NADPH Oxidase Inhibitor Apocynin Research Articles

NADPH oxidase-derived reactive oxygen species regulates adipose tissue lipolysis in humans

There is mounting evidence suggesting that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) regulate physiological processes, as NOX has been shown to promote increases in lipolysis in cultured adipocytes. PURPOSE: To determine the mechanisms by which NOX stimulates adipose tissue lipolysis. METHODS: Young healthy females (n = 17; mean ± SD; age: 21.7 ± 5 years; body mass index: 22.2 ± 3.8 kg/m2) participated in this study. Participants underwent microdialysis and hyperinsulinemic-euglycemic clamp (clamp) procedures. Three microdialysis probes were inserted into subcutaneous abdominal adipose tissue to monitor local adipose tissue ROS (H2O2) production and lipolysis (as indicated by dialysate glycerol). To determine whether NOX stimulates lipolysis via distinct signaling pathways, microdialysis probes were perfused with either isoproterenol (Iso; β-adrenergic agonist to stimulate canonical lipolysis signaling) or atrial natriuretic peptide (ANP to stimulate non-canonical lipolysis signaling), then perfused with a local NOX inhibitor (apocynin). A control probe was perfused with apocynin but without any agonists of lipolysis. The microdialysis procedures were then repeated under hyperinsulinemic conditions during the same study visit. RESULTS: In the fasted state, dialysate H2O2 concentrations were lower in the control and Iso probes upon apocynin perfusion (control probe without apocynin: 1.06 ± 0.5 μM, with apocynin: 0.62 ± 0.2 μM; p = 0.005; Iso probe without apocynin: 1.01 ± 0.4 μM, with apocynin: 0.66 ± 0.2 μM; p = 0.003). Apocynin perfusion did not alter dialysate H2O2 concentrations in the ANP probe under the fasted state (p = 0.11). Dialysate glycerol concentrations did not change upon apocynin perfusion under any of the microdialysis probe conditions in the fasted state (p > 0.05). During the clamp, apocynin perfusion resulted in lower dialysate H2O2 concentrations in the three conditions (control probe without apocynin: 1.22 ± 0.6 μM, with apocynin: 0.75 ± 0.3 μM; p = 0.03; Iso probe without apocynin: 1.55 ± 0.8 μM, with apocynin: 0.79 ± 0.3 μM; p = 0.01; ANP probe without apocynin: 1.55 ± 0.6 μM, with apocynin: 0.92 ± 0.3 μM; p = 0.03). Dialysate glycerol concentrations were lower in the Iso probe upon apocynin perfusion during the clamp (Iso probe without apocynin: 124.7 ± 38 μM, with apocynin: 90.9 ± 25 μM; p = 0.003). CONCLUSIONS: This study presents adipose tissue NOX as a source of in vivo ROS production in human participants. In addition, we reveal a novel mechanism of NOX in adipose tissue function whereby NOX exerts increases in β-adrenergic-mediated lipolysis during hyperinsulinemia. Our findings highlight the roles of ROS as signaling mediators and suggest that an imbalance between NOX activity and insulin action on adipose tissue could negatively affect systemic fatty acid metabolism. Porter Pre-doctoral Fellowship from the American Physiological Society and an NIH National Research Service Award (F31HL154642). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

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

GLP-1 in the Hypothalamic Paraventricular Nucleus Promotes Sympathetic Activation and Hypertension.

