Dihydroethidium Staining Research Articles

Placental oxidative stress and monoamine oxidase expression are increased in severe preeclampsia: a pilot study.

Preeclampsia (PE) is the most severe complication of pregnancy with substantial burden of morbidity and mortality for mother and neonate. The increased placental oxidative stress (OS) has been involved as central pathomechanism, yet the sources of reactive oxygen species (ROS) are partially elucidated. Monoamine oxidase (MAO) with 2 isoforms, A and B, at the outer mitochondrial membrane has emerged as a constant source of ROS in cardiometabolic pathologies. The present pilot study was purported to assess as follows: (i) the magnitude of placental OS in relation to the site of sampling and (ii) the expression of placental MAO in the setting of PE. To this aim, central and placental samples were harvested during cesarean section from mild and severe PE versus healthy pregnancies. ROS generation (dihydroethidium staining) and MAO expression were assessed (confocal microscopy). MAO gene transcript was evaluated by RT-PCR. The main findings are as follows: (i) a significant increase in placental OS was found in severe (but not in mild) PE with no regional differences between central and peripheral areas and (ii) placental MAO-A and B (gene and protein) were significantly increased in severe preeclampsia. The signal transduction of the latter finding, particularly in relation with mitochondrial dysfunction, is worth further studying.

Activating cGAS–STING axis contributes to neuroinflammation in CVST mouse model and induces inflammasome activation and microglia pyroptosis

BackgroundNeuroinflammation-induced injury is intimately associated with poor prognosis in patients with cerebral venous sinus thrombosis (CVST). The cyclic GMP-AMP synthase–stimulator of interferon gene (cGAS–STING) axis is a cytoplasmic double-stranded DNA (dsDNA) sensing pathway has recently emerged as a crucial mediator of neuroinflammation in ischemic stroke. However, the role of the cGAS–STING pathway in modulating post-CVST inflammation and the underlying mechanisms involved remain unclear.MethodsA CVST model was induced by ferric chloride in male C57BL/6J mice. The selective cGAS inhibitor RU.521, STING agonist 2′3′-cGAMP, and STING siRNA were delivered by intranasal administration or intraventricular injection. Post-CVST assessments included rotarod test, TUNEL staining, Fluoro-Jade C staining, dihydroethidium staining, western blotting, qPCR, immunofluorescence, immunohistochemistry, ELISA and flow cytometry.ResultscGAS, STING, NLRP3 and GSDMD were significantly upregulated after CVST and mostly in the microglia of the mouse brain. CVST triggered the release of dsDNA into the cytoplasm and elicited an inflammatory response via activating the cGAS–STING axis. RU.521 decreased the levels of 2′3′-cGAMP, STING and downstream inflammatory cytokines, and suppressed the expressions of NLRP3 inflammasome and pyroptosis-pertinent components containing cleaved caspase-1, GSDMD, GSDMD-C, pro- and cleaved IL-1β, and cleaved IL-1β/pro-IL-1β. Besides, RU.521 treatment also reduced oxidative stress, lessened the numbers of microglia and neutrophils, and ameliorated neuronal apoptosis, degeneration along with neurological deficits post-CVST. 2′3'-cGAMP delivery enhanced the expressions of STING and related inflammatory mediators, NLRP3 inflammasome and pyroptosis-relevant proteins, whereas these alterations were significantly abrogated by the silencing of STING by siRNA.ConclusionsOur data demonstrate that repression of the cGAS–STING pathway diminishes the neuroinflammatory burden of CVST and highlight this approach as a potential therapeutic tactic in CVST-mediated pathologies.

Sirtuin1 attenuates acute liver failure by reducing reactive oxygen species via hypoxia inducible factor 1α

