NADPH Oxidase Inhibitor Research Articles

ROS scavenging, DNA binding and NADPH oxidase inhibition potential of N’-Ferrocenylmethyl-N’- phenylpropionohydrazide using cyclic voltammetry and molecular docking

Overactivation of the NOX results in an excess production of ROS, which is linked to oxidative stress and deoxyribonucleic acid (DNA) damage. Free radical formation is a physiological event mainly coming from cellular respiration. Inflammatory diseases, cardiovascular diseases, and cancer are only some illnesses that this could trigger. Therefore, chemicals that can block NADPH oxidase (NOX) are essential in maintaining a healthy level of oxidative stress. However, interacting with DNA can alter its shape and slow replication, protecting cells from death. In this study, cyclic voltammetry (CV) techniques were employed to assess the scavenging activity of N’-Ferrocenylmethyl-N’- phenylpropionohydrazide (NFPP) towards the superoxide anion radical (O2.−). Additionally, the DNA binding modes and binding free energies of NFPP were investigated. To support the experimental results, molecular docking was performed with DNA (PDB ID: 5VY6) and the enzyme NOX1 (PDB ID: 7CFZ). The results were consistent between the two approaches, showing that the chemical investigated had an IC50 lower than that of ascorbic acid (AA) and a spontaneous form of binding with (O2.−) and DNA due to the negative sign of binding energies. Minor groove binding with two conventional hydrogen bonds was the type of interaction with the DNA. The binding energy between NFPP and NADPH exhibited a significant correlation with the experimental findings, indicating a strong affinity and a reduced inhibition concentration compared to ascorbic acid.

The roles of NADPH oxidase and PKC pathways in the modulation of GLP-1 induced-vasorelaxation in diabetic rat aorta

A hormone of the incretin system known as glucagon-like peptide-1 (GLP-1) is important for a number of glucoregulatory functions. This study aimed to investigate the effect of inhibitors on the vasorelaxant response of GLP-1 in T1D rats. A single subcutaneous injection of 50 mg/kg of streptozotocin (STZ) was used to develop diabetes mellitus. Eighty aortic rings from twenty male albino rats were prepared. An Automatic Organ Bath was used. In our study, apocynin increased vascular response to GLP- 1 in the non-diabetic group with (Emax:72.40±0.350) and (pD2: -9.923±0.444). In induced diabetes, the NADPH oxidase inhibitor decreased GLP-1 vasodilatation property with (Emax; 68.91±2.002) and (pD2: -9.480±0.138). The results show that bisindolylmaleimide IX (RO31-8220) has an increased effect on GLP-1 vasodilation in non-diabetic groups with (Emax:88.45±38.18) and (pD2: -10.81±0.281). While in induced diabetic rat’s vascular relaxation of GLP-1 decreased with (Emax: 77.73±2.801) and (pD2: - 10.28±0.203). The data analysis demonstrated that rotenone with (Emax: 63.69±35.10) (pD2: - 9.612±0.246), and in diabetic rats with (Emax:69.98±22.94) and (pD2: -9.612±0.246). In our study oxypurinol with (Emax: 82.16±16.10) and (pD2: - 9.434±0.443), and in diabetic rats with (Emax: 58.03±8.350) and (pD2: -9.612±0.246). We concluded that inhibitors could increase the vasorelaxant response of GLP-1 in non-diabetic rats, while this vasorelaxant response of GLP-1 diminished in diabetic-induced rats.

NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

The Role of Reactive Oxygen Species in the In Vitro Germination and Growth of the Petunia (Petunia hybrida E. Vilm.) Male Gametophyte