Glucagon-like peptide-1 (GLP-1) and its analogs are widely used for diabetes treatment. The paraventricular nucleus (PVN) is crucial for regulating cardiovascular activity. This study aims to determine the roles of GLP-1 and its receptors (GLP-1R) in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male normotensive rats and spontaneously hypertensive rats (SHR). Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. GLP-1 and GLP-1R expressions were present in the PVN. PVN microinjection of GLP-1R agonist recombinant human GLP-1 (rhGLP-1) or EX-4 increased RSNA and MAP, which were prevented by GLP-1R antagonist exendin 9-39 (EX9-39) or GLP-1R antagonist 1, superoxide scavenger tempol, antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor apocynin, adenylyl cyclase (AC) inhibitor SQ22536 or protein kinase A (PKA) inhibitor H89. PVN microinjection of rhGLP-1 increased superoxide production, NADPH oxidase activity, cAMP level, AC, and PKA activity, which were prevented by SQ22536 or H89. GLP-1 and GLP-1R were upregulated in the PVN of SHR. PVN microinjection of GLP-1 agonist increased RSNA and MAP in both WKY and SHR, but GLP-1 antagonists caused greater effects in reducing RSNA and MAP in SHR than in WKY. The increased superoxide production and NADPH oxidase activity in the PVN of SHR were augmented by GLP-1R agonists but attenuated by GLP-1R antagonists. These results indicate that activation of GLP-1R in the PVN increased sympathetic outflow and blood pressure via cAMP-PKA-mediated NADPH oxidase activation and subsequent superoxide production. GLP-1 and GLP-1R upregulation in the PVN partially contributes to sympathetic overactivity and hypertension.

Chronic SIRT1 supplementation in diabetic mice improves endothelial function by suppressing oxidative stress.

Enhancing SIRT1 activity exerts beneficial cardiovascular effects. In diabetes, plasma SIRT1 levels are reduced. We aimed to investigate the therapeutic potential of chronic recombinant murine SIRT1 (rmSIRT1) supplementation to alleviate endothelial and vascular dysfunction in diabetic mice (db/db). Left internal mammary arteries obtained from patients undergoing coronary artery bypass grafting with or without a diagnosis of diabetes were assayed for SIRT1 protein levels. Twelve-week-old male db/db mice and db/+ controls were treated with vehicle or rmSIRT1 intraperitoneally for 4 weeks, after which carotid artery pulse wave velocity (PWV) and energy expenditure/activity were assessed by ultrasound and metabolic cages, respectively. Aorta, carotid, and mesenteric arteries were isolated to determine endothelial and vascular function using the myograph system.Arteries obtained from diabetic patients had significantly lower levels of SIRT1 relative to non-diabetics. In line, aortic SIRT1 levels were reduced in db/db mice compared to db/+ mice, while rmSIRT1 supplementation restored SIRT1 levels. Mice receiving rmSIRT1 supplementation displayed increased physical activity and improved vascular compliance as reflected by reduced PWV and attenuated collagen deposition. Aorta of rmSIRT1-treated mice exhibited increased endothelial nitric oxide (eNOS) activity, while endothelium-dependent contractions of their carotid arteries were significantly decreased, with mesenteric resistance arteries showing preserved hyperpolarization. Ex vivo incubation with reactive oxygen species (ROS) scavenger Tiron and NADPH oxidase inhibitor apocynin revealed that rmSIRT1 leads to preserved vascular function by suppressing NADPH oxidase (NOX)-related ROS synthesis. Chronic rmSIRT1 treatment resulted in reduced expression of both NOX1 and NOX4, in line with a reduction in aortic protein carbonylation and plasma nitrotyrosine levels. In diabetic conditions, arterial SIRT1 levels are significantly reduced. Chronic rmSIRT1 supplementation improves endothelial function and vascular compliance by enhancing eNOS activity and suppressing NOX-related oxidative stress. Thus, SIRT1 supplementation may represent novel therapeutic strategy to prevent diabetic vascular disease.

