Nox4 Expression Research Articles

Prolactin protects hippocampal neurons against H2O2-induced neurotoxicity by suppressing BAX and NOX4 via the NF-κB signaling pathway.

Reactive oxygen species (ROS) are physiological byproducts of neuronal metabolism. However, an imbalance between ROS generation and antioxidant capacity, often driven by dysregulated pro-oxidant enzymes like nicotinamide adenine dinucleotide phosphate oxidases (NOX), can result in deleterious oxidative stress. This oxidative stress is a critical factor in the pathogenesis of neurodegenerative diseases. While interventions with broad-spectrum antioxidants have demonstrated limited efficacy, the modulation of endogenous antioxidant mechanisms presents a promising therapeutic avenue. Here, we investigated the potential of the neuroprotective hormone prolactin to mitigate oxidative stress and subsequent neuronal cell death. Prolactin protected primary mouse hippocampal neurons from hydrogen peroxide (H2O2)-induced oxidative damage. Prolactin reduced ROS levels, lipid peroxidation, and apoptosis, and its effects were occluded by a specific prolactin receptor antagonist (G129R-hPRL). Mechanistically, prolactin suppressed H2O2-induced mRNA upregulation of pro-oxidative Nox4 and pro-apoptotic Bax. Moreover, prolactin induced nuclear factor kappa B (NF-κB) nuclear translocation, and the inhibition of the NF-κB signaling pathway abolished the neuroprotective and transcriptional effects of prolactin, indicating its central role in prolactin-mediated protection. Our findings indicate that prolactin exerts potent antioxidant and neuroprotective effects by modulating the expression of Nox4 and Bax, thereby reducing ROS generation and neuronal apoptosis. This study underscores the therapeutic potential of prolactin in attenuating oxidative stress and suggests a possible role in the treatment of neurodegenerative diseases.

Candida albicans infection model in Drosophila melanogaster suggests a strain-specific virulent factor boosting a stormy innate immune response

IntorductionPathogens drive the evolution of host defence strategies, with both innate and adaptive immune systems playing key roles. Priming enhances the innate immune system’s readiness by functionally reprogramming immune cells after initial exposure to stimuli, like β-glucans. In this sense, Drosophila melanogaster is a valuable model to evaluate the role of innate immunity to control infections.ObjectivesIn this study we aimed to set light on the immune priming effect of oral treatment with heat-killed M. manresensis and two different heat-killed C. albicans isolates upon systemic infection by C. albicans in the D. melanogaster model.MethodsA clinical and a control ATCC 90028 Candida albicans strain were used. Flies were primed through oral administration of heat-killed C. albicans (hkCa), both clinical and control, and hk-Mycolicibacterium manresensis. After priming, flies were systemically infected with both C. albicans isolates. Host survival, pathogen load, and immune response in response to treatment and infection were evaluated.ResultsBoth treatments showed a significant capacity to enhance the expression of antimicrobial peptides, in particular Diptericin, and Drosomycin in males. This response had a marked sexual dimorphism due to the difference in Upd3, Nox, and Duox expression. Surprisingly, even when priming was able to avoid the growth of both C. albicans strains, survival was not improved in the case of the clinical isolate, causing an unexpected mortality rate in hours, regardless of the host’s sex. Gene expression analysis 24 hours post-infection showed an exacerbated increase in Diptericin, Drosomycin and Upd3 expression upon infection with the clinical strain.ConclusionData herein suggests the presence of a strain-specific component in C. albicans as the booster of a “stormy” innate immune response, which must be further investigated, and position D. melanogaster as a useful model for evaluating virulent factors related to the modulation of the innate immunity.

Interaction of three weeks HIIT and vitamin D supplementation on some oxidant and inflammatory factors in heart tissue of middle-aged rats

Abstract Aging causes changes in the structure and function of the heart, which increases the number of free radicals and provides the basis for the increase of inflammatory cytokines. Therefore, this study aimed to investigate the interaction of three weeks of high-intensity interval training and vitamin D supplementation on NOX, SOD, NF-κB, and Nrf-2 in the heart tissue of middle-aged rats. In this experimental study, 21 middle-aged rats in the age range of 10-12 months were purchased from the Pasteur Institute and randomly divided into high-intensity interval training (HIIT) (n = 8), HIIT+VitD (n = 8), and Sham (n = 6) groups. After two weeks of adaptation, HIIT was performed for three week and vitamin D was gavage with a dose of 500 units/kg. Finally, the variables were measured through real-time PCR, and a one-way analysis of variance was used for statistical analysis. The results of data analysis showed that after three weeks, the expression of NOX () and SOD () genes increased significantly in the HIIT group compared to the HIIT+VitD group. The interaction of three weeks of high-intensity interval training with vitamin D supplementation can improve oxidative indices, but this effect was more significant in the exercise group.

