Nox4 Expression Research Articles

CD4+ T Cells Expressing Viral Proteins Induce HIV-Associated Endothelial Dysfunction and Hypertension Through Interleukin 1α-Mediated Increases in Endothelial NADPH Oxidase 1.

Although combination antiretroviral therapy has increased life expectancy in people living with HIV, it has led to a marked increase in the prevalence of hypertension, the cause of which is unknown. Despite combination antiretroviral therapy, HIV-derived proteins remain expressed and produced by CD4+ T lymphocytes in people living with HIV. However, their contribution to HIV-associated hypertension and impaired endothelium-dependent relaxation remains ill defined. Here, we tested the hypothesis that CD4+ T cells expressing viral proteins contribute to endothelial dysfunction and hypertension using the Tg26 mouse model of HIV that expresses 7 of the 9 HIV proteins under the long terminal repeat promoter. We used male and female mice, bone marrow transplantation (BMT), adoptive transfer of CD4+ T cells, and aorta specimen discarded from people living with HIV. We reported that intact Tg26 mice and mice receiving BMT (Tg26→WT) or CD4+ T cells from Tg26 mice display impaired endothelium-dependent relaxation and hypertension. Conversely, BMT from WT mice into Tg26 mice, inhibition of T cell activation, and CD4+ T cell depletion restored endothelial function and blood pressure in Tg26 mice. Cytokine profiling revealed that Tg26 mice, Tg26→WT, and Tg26 CD4+ T cells consistently exhibit high interleukin 1α (IL-1α) levels with no significant increase in other cytokines, whereas BMT from WT mice into Tg26 mice reduced IL-1α levels. IL-1α neutralization reduced blood pressure and restored endothelial function in Tg26 mice. To investigate the role of CD4+ T cells and IL-1α in endothelial dysfunction, we developed an aorta-immune cell coculture system. Exposure of WT aortas to Tg26 CD4+ T cells impaired endothelium-dependent relaxation, which was blocked by IL-1α-neutralizing antibody. While investigating the mechanisms of endothelial dysfunction, we reported that Tg26 mice, Tg26→WT aorta exhibit high NADPH oxidase (NOX) 1 expression. IL-1α exposure increased NOX1 in human microvascular endothelial cells, and NOX1 blockade restored endothelial function in Tg26 and Tg26→WT arteries, whereas NOX1 deficiency protected against Tg26 BMT-induced impaired endothelium-dependent relaxation and hypertension. Aortas from people living with HIV exhibit high NOX1 levels, and exposure of human aorta to Tg26 T cells increased NOX1 expression. We provide the first evidence that CD4+ T cells expressing HIV viral proteins induced hypertension through IL-1α-mediated increases in vascular NOX1, which impairs endothelial function in males and females.

Spinal AT1R contributes to neuroinflammation and neuropathic pain via NOX2-dependent redox signaling in microglia

Microglia-mediated neuroinflammation demonstrates a crucial act in the progression of neuropathic pain. Oxidative damage induced by reactive oxygen species (ROS) derived from NADPH oxidase (NOX) in microglia drives proinflammatory microglia activation. Recent evidence points to the central renin angiotensin system (RAS) is involved in oxidative stress and neuroinflammation, with the angiotensin converting enzyme/angiotensin II/angiotensin receptor-1 (ACE/Ang II/AT1R) axis promoting inflammation through increased ROS production, counteracted by the ACE2/Ang (1-7)/ Mas receptor (MasR) axis. While interventions targeting spinal AT1R have been shown to alleviate nociceptive hypersensitivity; yet the mechanisms remain elusive. Here, we discovered that spared nerve injury (SNI)-induced mechanical allodynia in rats were associated with M1-like microglia activation, oxidative stress and overactivity of ACE/Ang II/AT1R axis in the spinal cord. Increased AT1R and NOX2 expression were observed in activated dorsal horn microglia following SNI. Blockade of AT1R with losartan potassium (LOP) suppressed NOX2-mediated oxidative stress, and promoted a shift in microglia from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype in LPS-treated BV-2 cells. Additionally, NOX2 overexpression triggered the activation of the high-mobility group box 1/ nuclear factor-kappa B (HMGB1/NF-κB) signaling pathway. Intrathecal administration of LOP effectively inhibited SNI-induced NOX2 overactivation in microglia and suppressed the HMGB1/NF-kB pathway, reducing oxidative stress and shifting the microglia polarization from M1 to M2 in the spinal cord, thereby attenuating neuroinflammation and pain hypersensitivity. Collectively, these findings underscore the neuroimmune-modulating effects of spinal AT1R in neuropathic pain, highlighting the regulation of redox homeostasis in microglia via a NOX2 dependent mechanism.