The in vitro growth of the pollen tube (PT), an object of comprehensive and intensive research, is a model for studying the mechanisms of sexual reproduction in higher plants. We have studied the potential role of reactive oxygen species (ROS) in the in vitro germination and growth maintenance of the petunia (Petunia hybrida E. Vilm.) male gametophyte. The exogenous treatment with H2O2 influences the PT germination and polar growth in vitro. The addition of H2O2 to culture medium increases both the percentage of pollen grain germination and the PT length in the case of long cultivation, but inhibits both processes during the first hour of cultivation. This suggests that endogenous ROS play a decisive role in the early stages of pollen germination, with the sensitivity to endogenous ROS emerging later over the course of their growth. The addition of diphenylene iodonium chloride (DPI), a NADPH oxidase inhibitor, considerably decreases both the germination and the growth of the petunia male gametophyte at low concentrations (0.1 μM), and completely arrests the growth at high concentrations (1 μM). ROS are necessary for polar growth of the petunia male gametophyte; they are secreted in the early stages of pollen grain activation and are further localized to the initiation of the PT, mainly in the PT apical part, during polar growth, as confirmed with the help of intravital fluorescence microscopy.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Identifying potential phytoactives from Glycine max (L.) Merr. and their role in NADPH oxidase inhibition

Identifying potential phytoactives from Glycine max (L.) Merr. and their role in NADPH oxidase inhibition

The NADPH Oxidase Inhibitor, Mitoapocynin, Mitigates DFP-Induced Reactive Astrogliosis in a Rat Model of Organophosphate Neurotoxicity.

NADPH oxidase (NOX) is a primary mediator of superoxides, which promote oxidative stress, neurodegeneration, and neuroinflammation after diisopropylfluorophosphate (DFP) intoxication. Although orally administered mitoapocynin (MPO, 10 mg/kg), a mitochondrial-targeted NOX inhibitor, reduced oxidative stress and proinflammatory cytokines in the periphery, its efficacy in the brain regions of DFP-exposed rats was limited. In this study, we encapsulated MPO in polyanhydride nanoparticles (NPs) based on 1,6-bis(p-carboxyphenoxy) hexane (CPH) and sebacic anhydride (SA) for enhanced drug delivery to the brain and compared with a high oral dose of MPO (30 mg/kg). NOX2 (GP91phox) regulation and microglial (IBA1) morphology were analyzed to determine the efficacy of MPO-NP vs. MPO-oral in an 8-day study in the rat DFP model. Compared to the control, DFP-exposed animals exhibited significant upregulation of NOX2 and a reduced length and number of microglial processes, indicative of reactive microglia. Neither MPO treatment attenuated the DFP effect. Neurodegeneration (FJB+NeuN) was significantly greater in DFP-exposed groups regardless of treatment. Interestingly, neuronal loss in DFP+MPO-treated animals was not significantly different from the control. MPO-oral rescued inhibitory neuronal loss in the CA1 region of the hippocampus. Notably, MPO-NP and MPO-oral significantly reduced astrogliosis (absolute GFAP counts) and reactive gliosis (C3+GFAP). An analysis of inwardly rectifying potassium channels (Kir4.1) in astroglia revealed a significant reduction in the brain regions of the DFP+VEH group, but MPO had no effect. Overall, both NP-encapsulated and orally administered MPO had similar effects. Our findings demonstrate that MPO effectively mitigates DFP-induced reactive astrogliosis in several key brain regions and protects neurons in CA1, which may have long-term beneficial effects on spontaneous seizures and behavioral comorbidities. Long-term telemetry and behavioral studies and a different dosing regimen of MPO are required to understand its therapeutic potential.

Involvement of NOX2-derived ROS in human hepatoma HepG2 cell death induced by Entamoeba histolytica.

Entamoeba histolytica is an enteric tissue-invasive protozoan parasite causing amoebic colitis and liver abscesses in humans. Amoebic contact with host cells activates intracellular signaling pathways that lead to host cell death via generation of caspase-3, calpain, Ca2+ elevation, and reactive oxygen species (ROS). We previously reported that various NADPH oxidases (NOXs) are responsible for ROS-dependent death of various host cells induced by amoeba. In the present study, we investigated the specific NOX isoform involved in ROS-dependent death of hepatocytes induced by amoebas. Co-incubation of hepatoma HepG2 cells with live amoebic trophozoites resulted in remarkably increased DNA fragmentation compared to cells incubated with medium alone. HepG2 cells that adhered to amoebic trophozoites showed strong dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, suggesting intracellular ROS accumulation within host cells stimulated by amoebic trophozoites. Pretreatment of HepG2 cells with the general NOX inhibitor DPI or NOX2-specific inhibitor GSK 2795039 reduced Entamoeba-induced ROS generation. Similarly, Entamoeba-induced LDH release from HepG2 cells was effectively inhibited by pretreatment with DPI or GSK 2795039. In NOX2-silenced HepG2 cells, Entamoeba-induced LDH release was also significantly inhibited compared with controls. Taken together, the results support an important role of NOX2-derived ROS in hepatocyte death induced by E. histolytica.