Downregulation of SODD mediates carnosol-induced reduction in cell proliferation in esophageal adenocarcinoma cells

Esophageal adenocarcinoma carries a poor prognosis associated with a 5-year survival rate of 12.5–20%. Therefore, a new therapeutic modality is needed for this lethal tumor. Carnosol is a phenolic diterpene purified from the herbs such as rosemary and Mountain desert sage and has been shown to have anticancer activities in multiple cancers. In this study we examined the effect of carnosol on cell proliferation in esophageal adenocarcinoma cells. We found that carnosol dose-dependently decreased cell proliferation in FLO-1 esophageal adenocarcinoma cells and significantly increased caspase-3 protein, indicating that carnosol decreases cell proliferation and increases cell apoptosis in FLO-1 cells. Carnosol significantly increased H2O2 production and N-acetyl cysteine, a reactive oxygen species (ROS) scavenger, significantly inhibited carnosol-induced decrease in cell proliferation, indicating that ROS may mediate carnosol-induced decrease in cell proliferation. Carnosol-induced decrease in cell proliferation was partially reversed by NADPH oxidase inhibitor apocynin, suggesting that NADPH oxidases may be partially involved in carnosol’s effect. In addition, carnosol significantly downregulated SODD protein and mRNA expression and knockdown of SODD significantly inhibited the carnosol-induced reduction in cell proliferation, suggesting that downregulation of SODD may contribute to carnosol-induced reduction in cell proliferation. We conclude that carnosol dose-dependently decreased cell proliferation and significantly increased caspase-3 protein. Carnosol’s effect may be through the overproduction of ROS and the downregulation of SODD. Carnosol might be useful for the treatment of esophageal adenocarcinoma.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

Effects of Apocynin, a NADPH Oxidase Inhibitor, in the Protection of the Heart from Ischemia/Reperfusion Injury.

Ischemia and perfusion (I/R) induce inflammation and oxidative stress, which play a notable role in tissue damage. The aim of this study was to investigate the role of an NADPH oxidase inhibitor (apocynin) in the protection of the heart from I/R injury. Hearts isolated from Wistar rats (n = 8 per group) were perfused with a modified Langendorff preparation. Left ventricular (LV) contractility and cardiovascular hemodynamics were evaluated by a data acquisition program, and infarct size was evaluated by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining. Furthermore, the effect of apocynin on the pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and anti-inflammatory cytokine (IL-10) was evaluated using an enzyme linked immunosorbent assay (ELISA). Hearts were subjected to 30 min of regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery, followed by 30 min of reperfusion. Hearts were infused with apocynin before ischemia, during ischemia or at reperfusion. To understand the potential pathways of apocynin protection of the heart, a nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP), nitric oxide blocker (N (gamma)-nitro-L-arginine methyl ester, L-Name), nicotinic acid adenine dinucleotide phosphate (NAADP) inhibiter (Ned-K), cyclic adenosine diphosphate ribose (cADPR) agonist, or CD38 blocker (Thiazoloquin (az)olin (on)e compound, 78c) was infused with apocynin. Antioxidants were evaluated by measuring superoxide dismutase (SOD) and catalase (CAT) activity. Apocynin infusion before ischemia or at reperfusion protected the heart by normalizing cardiac hemodynamics and decreasing the infarct size. Apocynin treatment resulted in a significant (p < 0.05) decrease in pro-inflammatory cytokine levels and a significant increase (p < 0.05) in anti-inflammatory and antioxidant levels. Apocynin infusion protected the heart by improving LV hemodynamics and coronary vascular dynamics. This treatment decreased the infarct size and inflammatory cytokine levels and increased anti-inflammatory cytokine and antioxidant levels. This protection follows a pathway involving CD38, nitric oxide and acidic stores.

The NADPH Oxidase Inhibitors Apocynin and Diphenyleneiodonium Protect Rats from LPS-Induced Pulmonary Inflammation