The inflammatory and oxidative phenotype of gestational diabetes is epigenetically transmitted to the offspring: role of methyltransferase MLL1-induced H3K4me3.

Hyperglycaemia during gestational diabetes (GD) predisposes women and their offspring to later cardiometabolic disease. The hyperglycaemia-mediated epigenetic changes remain to be elucidated. Methyltransferase MLL1-induced trimethylation of histone 3 at lysine 4 (H3K4me3) activates inflammatory and oxidative phenotype. This epigenetic mark in GD women and its transmission to the offspring were investigated. Peripheral blood mononuclear cells (PBMC) were collected from GD and control (C) women and also from adolescents born to women of both groups. Endothelial human umbilical vein endothelial cells (HUVEC) and cord blood mononuclear cells (CBMC) were from umbilical cords. The NF-κBp65 and NOX4 expressions were investigated by reverse transcription quantitative polymerase chain reaction and immunofluorescence (IF). MLL1 and H3K4me3 were investigated by immunoblotting and IF. H3K4me3 on NF-κBp65 and NOX4 promoters was studied by chromatin immunoprecipitation. Superoxide anion generation was measured by electron spin resonance spectroscopy. Plasma cytokines were measured by enzyme-linked immunosorbent assay. To investigate the role of MLL1, HUVEC were exposed to inhibitor MM102 or siRNA transfection. PBMC, CBMC, and HUVEC showed an increase of NF-κBp65, IL-6, ICAM-1, MCP-1, and VCAM-1 mRNAs. These findings were associated with H3K4me3 enrichment in the promoter of NF-κBp65. Elevated H3K4me3 and cytokine levels were observed in GD adolescents. MLL1 drives H3K4me3 not only on NF-kB p65, but also on NOX4 promoter. Inhibition of MLL1 blunted NF-κBp65 and NOX4 by modulating inflammatory and oxidative phenotype. Such proof-of-concept study shows persistence of MLL1-dependent H3K4me3 in offspring born to GD women, suggesting an epigenetic-driven transmission of maternal phenotype. These findings may pave the way for pharmacological reprogramming of adverse histone modifications to mitigate abnormal phenotypes underlying early ASCVD.

Takotsubo syndrome induced oxidative stress and endothelial dysfunction in left ventricle of rat model: association with NADPH oxidases/SGLT2 pro- oxidant pathway

Abstract Background Takotsubo syndrome (TTS) is characterized by apical ballooning predominantly affecting post-menopausal women following extreme physical and/or emotional stress. The phenotype of TTS is associated with akinetic apex and hyperkinetic base of the left ventricle (LV). Presently, there are no targeted therapies for TTS that can effectively reduce LV impairment. Empagliflozin (EMPA), an SGLT2 inhibitor, has shown considerable clinical benefits in managing heart failure. However, its specific effects and underlying mechanisms in treating TTS remain unclear. Purpose To explore the pathophysiology of TTS in the LV of rats, with a particular focus on the potential role of SGLT2, and to elucidate the underlying mechanisms involved. Methods Female Sprague Dawley rats (250-300 g) were allocated into four groups: a control group (n=8), a group treated with EMPA (30 mg/kg/day) (n=8), a group receiving ISO (Isoproterenol) at 100 mg/kg/day (n=18), and a group receiving both ISO and EMPA (IE) at 30 mg/kg/day (n=18). EMPA was administered orally through food two weeks prior to ISO injection. Echocardiography was conducted at 6 and 24 h, and on day 3. On day 3, rats were euthanized, and their organs were collected. Gene expression of markers was assessed using RT-qPCR, protein expression via immunofluorescence (IF) staining, and oxidative stress using dihydroethidium. Results In the ISO group, the success rate of induced Takotsubo syndrome (TTS) was 53% (10 out of 18 rats), with a mortality rate of 5%. Conversely, in the EMPA-treated group, TTS occurred in 22% of rats (4 out of 18) with no mortality observed. Significant reductions in the area of left ventricular (LV) ballooning and ejection fraction were noted in the IE group compared to the ISO group. Additionally, increased levels of reactive oxygen species (ROS) were observed in the LV of ISO rats, which were normalized by EMPA treatment in the IE rats. Furthermore, the ISO group exhibited higher protein expressions of CD68, SGLT2, and VCAM-1, which were attenuated by EMPA treatment. The mRNA expression of CYBA (p22phox) and NOX4, both representing subunits of NADPH oxidases, along with VCAM1, ICAM1, NOS2 (iNOS) and SELE (e-selectin) gene, endothelial activation marker, and TGF-β1, a fibrotic marker, showed elevated levels in the apex part of the left ventricle (LV) in the ISO group; these levels were significantly lower in the IE group. Conclusions These findings suggest that TTS is associated with oxidative stress-mediated endothelial dysfunction, inflammation, and pro-fibrotic responses. Moreover, SGLT2 inhibition appears to mitigate the pathology and progression of the syndrome. Therefore, SGLT2 inhibition emerges as an appealing and novel therapeutic approach for the treatment of TTS.