BACKGROUNDThe occurrence and development of acute liver failure (ALF) is closely related to a series of inflammatory reactions, such as the production of reactive oxygen species (ROS). Hypoxia inducible factor 1α (HIF-1α) is a key factor that regulates oxygen homeostasis and redox, and the stability of HIF-1α is related to the ROS level regulated by Sirtuin (Sirt) family. The activation of Sirt1 will lead to a powerful antioxidant defense system and therapeutic effects in liver disease. However, little is known about the relationship between HIF-1α and Sirt1 in the process of ALF and the molecular mechanism.AIMTo investigate whether HIF-1α may be a target of Sirt1 deacetylation and what the effects on ALF are.METHODSMice were administrated lipopolysaccharide (LPS)/D-gal and exposed to hypoxic conditions as animal model, and resveratrol was used as an activator of Sirt1. The cellular model was established with L02 cells stimulated by LPS. N-acetyl-L-cysteine was used to remove ROS, and the expression of Sirt1 was inhibited by nicotinamide. Western blotting was used to detect Sirt1 and HIF-1α activity and related protein expression. The possible signaling pathways involved were analyzed by immunofluorescent staining, co-immunoprecipitation, dihydroethidium staining, and Western blotting.RESULTSCompared with mice stimulated with LPS alone, the expression of Sirt1 decreased, the level of HIF-1α acetylation increased in hypoxic mice, and the levels of carbonic anhydrase 9 and Bcl-2-adenovirus E1B interacting protein 3 increased significantly, which was regulated by HIF-1α, indicating an increase of HIF-1α activity. Under hypoxia, the down-regulation of Sirt1 activated and acetylated HIF-1α in L02 cells. The inhibition of Sirt1 significantly aggravated this effect and the massive production of ROS. The regulation of ROS was partly through peroxisome proliferator-activated receptor alpha or AMP-activated protein kinase. Resveratrol, a Sirt1 activator, effectively relieved ALF aggravated by hypoxia, the production of ROS, and cell apoptosis. It also induced the deacetylation of HIF-1α and inhibited the activity of HIF-1α.CONCLUSIONSirt1 may have a protective effect on ALF by inducing HIF-1α deacetylation to reduce ROS.

Reduced expression of SOD2 in the Retrotrapezoid nucleus is associated with enhanced ventilatory chemoreception: implications for breathing disorders in heart failure

Enhanced chemoreflex drive and irregular breathing patterns are both hallmarks in heart failure (HF). The Retrotrapezoid nucleus (RTN), a main area for central chemoreception, contribute to maintain both heightened chemoreflex drive and irregular breathing in experimental HF. Recently, we found that high levels of reactive oxygen species (ROS) within the RTN of HF rats were closely linked to the enhanced central chemoreflex and the generation of breathing disorders. Despite this evidence, the effects of ROS on RTN‐mediated chemoreception and the precise mechanism(s) associated with ROS generation in the RTN has not been previously studied. However, pro/anti‐oxidant enzymes gene screening in RTN from HF rats revealed reduce expression of super oxide dismutase 2 (SOD2). Therefore, in the present study we aimed to determine whether reductions in SOD2 expression in healthy subjects resembles i) high RTN ROS levels, ii) heightened chemoreflex drive, and iii) generation of breathings disorders observed in HF animals. Accordingly, breathing function and central chemoreflex drive were assessed in freely moving heterozygous SOD2+/‐ mice by unrestrained whole‐body plethysmography. ROS levels were determined in brainstem sections containing the RTN using dihydroethidium staining. SOD2 protein expression and tissue localization were determined by immunoblot and immunofluorescence, respectively. SOD2+/‐ mice displayed reduced SOD2 protein expression (65.6 ± 3.2 vs. 100.0 ± 5.8 %; SOD2+/‐ vs. WT, respectively) and high ROS levels at the RTN compared to WT littermates (2.50 ± 0.46 vs. 1.00 ± 0.05 a.u.; SOD2+/‐ vs. WT, respectively). In addition, SOD2+/‐mice displayed an exacerbated ventilatory response to hypercapnia compared to WT mice (3.5 ± 0.2 vs. 1.9 ± 0.2 ml/min/10g; SOD2+/‐ vs. WT, respectively). Importantly, the latter was closely linked to RTN ROS production since a positive correlation (p<0.05) between ROS levels and the hypercapnic ventilatory response was found only in SOD2+/‐animals. Furthermore, SOD2+/‐ mice showed overt signs of disordered breathing while resting at eupneic conditions compared to WT mice as evidenced by large increases in ventilatory instability and apneas/hypoapneas incidence (AHI: 7.7 ± 0.9 vs. 3.4 ± 0.6 events/hr; SOD2+/‐ vs. WT, respectively). Lastly, we found that SOD2 expression is preferentially confined to RTN neurons rather than to medullary astrocytes. Our results shows that reductions in the SOD2 gene expression in healthy rodents mimic the respiratory phenotype observed in experimental HF and suggests that reduced expression of SOD2 within the RTN of HF animals may account for the local increases in ROS production, the enhanced central chemoreflex drive and the generation/maintenance of irregular breathing patterns.