Inflammation is the body's response to insults, for instance, lung inflammation is generally caused by pathogens or by exposure to pollutants, irritants and toxins. This process involves many inflammatory cells such as epithelial cells, monocytes, macrophages and neutrophils. These cells produce and release inflammatory mediators such as pro-inflammatory cytokines, lipids and reactive oxygen species (ROS). Lung epithelial cells and phagocytes (monocytes, macrophages and neutrophils) produce ROS mainly by the NADPH oxidase NOX1 and NOX2, respectively. The aim of this study was to investigate the effects of two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium (DPI), on lipopolysaccharide (LPS)-induced lung inflammation in rats. Our results showed that apocynin and DPI attenuated the LPS-induced morphological and histological alterations of the lung, reduced edema and decreased lung permeability. The evaluation of oxidative stress markers in lung homogenates showed that apocynin and DPI inhibited LPS-induced NADPH oxidase activity, and restored superoxide dismutase (SOD) and catalase activity in the lung resulting in the reduction in LPS-induced protein and lipid oxidation. Additionally, apocynin and DPI decreased LPS-induced MPO activity in bronchoalveolar liquid and lung homogenates, TNF-α and IL-1β in rat plasma. NADPH oxidase inhibition could be a new therapeutic strategy for the treatment of inflammatory lung diseases.

Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells

Ovarian follicles are the fundamental structures that support oocyte development, and communications between oocytes and follicle somatic cells are crucial for oogenesis. However, it is unknown that whether exposure to microgravity influences cellular communications and ovarian follicle development, which might be harmful for female fertility. By 3D culturing of ovarian follicles under simulated microgravity (SMG) conditions in a rotating cell culture system, we found that SMG treatment did not affect the survival or general growth of follicles but decreased the quality of cultured follicles released oocytes. Ultrastructure detections by high-resolution imaging showed that the development of cellular communicating structures, including granulosa cell transzonal projections and oocyte microvilli, were markedly disrupted. These abnormalities caused chaotic polarity of granulosa cells (GCs) and a decrease in oocyte-secreted factors, such as Growth Differentiation Factor 9 (GDF9), which led to decreased quality of oocytes in these follicles. Therefore, the quality of oocytes was dramatically improved by the supplementations of GDF9 and NADPH-oxidase inhibitor apocynin. Together, our results suggest that exposure to simulated microgravity impairs the ultrastructure of ovarian follicles. Such impairment may affect female fertility in space environment.

Dexmedetomidine Attenuates Lipopolysaccharide-Induced Sympathetic Activation and Sepsis via Suppressing Superoxide Signaling in Paraventricular Nucleus.

Sympathetic overactivity contributes to the pathogenesis of sepsis. The selective α2-adrenergic receptor agonist dexmedetomidine (DEX) is widely used for perioperative sedation and analgesia. We aimed to determine the central roles and mechanisms of DEX in attenuating sympathetic activity and inflammation in sepsis. Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide (LPS) in rats. Effects of DEX were investigated 24 h after injection of LPS. Bilateral microinjection of DEX in the paraventricular nucleus (PVN) attenuated LPS-induced sympathetic overactivity, which was attenuated by the superoxide dismutase inhibitor DETC, cAMP analog db-cAMP or GABAA receptor antagonist gabazine. Superoxide scavenger tempol, NADPH oxidase inhibitor apocynin, adenylate cyclase inhibitor SQ22536 or PKA inhibitor Rp-cAMP caused similar effects to DEX in attenuating LPS-induced sympathetic activation. DEX inhibited LPS-induced superoxide and cAMP production, as well as NADPH oxidase, adenylate cyclase and PKA activation. The roles of DEX in reducing superoxide production and NADPH oxidase activation were attenuated by db-cAMP or gabazine. Intravenous infusion of DEX inhibited LPS-induced sympathetic overactivity, NOX activation, superoxide production, TNF-α and IL-1β upregulation in the PVN and plasma, as well as lung and renal injury, which were attenuated by the PVN microinjection of yohimbine and DETC. We conclude that activation of α2-adrenergic receptors with DEX in the PVN attenuated LPS-induced sympathetic overactivity by reducing NADPH oxidase-dependent superoxide production via both inhibiting adenylate cyclase-cAMP-PKA signaling and activating GABAA receptors. The inhibition of NADPH oxidase-dependent superoxide production in the PVN partially contributes to the roles of intravenous infusion of DEX in attenuating LPS-induced sympathetic activation, oxidative stress and inflammation.

Treponema pallidum membrane protein Tp47 promotes angiogenesis through ROS-induced autophagy.

