Oxygen Species Production Research Articles

Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension

Pulmonary endarterectomy (PEA) resected material offers a unique opportunity to develop an in vitro endothelial cell model of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to comprehensively analyze the endothelial function, molecular signature, and mitochondrial profile of CTEPH-derived endothelial cells to better understand the pathophysiological mechanisms of endothelial dysfunction behind CTEPH, and to identify potential novel targets for the prevention and treatment of the disease. Isolated cells from specimens obtained at PEA (CTEPH-EC), were characterized based on morphology, phenotype, and functional analyses (in vitro and in vivo tubule formation, proliferation, apoptosis, and migration). Mitochondrial content, morphology, and dynamics, as well as high-resolution respirometry and oxidative stress, were also studied. CTEPH-EC displayed a hyperproliferative phenotype with an increase expression of adhesion molecules and a decreased apoptosis, eNOS activity, migration capacity and reduced angiogenic capacity in vitro and in vivo compared to healthy endothelial cells. CTEPH-EC presented altered mitochondrial dynamics, increased mitochondrial respiration and an unbalanced production of reactive oxygen species and antioxidants. Our study is the foremost comprehensive investigation of CTEPH-EC. Modulation of redox, mitochondrial homeostasis and adhesion molecule overexpression arise as novel targets and biomarkers in CTEPH.

Characterization of the SPI-1 Type III Secretion System in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a plant growth-promoting rhizobacterium (PGPR) isolated from wheat take-all decline soil. Genomic analysis of strain 2P24 revealed the presence of a complete SPI-1 type III secretion system (T3SS) gene cluster on the chromosome with an organization and orientation similar to the SPI-1 T3SS gene clusters of Salmonella enterica and P. kilonensis F113. Phylogenetic analysis revealed that the SPI-1 T3SS gene cluster of strain 2P24 might be obtained from Salmonella and Shigella by horizontal gene transfer. Two transcriptional regulator homologs of HilA and InvF were found from the SPI-1 T3SS gene cluster of strain 2P24. HilA regulated the expression of the structural genes positively, such as invG, sipB, sipD, prgI, and prgK. Prediction of transcriptional binding sites and RNA-seq analysis revealed 14 genes were up-regulated by InvF in strain 2P24. Exploring potential roles of SPI-1 T3SS revealed that it was not associated with motility. However, 2P24ΔinvF reduced resistance against Fusarium graminearum significantly. 2P24ΔhilA enhanced formation of biofilm significantly at 48 h. All three mutants 2P24ΔhilA, 2P24ΔinvF, and 2P24ΔinvE-C reduced the chemotactic responses to glucose significantly. Finally, the determination of SPI-1 mutants to trigger innate immunity in Nicotiana benthamiana showed that 2P24ΔinvE-C reduced the ability to induce the production of reactive oxygen species compared with the wild type strain 2P24.

LINC00473 protects against cerebral ischemia reperfusion injury via sponging miR-15b-5p and miR-15a-5p to regulate SRPK1 expression

ABSTRACT Background Cerebral ischemia is associated with a high burden of neurological disability. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) are crucial regulators in cerebral ischemia reperfusion (I/R) injury. Herein, we investigated the function and potential mechanism of long intergenic non-protein coding RNA 473 (LINC00473) in cerebral I/R injury. Methods We established oxygen glucose deprivation/reperfusion (OGD/R) model in Neuro-2a (N2a) cells to mimic the cerebral I/R injury in vitro. RT-qPCR and Western blot assays were conducted to detect target gene expression. Functional assays measured the effects of LINC00473 on cell viability, apoptosis and reactive oxygen species (ROS) production. A series of mechanism assays were carried out to detect the potential mechanism of LINC00473 in cerebral I/R injury. Results LINC00473 was significantly down-regulated in OGD/R-induced injury model. LINC00473 overexpression reversed the reduced cell viability as well as the enhanced apoptosis and ROS level induced by OGD/R. Moreover, LINC00473 functioneds as a competing endogenous RNA (ceRNA) to sponge miR-15b-5p and miR-15a-5p and thereby regulated SRSF protein kinase 1 (SRPK1) expression. Conclusions Our findings confirmed the protective role of LINC00473 in cerebral I/R injury, which might provide a novel target for treating ischemic brain injury.