Screening of m6A-associated ferroptosis-related genes in atherosclerosis based on WGCNA

BackgroundN6-methyladenosine (m6A) has been shown to mediate ferroptosis but its role in atherosclerosis (AS) is unclear.MethodsDifferentially expressed m6A-associated ferroptosis-related genes (DE-m6A-Ferr-RGs) were obtained using differential expression analysis and Pearson correlation analysis. Weighted gene co-expression network analysis (WGCNA) was also performed. The intersection of the module genes and the DE-m6A-Ferr-RGs were recorded as candidate m6A-Ferr-related signature genes. Finally, the m6A-Ferr-related signature genes were screened using least absolute shrinkage and selection operator (LASSO) analysis. Expression validation, receiver operating characteristic ( mapping, and immune correlation analysis were also performed based on the m6A-Ferr-related signature genes. The expression of m6A-Ferr-related signature genes was further validated using a real-time polymerase chain reaction (RT-qPCR).ResultsIn total, 6,167 differentially expressed genes were intersected with 24 m6A- and 259 ferroptosis-related genes, respectively, resulting in 113 DE-m6A-Ferr-RGs obtained using Pearson’s correlation analysis. The module genes obtained from the WGCNA and the 113 DE-m6A-Ferr-RGs were intersected to obtain 48 candidate m6A-Ferr-related signature genes. LASSO analysis was performed and six m6A-Ferr-related signature genes were screened. In addition, the area under the curve values of all six m6A-Ferr-related signature genes were greater than 0.7, indicating that they had potential diagnostic value. Furthermore, the RT-qPCR results revealed that the expression of SLC3A2, NOX4, and CDO1 was consistent with the transcriptome level. Moreover, there was a significant difference in two types of immune cells between the AS and control groups. Naive B cells, CD8+ T cells, regulatory T cells, and activated natural killer cells were positively correlated with CDO1 and NOX4 but negatively correlated with ATG7, CYBB, and SLC3A2.ConclusionIn total, three m6A-Ferr-related signature genes (NOX4, CDO1, and SLC3A2) were obtained through a series of bioinformatics analyses and an RT-qPCR.

Astaxanthin Supplementation Effects in Right Ventricle of Rats Exposed to Chronic Intermittent Hypobaric Hypoxia.

Thirty two male Wistar rats were randomly assigned to the following groups (n = 8 per group): the normoxia with vehicle (NX), normoxia with astaxanthin (NX + AS), chronic intermittent hypobaric hypoxia with vehicle (CIHH), and chronic intermittent hypobaric hypoxia with astaxanthin (CIHH + AS) groups. CIHH was simulated by 2 days in a hypobaric chamber followed by 2 days at sea level for 29 days. Exposure to CIHH induced RVH and increased lipid peroxidation (MDA), Nox2 expression, and SOD activity, however, it decreased pro-IL-1β expression. Astaxanthin restored oxidative stress markers (Nox2 and MDA), increased GPx activity, and decreased RVH compared to CIHH. Astaxanthin alleviates RVH and reduces Nox2 and MDA levels while increasing GPx activity in rats subjected to CIHH. These findings provide new insights of astaxanthin as a new nutraceutical against high-altitude effects.

Cinobufotalin inhibits proliferation, migration and invasion in hepatocellular carcinoma by triggering NOX4/NLRP3/GSDMD-dependent pyroptosis

IntroductionPyroptosis is an inflammatory form of programmed cell death that plays a significant role in tumorigenesis. Cinobufotalin (CB), a bufadienolide extracted from toad venom, is associated with antitumor effects in various cancers, including liver cancer. However, the role of CB in pyroptosis and its underlying mechanisms have not been well characterized.MethodsMTT, Colony formation, EdU, Wound healing and Transwell migration and invasion assays were applied to determine the effects of CB on the proliferation, migration, and invasion ability of hepatocellular carcinoma (HCC) cells in vitro. The subcutaneous xenograft mouse model and pulmonary metastasis model were used to evaluate the effect of CB on HCC cells in vivo. PCR, western blot, immunohistochemistry, immunofluorescence, and ELISA were used to verify the expression of proliferation, migration, pyroptosis, and inflammation related molecules after CB treatment. Using si-RNA and inhibitors to interfere with NOX4 and HLRP3 expression to validate the key signaling pathways of pyroptosis induced by CB treatment.ResultsIn vivo experiments using nude mice with xenografted HCC cells and in vitro experiments with HCC cell lines demonstrated that CB treatment significantly inhibited the proliferation, migration, and invasiveness of HCC cells. CB treatment also showed dose-dependent activation of the NLRP3 inflammasome complex in HCC cells, leading to gasdermin D-induced pyroptosis. However, these effects were abrogated via the pretreatment of HCC cells with VX-765, a caspase-1 inhibitor. Additionally, CB increased the production of reactive oxygen species (ROS) and H₂O₂, along with upregulating NOX4 protein expression in HCC cells. Conversely, NOX4 silencing or pretreatment with VAS2870 (an NOX4 inhibitor) or NAC (an ROS scavenger) suppressed the activation of the NLRP3 inflammasome complex and pyroptosis in CB-treated HCC cells.DiscussionOur study demonstrated that CB suppressed the proliferation, migration, and invasiveness of HCC cells by inducing pyroptosis through the activation of the NOX4/NLRP3/GSDMD signaling pathway. Therefore, our results suggest that CB is a promising therapeutic agent for HCC.