Galectin‐3 Regulates Oxidant Stress in Obesity

ObjectiveInvestigate the mechanism by which overexpression of Galectin‐3 (GAL3) in the endothelium of obese mice contributes to excess reactive oxygen species (ROS) production and subsequent vascular dysfunction.Methodsdb/db mice, a well‐characterized model of human obesity, were crossed with mice lacking GAL3, a receptor for advanced glycation end products, to generate lean and obese mice with and without GAL3 deletion. Metabolic and cardiovascular endpoints were assessed by mRNA and protein expression in freshly isolated microvascular endothelial cells, microvascular reactivity assessed by pressure myography, and ROS production assessed by dihydroethidium (DHE) staining. Other previously characterized db/db colonies were utilized to further elucidate the mechanistic contributions of GAL3, including mice harboring deletions of NADPH Oxidase I (NOX1), Myostatin (MSTN), and Protein Tyrosine Phosphatase 1B (PTP1B).ResultsDeletion of GAL3 in obese mice had no observable effect on anatomical, metabolic, or plasma phenotype, except for normalizing levels of plasma ROS as evidenced by decrease in thiobarbituric acid reactive substances (TBARS). Obese mice exhibited profound endothelial dysfunction, which was rescued by deletion of GAL3. Our data demonstrate that NOX1, the predominant source of endothelial superoxide production, is significantly downregulated by GAL3 deletion, thereby restoring endothelial function and ameliorating endothelial ROS production in obesity. Furthermore, we demonstrate that GAL3‐mediated NOX1 overexpression is amenable to improvements in metabolic status, such as lowering blood glucose with metformin, improving glucose handling by augmenting muscle mass through deletion of MSTN, or improving insulin signaling through deletion of PTP1B.ConclusionTaken together, these data demonstrate that the overproduction of superoxide by endothelial NOX1 is regulated by the metabolic sensor GAL3 in obesity, leading to endothelial dysfunction and subsequent cardiovascular disease. This pathway presents an attractive target for therapeutic intervention to break the link between aberrant metabolism in obesity and its corresponding vascular pathologies.

Edaravone Attenuated Particulate Matter-Induced Lung Inflammation by Inhibiting ROS-NF-κB Signaling Pathway.

Background Particulate matter (PM) exposure is related to mitochondria dysfunction and airway inflammation. Antioxidant drug edaravone (EDA) is reported to improve the occurrence and development of oxidative stress-related diseases. At present, there is no data on whether EDA can alleviate lung inflammation caused by PM. Methods The anti-inflammatory effects of EDA were investigated in urban PM-induced human bronchial epithelial cells (HBECs) and C57/BL6J mouse models. In vitro, its effects on the production of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory cytokines were assessed by DCFH-DA staining, JC-1 assay, and real-time PCR, respectively. In vivo, the oxidant stress in lung tissues was assessed by dihydroethidium (DHE) staining and malondialdehyde (MDA) activity, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were assessed by ELISA, respectively. Furthermore, the potential signaling pathways were studied by siRNA transfection and western blot. Results PM increased the expression of inflammatory cytokines and protein, including IL-6, IL-1α, IL-1β, and COX-2, while these alternations were significantly alleviated following EDA treatment in a dose-dependent manner. EDA treatment also alleviated the inflammatory responses in lung tissues of PM-exposed mice. We further showed mitochondrial dysfunction in PM-exposed HBECs and mice, which were reversed by EDA treatment. Moreover, the phosphorylation of NF-κB p65 in PM-exposed HBECs and mice was weakened by EDA. Transfection with NF-κB p65 siRNA further inhibited PM-induced inflammation in HBECs. Conclusion We demonstrated that EDA treatment had a protective role in PM-induced lung inflammation through maintaining mitochondrial balance and regulating the ROS-NF-κB p65 signaling pathway. This provided a new therapeutic method for PM-induced lung inflammation in the future.

Circ_0050908 up-regulates TRAF3 by sponging miR-324-5p to aggravate myocardial ischemia-reperfusion injury