Resistance exercise affects catheter-related thrombosis in rats through miR-92a-3p, oxidative stress and the MAPK/NF-\u03baB pathway

BackgroundMiR-92a-3p and oxidative stress are associated with catheter-related thrombosis (CRT). As a kind of physical intervention, resistance exercise can effectively promote blood circulation. In this study, we investigated the roles of miR-92a-3p, oxidative stress and the P38 mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) pathway in CRT during resistance exercise.MethodsThe rat CRT model was used for resistance exercise intervention. Moreover, pathological changes from the right jugular vein to the right auricle were observed under an electron microscope. In addition, reactive oxygen species (ROS) production, malondialdehyde (MDA) activity and heme oxygenase (HO-1) level in rat serum were detected via ELISA. The expression levels of miR-92A-3p and HO-1 in the vascular tissues of the rats were determined via real-time quantitative PCR. Additionally, the expression levels of HO-1, NF-κB P65, p38MAPK and IκBa in the venous tissues of the rats were analysed by Western blot analysis.ResultsThe pathological results showed that the thrombosis incidence rate in the CRT + RE group was lower than that in the CRT group. In the CRT group, the expression levels of ROS and MDA, which are markers related to oxidative stress in serum, significantly increased whilst the expression of HO-1 decreased. In the venous tissue, the expression of miR-92a-3p increased, the level of HO-1 decreased, the levels of p38MAPK and NF-κB p65 significantly increased but that of P-IκBa and IκBa significantly decreased. In the CRT + RE group, after administering the resistance exercise intervention, ROS production and MDA activity in serum significantly decreased, the expression level of HO-1 increased and the expression level of miR-92a-3p in the venous tissues significantly decreased and was negatively correlated with that of HO-1. The levels of p38MAPK and NF-κB p65 significantly decreased but that of P- IκBa and IκBa significantly increased.ConclusionResistance exercise intervention downregulated miR-92a-3p expression, repaired oxidative stress injury and prevented CRT formation.

Shikonin induces programmed death of fibroblast synovial cells in rheumatoid arthritis by inhibiting energy pathways

Shikonin is the main component of the traditional Chinese medicine comfrey, which can inhibit the activity of PKM2 by regulating glycolysis and ATP production. Rheumatoid arthritis synovial cells (RA-FLSs) have been reported to increase glycolytic activity and have other similar hallmarks of metabolic activity. In this study, we investigated the effects of shikonin on glycolysis, mitochondrial function, and cell death in RA-FLSs. The results showed that shikonin induced apoptosis and autophagy in RA-FLSs by activating the production of reactive oxygen species (ROS) and inhibiting intracellular ATP levels, glycolysis-related proteins, and the PI3K-AKT-mTOR signaling pathway. Shikonin can significantly reduce the expression of apoptosis-related proteins, paw swelling in rat arthritic tissues, and the levels of inflammatory factors in peripheral blood, such as TNF-α, IL-6, IL-8, IL-10, IL-17A, and IL-1β while showing less toxicity to the liver and kidney.

Evaluation of neutrophil functional activity for rhinosinusitis diagnosis.

Bacterial rhinosinusitis (BRS) is a complicated form of rhinosinusitis, characterized by its acute course and tendency to chronicity. A significant role in pathogenetic mechanisms of HRS is played by functional state of neutrophils responsible for inflammation development at HRS. Investigation of neutrophils metabolic activity on external stimuli by chemiluminescence method in patients with acute and chronic forms of the disease will enable to develop method of differential diagnostics of BRS forms. The examination of 65 patients with BRS which were divided into patients with acute form (n=35) and chronic form of the disease (n=30) was carried out. The control group consisted of 30 healthy subjects. Functional activity of neutrophils was studied in whole blood by chemiluminescence with double stimulation (phorbol-12-myristate-13-acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (fMLP). In patients with the acute form of BRS we found an increase in parameters of specific maximum intensity of neutrophils luminescence, light sum of spontaneous and stimulated luminol-dependent chemiluminescence and a decrease in time to maximum output. Patients with the chronic form of BRS revealed a decrease in the luminescence of spontaneous, stimulated neutrophil production and maximum luminescence intensity. On the basis of significant differences in chemiluminescence indices of neutrophils, a method of differential diagnosis of the BRS forms was developed. The results obtained testify to increased production of reactive oxygen species (ROS) by neutrophils in the process of killing in patients with acute form of LRS and depletion of cell function and reserve in patients with the chronic form. Developed on the basis of the studied indicators method of diagnosing the forms of BRS by chemiluminescence will allow to differentiate acute and chronic forms of the disease, which will increase the effectiveness of treatment of patients.