Contribution of membrane raft redox signalling to visfatin-induced inflammasome activation and podocyte injury.

Recently we have shown that adipokine visfatin-induced NLRP3 inflammasome activation contributes to podocyte injury. However, the molecular mechanisms of how visfatin-induces the Nlrp3 inflammasome activation and podocyte damage is still unknown. The present study tested whether membrane raft (MR) redox signalling pathway plays a central role in visfatin-induced NLRP3 inflammasomes formation and activation in podocytes. Upon visfatin stimulation an aggregation of NADPH oxidase subunits, gp91phox and p47phox was observed in the membrane raft (MR) clusters, forming a MR redox signalling platform in podocytes. The formation of this signalling platform was blocked by prior treatment with MR disruptor MCD or NADPH oxidase inhibitor DPI. In addition, visfatin stimulation significantly increased the colocalization of Nlrp3 with Asc or Nlrp3 with caspase-1, IL-β production, cell permeability in podocytes compared to control cells. Pretreatment with MCD, DPI, WEHD significantly abolished the visfatin-induced colocalization of NLRP3 with Asc or NLRP3 with caspase-1, IL-1β production and cell permeability in podocytes. Furthermore, Immunofluorescence analysis demonstrated that visfatin treatment significantly decreased the podocin and nephrin expression (podocyte damage) and prior treatments with DPI, WEHD, MCD attenuated this visfatin-induced podocin and nephrin reduction. In conclusion, our results suggest that visfatin stimulates membrane raft clustering in the membrane of podocytes to form redox signaling platforms by aggregation and activation of NADPH oxidase subunits enhancing O2·- production and leading to NLRP3 inflammasome activation in podocytes and ultimate podocyte injury.

Anti-epileptic and Neuroprotective Effects of Ultra-low Dose NADPH Oxidase Inhibitor Dextromethorphan on Kainic Acid-induced Chronic Temporal Lobe Epilepsy in Rats.

Neuroinflammation mediated by microglia and oxidative stress play pivotal roles in the development of chronic temporal lobe epilepsy (TLE). We postulated that kainic acid (KA)-Induced status epilepticus triggers microglia-dependent inflammation, leading to neuronal damage, a lowered seizure threshold, and the emergence of spontaneous recurrent seizures (SRS). Extensive evidence from our laboratory suggests that dextromethorphan (DM), even in ultra-low doses, has anti-inflammatory and neuroprotective effects in many animal models of neurodegenerative disease. Our results showed that administration of DM (10 ng/kg per day; subcutaneously via osmotic minipump for 4 weeks) significantly mitigated the residual effects of KA, including the frequency of SRS and seizure susceptibility. In addition, DM-treated rats showed improved cognitive function and reduced hippocampal neuronal loss. We found suppressed microglial activation-mediated neuroinflammation and decreased expression of hippocampal gp91phox and p47phox proteins in KA-induced chronic TLE rats. Notably, even after discontinuation of DM treatment, ultra-low doses of DM continued to confer long-term anti-seizure and neuroprotective effects, which were attributed to the inhibition of microglial NADPH oxidase 2 as revealed by mechanistic studies.

Metformin Attenuates TGF-β1-Induced Fibrosis in Salivary Gland: A Preliminary Study.