BackgroundAberrant circular RNAs (circRNAs) expression is closely associated with cardiovascular diseases. However, the regulatory functions of circRNAs in myocardial ischemia-reperfusion (I/R) injury remain largely undefined. MethodsWe established myocardial I/R model in vitro by oxygen and glucose deprivation and reperfusion in cardiomyocytes. The expression of circ_0050908, microRNA (miR)-324-5p, and TNF receptor-associated factor (TRAF3) was detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The apoptosis was assayed by flow cytometry and Western blot. The activity of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF-α), lactate dehydrogenase (LDH), creatine kinase myocardial band (CK-MB), CK, and superoxide dismutase (SOD) was evaluated using the relative commercial kits. Reactive oxygen species (ROS) detection was conducted using Dihydroethidium (DHE) staining. The interactions between miR-324-5p and circ_0050908 or TRAF3 were determined by dual-luciferase activity, RNA immunoprecipitation (RIP), and pull-down assays. ResultsI/R stimulation up-regulated circ_0050908 expression in cardiomyocytes. Functional experiments suggested that circ_0050908 knockdown led to the rescue of apoptosis enhancement, inflammation, and oxidative stress induced by I/R in cardiomyocytes. Mechanistically, circ_0050908 directly targeted miR-324-5p, and miR-324-5p inhibition reversed the inhibitory action of circ_0050908 knockdown on myocardial I/R injury. TRAF3 was verified to be a target of miR-324-5p, and miR-324-5p suppressed I/R-induced apoptosis, inflammatory response, and oxidative stress in cardiomyocytes through TRAF3. Besides that, circ_0050908 could regulate TRAF3 expression by miR-324-5p. ConclusionCirc_0050908 knockdown protects cardiomyocytes against I/R injury by reducing apoptosis, inflammatory response, and oxidative stress through miR-324-5p/TRAF3 axis, revealing a novel therapeutic strategy for preventing myocardial I/R injury.

Intake of coated EPA:DHA 6:1 nanoparticles improves age-related endothelial dysfunction by restoring the endothelial formation of NO and improving oxidative stress: Role of the local angiotensin system

Omega-3 polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce cardiovascular risk. This study investigated whether nanoencapsulation of EPA:DHA 6:1 is associated with an improved beneficial effect on age-related endothelial dysfunction. Thoracic aorta and main mesenteric artery from young and middle-aged rats that received daily by gavage 100 mg/kg of either native or coated form for 1-week were investigated. Vascular reactivity and level of oxidative stress and target proteins were assessed using organ chambers, dihydroethidium and immunofluorescence staining, respectively. Compared to the native form, the coated EPA:DHA 6:1 nanoparticles treatment improved to a greater extent NO-mediated relaxation and contractile response, and the level of vascular oxidative stress and expression of ACE/AT1R in the aged artery. The nanoencapsulation of EPA:DHA 6:1 promotes a greater beneficial effect on age-related endothelial dysfunction involving improved NO-mediated relaxations and normalization of the pro-oxidant local angiotensin system.

Ginsenoside Rh1 Inhibits Angiotensin II-Induced Vascular Smooth Muscle Cell Migration and Proliferation through Suppression of the ROS-Mediated ERK1/2/p90RSK/KLF4 Signaling Pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway.

EPC-EXs improve astrocyte survival and oxidative stress through different uptaking pathways in diabetic hypoxia condition

BackgroundHyperglycemia contributes to cardiovascular complications in patients with type 2 diabetes. We confirmed that high glucose (HG) induces endothelial dysfunction and cerebral ischemic injury is enlarged in diabetic mice. Stem cell-released exosomes have been shown to protect the brain from ischemic stroke. We have previously shown that endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) can protect endothelial cells from hypoxia/reoxygenation (H/R) and HG-induced injury. Here, we aim to investigate the effects of EPC-EXs on astrocytes under H/R and HG-induced injury and whether miR-126 enriched EPC-EXs (miR126-EPC-EXs) have enhanced efficacy.MethodsEPC-EX uptake and co-localization were measured by fluorescent microscopy using PKH26 and DAPI staining. miR-126 enrichment was achieved by transfecting with miR-126 mimics and quantified with real-time PCR. After co-incubation, cell death or injury was measured by using LDH (Lactate Dehydrogenase) assay. Oxidative stress/ROS (reactive oxygen species) generation was measured by DHE (Dihydroethidium) staining and lipid peroxidation assay.ResultsThe EPC-EXs were effectively taken up by the astrocytes in a concentration as well as time-dependent manners and were co-localized within the nucleus as well as the cytoplasm. Pathway uptake inhibitors revealed that the EPC-EXs are effectively taken up by the clathrin-mediated, caveolin-dependent, and micropinocytosis via PI3K/Akt pathway. H/R and HG-induced a cell injury which could be protected by EPC-EXs evidenced by decreased cell cytotoxicity, oxidative stress, and lipid peroxidation. Moreover, miR-126 overexpression could increase the level of miR-126 in astrocytes and enhance the protective effects of EPC-EXs.ConclusionsThese results collectively indicate that the EPC-EXs could protect astrocytes against the HG plus H/R-induced damage.

Vasoactive Properties of a Cocoa Shell Extract: Mechanism of Action and Effect on Endothelial Dysfunction in Aged Rats.