Abstract MP250: Reduction In Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator (arnt) Mediates Vascular Dysfunction In Mice With Type 2 Diabetes Mellitus

Background: Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood. Results and method: We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation ( Arnt ΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia, delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow. In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in HUVECs cultured under high-glucose conditions. The Arnt ΔEC, ERT2 mutation also reduced measures of cell viability while increasing the production of reactive oxygen species (ROS) in MVECs isolated from mouse skeletal muscle, and the viability of Arnt ΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor. Conclusion: Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.

Modeling Alleviative Effects of Ca, Mg, and K on Cu-Induced Oxidative Stress in Grapevine Roots Grown Hydroponically.

The aim of this study was to determine the pattern of alleviation effects of calcium (Ca), magnesium (Mg), and potassium (K) on copper (Cu)-induced oxidative toxicity in grapevine roots. Root growth, Cu and cation accumulation, reactive oxygen species (ROS) production, and antioxidant activities were examined in grapevine roots grown in nutrient solutions. The experimental setting was divided into three sets; each set contained a check (Hoagland solution only) and four treatments of simultaneous exposure to 15 μM Cu with four cation levels (i.e., Ca set: 0.5, 2.5, 5, and 10 mM Ca; Mg set: 0.2, 2, 4, and 8 mM Mg; K set: 0.6, 2.4, 4.8, and 9.6 mM K). A damage assessment model (DAM)-based approach was then developed to construct the dose-effect relationship between cation levels and the alleviation effects on Cu-induced oxidative stress. Model parameterization was performed by fitting the model to the experimental data using a nonlinear regression estimation. All data were analyzed by a one-way analysis of variance (ANOVA), followed by multiple comparisons using the least significant difference (LSD) test. The results showed that significant inhibitory effects on the elongation of roots occurred in grapevine roots treated with 15 μM Cu. The addition of Ca and Mg significantly mitigated phytotoxicity in root growth, whereas no significant effect of K treatment on root growth was found. With respect to oxidative stress, ROS and malondialdehyde (MDA) contents, as well as antioxidant (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) activities, were stimulated in the roots after exposure to 15 μM Cu for three days. Moreover, H2O2 levels decreased significantly as Ca, Mg, and K concentrations increased, indicating that the coexistence of these cations effectively alleviated Cu-induced oxidative stress; however, alleviative effects were not observed in the assessment of the MDA content and antioxidant enzyme activities. Based on the DAM, an exponential decay equation was developed and successfully applied to characterize the alleviative effects of Ca, Mg, and K on the H2O2 content induced by Cu in the roots. In addition, compared with Mg and K, Ca was the most effective cation in the alleviation of Cu-induced ROS. Based on the results, it could be concluded that Cu inhibited root growth and Ca and Mg absorption in grapevines, and stimulated the production of ROS, lipid peroxidation, and antioxidant enzymes. Furthermore, the alleviation effects of cations on Cu-induced ROS were well described by the DAM-based approach developed in the present study.

Abstract P423: Nox2 Mediates Increased Oxidative Stress And Ros Production Leading To Atrial Ion Channel And Structural Remodeling In Obesity-induced Af.

Background: Inducible atrial fibrillation (AF) in diet-induced obese (DIO) mice is mediated in part by a combined effect of ion channel remodeling and atrial fibrosis. NADPH oxidase 2 (NOX2), a major source of reactive oxygen species (ROS) production in human atria, is increased in DIO mice. Although a mitochondrial antioxidant (MitoTEMPO) reduced AF burden, and reversed ion channel and structural remodeling, the role of NOX2 in increased ROS production and atrial remodeling in obesity-induced AF remains unclear. Objective: To test the hypothesis that increased NOX2 modulates atrial remodeling in obesity-induced AF, we treated DIO mice with apocynin, NOX2 inhibitor, and fed a 60% high fat diet (HFD) to Nox2 -KO mice. Methods: Weight, BP, plasma glucose, trans-esophageal rapid (TE) pacing and F 2 -isoprostanes were measured in DIO mice and compared to controls. Echocardiography, electrophysiology (EP), immunohistochemistry, Western blotting, cellular patch clamping and optical mapping studies were performed. Results: The average weight of DIO mice treated with apocynin (DIO-A) and vehicle control mice was 38.4 ± 3.8 g versus 44.0 ± 7.5 g versus 31.7 ± 1.19 g respectively (P≤0.0001). Both groups of DIO mice displayed progressive increase in weight over 10 weeks of HFD + drug treatment compared to controls (P≤0.0001). After TE pacing, DIO mice treated with apocynin showed significantly reduced pacing-induced AF burden when compared to DIO mice treated with vehicle. DIO mice treated with apocynin displayed 20.2 ± 26.1 sec versus 162.3 ± 133.7 sec and 18.3 ± 18.1 sec in DIO mice treated with vehicle and control mice respectively (P≤0.0001). Western blotting experiments showed that potassium channels, Kv7.1 and Kv1.5 protein expression is restored in Nox2 -KO HFD mice compared to DIO mice thus indicating restoration of ion channel remodeling upon Nox2 inhibition. Conclusions: We showed that genetic and pharmacological inhibition of NOX2 abrogates ion channel remodeling and reverses obesity-induced AF burden. Our findings may have important implications for the management of obesity-mediated AF in patients.