Fibrosis commonly arises from salivary gland injuries induced by factors such as inflammation, ductal obstruction, radiation, aging, and autoimmunity, leading to glandular atrophy and functional impairment. However, effective treatments for these injuries remain elusive. Transforming growth factor-beta 1 (TGF-β1) is fundamental in fibrosis, advancing fibroblast differentiation into myofibroblasts and enhancing the extracellular matrix in the salivary gland. The involvement of the SMAD pathway and reactive oxygen species (ROS) in this context has been postulated. Metformin, a type 2 diabetes mellitus (T2DM) medication, has been noted for its potent anti-fibrotic effects. Through human samples, primary salivary gland fibroblasts, and a rat model, this study explored metformin's anti-fibrotic properties. Elevated levels of TGF-β1 (p < 0.01) and alpha-smooth muscle actin (α-SMA) (p < 0.01) were observed in human sialadenitis samples. The analysis showed that metformin attenuates TGF-β1-induced fibrosis by inhibiting SMAD phosphorylation (p < 0.01) through adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-independent pathways and activating the AMPK pathway, consequently suppressing NADPH oxidase 4 (NOX4) (p < 0.01), a main ROS producer. Moreover, in rats, metformin not only reduced glandular fibrosis post-ductal ligation but also protected acinar cells from ligation-induced injuries, thereby normalizing the levels of aquaporin 5 (AQP5) (p < 0.05). Overall, this study underscores the potential of metformin as a promising therapeutic option for salivary gland fibrosis.

Increased pro-remodeling and pro-thrombotic responses in the left compared to the right atrial appendage: role of low-grade inflammation and the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction In atrial fibrillation (AF), there is a complex interplay between arrhythmia burden and cardiovascular risk factors leading to left atrial remodeling, an increased risk of stroke and heart failure. Most thrombi get formed in the atrial appendage. Several studies suggested that sodium-glucose cotransporter 2 inhibitors (SGLT2i), besides showing major benefits on heart failure, might lower the risk of incident AF. Therefore, we examined the expression level of SGLT2 in human right and left atrial appendages (RAA, LAA) and determined its role in pro-oxidant, pro-fibrotic and pro-thrombotic responses. Methods Human RAA and LAA were collected from patients undergoing cardiac surgery at a university hospital. The LAA was cut into a proximal part (low stasis, high shear) and a distal part (high stasis, low shear). The level of reactive oxygen species (ROS) was determined using dihydroethidium, mRNA and protein expression levels by RT-qPCR, and Western blot analysis and immunofluorescence, respectively, and the level of fibrosis by Sirius red staining. Results The distal LAA had higher mRNA levels of ICAM-1 and p53, and protein levels of ICAM-1, AT1R, p53, p21 and tissue factor than the proximal LAA. LAA displayed higher mRNA and protein levels of SGLT2 and SGLT1, pro-adhesive, pro-thrombotic, pro-remodeling, pro-fibrotic and senescence markers, and components of the angiotensin system, in addition to IL-1 mRNA levels and p-p65 NF-kB protein levels compared to RAA. These responses were associated with higher levels of oxidative stress, nitrotyrosine and fibrosis. SGLT2 immunofluorescence signals in distal LAA were colocalized with those of CD31, CD68, TNF-α and troponin T. Oxidative stress levels in distal and proximal LAA and RAA were reduced by inhibitors of NADPH oxidases, ACE1 and SGLT2, an AT1R antagonist, and by a TNF-α neutralizing antibody whereas NG-nitro-L-Arginine methyl ester, the non-selective NO synthase inhibitor, inhibited ROS in distal and proximal LAA without affecting RAA. In addition, the level of TNF-α in LAA was positively correlated to the indexed LA volume indicating a link between inflammation and remodeling. Conclusions The distal part of the LAA of patients undergoing heart surgery showed greater impaired endothelial function and senescence associated with more pronounced pro­-oxidant, pro­-remodeling and pro-­inflammatory responses than the proximal part and the RAA. These responses were associated with an increased expression level of SGLT 2 in endothelial cells, cardiomyocytes, and macrophages in areas showing an inflammatory response. Moreover, the pro-oxidant signal was sensitive to inhibitors of the local angiotensin system, NO synthase, SGLT2 and TNF-α. Thus, the AT1R/NADPH oxidases/SGLT2 pathway appears as an interesting target to blunt the stimulatory pro-oxidant signal in cardiac tissues affected by low-grade inflammation that leads to remodeling and fibrosis.