Cocoa has cardiovascular beneficial effects related to its content of antioxidant phytochemicals. Cocoa manufacturing produces large amounts of waste, but some by-products may be used as ingredients with health-promoting potential. We aimed to investigate the vasoactive actions of an extract from cocoa shell (CSE), a by-product containing theobromine (TH), caffeine (CAF) and protocatechuic acid (PCA) as major phytochemicals. In carotid and iliac arteries from 5-month and 15-month-old rats, we investigated CSE vasoactive properties, mechanism of action, and the capacity of CSE, TH, CAF and PCA to improve age-induced endothelial dysfunction. Vascular function was evaluated using isometric tension recording and superoxide anion production by dihydroethidium (DHE) staining and confocal microscopy. CSE caused endothelium-dependent vasorelaxation, blocked by L-NAME, but not indomethacin, regardless of sex, age, or vessel type. CSE maximal responses and EC50 were significantly lower compared to acetylcholine (ACh). Arterial preincubation with CSE, TH, CAF or PCA, significantly reduced the number of vascular DHE-positive cells. Compared to adult males, iliac arteries from aged males exhibited reduced ACh concentration-dependent vasodilatation but larger CSE responses. In iliac arteries from aged male and female rats, preincubation with 10−4 M CSE and PCA, but not TH or CAF, improved ACh-relaxations. In conclusion, CSE has vasodilatory properties associated with increased nitric oxide bioavailability, related to its antioxidant phytochemicals, being particularly relevant PCA. Therefore, CSE is a potential food ingredient for diseases related to endothelial dysfunction.

Relationship between oxidative stress and three-dimensional echocardiographic changes in myxomatous mitral valves with severe regurgitation - a pilot study

Abstract Funding Acknowledgements Type of funding sources: None. Background. Reactive oxygen species (ROS) have been reported to contribute to the molecular pathogenesis of severe mitral regurgitation induced by myxomatous degeneration. Data about oxidative stress and the possible relationship with the extent of prolapsing and/or flail elements is scarce in the literature. The pathogenic mechanism may prompt the development of therapeutic approaches aimed at delaying extensive valvular alteration. Purpose. The aim of the study is to investigate the possible correlation between ROS expression in myxomatous mitral valves with severe regurgitation and the extent of valvular alteration. Material and methods. Samples from myxomatous mitral valves with ruptured chordae and severe regurgitation were harvested during surgical intervention for valve repair/replacement from 9 patients. Besides transthoracic echocardiography, all patients were evaluated before surgery by transesophageal echocardiography (TEE) with three-dimensional (3D) reconstruction of the mitral valve, using 3D zoom mode acquisition with qualitative and quantitative analysis through the 4D Auto MVQ technology. Tissue from the valvular samples was further analysed for the assessment of ROS by 2 methods: spectrophotometry (ferrous oxidation xylenol orange - FOX assay) and confocal microscopy (dihydroethidium staining). Results. Patients had a mean age of 51.88 ± 13.54 years, with a left ventricular ejection fraction of 62.55 ± 7.33%, left ventricular end-diastolic diameter of 5.4 ± 0.72cm and end-diastolic volume of 168.55 ± 62.57ml, a mitral annulus area of 17.91 ± 5.74 cm2 and a number of prolapsing/flail scallops varying between 1 and 5 (average of 2 ± 1 scallops). All patients had diffuse thickening of the mitral leaflets with at least one primary and one secondary ruptured chordae and flail of the P2 scallop. Two patients had significant prolapse of at least 2 anterior mitral leaflet scallops and three patients had supplemental significant prolapse and/or flail of at least 1 posterior scallop, besides flail of P2. The mean value for ROS in the valvular tissue was 9.46 ± 2.03nM H2O2/mg tissue/h at FOX assay. A significant positive correlation between the ROS values and the extent of prolapsing/flail segments was found (R = 0.67, p = 0.04). Conclusion. Besides the diagnosis and accurate preoperatory valve description, 3D TEE along with molecular investigations contribute to the understanding of myxomatous mitral valves’ pathogenesis. Oxidative stress increased in the mitral valves with advanced myxomatous degeneration. Whether this occurs as a cause or a consequence contributing to the disease progression as well as the sources of ROS are worth further investigation.

Long-Term Effects of Aircraft Noise Exposure on Vascular Oxidative Stress, Endothelial Function and Blood Pressure: No Evidence for Adaptation or Tolerance Development.