Effect of Vitamin B2 and Vitamin E on Cancer-Related Sarcopenia in a Mouse Cachexia Model

Cancer-related sarcopenia is associated with impaired energy metabolism and increased oxidative stress production in skeletal muscles. With an aim to treat cancer-related sarcopenia using dietary intervention, we investigated the effects of vitamin B2 (VB2) and vitamin E (VE), which are recognized to have antioxidant effects, on CT26 mouse colon cancer cells and skeletal muscles in vitro and in vivo. VB2 suppressed tumor growth by suppressing cell proliferation and inducing more pronounced apoptosis by increasing the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS). VE suppressed tumor growth by suppressing cell proliferation and increasing apoptosis by decreasing the production of ATP and ROS. In C2C12 mouse skeletal myoblast cells, VB2 treatment increased the production of ATP and ROS and VE treatment decreased the production of ATP and ROS; both treatments suppressed skeletal myoblast maturation. In the mouse model, intraperitoneal inoculation (peritoneal model) resulted in marked macrophage infiltration and elevated blood tumor necrosis factor-α and high-mobility group box-1 inflammatory cytokine levels, leading to cachexia. In contrast, subcutaneous inoculation (subcutaneous model) showed poor macrophage infiltration and low inflammatory cytokine levels, without cachexia. VB2 and VE activated macrophages and exacerbated cancer-related sarcopenia in the peritoneal model, whereas VB2 and VE treatment did not exhibit significant changes in sarcopenia in the subcutaneous model. In order to improve cancer-related sarcopenia by dietary intervention, it is important to consider the effect on inflammatory cytokines.

The Arabidopsis pattern recognition receptor EFR enhances fire blight resistance in apple

Fire blight disease, caused by the bacterium Erwinia amylovora (E. amylovora), is responsible for substantial losses in cultivated apples worldwide. An important mechanism of plant immunity is based on the recognition of conserved microbial molecules, named pathogen-associated or microbe-associated molecular patterns (PAMPs or MAMPs), through pattern recognition receptors (PRRs), leading to pattern-triggered immunity (PTI). The interspecies transfer of PRRs represents a promising strategy to engineer broad-spectrum and durable disease resistance in crops. EFR, the Arabidopsis thaliana PRR for the PAMP elf18 derived from the elongation factor thermal unstable (EF-Tu) proved to be effective in improving bacterial resistance when expressed into Solanaceae and other plant species. In this study, we tested whether EFR can affect the interaction of apple with E. amylovora by its ectopic expression in the susceptible apple rootstock M.26. Stable EFR expression led to the activation of PAMP-triggered immune response in apple leaves upon treatment with supernatant of E. amylovora, as measured by the production of reactive oxygen species and the induction of known defense genes. The amount of tissue necrosis associated with E. amylovora infection was significantly reduced in the EFR transgenic rootstock compared to the wild-type. Our results show that the expression of EFR in apple rootstock may be a valuable biotechnology strategy to improve the resistance of apple to fire blight.