Preventive Effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole on Pulmonary Fibrosis through Inhibition of Inflammation and Down-regulation of TGF-b Signaling Pathway.

In the present study effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on plmonary fibrosis development was investigated in vitro in primary lung fibroblasts as well as in vivo in mice model. The results demonstrated that STT treatment effectively inhibited the TGF-β1 induced increase in expression of α-SMA and collagen I proteins in PLFs. STT treatment effectively reversed the TGF-β1 induced increase in expression of LOXL2 protein and phosphorylation of Smad2/3 proteins. Treatment of PLFs with STT reversed the TGF-β1-induced increase in expression of NOX4 and suppression of p-AMPK protein. In mice model of pulmonary fibrosis STT treatment significantly inhibited the BLM-mediated decrease in body weight and survival rate. The BLM induced increase in pulmonary index in mice was also effectively inhibited on treatment with STT. Treatment of the mice with STT inhibited the BLM-induced increase in α-SMA and Col I protein expression in pulmonary tissues. The BLM-induced increase in TGF-β1 protein expression in pulmonary tissues of the mice was inhibited on treatment with STT. Treatment with STT effectively promoted the AMPK activation in lung tissues of the BLM administered mice. In summary, the present study demonstrates that STT treatment prevents TGF-β1 induced up-regulation of α-SMA, collagen I, LOXL2 protein expression and targets phosphorylation of Smad2/3 proteins in PLFs. Moreover, it inhibits TGF-β1-induced increase in expression of NOX4 and reverses TGF-β1-mediated suppression in expression of p-AMPK protein. Therefore, STT inhibits fibrosis development in vitro as well as in vivo and therefore can be investigated further as a therapeutic agent for the treatment of lung fibrosis.

Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

Microplastics (MPs) accumulate in tissues, including kidney tissue, while Bisphenol A (BPA) is a plasticizer of particular concern. At present, the combined effects of MPs and BPA are unexplored in human renal cells. Therefore, we exposed a proximal tubular cell line (PTECs) to polyethylene (PE)-MPs and BPA, both separately and in combination. When co-exposed, cells showed a significantly reduced cell viability (MTT test) and a pronounced pro-oxidant (MDA levels, NRF2 and NOX4 expression by Western blot) and pro-inflammatory response (IL1β, CCL/CCR2 and CCL/CCR5 mRNAs by RT-PCR), compared to those treated with a single compound. In addition, heat shock protein (HSP90), a chaperone involved in multiple cellular functions, was reduced (by Western Blot and immunocytochemistry), while aryl hydrocarbon receptor (AHR) expression, a transcription factor which binds environmental ligands, was increased (RT-PCR and immunofluorescence). Our research can contribute to the study of the nephrotoxic effects of pollutants and MPs and shed new light on the combined effects of BPA and PE-MPs.

Comparative effect of atorvastatin and risperidone on modulation of TLR4/NF-κB/NOX-2 in a rat model of valproic acid-induced autism

BackgroundAutism spectrum disorder (ASD) is a complex neurodevelopmental condition that is significantly increasing, resulting in severe distress. The approved treatment for ASD only partially improves the sympoms, but it does not entirely reverse the symptoms. Developing novel disease-modifying drugs is essential for the continuous improvement of ASD. Because of its pleiotropic effect, atorvastatin has been garnered attention for treating neuronal degeneration. The present study aimed to investigate the therapeutic effects of atorvastatin in autism and compare it with an approved autism drug (risperidone) through the impact of these drugs on TLR4/NF-κB/NOX-2 and the apoptotic pathway in a valproic acid (VPA) induced rat model of autism.MethodsOn gestational day 12.5, pregnant rats received a single IP injection of VPA (500 mg/kg), for VPA induced autism, risperidone and atorvastatin groups, or saline for control normal group. At postnatal day 21, male offsprings were randomly divided into four groups (n = 6): control, VPA induced autism, risperidone, and atorvastatin. Risperidone and atorvastatin were administered from postnatal day 21 to day 51. The study evaluated autism-like behaviors using the three-chamber test, the dark light test, and the open field test at the end of the study. Biochemical analysis of TLR4, NF-κB, NOX-2, and ROS using ELISA, RT-PCR, WB, histological examination with hematoxylin and eosin and immunohistochemical study of CAS-3 were performed.ResultsMale offspring of prenatal VPA-exposed female rats exhibited significant autism-like behaviors and elevated TLR4, NF-κB, NOX-2, ROS, and caspase-3 expression. Histological analysis revealed structural alterations. Both risperidone and atorvastatin effectively mitigated the behavioral, biochemical, and structural changes associated with VPA-induced rat model of autism. Notably, atorvastatin group showed a more significant improvement than risperidone group.ConclusionsThe research results unequivocally demonstrated that atorvastatin can modulate VPA-induced autism by suppressing inflammation, oxidative stress, and apoptosis through TLR4/NF-κB/NOX-2 signaling pathway. Atorvastatin could be a potential treatment for ASD.Graphical