GLP1 receptor agonists exhibit antioxidant and anti-inflammatory properties in human heart tissue via the canonical GLP1 receptor expression through a AMPK-mediated pathway

Abstract Background Clinical research has demonstrated that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) can decrease the occurrence of major cardiovascular events in diabetic patients, regardless of their glycemic control. However, a significant challenge in understanding the positive effects of GLP1RA has been the lack of knowledge regarding the specific cellular types that express GLP1R. Despite the established cardiovascular benefits of GLP1RA, previous studies have been unable to confirm the expression of GLP1R in the heart. Aim The study aimed to investigate the expression and function of GLP1R in heart tissue. Methods We collected human cardiac tissue samples from 30 patients who underwent left ventricle (LV) biopsies (n=21) or right atrial (RA) appendage closure (n=9) at a university hospital. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results LV biopsies and RA appendages showed a substantial heterogeneity in GLP1R mRNA levels. GLP1R mRNA levels exhibited a positive correlation with IL1B, IL6, TNFα, CCL2, CD68, ACE1, AT1R, and VCAM1 mRNA levels, but a negative correlation with NOS3 mRNA levels. High GLP1R expression in both RA appendages and LV biopsies was associated with increased oxidative stress levels, which were mitigated by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50-60 %. Exendin, a GLP1R antagonist reversed the effect of liraglutide and semaglutide which accounted for a receptor-mediated antioxidant effect. H-89, a PKA inhibitor equally reversed liraglutide and semaglutide effect on ROS generation suggesting a canonical GLP1R mediated effect involving the AMPK pathway. Conclusion These findings indicate that human cardiac GLP1R expression levels are correlated to low-grade inflammation and endothelial dysfunction. Increased expression levels of GLP1R were associated with higher levels of oxidative stress sensitive to GLP1RA in a canonical GLP1R AMPK-mediated manner. Therefore, the cardiovascular beneficial effects of GLP1RA might be attributable to their antioxidant and anti-inflammatory properties, specifically within the cardiac system.

Human calcified aortic valves release procoagulant microparticles inducing endothelial dysfunction and thrombogenicity: Role of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Background Aortic stenosis (AS) pathogenesis is a dynamic process with interplay between the hemostatic system and the valve leading to inflammation, oxidative stress, endothelial dysfunction, and calcification. Within the pathological valve, mechanical strain induces the release of cell-derived microparticles (MPs) that promote mineralization and carry a pro-oxidant activator signal. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) showed cardiovascular benefit and anti-inflammatory responses in patients with heart failure. Oxidative stress has been shown to trigger SGLT2 expression in endothelial cells (ECs) to induce endothelial dysfunction. Therefore, this study evaluated calcified AS valves and the effect of AS-derived MPs on ECs and, if so, the role of the SGLT2. Methods Pathological valves were collected from 90 AS and 15 aortic insufficiency (AI) patients. MPs were extracted from human pathological valves, and quantified through their prothrombinase activity. Primary cultures of porcine valvular endothelial cells (VECs) were treated with MPs (10 nM) or thrombin (1 U/ml). Protein expression levels were assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-qPCR, oxidative stress using dihydroethidium, and nitric oxide (NO) generation by DAF-FM diacetate. THP1 cells were used to evaluate the adhesiveness of VECs, and the generation of thrombin using a specific chromogenic substrate. Results Calcified AS valves showed increased levels of thrombogenic (TF, PAI-1), adhesive (VCAM-1, ICAM-1) and inflammatory markers (CD68, p-p65 NF-κB) and SGLT2 compared to non-calcified valves. These responses were associated with a higher level of oxidative stress that was reduced by inhibitors of the angiotensin system (ACEi, AT1R) and SGLT2, and an increased release of procoagulant MPs. Exposure of VECs to AS-MPs increased the expression of markers of the angiotensin system (ACE, p67 phox), inflammation (CXCL10, CCL11, CXCL8), thrombogenicity (TF, PAI-1), angiogenesis (VEGFR2, ANGPTL4), matrix remodeling (BMP-2, MMP-1), and SGLT2 and caused the down-regulation of eNOS. These responses were associated with a sustained pro-oxidant response, the nuclear translocation of NF-kB and blunted bradykinin-induced NO formation, which were prevented by inhibition of AT1R, NADPH oxidases, SGLT2, and NF-kB. AS-MPs-treated VECs showed also increased adhesion of THP-1 cells, and generation of thrombin. Conclusions Calcified AS valve is a potent reservoir of MPs, acting as a pro-thrombogenic source per se and prompting valvular endothelial cells dysfunction through activation of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. The observed protective effect of empagliflozin suggests SGLT2 inhibitors as a novel therapeutic option to curb valve dysfunction.