Pathological angiogenesis is an important manifestation of syphilis, but the underlying mechanism of Treponema pallidum subspecies pallidum (T.pallidum)-induced angiogenesis is poorly understood. The objective of this study is to investigate the role and related mechanism of the T.pallidum membrane protein Tp47 in angiogenesis. The proangiogenic activity of recombinant T.pallidum membrane protein Tp47 in human umbilical vein endothelial cells (HUVECs) was assessed by tube formation assay, three-dimensional angiogenesis analysis and experiments with a zebrafish embryo model. The effects of mitochondrial ROS and NADPH oxidase on intracellular ROS induced by Tp47 were further investigated. Furthermore, the levels of autophagy-related proteins and autophagic flux were measured. Finally, the role of ROS-induced autophagy in angiogenesis was studied. Tp47 promoted tubule formation and the formation of angiogenic sprouts in vitro. In addition, a significant increase in the number of subintestinal vessel branch points in zebrafish injected with Tp47 was observed using a zebrafish embryo model. Tp47 also significantly increased intracellular ROS levels in a dose-dependent manner. Tp47-induced tube formation and angiogenic sprout formation were effectively prevented by the ROS inhibitor NAC. In addition, Tp47 enhanced the production of mitochondrial ROS and expression of the NADPH oxidase-related proteins Nox2 and Nox4. The production of mitochondrial ROS and intracellular ROS was reduced by the NADPH oxidase inhibitors DPI and apocynin. Furthermore, Tp47 significantly increased expression of the autophagy-related proteins P62 and Beclin 1 and the LC3-II/LC3-I ratio and promoted an increase in autophagic flux, which could be effectively rescued by coincubation with the ROS inhibitor NAC. Further intervention with the autophagy inhibitor BafA1 significantly inhibited tube formation and angiogenic sprout formation. Tp47-induced NADPH oxidase enhanced intracellular ROS production via mitochondrial ROS and promoted angiogenesis through autophagy mediated by ROS. These findings may contribute to our understanding of pathological angiogenesis in syphilis.

Chemerin-9 in paraventricular nucleus increases sympathetic outflow and blood pressure via glutamate receptor-mediated ROS generation

Chemerin is an adipokine involved in regulating energy homeostasis and reproductive function. Excessive sympathetic activity contributes to hypertension, chronic heart failure and chronic renal disease. Hypothalamic paraventricular nucleus (PVN) is crucial in regulating sympathetic activity and blood pressure. The present study is designed to investigate the roles of chemerin in the PVN in regulating sympathetic activity and blood pressure and underlying mechanisms. Microinjections were performed in the bilateral PVN in male adult rats under anesthesia. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. The PVN microinjection of chemerin-9, an active fragment of chemerin, increased RSNA and MAP, which were abolished by chemokine-like receptor 1 (CMKLR1) antagonist α-NETA, a superoxide scavenger tempol, antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and apocynin. Immunofluorescence analyses showed that N-methyl-D-aspartate (NMDA) receptors existed in most of cells of the PVN, and some of them co-existed with chemerin. The effects of chemerin-9 on RSNA and MAP were prevented by glutamate-binding site antagonist L-phenylalanine, NMDA receptor antagonist MK-801, and calcium channel blocker verapamil or nifedipine, but only attenuated by non-NMDA receptor antagonist CNQX. Moreover, chemerin-9 increased NADPH oxidase activity and superoxide production, which were prevented by α-NETA, MK-801, or verapamil. These results indicate that chemerin-9 in the PVN increases sympathetic activity and blood pressure via CMKLR1-dependent calcium influx, and glutamate receptor-mediated NADPH oxidase activation and subsequent superoxide production.

Asprosin in the Paraventricular Nucleus Induces Sympathetic Activation and Pressor Responses via cAMP-Dependent ROS Production.