LncRNA C2orf27A Promotes Gastric Cancer by Sponging MiR-610 and Elevating NOX4 Expression

LncRNA C2orf27A Promotes Gastric Cancer by Sponging MiR-610 and Elevating NOX4 Expression

Alleviation of Acute Heat Stress in Broiler Chickens by Dietary Supplementation of Polyphenols from Shredded, Steam-Exploded Pine Particles

Reducing the detrimental effects of heat stress (HS) in poultry is essential to minimize production losses. The present study evaluates the effects of dietary polyphenols prepared from underutilized wood byproducts on the growth, gut health, and cecal microbiota in broilers subjected to acute heat stress (AHS). One hundred eight one-day-old Indian River broilers were fed with 0%, 0.5%, or 1% polyphenols from shredded, steam-exploded pine particles (PSPP) in their diet. On the 37th day, forty birds were equally distributed among four groups containing either a control diet at thermoneutral temperatures (NT0%) or AHS temperatures with 0% (AHS0%), 0.5% (AHS0.5%), and 1% (AHS1%) PSPP-supplemented diets. The temperature in the NT room was maintained at 21.0 °C, while, in the AHS room, it was increased to 31 °C. AHS negatively influenced performance parameters and increased rectal temperature (RT) in broilers. The AHS0% group showed a higher expression of NOX4, HSP-70, and HSP-90 genes, while the expression was lower in PSPP-supplemented birds. In the jejunum, mRNA expression of SOD was increased in all the birds under AHS compared to NT. The expression of the CLDN1 and ZO2 genes was increased in AHS0%, while that of the ZO1 and MUC2 genes was increased in PSPP-supplemented birds. HS tends to increase TLR2 and TLR4 gene expression in chickens. The significantly modified genera were Bariatricus, Sporobacter, Sporanaerobacter, and Natranaerovirga. Concludingly, AHS negatively influences the performance parameters, RT, stress, gut-health-related genes, and pathogenic penetration, but PSPP supplementation reduces its bad impact by overcoming the stress and gut-health-related genes, increasing favorable bacterial abundance and reducing pathogenic penetration in chickens.

The histone demethylase KDM6B links obstructive sleep apnea to idiopathic pulmonary fibrosis.

Obstructive sleep apnea (OSA) is increasingly recognized for its link to idiopathic pulmonary fibrosis (IPF), though the underlying mechanisms remain poorly understood. Histone lysine demethylase 6B (KDM6B) may either prevent or promote organ fibrosis, but its specific role in IPF is yet to be clarified. This study aimed to investigate the function and mechanisms of KDM6B in IPF and the exacerbating effects of OSA. We assessed KDM6B levels in lung tissues from IPF patients, IPF mouse models, and a dual-hit model combining OSA-associated intermittent hypoxia (IH) with bleomycin (BLM) or TGF-β1. We evaluated pulmonary fibrosis, myofibroblast activation, and oxidative stress. KDM6B levels were elevated in lung tissues from IPF patients and BLM-treated mice, as well as in TGF-β1-stimulated myofibroblasts. Importantly, IH significantly worsened BLM-induced pulmonary fibrosis and TGF-β1-induced myofibroblast activation, further amplifying KDM6B expression both invivo and invitro. Inhibition of KDM6B reduced pulmonary fibrosis and decreased fibroblast activation and migration in IPF and dual-hit models. Mechanistically, KDM6B inhibition led to decreased NOX4 expression and reduced oxidative stress. KDM6B plays a critical role in promoting pulmonary fibrosis and mediating the exacerbating effects of OSA on this condition. Our findings identify KDM6B as a novel potential therapeutic target for IPF.

Exercise-Intervened Circulating Extracellular Vesicles Alleviate Oxidative Stress in Cerebral Microvascular Endothelial Cells Under Hypertensive Plus Hypoxic Conditions.