Chronic sleep deprivation induces erectile dysfunction through increased oxidative stress, apoptosis, endothelial dysfunction, and corporal fibrosis in a rat model.

Sleep is foundational for nocturnal erections, facilitating nutrient exchange and waste removal, which has brought widespread attention to the relationship between sleep and erectile dysfunction (ED). However, there is currently a lack of basic research confirming whether chronic sleep deprivation (CSD) leads to erectile impairment and its underlying pathological mechanisms. The study sought to investigate whether CSD impairs erectile function in rats and the potential tissue damage it may cause in rats. The modified multiple platform method was employed to induce CSD in 14 rats, randomly divided into a platform control group and a CSD group. After 3 weeks, erectile function was evaluated by measuring intracavernosal pressure following cavernous nerve stimulation. Arterial blood samples were then analyzed for testosterone levels, and cavernous tissues were processed for advanced molecular biology assays, including Western blotting and immunofluorescence. After inducing CSD, rats exhibited a marked reduction in erectile function, yet their serum testosterone levels remained statistically unchanged when compared with the control group. More importantly, rats in the CSD group exhibited a significant increase in oxidative stress levels, accompanied by low expression of HO-1 and high expression of NOX1 and NOX4. Subsequently, elevated oxidative stress induced increased apoptosis in smooth muscle and endothelial cells, as evidenced by significant decreases in CD31 and α-smooth muscle actin expression in the CSD group, demonstrated through Western blotting and immunofluorescence assays. Endothelial cell apoptosis led to a significant decrease in endothelial nitric oxide synthase, resulting in lowered levels of nitric oxide and cyclic guanosine monophosphate, which severely impaired the erectile mechanism. Additionally, activation of the transforming growth factor β1 fibrotic pathway led to increased levels of tissue fibrosis, resulting in irreversible damage to the penile tissue in the CSD group. Our study lacks further exploration of the molecular mechanisms linking CSD and ED, representing a future research focus for potential targeted therapies. Our findings demonstrated that CSD significantly impairs erectile function in rats. CSD severely impairs erectile function in rats. When exposed to CSD, rats exhibit significantly elevated oxidative stress levels, which lead to increased tissue apoptosis, endothelial dysfunction, and ultimately irreversible fibrotic changes in the tissues. Further researches into the potential molecular mechanisms are needed to identify possible therapeutic targets for ED related to CSD.

Tumor Microenvironment-Activated In Situ Synthesis of Peroxynitrite for Enhanced Chemodynamic Therapy.

Chemodynamic therapy (CDT) can induce cancer cell death through hydroxyl radicals (·OH) generated from Fenton or Fenton-like reactions. Compared with traditional therapies, CDT effectively overcomes inevitable drug resistance and exhibits low side effects. However, clinical application still faces challenges, primarily due to insufficient ·OH generation and the short-lifetime of ·OH in vivo. To address these challenges, we developed a peroxynitrite (ONOO-)-based CDT nanodrug (DOX@PMOF) composed of MOF-199, NO donor (PArg), and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) activator (doxorubicin, DOX). In DOX@PMOF, MOF-199 serves as both a carrier for loading DOX and a source of Cu+ for triggering CDT. Upon uptake by cancer cells, the high concentration of glutathione (GSH) reduces MOF-199 to Cu+, which then reacts with H2O2 to generate ·OH. Moreover, the released DOX upregulates NOX4 expression, leading to the elevated H2O2 level and thereby promoting a high-efficiency Fenton-like reaction for sufficient ·OH generation. Subsequently, PArg generates abundant NO in response to the tumor microenvironment, leading to a cascade of NO and ·OH for the in situ synthesis of ONOO-. ONOO- is more toxic and has a longer lifetime and diffusion distance than ·OH, resulting in a more effective CDT treatment. To further enhance the in vivo therapeutic effect, we coated DOX@PMOF with a homologous cell membrane to form an active tumor-targeting nanodrug (DOX@MPMOF), which has demonstrated the ability to effectively inhibit tumor growth and metastasis while exhibiting good biosafety.