Abstract 14079: Inhibition of NOX2 Prevents Obesity-Mediated Atrial Fibrillation

Introduction: Obesity is an independent risk factor for atrial fibrillation (AF). While oxidative stress plays an important role in obesity-mediated AF, the specific pathways that cause atrial remodeling remain unclear. NADPH oxidase 2 (NOX2), a major source of cytosolic reactive oxygen species (ROS) production in the heart, is increased in diet-induced obese (DIO) mice and can independently predispose to post-operative AF via atrial remodeling. Hypothesis: We used a Nox2 -knock-out (KO) mouse model and mature human iPSC-derived atrial cardiomyocytes (hiPSC-aCMs) to test the hypothesis that NOX2 increase modulates ion channel remodeling in obesity-mediated AF. Methods: DIO mice and palmitic acid (PA)-treated hiPSC-aCMs were given a NOX blocker (apocynin), and GSK2793059, a NOX2-specific small molecule inhibitor respectively. Trans-esophageal rapid (TE) pacing was used to examine changes to the AF phenotype. Whole-cell patch clamping, qPCR, Western blotting, and electrical mapping were performed to study ion channel remodeling in both models. Results: DIO, DIO-Apocynin, and DIO Nox2 -KO mice displayed increased weight compared to control and Nox2 -KO mice (Figure A). After TE pacing, DIO-Apocynin mice displayed 28.2 ± 25.4 s and DIO Nox2 -KO mice displayed 17.4 ± 31.8 s compared to 167.3 ± 168.9 s in DIO mice (Figure B). Electrophysiological studies show that Nox2 inhibition reverses ion channel remodeling in I Na , I Ks , and I Ca,L (Figure D-F), and atrial fibrosis (Figure H) and prolongs the atrial action potential (AP; Figure C) and increases conduction velocity (CV; Figure G-I) in both DIO mice (Figure C-I) and PA-hiPSC-aCMs (Figure J-L). Conclusions: Collectively, genetic and pharmacological inhibition of NOX2 in DIO mice and PA-treated hiPSC-aCMs abrogate ion channel and structural remodeling and prevents the development of obesity-mediated AF. These findings have important implications for targeted antioxidant therapy for obese patients with AF.

Targeting NADPH Oxidase and Integrin α5β1 to Inhibit Neutrophil Extracellular Traps-Mediated Metastasis in Colorectal Cancer.

Metastasis leads to a high mortality rate in colorectal cancer (CRC). Increased neutrophil extracellular traps (NETs) formation is one of the main causes of metastasis. However, the mechanism of NETs-mediated metastasis remains unclear and effective treatments are lacking. In this study, we found neutrophils from CRC patients have enhanced NETs formation capacity and increased NETs positively correlate with CRC progression. By quantitative proteomic analysis of clinical samples and cell lines, we found that decreased secreted protein acidic and rich in cysteine (SPARC) results in massive NETs formation and integrin α5β1 is the hub protein of NETs-tumor cell interaction. Mechanistically, SPARC regulates the activation of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) pathway by interacting with the receptor for activated C kinase 1 (RACK1). Over-activated NADPH oxidase generates more reactive oxygen species (ROS), leading to the release of NETs. Then, NETs upregulate the expression of integrin α5β1 in tumor cells, which enhances adhesion and activates the downstream signaling pathways to promote proliferation and migration. The combination of NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and integrin α5β1 inhibitor ATN-161 (Ac-PHSCN-NH2) effectively suppresses tumor progression in vivo. Our work reveals the mechanistic link between NETs and tumor progression and suggests a combination therapy against NETs-mediated metastasis for CRC.