Our group has recently demonstrated that exercise intervention affects the release and function of bone marrow endothelial progenitor cell-derived extracellular vesicles (EVs) in transgenic hypertensive mice. Whether such an exercise regimen can impact circulating EVs (cEVs) remains unknown. In this study, we investigated the influence of exercise on cEV level and function. Transgenic hypertensive mice (Alb1-Ren) underwent 8-week treadmill exercise (10 m/min for 1 h, 5 days per week). Age- and sex-matched sedentary Alb1-Ren mice served as controls. cEVs were isolated from the blood of exercised and sedentary mice and are denoted as ET-cEV and nET-cEV, respectively. cEVs were labeled to determine their uptake efficiency and pathways. The functions of cEVs were assessed in an Angiotensin II (Ang II) plus hypoxia-injured cerebral microvascular endothelial cell (mBMEC) injury model. Cellular migration ability and oxidative stress were evaluated. We found that treadmill exercise stimulated cEV release, and ET-cEVs were more prone to be internalized by mBMECs. The ET-cEV internalization was mediated by macropinocytosis and endocytosis pathways. Functional studies showed that ET-cEVs can improve the compromised migration capability of mBMECs challenged by Ang II plus hypoxia. Additionally, ET-cEV treatment upregulated the expression of p-Akt/Akt in mBMECs. Compared to nET-cEVs, ET-cEVs significantly reduced ROS overproduction in Ang II plus hypoxia-injured mBMECs, associated with decreased Nox2 expression. All these findings suggest that exercise-intervened cEVs can protect cerebral microvascular endothelial cells against hypertensive and hypoxic injury.

Lipid Coating Modulates Effects of Nanoceria on Oxidative Metabolism in Human Embryonic Lung Fibroblasts: A Case of Cardiolipin.

The unique redox properties of nanoscale cerium dioxide determine its diverse application in biology and medicine as a regulator of oxidative metabolism. Lipid modifiers of the nanoparticle surface change their biochemical properties and bioavailability. Complexes with lipids can be formed upon contact of the nanoparticles with the membrane. The effects of lipid coating on nanoceria have not been studied yet. Here, we assessed the effect of bare and cardiolipin-coated CeO2 on the expression of oxidative metabolism genes in human embryonic lung fibroblasts. Cell viability, mitochondrial activity, intracellular reactive oxygen species, NOX4, NRF2, and NF-κB expression, oxidative DNA damage/repair, autophagy, and cell proliferation were studied. We used an MTT assay, fluorescence microscopy, real-time reverse transcription polymerase chain reaction, and flow cytometry. At a concentration of 1.5 μM, bare and cardiolipin-coated nanoceria penetrated into cells within 1-3 h. Cell survival, mitochondrial activity, and the proliferative effect were similar for bare and cardiolipin-coated nanoceria. Intracellular ROS, activation of NOX4, NRF2, and NF-kB, DNA oxidative damage, and DNA break/repair were different. Cardiolipin-coated nanoceria induced intracellular oxidative stress and short-term activation of these genes and DNA damage/break/repair. Unlike bare nanoceria, cardiolipin-coated nanoceria induced autophagy. Thus, the effects of cardiolipin-coated nanoceria are determined by both the nanoceria itself and cardiolipin. Presumably, the differences in properties are due to lipid peroxidation of cardiolipin. This effect needs to be taken into account when developing nanoceria-based drugs targeting mitochondria.

Extractable organic matter from PM2.5 inhibits cardiomyocyte differentiation via AHR-mediated m6A RNA methylation.

An ever-increasing body of research has established a link between maternal PM2.5 exposure and congenital heart diseases in the offspring, but the underlying mechanisms remain elusive. We recently reported that activation of the aryl hydrocarbon receptor (AHR) by PM2.5 causes aberrant m6A RNA methylation, leading to cardiac malformations in zebrafish embryos. We hypothesized that PM2.5 can disrupt heart development by inducing m6A methylation changes through AHR in mammals. In this study, we observed that extractable organic matters (EOM) from PM2.5 significantly impaired cardiomyocyte differentiation in embryonic rat cardiomyoblasts H9c2. Importantly, EOM exposure reduced global m6A methylation levels, which was reversed by AHR inhibition. Moreover, AHR, activated by EOM directly promoted the transcription of the demethylase, FTO, leading to global m6A hypomethylation. Specifically, AHR-induced FTO overexpression decreased the m6A methylation levels of Nox4 mRNA, resulting in NOX4 overexpression and subsequent oxidative stress in EOM samples. We then demonstrated that oxidative stress contributes to the inhibition of cardiomyocyte differentiation by EOM through suppression of Wnt/β-catenin signaling. In summary, our findings indicate that AHR activation by PM2.5 directly enhances the expression of the demethylase, FTO, which increases NOX4 expression by reducing its m6A methylation. The oxidative stress caused by NOX4 overexpression inhibits Wnt/β-catenin signaling, thereby compromising cardiomyocyte differentiation.