NoxO1 Determines the Level of ROS Formation by the Nox1-Centered NADPH Oxidase.

The Nox1-centered NADPH oxidase complex facilitates the transfer of electrons from intracellular NADPH across the cell membrane to extracellular molecular oxygen, resulting in the formation of superoxide. The complex is comprised of two membrane-bound subunits, namely Nox1 and p22phox, and the cytosolic subunits, namely NoxA1 and NoxO1. The presence of NoxO1 facilitates the proximity of all components, thereby enabling the complex to exhibit constitutive activity. Despite the theoretical sufficiency of all subunits in a 1:1 ratio, the precise composition of the Nox1-centered NADPH oxidase remains unknown. Analyses of mRNA expression in different cell lines revealed an unequal expression of the components, with an excess of NoxO1. Furthermore, plasmid-based overexpression of individual components of the Nox1-centered NADPH oxidase resulted in an excess of NoxO1 mRNA. The objective of this study was to analyze the ability of NoxO1 to control the level of ROS formation by the Nox1 complex. To this end, we generated Hek293 cells for constitutive expression of Nox1 and NoxA1, which were then transfected with increasing concentrations of NoxO1. The data presented herein suggests that ROS formation by the Nox1-centered NADPH oxidase is dependent on the concentration of NoxO1. A surplus of NoxO1 has been observed to exert control over the activity of the complex in accordance with a dose-dependent mechanism. We thus conclude that the ratio of Nox1, NoxA1, and NoxO1 complexes does not adhere to a 1:1 ratio. Conversely, the availability of NoxO1 serves to regulate the formation of ROS by the Nox1-centered NADPH oxidase.

Unravelling bisphenol A-induced hepatotoxicity: Insights into oxidative stress, inflammation, and energy dysregulation

Bisphenol A (BPA), a prevalent plastic monomer and endocrine disruptor, negatively impacts metabolic functions. This study examines the chronic effects of eco-relevant BPA concentrations on hepatotoxicity, focusing on redox balance, inflammatory response, cellular energy sensors, and metabolic homeostasis in male Swiss albino mice. Chronic BPA exposure resulted in reactive oxygen species (ROS) accumulation, altered hepatic antioxidant defense, lipid peroxidation, and NOX4 expression, leading to reduced cell viability. Additionally, BPA exposure significantly upregulated hepatic pro-inflammatory cytokine genes (Tnf-α, Il-1β, Il-6), NOS2, and arginase II, correlating with increased TLR4 expression, NF-κB phosphorylation, and a dose-dependent decrease in IκBα levels. BPA-induced NF-κB nuclear localization and inflammasome activation (NLRP3, cleaved caspase-1, IL-1β) established an inflammatory milieu. Perturbations in hepatic AMPKα phosphorylation, SIRT1, and PGC-1α, along with elevated p38 MAPK phosphorylation and ERα expression, indicated BPA-induced energy dysregulation. Furthermore, increased PLA2G4A, COX1, COX2, and PTGES2 expression in BPA-treated liver correlated with hyperlipidemia, hepatic FASN expression, steatosis, and visceral adiposity, likely due to disrupted energy sensors, oxidative stress, and inflammasome activation. Elevated liver enzymes (ALP, AST, ALT) and apoptotic markers indicated liver damage. Notably, N-acetylcysteine (NAC) priming reversed BPA-induced hepatocellular ROS accumulation, NF-κB-inflammasome activation, and intracellular lipid accumulation, while upregulating cellular energy sensors and attenuating ERα expression, suggesting NAC's protective effects against BPA-induced hepatotoxicity. Pharmacological inhibition of the NF-κB/NLRP3 cascade in BAY11-7082 pretreated, or NLRP3 immunodepleted hepatocytes reversed BPA's negative impact on SIRT1/p-AMPKα/PGC-1α and intracellular lipid accumulation, providing mechanistic insights into BPA-induced metabolic disruption.

Apocynin alleviates thioacetamide-induced acute liver injury: Role of NOX1/NOX4/NF-κB/NLRP3 pathways