Abstract 33: Impaired Mitochondrial Metabolism Contributes To Endothelial Dysfunction And Hypertension Independently From Oxidative Stress

Metabolic disorders contribute to pathogenesis of endothelial dysfunction, and mitochondrial deacetylase Sirt3 is critical in regulation of metabolic and antioxidant functions, but the role of Sirt3 has been largely ignored. We have recently shown that Sirt3 level is reduced in human hypertension. We propose Sirt3 as a novel target to improve the treatment of endothelial dysfunction and hypertension. Oxidative stress promotes metabolic dysregulation while metabolic conditions increase production of reactive oxygen species. In this work we have discerned the pathological role of impaired mitochondrial metabolism and oxidative stress using our new SOD2K68R deacetylation mimic mouse model. SOD2K68R mice are protected from oxidative stress because mutation of lysine 68 to arginine prevents SOD2 inactivation by acetylation. To define the specific role of impaired mitochondrial metabolism in endothelial dysfunction and hypertension, we have crossed SOD2K68R with Sirt3 -/- mice. Resultant SOD2K68R-Sirt3 -/- mice are protected from mitochondrial oxidative stress but have impaired Sirt3-mediated mitochondrial metabolism. We have tested the hypothesis that impaired Sirt3-mediated mitochondrial metabolism in SOD2K68R-Sirt3 -/- mice promotes vascular dysfunction and hypertension. SOD2K68R-Sirt3 -/- mice are protected from AngII-induced superoxide measured by mitoSOX and DHE. Meanwhile, systolic blood pressure in AngII-infused SOD2K68R-Sirt3 -/- mice was substantially higher than in SOD2K68R mice but lower than in Sirt3 -/- mice. To define the role of mitochondrial metabolism in vascular homeostasis we analyzed metabolic, inflammatory, and cellular pathways. LCAD hyperacetylation and increased HIF1α in SOD2K68R-Sirt3 -/- mice indicates impaired mitochondrial fatty acid oxidation and increased glycolysis which were further exacerbated by AngII-infusion and NFkB/ICAM increase promoting vascular inflammation. The loss of VeCAD in SOD2K68R-Sirt3 -/- Sham mice was further aggravated in AngII-infusion supporting alteration of endothelial phenotype in SOD2K68R-Sirt3 -/- mice. These data indicate that impaired mitochondrial metabolism contributes to endothelial dysfunction and hypertension independently from oxidative stress.

Abstract T4: Nox2-derived Reactive Oxygen Species Contribute To The Impaired Renal Function And Increased Maternal Mortality Observed In Dahl SS Rats Following Multiple Pregnancies

We recently demonstrated that female Dahl Salt-Sensitive (SS) rats develop significant proteinuria, reduced renal function, and increased mortality (greater than 50% compared to virgin controls) following multiple pregnancies while fed a low salt diet. While the mechanisms are unclear, it is proposed that reactive oxygen species (ROS) production contributes to this renal disease phenotype. Previous experiments demonstrated that NADPH oxidase (NOX) 2-derived ROS production mediates the development of maternal syndrome (hypertension and proteinuria) in Dahl SS rat during a singular pregnancy, yet the role of NOX 2-derived ROS on long term renal health and survival following multiple pregnancies has not been explored. Therefore, we tested the hypothesis that genetic deletion of the p67phox subunit of NOX2 (SS p67phox-/- ) will result in the protection from reduced renal function and maternal death following multiple pregnancies. There were no maternal deaths observed in the SS p67phox-/- virgin or mated rats (n=3-5/group) following three pregnancies, suggesting that the production of NOX2-derived ROS contributes to the increased mortality seen in the SS rats. In terms of renal damage, protein excretion levels were comparable at baseline between virgin and mated SS p67phox-/- rats (26.3±2.8 vs 19.6±3.8 mg/day, p >0.05). Unlike the SS rats that develop significant proteinuria at the end of three pregnancies (217±53 mg/day, n=7), the SS p67phox-/- rats do not develop a pregnancy-specific increase in proteinuria as they remain comparable to the virgin controls following three pregnancies (31.8±10 vs 50.7±14 mg/day, p >0.05). Furthermore, the mated SS p67phox-/- do not exhibit changes in creatinine clearance (0.65±0.1 vs 0.62±0.1 ml/min/g, p >0.05) or blood urea nitrogen (BUN) levels (16±2 vs 15.7±1.3 mg/dl, p >0.05) relative to virgin controls. In contrast, SS rats develop significant impairment in renal function (creatinine clearance: 0.39±0.06 ml/min/g and BUN: 26.9±2.9) following multiple pregnancies, suggesting that NOX2-derived ROS contribute to the significant renal damage phenotype observed in the SS rats. Further studies are necessary to identify the cell type responsible for producing the detrimental ROS contributing to this phenotype.