Transportation noise is recognized as an important cardiovascular risk factor. Key mechanisms are noise-triggered vascular inflammation and oxidative stress with subsequent endothelial dysfunction. Here, we test for adaptation or tolerance mechanisms in mice in response to chronic noise exposure. C57BL/6J mice were exposed to aircraft noise for 0, 4, 7, 14 and 28d at a mean sound pressure level of 72 dB(A) and peak levels of 85 dB(A). Chronic aircraft noise exposure up to 28d caused persistent endothelial dysfunction and elevation of blood pressure. Likewise, reactive oxygen species (ROS) formation as determined by dihydroethidium (DHE) staining and HPLC-based measurement of superoxide formation in the aorta/heart/brain was time-dependently increased by noise. Oxidative burst in the whole blood showed a maximum at 4d or 7d of noise exposure. Increased superoxide formation in the brain was mirrored by a downregulation of neuronal nitric oxide synthase (Nos3) and transcription factor Foxo3 genes, whereas Vcam1 mRNA, a marker for inflammation was upregulated in all noise exposure groups. Induction of a pronounced hearing loss in the mice was excluded by auditory brainstem response audiometry. Endothelial dysfunction and inflammation were present during the entire 28d of aircraft noise exposure. ROS formation gradually increases with ongoing exposure without significant adaptation or tolerance in mice in response to chronic noise stress at moderate levels. These data further illustrate health side effects of long-term noise exposure and further strengthen a consequent implementation of the WHO noise guidelines in order to prevent the development of noise-related future cardiovascular disease.

Gut microbiota modulates osteoclast glutathione synthesis and mitochondrial biogenesis in mice subjected to ovariectomy.

ObjectivesOsteoporosis is a common bone disease in the elderly mainly regulated by osteoblasts (OBs) and osteoclasts (OCs). The gut microbiota has been recognized as an important factor in many physiological and pathological processes in the host. Thus, we hypothesize that the gut microbiota is necessary for postmenopausal osteoporosis and that germ‐free (GF) mice are protected from osteoporosis.Material and MethodsOsteoporosis models were established by performing ovariectomy (OVX) in mice. Bone mass was measured by micro‐CT, and gut microbiota were assessed by 16s rDNA sequencing. Reactive oxygen species (ROS) were detected by dihydroethidium (DHE) staining in vivo and 2’,7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining in vitro.ResultsFirmicutes and Bacteroidetes in the intestine are pivotal in OC differentiation, and the Firmicutes/Bacteroidetes ratio (F/B ratio) is a specific indicator of osteoporosis. Furthermore, we found that Firmicutes and Bacteroidetes affect the de novo synthesis of glutathione (GSH) by regulating its key enzyme glutamate–cysteine ligase catalytic subunit (Gclc) and inhibiting mitochondrial biogenesis and ROS accumulation via the cAMP response element‐binding (CREB) pathway. In addition, supplementing OVX mice with the probiotic Lactobacillus salivarius LI01 from the Firmicutes phylum prevented osteoporosis.ConclusionsOur results reveal that GSH plays a vital role in OVX‐induced bone loss, and probiotics that affect GSH metabolism are potential therapeutic targets for overcoming osteoporosis.

FNDC5/irisin reduces ferroptosis and improves mitochondrial dysfunction in hypoxic cardiomyocytes by Nrf2/HO-1 axis.

Myocardial infarction is characterized by cardiomyocyte death and mitochondrial dysfunction induced by ischemia. Ferroptosis, a novel form of cell death, has been found to play critical roles under ischemic conditions. Recently, several studies have shown that fibronectin type III domain-containing 5 (FNDC5) and its cleaved form, irisin, protect the heart against injury. However, its protective effect on ferroptosis and mitochondrial impairments is still unclear. Thus, our aim was to investigate the role of irisin in ferroptosis and mitochondrial dysfunction in cardiomyocytes under hypoxic conditions. Cardiomyocytes were treated with FNDC5 overexpression and/or irisin under normoxic and hypoxic conditions. Cell viability was assessed by Cell Counting Kit-8 assay. Reactive oxygen species production was evaluated by dihydroethidium staining. In addition, the intracellular ferrous iron level (Fe2+ ) and the relative concentration of malondialdehyde and ATP content were determined using an Iron Assay Kit, Lipid Peroxidation Assay Kit, and ATP Bioluminescent Assay Kit. The superoxide dismutase level in cells was measured using an Enzyme-Linked ImmunosorbentAssay Kit. Furthermore, an immunoblotting assay was used to determine ferroptosis-related mitochondrial proteins. Hypoxia promoted cell death, increased ferroptosis, and caused mitochondrial dysfunction in cardiomyocytes. Interestingly, FNDC5 overexpression and/or irisin administration elevated cell viability, decreased ferroptosis, and reversed mitochondrial impairments induced by hypoxia. Mechanistically, FNDC5/irisin reduced ferroptosis and reversed mitochondrial impairments by Nrf2/HO-1 axis in hypoxic cardiomyocytes. Thus, we have demonstrated that FNDC5/irisin plays a protective role in ferroptosis and mitochondrial dysfunction in hypoxia-induced cardiomyocytes.