The liver has a distinctive capacity to regenerate, yet severe acute injury can be life-threatening if not treated appropriately. Inflammation and oxidative stress are central processes implicated in the pathophysiology of acute livery injury. NOX isoforms are important enzymes for ROS generation, NF-κB and NLRP3 activation, its inhibition could be vital in alleviating acute liver injury (ALI). Here in our study, we used apocynin, a natural occurring potent NOX inhibitor, to exploreits potential protective effect against thioacetamide (TAA)-induced ALI through modulating crucial oxidative and inflammatory pathways. Rats were injected once with TAA (500 mg/kg/i.p) and treated with apocynin (10 mg/kg/i.p) twice before TAA challenge. Sera and hepatic tissues were collected for biochemical, mRNA expression, western blot analysis and histopathological assessments. Pretreatment with apocynin improved liver dysfunction evidenced by decreased levels of aminotransferases, ALP, GGT and bilirubin. Apocynin reduced mRNA expression of NOX1 and NOX4 which in turn alleviated oxidative stress, as shown by reduction in MDA and NOx levels, and elevation in GSH levels andcatalase and SOD activities. Moreover, apocynin significantly reduced MPO gene expression. We also demonstrate that apocynin ameliorated inflammation through activating IκBα and suppressing IKKα, IKKβ, NF-κBp65 and p-NF-κBp65, IL-6 andTNF-α. Additionally, apocynin potentiated the gene expression of anti-inflammatory IL-10 and reduced levels of hepatic NLRP3, Caspase-1 and IL-1β. These results suggest that apocynin protects against ALI in association with the inhibition of NOX1 and NOX4 and regulating oxidative and inflammatory pathways.

Abstract 10: Adrenal Zona Glomerulosa Long Form Leptin Receptor (LepRb) Protects from Leptin-Mediated Vascular Disorders in Female Mice

Introduction: Previous work from our group demonstrated that leptin is a major contributor to obesity-associated cardiovascular disease, in females, by acting on the adrenal gland to promote aldosterone production. Hypothesis: This leptin-mediated aldosterone secretion is dependent on the presence of the long form leptin receptor (LepRb) in adrenal aldosterone synthase (AS) expressing cells. Methods: We generated AS LepRb deficient mice, ASKO and WT control (ASWT), by crossing LepRb flox/flox mice with AS-Cre mice to investigate the role of LepRb in AS expressing cells in leptin-mediated cardiovascular alteration in females and also generated db/AS + mice that have LepRb only in AS expressing cells by crossing LepR TBflox/flox (db/db) with AS-Cre mice to compare their phenotype. Results: Under baseline conditions, ASKO female mice exhibited no alteration in adrenal AS, plasma aldosterone, systolic blood pressure, and aortic endothelium-dependent relaxation. To mimic obese conditions, we submitted ASWT and ASKO to chronic leptin infusion for a week. Leptin increased adrenal AS expression, aldosterone levels, and blood pressure and impaired endothelial function, which were further elevated in ASKO compared to ASWT, suggesting protective effects of AS LeRb. Both mineralocorticoid receptor (MR) antagonist. eplerenone, or AS antagonist, fadrazole, rescued endothelial function in ASWT and ASKO mice treated with leptin, indicating that leptin-mediated endothelial dysfunction is still aldosterone-dependent in ASKO mice. Restoration of LepRb in AS expressing cells in db/db decreased adrenal AS and plasma aldosterone and improved endothelial function further indicating that AS LepRb blunts leptin-mediated aldosterone production. On a molecular level, leptin treatment elevated aorta NOX1 expression in ASWT, which was further increased in ASKO. Aldosterone treatment increased NOX1 expression in cultured microvascular endothelial cells. Incubation of aortic rings with a selective NOX1 inhibitor restored endothelial function in both ASWT and ASKO treated with leptin, suggesting leptin-mediated aldosterone increased NOX1 expression in the aorta, which causes endothelial dysfunction. Conclusion: These data indicate that AS LepRb protects from leptin-mediated aldosterone production and vascular dysfunction by mitigating the effects of leptin on aldosterone production and suggests that other isoforms of LepR are involved in stimulating aldosterone production.

SIRT1/NOX1 pathway mediated ameliorative effects of rosmarinic acid in folic acid-induced renal injury

Background and purpose: Renal injury is a serious disorder that can be caused by some diseases or agents. Rosmarinic acid (RA) is a natural and safe compound with powerful antioxidant and anti-inflammatory properties. In this study, the ameliorative effects of RA were assayed in folic acid (FA)-induced renal injury by involving the SIRT1/NOX1 pathway. Experimental approach: Thirty-six male C57/BL6 mice were divided into 6 groups (n = 6) including control, vehicle, FA, RA, FA + RA 50, and FA + RA 100. After 10 days, blood urea nitrogen (BUN), creatinine, and oxidative stress were measured. The expression of SIRT1 and NOX1 proteins was evaluated by western blot. Also, histopathological alterations were assayed by H&E and PAS staining methods. Findings/Results: BUN and creatinine were significantly higher in the FA group compared to the control group; however, their levels decreased after RA treatment in both doses. A significant decrease was observed in swelling, necrosis, and desquamation of tubular epithelial cells in the FA + RA 50 and FA + RA 100 groups compared to the FA group. RA in the animals receiving FA increased SIRT1 expression and the levels of GSH and SOD compared to the FA group. RA in the animals receiving FA showed a significant decrease in NOX1 expression and MDA level compared to the FA group. Conclusion and implications: The findings declared that the administration of RA has positive effects against renal damage induced by FA. The effect might result from involvement in the SIRT1/NOX1 pathway and thereby attenuation of oxidative stress.