High glucose enhances the activation of NLRP3 inflammasome by ambient fine particulate matter in alveolar macrophages

BackgroundEpidemiological studies have demonstrated that individuals with preexisting conditions, including diabetes mellitus (DM), are more susceptible to air pollution. However, the underlying mechanisms remain unclear. In this study, we proposed that a high glucose setting enhances ambient fine particulate matter (PM2.5)-induced macrophage activation and secretion of the proinflammatory cytokine, IL-1β, through activation of the NLRP3 inflammasome, altering the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs).ResultsExposure of mouse alveolar macrophages to non-cytotoxic doses of PM2.5 led to upregulation of IL-1β, activation of the NLRP3 inflammasome, increased nuclear translocation of the transcription factor NF-κB, increased generation of reactive oxygen species (ROS), and increased expression and enzymatic activity of MMP-9; these effects were enhanced when cells were pretreated with high glucose. However, pretreatment in a high glucose setting alone did not induce significant changes. ROS generation following PM2.5 exposure was abolished when cells were pretreated with ROS scavengers such as Trolox and superoxide dismutase (SOD), or with an NADPH oxidase inhibitor, DPI. Pretreatment of cells with DPI attenuated the effects of a high glucose setting on PM2.5-induced upregulation of IL-1β, activation of the NLRP3 inflammasome, and nuclear translocation of NF-κB. In addition, enhancement of PM2.5-induced expression and enzymatic activity of MMP-9 following high glucose pretreatment was not observed in primary alveolar macrophages obtained from NLRP3 or IL-1R1 knockout (KO) mice, where pro-IL-1β cannot be cleaved to IL-1β or cells are insensitive to IL-1β, respectively.ConclusionsThis study demonstrated that exposure of mouse alveolar macrophages to PM2.5 in a high glucose setting enhanced PM2.5-induced production of IL-1β through activation of the NLRP3 inflammasome and nuclear translocation of NF-κB due to PM2.5-induced oxidative stress, leading to MMP-9 upregulation. The key role of NADPH oxidase in PM2.5-induced ROS generation and activation of the IL-1β secretion pathway and the importance of IL-1β secretion and signaling in PM2.5-induced increases in MMP-9 enzymatic activity were also demonstrated. This study provides a further understanding of the potential mechanisms underlying the susceptibility of individuals with DM to air pollution and suggests potential therapeutic targets.

Inhibition of NADPH oxidase 2 improves cognitive abilities by modulating aquaporin-4 after traumatic brain injury in mice

Traumatic brain injury (TBI) is caused by acquired damage that includes cerebral edema after a mechanical injury and may cause cognitive impairment. We explored the role of nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2; NOX2) and aquaporin-4 (AQP4) in the process of edema and cognitive abilities after TBI in NOX2−/− and AQP4−/− mice by using the Morris water maze test (MWM), step-down test (STD), novel object recognition test (NOR) and western blotting. Knockout of NOX2 in mice decreased the AQP4 and reduce edema in the hippocampus and cortex after TBI in mice. Moreover, inhibiting AQP4 by 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020) or genetic deletion of AQP4 could attenuate neurological deficits without changing reactive oxygen species (ROS) levels after TBI in mice. Taken together, we suspected that inhibiting NOX2 could improve cognitive abilities by modulating ROS levels, then affecting AQP4 levels and brain edema after in TBI mice. Our study demonstrated that NOX2 play a key role in decreasing edema in brain and improving cognitive abilities by modulating AQP4 after TBI.

Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.

Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/β, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.

Targeting ROS production through inhibition of NADPH oxidases.

NADPH oxidases (NOXs) are transmembrane enzymes that are devoted to the production of reactive oxygen species (ROS). In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. Consequently, NOXs are a drug target for cancer therapeutics, although current therapies have off-target effects: there is a need for isoenzyme-selective inhibitors. Here, we describe fully validated human NOX inhibitors, obtained from an in silico screen, targeting the active site of Cylindrospermum stagnale NOX5 (csNOX5). The hits are validated by in vitro and in cellulo enzymatic and binding assays, and their binding modes to the dehydrogenase domain of csNOX5 studied via high-resolution crystal structures. A high-throughput screen in a panel of cancer cells shows activity in selected cancer cell lines and synergistic effects with KRAS modulators. Our work lays the foundation for the development of inhibitor-based methods for controlling the tightly regulated and highly localized ROS sources.