Klotho enhances stability of chronic kidney disease atherosclerotic plaques by inhibiting GRK2/PLC-β-mediated endoplasmic reticulum stress in macrophages via modulation of the ROS/SHP1 pathway

Klotho has been importantly linked to atherosclerosis, but little is known about its specific role. This study investigates the mechanism by which Klotho enhances the stability of atherosclerotic plaques in chronic kidney disease. apoE-/- knockout mice and C57BL/6 mice underwent 5/6 nephrectomy and then klotho-NC and klotho-mimic groups were set up to be fed a high-fat chow diet and a dummy group was created to be fed a normal chow diet. qPCR detected relative mRNA expression of klotho. Oil Red O and HE staining assessed lipid proportion in the aorta. Masson staining evaluated renal failure pathology in mice. Immunohistochemistry measured MAC-2 and α-SMA expression in the aorta. ELISA quantified urea, cholesterol, calcium ions, and triglycerides in mouse plasma. Western blotting detected associated protein expression, followed by cell-based experiments for validation. Compared with the Klotho-NC group, the plaque area and aortic lipid and renal fibrosis area were reduced in the Klotho-mimic group. Klotho-mimic reduced macrophage area, plasma urea, cholesterol, calcium ions, and triglyceride levels, and decreased the expression of p-PERK, NOX2, NOX4, Caspase-3, Caspase-9, Bax, p-GRK2, p-PLCβ, p-Src, and p-IP3R. Without ox-LDL stimulation, Klotho expression increased in the Klotho-mimic group, with no significant differences in NOX2, p-SHP1, p-Src, p-PERK, p-GRK2, and p-PLCβ. With ox-LDL in high-calcium medium, Klotho and p-SHP1 increased, while NOX2, p-Src, p-PERK, p-GRK2, and p-PLCβ decreased in the Klotho-mimic group. After ox-LDL and TPI-1 treatment, Klotho increased, NOX2 decreased, and other proteins showed no significant changes. Adding shRNA-GRK2 reduced NOX2, p-Src, and p-PERK, increased p-SHP1, with no changes in p-GRK2 and p-PLCβ. Differences in NOX2, p-GRK2, p-PLCβ, and p-PERK between groups were reduced in high-calcium medium, while p-SHP1 differences increased. Klotho enhances chronic kidney disease atherosclerotic plaque stability by inhibiting GRK2/PLC-β-mediated endoplasmic reticulum stress in macrophages via the ROS/SHP1 pathway.

Effects of 17β estradiol on blood pressureelevation in ovariectomized rats withcollagen-induced arthritisviamodulation of oxidative stress, inflammation, fibrosis, and apoptosis in the aorta involving TLR4/NOX4/NF-kβ and TGFβ1/fibronectin/α-SMA pathways.

Rheumatoid arthritis (RA) can cause blood pressure (BP) elevation in estrogen-deficient, post-menopausal women; however, the underlying mechanisms are not well understood. In this study, the aortic involvement and its underlying mechanisms that contribute to the BP elevation in estrogen-deficient, RA condition were identified. Ovariectomy was performed to create a state of estrogen deficiency and RA was then induced in ovariectomized rats by using incomplete Freund's adjuvant and immune-mediated collagen type-II. Ovariectomized, RA-induced rats (Ovx + RA) were given either 17β-estradiol, baricitinib, or losartan. Direct blood pressure (BP) monitoring was made via cannulation of the carotid artery. Rats were then sacrificed and the aorta was harvested followed by H&E and Picrosirius staining to evaluate histological changes and collagen deposition. Oxidative stress, inflammation, apoptosis, growth, and fibrosis levels in the aorta were assessed by using molecular biological techniques. Mean arterial pressure (MAP) was significantly elevated in Ovx + RA rats when compared to sham and Ovx rats (p < 0.05). 17β-estradiol and losartan treatment significantly reduced the MAP and heart rate in Ovx + RA rats when compared to untreated Ovx + RA rats. Expression of iNOS, Nox2 and Nox4, TLR4, NF-ĸB, TNF-α, VEGF, FGF-2, αSMA, eNOS, and caspase-3 were elevated in the aorta of Ovx + RA rats and were reduced upon 17β-estradiol treatment. However, expression of TGFβ1, Bax-2, fibronectin, and Smad2 in the aorta of Ovx + RA rats was increased following 17β-estradiol treatment (p < 0.05 compared to without treatment). The presence of RA with estrogen deficiency enhanced the BP elevation due to changes in the aorta which could be ameliorated by estrogen.