Abstract P100: TRPM7 deficiency amplifies aldosterone signalling cardiac fibroblasts

Introduction: Transient Receptor Potential Melastatin 7 (TRPM7) channel is a channel bound to a kinase domain that influences cellular Mg 2+ with an important role in inflammatory response and cell metabolism. TRPM7 is regulated by aldosterone. Here, we investigated the importance of TRPM7-Mg2+ in pro-fibrotic responses and oxidative stress in cardiac fibroblasts (CF) in the context of hypertension and organ damage induced by aldosterone. Methods: Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600µg/Kg/day) plus NaCl (1% drinking water), 4 weeks. Mechanisms were investigated in primary culture of CF. Ca 2+ and Mg 2+ influx was assessed by fluorescence microscopy and flow cytometry. Gene and protein expression was assessed by real-time PCR and immunoblotting. Proteomic analysis was performed in two-dimensional liquid chromatography and mass spectrometry (LC/LC-MS/MS). Results: M7+/Δ mice exhibited reduced tissue [Mg 2+ ] (28%) and increased cardiac collagen deposition (2-fold). Aldosterone-induced fibrosis and hypertension were enhanced in M7+/Δ. CF M7+/Δ had reduced aldosterone-induced calcium influx (136±6 vs WT:166±7) and Mg 2+ influx (1.05±0.05 vs WT 1.14±0.02). CF M7+/Δ exhibit increased expression of calpain-1(300%), α-SMA(80%), collagen-1 (50%), mineralocorticoid receptor target proteins ENaC(70%) and SGK1(30%) and reduced proliferation(33%) vs WT(p<0.05). Aldosterone reduced TRPM7 (44%) and increased expression of ENaC (150%) and collagen-1 (93%) in CF WT to similar levels observed in CF M7+/Δ. CF M7+/Δ exhibit increased Nox2(70%), Nox4(120%) and ROS production (1,362 vs WT: 899 RLU/mg protein). Increased mRNA for the antioxidant catalase, NQO1 and Prdx1 (1.7-2.6-fold) was found in M7+/Δ CF. Mg 2+ supplementation normalised Ca 2+ and Mg 2+ influx, proliferation and expression of SGK1, Nox4, catalase and NQO1, whereas other effects might be mediated by the kinase domain. Proteomic analysis showed that CF M7+/Δ exhibit reduced expression of proteins related to cell migration and proliferation (Tnc, Cdcc80, Crip1, Ggt5) cell cycle and differentiation (AnxA8, Rpl22, Prickle2) and cell metabolism (Marcks, Mtab, Ces1d, Serpina3n, Afap1l1). Conclusion: Our findings demonstrate that in aldosterone-treated mice TRPM7 expression and tissue Mg 2+ levels are decreased. These processes are associated with cardiac fibrosis and oxidative stress. Our findings suggest a cardio-protective role for TRPM7/Mg2+ in aldosterone-induced hypertension.

Oxidative stress and regulation of adipogenic differentiation capacity by sirtuins in adipose stem cells derived from female patients of advancing age

Patient age is critical for mesenchymal stem cell quality and differentiation capacity. We demonstrate that proliferation and adipogenic capacity of subcutaneous adipose stem cells (ASCs) from female patients declined with advanced age, associated with reduction in cell nucleus size, increase in nuclear lamina protein lamin B1/B2, and lamin A, upregulation of senescence marker p16INK4a and senescence-associated β-galactosidase activity. Adipogenic induction resulted in differentiation of adipocytes and upregulation of adipogenic genes CCAAT enhancer binding protein alpha, fatty acid binding protein 4, lipoprotein lipase, and peroxisome proliferator-activated receptor-γ, which was not affected by the Sirt-1 activator YK-3-237 or the Sirt-1 inhibitor EX-527. Protein expression of the stem cell markers Oct4 and Sox2 was not significantly downregulated with advanced patient age. Mitochondrial reactive oxygen species were increased in ASCs from old-aged patients, whereas protein expression of NADPH oxidases NOX1 and NOX4 was downregulated, and dual oxidase isoforms remained unchanged. Generation of nitric oxide and iNOS expression was downregulated. Protein expression of Sirt-1 and Sirt-3 decreased with patient age, whereas Sirt-2 and Sirt-5 remained unchanged. Induction of adipogenesis stimulated protein expression of Sirt-1 and Sirt-3, which was not affected upon pre-incubation with the Sirt-1-activator YK-3-237 or the Sirt-1-inhibitor EX-527. The Sirt-1 inhibitor Sirtinol downregulated adiponectin protein expression and the number of adipocytes, whereas YK-3-237 exerted stimulatory effects. In summary, our data demonstrate increased oxidative stress in ASCs of aging patients, and decline of adipogenic capacity due to Sirt-1- mediated adiponectin downregulation in elderly patients.