Asprosin is a newly discovered adipokine that is involved in regulating metabolism. Sympathetic overactivity contributes to the pathogenesis of several cardiovascular diseases. The paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in the regulation of sympathetic outflow and blood pressure. This study was designed to determine the roles and underlying mechanisms of asprosin in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male adult SD rats under anesthesia. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) were recorded, and PVN microinjections were performed bilaterally. Asprosin mRNA and protein expressions were high in the PVN. The high asprosin expression in the PVN was involved in both the parvocellular and magnocellular regions according to immunohistochemical analysis. Microinjection of asprosin into the PVN produced dose-related increases in RSNA, MAP, and HR, which were abolished by superoxide scavenger tempol, antioxidant N-acetylcysteine (NAC), and NADPH oxidase inhibitor apocynin. The asprosin promoted superoxide production and increased NADPH oxidase activity in the PVN. Furthermore, it increased the cAMP level, adenylyl cyclase (AC) activity, and protein kinase A (PKA) activity in the PVN. The roles of asprosin in increasing RSNA, MAP, and HR were prevented by pretreatment with AC inhibitor SQ22536 or PKA inhibitor H89 in the PVN. Microinjection of cAMP analog db-cAMP into the PVN played similar roles with asprosin in increasing the RSNA, MAP, and HR, but failed to further augment the effects of asprosin. Pretreatment with PVN microinjection of SQ22536 or H89 abolished the roles of asprosin in increasing superoxide production and NADPH oxidase activity in the PVN. These results indicated that asprosin in the PVN increased the sympathetic outflow, blood pressure, and heart rate via cAMP–PKA signaling-mediated NADPH oxidase activation and the subsequent superoxide production.

Fasting-mimicking diet cycles reduce neuroinflammation to attenuate cognitive decline in Alzheimer's models.

The effects of fasting-mimicking diet (FMD) cycles in reducing many aging and disease risk factors indicate it could affect Alzheimer's disease (AD). Here, we show that FMD cycles reduce cognitive decline and AD pathology in E4FAD and 3xTg AD mouse models, with effects superior to those caused by protein restriction cycles. In 3xTg mice, long-term FMD cycles reduce hippocampal Aβ load and hyperphosphorylated tau, enhance genesis of neural stem cells, decrease microglia number, and reduce expression of neuroinflammatory genes, including superoxide-generating NADPH oxidase (Nox2). 3xTg mice lacking Nox2 or mice treated with the NADPH oxidase inhibitor apocynin also display improved cognition and reduced microglia activation compared with controls. Clinical data indicate that FMD cycles are feasible and generally safe in a small group of AD patients. These results indicate that FMD cycles delay cognitive decline in AD models in part by reducing neuroinflammation and/or superoxide production in the brain.

Post-amputation reactive oxygen species production is necessary for axolotls limb regeneration.

Introduction: Reactive oxygen species (ROS) represent molecules of great interest in the field of regenerative biology since several animal models require their production to promote and favor tissue, organ, and appendage regeneration. Recently, it has been shown that the production of ROS such as hydrogen peroxide (H2O2) is required for tail regeneration in Ambystoma mexicanum. However, to date, it is unknown whether ROS production is necessary for limb regeneration in this animal model. Methods: forelimbs of juvenile animals were amputated proximally and the dynamics of ROS production was determined using 2′7- dichlorofluorescein diacetate (DCFDA) during the regeneration process. Inhibition of ROS production was performed using the NADPH oxidase inhibitor apocynin. Subsequently, a rescue assay was performed using exogenous hydrogen peroxide (H2O2). The effect of these treatments on the size and skeletal structures of the regenerated limb was evaluated by staining with alcian blue and alizarin red, as well as the effect on blastema formation, cell proliferation, immune cell recruitment, and expression of genes related to proximal-distal identity. Results: our results show that inhibition of post-amputation limb ROS production in the A. mexicanum salamander model results in the regeneration of a miniature limb with a significant reduction in the size of skeletal elements such as the ulna, radius, and overall autopod. Additionally, other effects such as decrease in the number of carpals, defective joint morphology, and failure of integrity between the regenerated structure and the remaining tissue were identified. In addition, this treatment affected blastema formation and induced a reduction in the levels of cell proliferation in this structure, as well as a reduction in the number of CD45+ and CD11b + immune system cells. On the other hand, blocking ROS production affected the expression of proximo-distal identity genes such as Aldha1a1, Rarβ, Prod1, Meis1, Hoxa13, and other genes such as Agr2 and Yap1 in early/mid blastema. Of great interest, the failure in blastema formation, skeletal alterations, as well as the expression of the genes evaluated were rescued by the application of exogenous H2O2, suggesting that ROS/H2O2 production is necessary from the early stages for proper regeneration and patterning of the limb.