The Effects of P53 in the Globular Heads of the C1q Receptor in Gastric Carcinoma Cell Apoptosis Are Exerted via a Mitochondrial-Dependent Pathway.

The globular heads of the C1q receptor (gC1qR), located in the B cell cytoplasm, perform important roles in many cellular processes. A recent studies reported a major role of mitochondrial apoptosis in several cancers, but there has been no report on gastric carcinoma (GC). In this study, the mechanism by which cell apoptosis is induced by gC1qR in GC was explored. Western blot showed that gC1qR and P53 protein levels were lower in GC tissues than in normal tissues. Cytotoxicity was dynamically increased in gC1qR-overexpressing GC cells compared to the control. CCK8 assay indicated that overexpression of gC1qR induced GC cell apoptosis, increased reactive oxygen species (ROS) production, decreased the mitochondrial transmembrane potential and promoted mitochondrial apoptosis. Moreover, the P53 level increased in response to gC1qR. The viability, migration, and mitochondrial transmembrane potential of GC cells increased in association with decreased levels of ROS and mitochondrial apoptosis in the P53-silenced group. Collectively, our findings indicate that apoptosis of GC cells is enhanced when gC1qR overexpression is induced by P53-mediated mitochondrial apoptosis.

Tournefortia sarmentosa Inhibits the Hydrogen Peroxide-Induced Death of H9c2 Cardiomyocytes.

Tournefortia sarmentosa is a traditional Chinese medicine used to reduce tissue swelling, to exert the antioxidant effect, and to detoxify tissue. T. sarmentosa is also used to promote development in children and treat heart dysfunction. However, many of the mechanisms underlying the effects of T. sarmentosa in the treatment of disease remain unexplored. In this study, we investigated the antioxidant effect of T. sarmentosa on rat H9c2 cardiomyocytes treated with hydrogen peroxide (H2O2). T. sarmentosa reduced the cell death induced by H2O2. T. sarmentosa inhibited H2O2-induced changes in cell morphology, activation of cell death-related caspases, and production of reactive oxygen species. In addition, we further analyzed the potential active components of T. sarmentosa and found that the compounds present in the T. sarmentosa extract, including caffeic acid, rosmarinic acid, salvianolic acid A, and salvianolic acid B, exert effects similar to those of the T. sarmentosa extract in inhibiting H2O2-induced H9c2 cell death. Therefore, according to the results of this study, the ability of the T. sarmentosa extract to treat heart disease may be related to its antioxidant activity and its ability to reduce the cellular damage caused by free radicals.

Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis

BackgroundsThe present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry.ResultsMilk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4− T, CD8− CD4− T, or B lymphocytes.ConclusionsThus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.

Effects of Naringin on Cardiomyocytes From a Rodent Model of Type 2 Diabetes.

Diabetic cardiomyopathy (DCM) is a primary disease in diabetic patients characterized by diastolic dysfunction leading to heart failure and death. Unfortunately, even tight glycemic control has not been effective in its prevention. We have found aberrant diastolic Ca2+ concentrations ([Ca2+]d), decreased glucose transport, elevated production of reactive oxygen species (ROS), and increased calpain activity in cardiomyocytes from a murine model (db/db) of type 2 diabetes (T2D). Cardiomyocytes from these mice demonstrate significant cell injury, increased levels of tumor necrosis factor-alpha and interleukin-6 and expression of the transcription nuclear factor-κB (NF-κB). Furthermore, decreased cell viability, and reduced expression of Kir6.2, SUR1, and SUR2 subunits of the ATP-sensitive potassium (KATP) channels. Treatment of T2D mice with the citrus fruit flavonoid naringin for 4 weeks protected cardiomyocytes by reducing diastolic Ca2+ overload, improving glucose transport, lowering reactive oxygen species production, and suppressed myocardial inflammation. In addition, naringin reduced calpain activity, decreased cardiac injury, increased cell viability, and restored the protein expression of Kir6.2, SUR1, and SUR2 subunits of the KATP channels. Administration of the KATP channel inhibitor glibenclamide caused a further increase in [Ca2+]d in T2D cardiomyocytes and abolished the naringin effect on [Ca2+]d. Nicorandil, a KATP channel opener, and nitric oxide donor drug mimic the naringin effect on [Ca2+]d in T2D cardiomyocyte; however, it aggravated the hyperglycemia in T2D mice. These data add new insights into the mechanisms underlying the beneficial effects of naringin in T2D cardiomyopathy, thus suggesting a novel approach to treating this cardiovascular complication.