Tyrosine phosphatase SHP2 promoted the progression of CRC via modulating the PI3K/BRD4/TFEB signaling induced ferroptosis

ObjectiveTo elucidate the mechanism by which tyrosine phosphatase SHP2 protects CRC through modulation of TFEB-mediated ferritinophagy, thereby suppressing ROS and ferroptosis.MethodsSW480 and SW620 cells, in the logarithmic growth phase, were treated with or without the SHP2 inhibitor PHPS1, the activator Trichomide A, EGF, or MMP inhibitors, and randomly assigned to four groups. Additionally, SW480 cells in the logarithmic phase underwent treatments with EGF, the ferroptosis inducer erastin, Trichomide A, or the curcumin analog C1, forming seven groups. Cell migration assessment in these groups employed scratch and Transwell assays. Protein expression analysis of total SHP2, total PI3K, p-SHP2, p-PI3K, p-TFEB, TFEB, SQSTM1, LC3, LAMP2, NCOA4, FTH1, GPX4, NOX4, and ACSL4 in the seven SW480 groups was conducted through Western blot and immunofluorescence. Apoptosis analysis was performed on these seven groups, while gene co-expression analysis utilized bioinformatics. SW480 and CCD-841CoN cells were categorized into four groups, undergoing treatment with saline, EGFR-OE lentivirus, SHP2-KD lentivirus, or SHP2-OE lentivirus. Western blot analysis in SW480 cells detected EGFR, total SHP2, p-SHP2, GPX4, and ACSL4 proteins, and tumor volume observations were conducted in a nude mouse xenograft model. Western blot also evaluated total SHP2, p-SHP2, GPX4, and ACSL4 protein expression in CCD-841CoN cells.ResultsBioinformatics analysis revealed correlations between EGFR and SHP2, SHP2 and PIK3CA, SHP2 and MAPK1, BRK4 and HIF1A, HIF1A and NCOA4, as well as TFEB and FTH1. Scratch and Transwell assays showed that SHP2 diminishes the migratory capacity of SW480 and SW620 cells. Western blot and immunofluorescence demonstrated that EGFR activation of SHP2 markedly elevated p-TFEB levels while reducing TFEB protein expression. EGF stimulation enhanced the expression of FTH1, GPX4, NOX4, and ACSL4. Combined stimulation with EGF and SHP2 further amplified the expression of p-SHP2, p-TFEB, and NCOA4 while reducing TFEB, SQSTM1, LC3, and LAMP2. Erastin augmented FTH1, GPX4, NOX4, and ACSL4 expression while decreasing p-SHP2, p-TFEB, TFEB, SQSTM1, LC3, LAMP2, and NCOA4. TFEB activation suppressed p-SHP2, p-TFEB, NCOA4, FTH1, and GPX4 expression, while promoting TFEB, SQSTM1, LC3, LAMP2, NOX4, and ACSL4 expression. Apoptosis assays indicated that SHP2 activation decelerated apoptosis in SW480 cells, whereas erastin under EGF stimulation accelerated apoptosis, as did TFEB activation. Western blot results in SW480 cells displayed that overexpression of EGFR or SHP2 significantly increased total SHP2, p-SHP2, and GPX4 expression while decreasing ACSL4 levels. SHP2 knockdown decreased total SHP2, p-SHP2, and GPX4 expression, with an increase in ACSL4 expression. In CCD-841CoN cells, overexpression of EGFR or SHP2 resulted in a decrease in p-SHP2 and an increase in total SHP2, more pronounced with SHP2 overexpression, while GPX4 and ACSL4 levels remained stable. SHP2 knockdown led to reduced EGFR, total SHP2, p-SHP2, and GPX4 expression, without a significant impact on ACSL4 levels. The nude mouse xenograft model demonstrated that EGFR overexpression significantly increased tumor size, whereas SHP2 overexpression markedly decreased tumor volume. SHP2 knockdown resulted in significantly larger tumors.ConclusionSHP2 advances CRC progression by modulating TFEB-mediated ferritinophagy, suppressing ROS and ferroptosis. Targeting SHP2 presents a promising therapeutic strategy for CRC.

C-Peptide Ameliorates Particulate Matter 2.5-Induced Skin Cell Apoptosis by Inhibiting NADPH Oxidation.

Connecting peptide (C-peptide), a byproduct of insulin biosynthesis, has diverse cellular and biological functions. Particulate matter 2.5 (PM2.5) adversely affects human skin, leading to skin thickening, wrinkle formation, skin aging, and inflammation. This study aimed to investigate the protective effects of C-peptide against PM2.5-induced damage to skin cells, focusing on oxidative stress as a key mechanism. C-peptide mitigated NADPH oxidation and intracellular reactive oxygen species (ROS) production induced by PM2.5. It also suppressed PM2.5-induced NADPH oxidase (NOX) activity and alleviated PM2.5-induced NOX1 and NOX4 expression. C-peptide protected against PM2.5-induced DNA damage, lipid peroxidation, and protein carbonylation. Additionally, C-peptide mitigated PM2.5-induced apoptosis by inhibiting intracellular ROS production. In summary, our findings suggest that C-peptide mitigates PM2.5-induced apoptosis in human HaCaT keratinocytes by inhibiting intracellular ROS production and NOX activity.