Prenatal hypothyroidism diminished exogenous NO-mediated diastolic effects in fetal rat thoracic aorta smooth muscle via increased oxidative stress

Maternal hypothyroidism is an important problem of modern healthcare and is reported to increase the risk of cardiovascular diseases in the offspring later in life. However, it is unclear whether hypothyroidism during pregnancy causes vascular damage in the fetal period. We established the prenatal hypothyroidism rat model and collected the fetuses at the 21th day of gestation (GD21). Thyroid hormone concentrations in maternal and offspring blood serum were assessed by enzyme-linked immunosorbent assay (ELISA). The thoracic aortas of the fetuses were isolated for microvessel functional testing and histochemical stainings. qPCR and Western blot were performed to access mRNA and protein expression. We found that the concentrations of thyroid hormones in the serum of pregnant rats and fetuses were significantly suppressed at GD21. The responses of the fetal thoracic aortas to SNP were significantly attenuated in the PTU group. However, no statistical difference was found between the two groups when treated with either inhibitor (ODQ) or activator (BAY58–2667) of sGC. The production of O2−• in the arterial wall was significantly increased in hypothyroid fetuses. Moreover, the level of NADPH oxidase (NOX) was increased, while superoxide dismutase 2 (SOD2) was down-regulated in the PTU group, ultimately contributing to the increased production of superoxide. Additionally, decreased SNP-mediated vasodilation found in fetal vessels was improved by either NOX inhibitor (Apocynin) or SOD mimic (Tempol). These results indicate that increased oxidative stress is probably the cause of the diminished diastolic effect of exogenous NO in the thoracic artery of prenatal hypothyroidism exposed fetuses.

Induction of reactive oxygen species by mechanical stretch drives endothelin production in neonatal pig renal epithelial cells

Vasoactive endothelin (ET) is generated by ET converting enzyme (ECE)-induced proteolytic processing of pro-molecule big ET to biologically active peptides. H2O2 has been shown to increase the expression of ECE1 via transactivation of its promoter. The present study demonstrates that H2O2 triggered ECE1-dependent ET1-3 production in neonatal pig proximal tubule (PT) epithelial cells. A uniaxial stretch of PT cells decreased catalase, increased NADPH oxidase (NOX)2 and NOX4, and increased H2O2 levels. Stretch also increased cellular ECE1, an effect reversed by EUK-134 (a synthetic superoxide dismutase/catalase mimetic), NOX inhibitor apocynin, and siRNA-mediated knockdown of NOX2 and NOX4. Short-term unilateral ureteral obstruction (UUO), an inducer of renal tubular cell stretch and oxidative stress, increased renal ET1-3 generation and vascular resistance (RVR) in neonatal pigs. Despite removing the obstruction, UUO-induced increase in RVR persisted, resulting in early acute kidney injury (AKI). ET receptor (ETR)-operated Ca2+ entry in renal microvascular smooth muscle (SM) via transient receptor potential channel 3 (TRPC3) channels reduced renal blood flow and increased RVR. Although acute reversible UUO (rUUO) did not change protein expression levels of ETR and TRPC3 in renal microvessels, inhibition of ECE1, ETR, and TRPC3 protected against renal hypoperfusion, RVR increase, and early AKI. These data suggest that mechanical stretch-driven oxyradical generation stimulates ET production in neonatal pig renal epithelial cells. ET activates renal microvascular SM TRPC3, leading to persistent vasoconstriction and reduction in renal blood flow. These mechanisms may underlie rUUO-induced renal insufficiency in infants.