Early high-dose vitamin C in post-cardiac arrest syndrome (VITaCCA): study protocol for a randomized, double-blind, multi-center, placebo-controlled trial

BackgroundHigh-dose intravenous vitamin C directly scavenges and decreases the production of harmful reactive oxygen species (ROS) generated during ischemia/reperfusion after a cardiac arrest. The aim of this study is to investigate whether short-term treatment with a supplementary or very high-dose intravenous vitamin C reduces organ failure in post-cardiac arrest patients.MethodsThis is a double-blind, multi-center, randomized placebo-controlled trial conducted in 7 intensive care units (ICUs) in The Netherlands. A total of 270 patients with cardiac arrest and return of spontaneous circulation will be randomly assigned to three groups of 90 patients (1:1:1 ratio, stratified by site and age). Patients will intravenously receive a placebo, a supplementation dose of 3 g of vitamin C or a pharmacological dose of 10 g of vitamin C per day for 96 h. The primary endpoint is organ failure at 96 h as measured by the Resuscitation-Sequential Organ Failure Assessment (R-SOFA) score at 96 h minus the baseline score (delta R-SOFA). Secondary endpoints are a neurological outcome, mortality, length of ICU and hospital stay, myocardial injury, vasopressor support, lung injury score, ventilator-free days, renal function, ICU-acquired weakness, delirium, oxidative stress parameters, and plasma vitamin C concentrations.DiscussionVitamin C supplementation is safe and preclinical studies have shown beneficial effects of high-dose IV vitamin C in cardiac arrest models. This is the first RCT to assess the clinical effect of intravenous vitamin C on organ dysfunction in critically ill patients after cardiac arrest.Trial registrationClinicalTrials.gov NCT03509662. Registered on April 26, 2018. https://clinicaltrials.gov/ct2/show/NCT03509662European Clinical Trials Database (EudraCT): 2017-004318-25. Registered on June 8, 2018. https://www.clinicaltrialsregister.eu/ctr-search/trial/2017-004318-25/NL

TPPU treatment of burned mice dampens inflammation and generation of bioactive DHET which impairs neutrophil function

Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

Biomineralization potential and biological properties of a new tantalum oxide (Ta2O5)\u2013containing calcium silicate cement

ObjectiveThe present study evaluated the biological effects and biomineralization potential of a new tantalum oxide (Ta2O5)–containing material designed for vital pulp therapy or perforation repair (NeoMTA 2), compared to NeoMTA Plus and Bio-C Repair.Material and methodsHuman dental pulp stem cells (hDPSCs) were exposed to different eluates from NeoMTA Plus, NeoMTA 2, and Bio-C Repair. Ion release from each material was determined using inductively coupled plasma-optical emission spectrometry (ICP-MS). The biological experiments performed were MTT assays, apoptosis/necrosis assays, adhesion assays, migration assays, morphology evaluation, and reactive oxygen species (ROS) production analysis. Biomineralization was assessed by Alizarin red S staining. Finally, osteo/odontogenic gene expression was determined by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test.ResultsNeoMTA 2 displayed a significantly higher calcium release compared to the other materials (p < 0.05). When hDPSCs were cultured in presence of the different material eluates, all groups exhibited similar hDPSC viability and migration rates when compared to untreated cells. Substantial cell attachment and spreading were observed in all materials’ surfaces, without significant differences. hDPSCs treated with NeoMTA 2 displayed an upregulation of ALP, Col1A1, RUNX2 (p < 0.001), ON, and DSPP genes (p < 0.05), and showed the highest mineralization potential compared to other groups (p < 0.001). Finally, the more concentrated eluates from these materials, specially NeoMTA Plus and NeoMTA 2, promoted higher ROS production in hDPSCs compared to Bio-C Repair and control cells (p < 0.001), although these ROS levels did not result in increased cell death.ConclusionsThe new tantalum oxide (Ta2O5)–containing material shows an adequate cytocompatibility and the ability to promote biomineralization without using chemical osteogenic inducers, showing great potential as a new material for vital pulp therapy.Clinical relevanceNeoMTA 2 seems to be a promising material for vital pulp therapy. Further studies considering its biocompatibility and biomineralization potential are necessary.