Reduced glutathione enhances adipose tissue‐derived mesenchymal stem cell engraftment efficiency for liver fibrosis by targeting TGFβ1/SMAD3/NOX4 pathway

AbstractReduced glutathione (GSH) could reduce oxidative stress to improve adipose tissue‐derived mesenchymal stem cell (ADSC) engraftment efficiency in vivo. However, the underlying mechanisms remain unclear. Our goal is to investigate whether GSH enhances ADSC engraftment through targeting the TGFβ/SMAD3/NOX4 pathway. Liver fibrotic male mice were administrated GSH, setanaxib (STX), and SIS3 during ADSC transplantation. ADSC engraftment efficiency and reactive oxygen species (ROS) level were detected both in vivo and ex vivo. Biochemical analysis was used to analyze the content of superoxide and nicotinamide adenine dinucleotide phosphate oxidases (NOXs) in liver tissues. Immunohistochemistry and western blotting were used to examine the protein level of NOX1, NOX2, NOX4, transforming growth factor‐β1 (TGFβ1), SMAD3, and p‐SMAD3 in liver tissues. Additionally, the therapeutic efficacy of the ADSC transplantation was further investigated. We found that GSH significantly improved ADSC engraftment efficiency, which was closely related to the reduced ROS generation in liver tissues. However, the enhanced cell engraftment was abolished after the combined treatment with STX or SIS3. GSH could effectively reduce superoxide and NOXs content, and selectively inhibit NOX4 expression in liver tissues. The co‐localization results showed that GSH could reduce NOX4 expressed in activated hepatic stellate cells. Mechanistically, GSH down‐regulated TGFβ/SMAD3 signaling. More importantly, GSH enhanced the therapeutic efficacy of ADSC therapy in liver fibrotic mice. Taken together, GSH could improve the engraftment efficiency of ADSCs in liver fibrosis by targeting TGFβ1/SMAD3/NOX4 signaling pathway, which provides a new theoretical basis for GSH enhancing ADSC engraftment efficiency in liver diseases.

Nitric oxide fumigation maintains cantaloupe fruit quality by regulating the production of reactive oxygen species

Cantaloupe (Cucumis melo L.) is a major crop cultivated in Northwest China. However, with the extension of storage periods, cantaloupes are increasingly prone to post-ripening senescence, quality deterioration, rot, and other phenomena, which in turn seriously limit shelf life. During postharvest storage, the quality of cantaloupes is affected by a large increase in ROS in the fruit. Herein, the effects of exogenous NO fumigation treatment on the quality of Xizhoumi NO.17 cantaloupe were investigated, with a focus on ROS generation and removal. NO fumigation treatment effectively reduced the rate of cantaloupe decay, inhibited fruit water loss and better maintained fruit firmness and relative conductivity. NO treatment induced O2·– and H2O2 production during early storage (within 24 h), whereas their accumulation decreased during later storage. NOX activity and expression of related genes in the treatment group were higher than those in the control group during the early storage but lower during later storage. NO fumigation also induced the antioxidant capacity of enzymatic and non-enzymatic systems in cantaloupe and helped to inhibit the accumulation of ROS. Taken together, NO fumigation triggers H2O2 production via the NOX/SOD system, activating the antioxidant response of cantaloupe to overcome excessive ROS production in the late stage of storage, thus maintaining fruit quality.

NADPH oxidase expression profile and PBMC immunophenotypic changes in anti-TNF-treated rheumatoid arthritis patients

The aim of this research was to prospectively evaluate the impact of NOX2 gene expression profile (including NCF1, NCF2 and NCF4 genes) in peripheral blood mononuclear cells (PBMCs) on immune signatures, clinical characteristics and responsiveness to anti-TNFα treatment in RA patients. Blood specimens were collected from 31 rheumatoid arthritis (RA) patients and 25 healthy controls, and 16 RA patients were followed at two timepoints during anti-TNF treatment. mRNA expression levels of selected genes and immunoregulatory cytokines concentrations were determined. We observed the significant upregulation of NCF4 and CD14 expression in RA group. The mRNA levels of NCF1 and CD14 positively correlated both in groups of RA patients and healthy controls. NOX2 gene expression profile was not associated with anti-TNFα responsiveness, nor with RA clinical features. TNFα inhibition has not influenced NOX2 expression either. Notably, this study indicate the novel links between expression levels of NCF1 and monocyte differentiation antigen CD14.

