantly increased (P = 0.003) in all HS-treated groups. A significant linear decrease in the final RT (P = 0.005) and a change in RT (P = 0.003) were detected with increasing AKG concentration. Total antioxidant capacity (P = 0.029) and antioxidant balance (P = 0.001) in plasma increased linearly with increasing AKG concentration whereas malondialdehyde concentrations were linearly decreased (P = 0.001). Hepatic gene expression of CAT (P = 0.026) and GPX1 (P = 0.001) were dose-dependently upregulated while nicotinamide adenine dinucleotide phosphate oxidase (NOX)1, NOX4, and heat shock protein (HSP)70 were linearly downregulated (P < 0.05). Hence, in ovo injection of AKG was effective in mitigating HS-induced oxidative stress without attenuating the adverse effects on broiler growth.

Pharmacokinetics and Pharmacodynamics of a Nanostructured Lipid Carrier Co-Encapsulating Artemether and miRNA for Mitigating Cerebral Malaria.

Cerebral malaria (CM), a severe neurological pathology caused by Plasmodium falciparum infection, poses a significant global health threat and has a high mortality rate. Conventional therapeutics cannot cross the blood-brain barrier (BBB) efficiently. Therefore, finding effective treatments remains challenging. The novelty of the treatment proposed in this study lies in the feasibility of intranasal (IN) delivery of the nanostructured lipid carrier system (NLC) combining microRNA (miRNA) and artemether (ARM) to enhance bioavailability and brain targeting. The rational use of NLCs and RNA-targeted therapeutics could revolutionize the treatment strategies for CM management. This study can potentially address the challenges in treating CM, allowing drugs to pass through the BBB. The NLC formulation was developed by a hot-melt homogenization process utilizing 3% (w/w) precirol and 1.5% (w/v) labrasol, resulting in particles with a size of 94.39 nm. This indicates an effective delivery to the brain via IN administration. The results further suggest the effective intracellular delivery of encapsulated miRNAs in the NLCs. Investigations with an experimental cerebral malaria mouse model showed a reduction in parasitaemia, preservation of BBB integrity, and reduced cerebral haemorrhages with the ARM+ miRNA-NLC treatment. Additionally, molecular discoveries revealed that nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) and Interleukin-6 (IL-6) levels were reduced in the treated groups in comparison to the CM group. These results support the use of nanocarriers for IN administration, offering a viable method for mitigating CM through the increased bioavailability of therapeutics. Our findings have far-reaching implications for future research and personalized therapy.

Effects of garlic (Allium sativum L) and Citrullus colocynthis (L.) Schrad individually and in combination on male reproductive damage due to diabetes: suppression of the AGEs/RAGE/Nox-4 signaling pathway

BackgroundDiabetes Mellitus is associated with disturbances in male reproductive function and fertility. Studies have shown that oxidative stress with the subsequent inflammation and apoptosis cause these complications in diabetes. Garlic (G) (Allium sativum L) and Citrullus colocynthis (L.) Schrad (C) both have antidiabetic and antioxidant properties. Recently, we demonstrated their synergistic effects in alleviating reproductive complications when administered concomitantly. However, as even medicinal plants in long term usage may lead to some unwanted side effects of their own, we examined whether with half the original doses of these two medicinal plants we could achieve the desired results.MethodsThirty-five male Wistar rats were divided into five groups (n = 7/group): Control, Diabetic, Diabetic + G (0.5 ml/100 g BW), Diabetic + C (5 mg/kg BW) and Diabetic + GC (0.5 ml/100 g BW of garlic and 5 mg/kg BW of C. colocynthis) groups. The experimental period was 30 days.ResultsOxidative stress, advanced glycation end products (AGEs), immunoexpression of caspase-3, and expression of mRNAs for receptor for advanced glycation end products (RAGE), NADPH oxidase-4 (NOX-4) and nuclear factor kappa B increased in testis of diabetic rats. Treatment with garlic and C. colocynthis alone showed some beneficial effects, but in the combination form the effectiveness was more profound.ConclusionsWe conclude that the combination therapy of diabetic rats with lower doses is still as efficient as higher doses; therefore, the way forward for reducing complications in long term consumption.