A combined metabolomics and molecular biology approach to reveal hepatic injury and underlying mechanisms after chronic l-lactate exposure in mice

This study aimed to explore whether chronic l-lactate exposure could affect the peripheral tissues of mice and to determine the underlying pathogenesis. Herein, male C57BL/6 mice were divided into control and l-lactate groups. After l-lactate treatment for eight weeks (1 g/kg), metabolic changes in liver, kidney, muscle, and serum samples were determined by 1H nuclear magnetic resonance (1H NMR)-based metabolomics. Additionally, organ function was evaluated by serum biochemical and histopathological examinations. Reactive oxygen species (ROS) levels were measured using dihydroethidium staining; levels of signals involved in lactate metabolism and ROS-related pathways were detected using western blotting or polymerase chain reaction. Apoptosis was detected by TUNEL-fluorescence staining. Metabolomic analysis revealed that l-lactate mice showed decreased levels of glutathione (GSH), taurine, ATP, and increased glucose content, compared to control mice. Furthermore, l-lactate mice presented significantly higher serum levels of alanine aminotransferase and aspartate aminotransferase and increased glycogen content in hepatic tissues, compared to control mice. l-lactate mice also had a greater number of apoptotic nuclei in the livers than controls. Moreover, l-lactate exposure reduced mRNA and protein levels of superoxide dismutase-2 and c-glutamylcysteine ligase, elevated levels of cytochrome P450 2E1 and NADPH oxidase-2, and increased the protein expressions of LDHB, Bax/Bcl-2, cleaved caspase-3, and sirtuin-1 in hepatic tissues. Together, these results indicate that chronic l-lactate exposure increases oxidative stress and apoptosis in hepatocytes via upregulation of Bax/Bcl-2 expression and the consequent mitochondrial cytochrome-C release and caspase-3 activation, which contributes to the pathogenesis of hepatic dysfunction.

Prazosin improves neurogenic acute heart failure through downregulation of fibroblast growth factor 23 in rat hearts.

Bilateral nucleus tractus solitarii (NTS) lesions, possibly caused by enterovirus 71 infection, cause severe neurogenic hypertension, leading to acute heart failure (HF), pulmonary edema, and death within hours. Alpha-adrenergic blockers attenuate blood pressure and ameliorate HF and pulmonary edema, thereby prolonging survival time. However, the molecular mechanisms of these blockers are not clear. In this study, we investigated these mechanisms in a rat model of 6-hydroxydopamine (6-OHDA)-induced HF. Sprague-Dawley rats were treated with prazosin 10 min after the microinjection of 6-OHDA into the NTS. Immunohistochemistry and dihydroethidium (DHE) staining were used for analysis. In the cardiac tissue of 6-OHDA-induced HF, in situ expression of tumor necrosis factor-alpha (TNF-α), fibroblast growth factor-23 (FGF23), and FGF receptor 1 (FGFR1) increased, but in situ expression of Vitamin D receptor (VDR) decreased. DHE staining revealed several heart cells with high reactive oxygen species production. Prazosin treatment decreased TNF-α, FGF23, and FGFR1 expression in the heart of rats with 6-OHDA-induced HF. It also prevented cardiomyopathy caused by 6-OHDA-induced bilateral NTS lesions by inhibiting the FGF23-FGFR1 pathway and downregulating TNF-α expression. In situ, FGF23, FGFR1, VDR, superoxide, and TNF-α in the heart were found to be involved in acute HF in our rat model of 6-OHDA-induced bilateral NTS lesions. These findings are potentially useful for treating fatal enterovirus 71 infection-induced NTS lesions and HF.

Genistein mitigates oxidative stress and inflammation by regulating Nrf2/HO-1 and NF-κB signaling pathways in hypoxic-ischemic brain damage in neonatal mice.