CRGD-Conjugated Bilirubin Nanoparticles Alleviate Dry Eye Disease Via Activating the PINK1-Mediated Mitophagy.

The purpose of this study was to evaluate the cytoprotective effect and the mechanism of cRGD-conjugated bilirubin nanoparticles (cNPs@BR) in dry eye disease (DED). The binding capacity and cellular uptake of cNPs@BR in human corneal epithelial cells (HCECs) were assessed by immunofluorescence. The anti-inflammation and anti-oxidative stress effects of cNPs@BR were determined by flow cytometry, immunofluorescence, Western blot, chromatin immunoprecipitation (ChIP), and ELISA assay in LPS-stimulated RAW264.7 cells and hypertonic HCECs. The function of ocular surface barrier, tear production, and the number of goblet cells after cNPs@BR treatment were further assessed by fluorescein sodium staining, phenol red cotton threads, quantitative PCR (qPCR), hematoxylin and eosin (H&E) staining, and Periodic Acid-Schiff (PAS) staining in a 0.2% BAC-induced DED mouse model. Furthermore, the mechanism of cNPs@BR in treating DED was explored by RNA sequencing and RNA interference. The cRGD peptide prolonged the retention time of nanoparticles on HCECs and enhanced the cellular uptake efficiency. In both cell models, 20 µM cNPs@BR pretreatment ameliorated oxidative stress by decreasing the intracellular reactive oxygen species (ROS) levels and the expression of NOX4 and 4-HNE, while promoting HO-1 and nuclear Nrf2 levels. Moreover, cNPs@BR alleviated the inflammatory response by inhibiting NF-κB p65 nuclear translocation and decreasing the expression of iNOS and the secretion of IL-1β, IL-6, and TNF-α. In addition, cNPs@BR protected ocular surface epithelium against oxidative stress and inflammation and restored conjunctival goblet cells in the mouse model of DED by activating PINK1-mediated mitophagy. The cNPs@BR suppressed oxidative stress and inflammatory response in the ocular surface epithelium and restored goblet cells by activating PINK1-mediated mitophagy.

Estrogen hindrance escalates inflammation and neurodegeneration in the hippocampal regions of collagen-induced arthritis female Sprague-Dawley rats.

This study aims to investigate the effect of estrogen hindrance, i.e., menopause in women for instance with rheumatoid arthritis on the brain hippocampal region by using collagen-induced arthritis (CIA) female rat model (RA). CIA was induced in female rats by injecting bovine type II collagen and incomplete Freund's adjuvant. Estrogen receptor antagonist, fulvestrant (Ful), was given to RA rats to create estrogen hindrance. Control (C) and RA rats were injected with saline and DMSO, respectively, while RA + Ful rats received a 7-day fulvestrant injection. Following experiment completion, rats were sacrificed, and brains were harvested. Brains were stained with H&E and cresyl violet staining and morphological changes in the hippocampus were identified. Additionally, oxidative stress, inflammatory, and apoptosis markers' levels in the hippocampus were analyzed by qPCR, ELISA, and immunohistochemistry techniques. RA + Ful rats showed neuronal atrophy and reduced neurogenesis in the hippocampal regions. NOX4, NF-κB, IL-1β, IL-6, TNF-α, IKK-β, and Bax protein expression levels in the hippocampus were increased, whereas hippocampal Bcl-2, caspase-3, caspase-9, and IGF-1R protein expression levels were decreased. Furthermore, RA + Ful rats had lower levels of antioxidants PON-1 and catalase in the hippocampal regions. The changes in these molecular markers were statistically significant when compared to RA rats without Ful treatment (p < 0.05). Estrogen hindrance exaggerated oxidative stress, inflammation, and apoptosis which resulted in neuronal degeneration in the hippocampal regions in rheumatoid arthritis.

Association of NOX5 Expression with Sperm Activity and Motility in Pathospermic Infertile Men.