Accumulation of advanced oxidation protein products contributes to age-related impairment of gap junction intercellular communication in osteocytes of male mice.

AimsGap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism.MethodsChanges in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks.ResultsThe AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo.ConclusionOur study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing.Cite this article: Bone Joint Res 2022;11(7):413–425.

Effects of polystyrene nanoplastics on endothelium senescence and its underlying mechanism.

Global plastic use has increased rapidly, and environmental pollution associated with nanoplastics (NPs) has been a growing concern recently. However, the impact and biological mechanism of NPs on the cardiovascular system are not well characterized. This study aimed to assess the possibility that NPs exposure promotes premature endothelial cell (EC) senescence in porcine coronary artery ECs and, if so, to elucidate the underlying mechanism. Treatment of ECs with NPs promoted the acquisition of senescence markers, senescence-associated β-galactosidase activity, and p53, p21, and p16 protein expression, resulting in the inhibition of proliferation. In addition, NPs impaired endothelium-dependent vasorelaxation associated with decreased endothelial nitric oxide synthase (eNOS) expression. NPs enhanced reactive oxygen species formation in ECs, and increased oxidative stress levels were associated with the induction of NADPH oxidases expression, followed by the subsequent downregulation of Sirt1 expression. The characteristics of EC senescence and dysfunction caused by NPs are prevented by an antioxidant (N-acetylcysteine), an NADPH oxidase inhibitor (apocynin), and a Sirt1 activator (resveratrol). These findings indicate that NPs induced premature EC senescence, at least in part, through the redox-sensitive eNOS/Sirt1 signaling pathway. This study suggested the effects and underlying mechanism of NPs on the cardiovascular system, which may provide pharmacological targets to prevent NPs-associated cardiovascular diseases.

Mechanical stretch‐driven ROS generation stimulates endothelin production in neonatal pig renal epithelial and vascular endothelial cells

The circulating level of endothelin (ET), a potent vasoactive peptide, decreases with postnatal age. ET is also of pathophysiological significance in the kidney. ET is generated via endothelin converting enzyme (ECE)‐induced proteolytic processing of pro‐molecule big ET to biologically active peptides. Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), transactivates the promoter of ECE1. Oxyradicals contribute to kidney pathophysiology, but whether ROS regulates ET production in the kidneys remains unresolved. The present study shows that ECE1 is the predominant isoform in neonatal pig kidneys. We also demonstrated that H2O2 induces ECE1‐dependent ET1‐3 production in the neonatal pig renal proximal tubules (PT) and vascular endothelial cells (EC). A uniaxial stretch of neonatal pig PT and EC increased NADPH oxidase (NOX) 2 and 4 levels and stimulated H2O2production. Mechanical stretch increased cellular ECE1 production in the PC and EC cells, which catalase mimetic EUK 134 and NOX inhibitor apocynin reversed. Mechanical stretch also triggered cellular ET1‐3 generation in PT and EC. Mechanical stretch‐induced ET1‐3 production was attenuated by EUK 134, apocynin, and a selective ECE1 inhibitor CGS 35066. Short‐term unilateral urinary tract obstruction (UUO), an inducer of the mechanical stretch of kidney cells and oxyradical production, increased ECE1‐dependent ET1‐3 production in neonatal pig kidneys. Despite removing the obstruction, acute UUO generated ROS, reduced kidney perfusion, and increased renal vascular resistance (RVR). UUO also elevated early biomarkers of acute kidney injury (AKI). Inhibition of ECE1 and ET receptors protected against UUO‐induced increase in RVR and early AKI. These data suggest that mechanical stretch‐driven ROS generation stimulates ET production in neonatal pig renal epithelial and vascular endothelial cells. ET then promotes AKI by causing renal hypoperfusion. This signaling pathway may underlie UUO‐induced renal insufficiency in infants.