BackgroundOxidative stress and neuroinflammation play crucial roles in the progression of neonatal hypoxic-ischemic brain damage (HIBD). Genistein, a natural phytoestrogen, has been found to protect against ischemic brain injury. However, its effects and potential mechanisms in HIBD have not yet been explored.MethodsA neonatal mouse model of hypoxia-ischemia (HI) and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) were employed. In the in vivo study, genistein (10 mg/kg; ip) was administered in mice once daily for 3 consecutive days before the operation and once immediately after HI. The effects of genistein treatment on acute brain damage and long-term responses were evaluated. Neuronal injury and apoptosis were estimated using hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. The expression of apoptosis-related proteins were also measured by Western blot analysis. Dihydroethidium (DHE) staining and glutathione (GSH) and malondialdehyde (MDA) production were determined to assess the extent of oxidative stress. The messenger RNA (mRNA) levels of proinflammatory cytokines were detected using real-time quantitative polymerase chain reaction (RT-qPCR) to evaluate the extent of neuroinflammation. In the in vitro study, cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays, as well as propidium iodide (PI) staining, were performed to analyse the neuroprotective effects of genistein on primary cortical neurons. Western blot assays were used to detect the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), phosphorylated inhibitor kappa B-α (p-IκB-α) and phosphorylated nuclear factor-kappa B (p-NF-κB) both in vivo and in vitro.ResultsOur results showed that genistein treatment effectively reduced cerebral infarction, attenuated neuronal injury and apoptosis, and contributed to the long-term recovery of neurological outcomes and brain atrophy in neonatal HIBD mice. Moreover, genistein ameliorated HIBD-induced oxidative stress and neuroinflammation. Meanwhile, genistein significantly increased cell viability, reversed neuronal injury and decreased cell apoptosis after OGD/R injury. Finally, the activation of the Nrf2/HO-1 pathway and inhibition of the NF-κB pathway by genistein were verified in the brain tissues of neonatal mice subjected to HIBD and in primary cortical neurons exposed to OGD/R.ConclusionsGenistein exerted neuroprotective effects on HIBD by attenuating oxidative stress and neuroinflammation through the Nrf2/HO-1 and NF-κB signalling pathways.

Cyclosporin A alleviates trophoblast apoptosis and senescence by promoting autophagy in preeclampsia.

Abnormal extravillous trophoblast (EVT) function is closely related to preeclampsia (PE) and may be caused by inadequate autophagy, apoptosis, and senescence. Cyclosporin A (CsA) is an effective immunosuppressant that has been reported to stimulate autophagy and exert benign biological effects on EVTs. Therefore, we hypothesized that CsA may display therapeutic efficacy against PE by activating autophagy. We established the nitro-l-arginine methyl ester (l-NAME)-induced preeclamptic mice model and a hypoxia-reoxygenation (H/R) model in vitro. The effects of CsA on autophagy were evaluated by western blotting (WB). The effects of CsA on apoptosis were analyzed by Hematoxylin-eosin (H&E) staining, cell apoptosis assay and WB. Senescence-associated β-galactosidase (SA-β-gal) staining, RT-qPCR and WB were used to examine the senescence level. RT-qPCR were used to detect the senescence-associated secretory phenotype (SASP) level. DCFH-DA fluorescent probe, dihydroethidium (DHE) staining and mitochondrial membrane potential (ΔΨm) were used to detect senescence-associated mitochondrial dysfunction (SAMD). CsA alleviated PE-like symptoms and reduced placental necrosis and senescence in mice injected with l-NAME. CsA ameliorated placental SASP and SAMD level induced by l-NAME. CsA also upregulated the expression of autophagic proteins in mouse placentas disrupted using l-NAME. In vitro, we found that CsA reversed H/R-induced apoptosis and senescence, as well as decreasing SASP and SAMD levels and upregulating autophagic proteins levels. Notably, 3-methyladenine (3-MA), an early phase inhibitor of autophagosome formation, abolished the protective effects of CsA against H/R. CsA may display some therapeutic effects against PE by activating autophagy in vivo and in vitro.

Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis.

Paraquat (PQ)-induced acute lung injury (ALI) and pulmonary fibrosis are common diseases with high mortality but without effective antidotes in emergency medicine. Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ. We wondered whether arctigenin could also have a protective effect on PQ-induced ALI. A PQ-induced A549 cell injury model was used, and the effect of arctigenin was determined by a cell counting kit-8 (CCK-8) cell viability assay. In addition, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis. The generation of reactive oxygen species (ROS) was reflected by dihydroethidium (DHE) staining and a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Moreover, immunoblotting studies were used to assess the expression of mitogen-activated protein kinases (MAPKs) and p38 MAPK. Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner. Arctigenin also significantly reduced PQ-induced A549 cell apoptosis, as reflected by the TUNEL assay and mitochondrial membrane potential assay, which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation. Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis, and arctigenin might be considered a potential candidate drug for PQ-induced ALI.