The newest NOX isoform, NOX5, has been found in mammalian spermatozoa. Many physiological and pathological situations in spermatozoa are mediated by reactive oxygen species (ROS). NOX5 is the main source of ROS in spermatozoa. Our purpose was to investigate the changes in NOX5 expression and the effect of NOX5 expression on sperm motility, chromatin integrity, and oxidative status in oligoasthenozoospermic compared to normozoospermic men. Semen samples were collected from 30 normozoospermic (NS) and 30 oligoasthenozoospermic (OAS) men. NOX5 protein expression in sperm samples was evaluated by immunohistochemistry and western blot. Oxidative stress status was evaluated by total antioxidant capacity (TAC), total oxidant capacity (TOC), and oxidative stress index (OSI) parameters. Chromatin integrity in spermatozoa was evaluated by toluidine blue staining. NOX5 expression levels were significantly higher in OAS group than in NS group (p<0.001). In addition, chromatin integrity was significantly higher in the OAS group in comparison to NS group (p<0.001). TAC levels were higher in the NS group, but OSI and TOC levels were significantly higher in OAS group (p<0.001). It was found that NOX5 protein expression was positively correlated with oxidative stress and chromatin integrity and negatively correlated with motility (p<0.01). These results suggest that overexpression of NOX5 may be the source of excessive ROS production and oxidative stress injuries in oligoasthenozoospermic men. Considering that NOX5 expression is positively correlated with oxidative stress and chromatin integrity but negatively correlated with motility, it can be considered a biomarker to be used in assisted reproductive procedures.

LRRC8A drives NADPH oxidase-mediated mitochondrial dysfunction and inflammation in allergic rhinitis

ObjectivesAllergic rhinitis (AR) is a complex disorder with variable pathogenesis. Increasing evidence suggests that the LRRC8A is involved in maintaining cellular stability, regulating immune cell activation and function, and playing significant roles in inflammation. However, the involvement of LRRC8A in AR inflammation and its underlying mechanisms remain unclear.MethodsLRRC8A expression in AR patients, confirmed by qRT-PCR and Western blotting, was analyzed to investigate its relationship with the clinical characteristics of AR patients. In vitro, IL-13 stimulated HNEpCs to establish a Th2 inflammation model, with subsequent LRRC8A knockout or overexpression. NOX1/NOX4 inhibitor (GKT137831) and chloride channel inhibitor (DCPIB) were utilized to investigate AR development mechanisms during LRRC8A overexpression. An OVA-induced AR model with nasal mucosa LRRC8A knockdown confirmed LRRC8A's regulatory role in AR inflammation.ResultsLRRC8A mRNA and protein levels were significantly elevated in AR patients, positively correlating with NADPH oxidase subunits and Th2 inflammatory markers. In vitro, IL-13 stimulation of HNEpCs resulted in upregulation of LRRC8A and increased expression of NOX1, NOX4, and p22phox, along with mitochondrial dysfunction and NF-κB pathway activation. The knockout of LRRC8A reversed these effects. In nasal mucosal epithelial cells, DCPIB and GKT137831 completely blocked mitochondrial dysfunction caused by the overexpression of LRRC8A, which led to up-regulation of NOX1, NOX4, and p22phox. In vivo, knocking down LRRC8A reduced eosinophil infiltration, downregulated the expression of NOX1, NOX4, p22phox IL-4, IL-5, and IL-13, and decreased NF-κB pathway activation.ConclusionLRRC8A drives the upregulation of NOX1, NOX4, and p22phox, leading to ROS overproduction and mitochondrial dysfunction. It also activates NF-κB, ultimately leading to nasal mucosal epithelial inflammation. LRRC8A may be a potential target for the treatment of AR.

P75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome.

Background: Rett syndrome (RTT) is an early-onset neurological disorder primarily affecting females, leading to severe cognitive and physical disabilities. Recent studies indicate that an imbalance of redox homeostasis and exacerbated inflammatory responses are key players in the clinical manifestations of the disease. Emerging evidence highlights that the p75 neurotrophin receptor (p75NTR) is implicated in the regulation of oxidative stress (OS) and inflammation. Thus, this study is aimed at investigating the effects of p75NTR modulation by LM11A-31 on fibroblasts derived from RTT donors. Methods: RTT cells were treated with 0.1 µM of LM11A-31 for 24 h, and results were obtained using qPCR, immunofluorescence, ELISA, and Western blot techniques. Results: Our findings demonstrate that LM11A-31 reduces OS markers in RTT fibroblasts. Specifically, p75NTR modulation by LM11A-31 restores protein glutathionylation and reduces the expression of the pro-oxidant enzyme NOX4. Additionally, LM11A-31 significantly decreases the expression of the pro-inflammatory mediators interleukin-6 and interleukin-8. Additionally, LM11A-31 normalizes the expression levels of transcription factors involved in the regulation of the antioxidant response and inflammation. Conclusions: Collectively, these data suggest that p75NTR modulation may represent an effective therapeutic target to improve redox balance and reduce inflammation in RTT.