NADPH Oxidase Subunits P47phox Research Articles

Effects of concurrent training and N-acetylcysteine supplementation on cardiac remodeling and oxidative stress in middle-aged spontaneously hypertensive rats

BackgroundThis study evaluated the effects of concurrent isolated training (T) or training combined with the antioxidant N-acetylcysteine (NAC) on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR).MethodsSix-month-old male SHR were divided into sedentary (S, n = 12), concurrent training (T, n = 13), sedentary supplemented with NAC (SNAC, n = 13), and concurrent training with NAC supplementation (TNAC, n = 14) groups. T and TNAC rats were trained three times a week on a treadmill and ladder; NAC supplemented groups received 120 mg/kg/day NAC in rat chow for eight weeks. Myocardial antioxidant enzyme activity and lipid hydroperoxide concentration were assessed by spectrophotometry. Gene expression of NADPH oxidase subunits Nox2, Nox4, p22 phox, and p47 phox was evaluated by real time RT-PCR. Statistical analysis was performed using ANOVA and Bonferroni or Kruskal–Wallis and Dunn.ResultsEchocardiogram showed concentric remodeling in TNAC, characterized by increased relative wall thickness (S 0.40 ± 0.04; T 0.39 ± 0.03; SNAC 0.40 ± 0.04; TNAC 0.43 ± 0.04 *; * p < 0.05 vs T and SNAC) and diastolic posterior wall thickness (S 1.50 ± 0.12; T 1.52 ± 0.10; SNAC 1.56 ± 0.12; TNAC 1.62 ± 0.14 * mm; * p < 0.05 vs T), with improved contractile function (posterior wall shortening velocity: S 39.4 ± 5.01; T 36.4 ± 2.96; SNAC 39.7 ± 3.44; TNAC 41.6 ± 3.57 * mm/s; * p < 0.05 vs T). Myocardial lipid hydroperoxide concentration was lower in NAC treated groups (S 210 ± 48; T 182 ± 43; SNAC 159 ± 33 *; TNAC 110 ± 23 *# nmol/g tissue; * p < 0.05 vs S, #p < 0.05 vs T and SNAC). Nox 2 and p22 phox expression was higher and p47 phox lower in T than S [S 1.37 (0.66–1.66); T 0.78 (0.61–1.04) *; SNAC 1.07 (1.01–1.38); TNAC 1.06 (1.01–1.15) arbitrary units; * p < 0.05 vs S]. NADPH oxidase subunits did not differ between TNAC, SNAC, and S groups.ConclusionN-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.

Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Naringenin Inhibits Acid Sphingomyelinase-Mediated Membrane Raft Clustering to Reduce NADPH Oxidase Activation and Vascular Inflammation.

Macrophage inflammation and oxidative stress promote atherosclerosis progression. Naringenin is a naturally occurring flavonoid with antiatherosclerotic properties. Here, we elucidated the effects of naringenin on monocyte/macrophage endothelial infiltration and vascular inflammation. We found naringenin inhibited oxidized low-density lipoprotein (oxLDL)-induced pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α toward an M2 macrophage phenotype and inhibited oxLDL-induced TLR4 (Toll-like receptor 4) membrane translocation and downstream NF-κB transcriptional activity. Results from flow cytometric analysis showed that naringenin reduced monocyte/macrophage infiltration in the aorta of high-fat-diet-treated ApoE-deficient mice. The aortic cytokine levels were also inhibited in naringenin-treated mice. Further, we found that naringenin reduced lipid raft clustering and acid sphingomyelinase (ASMase) membrane gathering and inhibited the TLR4 and NADPH oxidase subunit p47phox membrane recruitment, which reduced the inflammatory response. Recombinant ASMase treatment or overexpression of ASMase abolished the naringenin function and activated macrophage and vascular inflammation. We conclude that naringenin inhibits ASMase-mediated lipid raft redox signaling to attenuate macrophage activation and vascular inflammation.

Comparison effect of high-intensity interval training and moderate intensity continuous training on vascular function in a mouse model of lower extremity peripheral artery disease

Introduction&#x0D; Lower extremity peripheral artery disease (PAD), due to narrowing of the arteries supplying the legs, represents a major cause of physical disability. Exercise training is an important part of PAD treatment, improving functional capacity. Recent research suggests that exercise training-induced endothelial function improvement is involved in the benefits of exercise in PAD (Lee et al., 2023); however, the underlying molecular mechanisms remain unknown. In addition, the potential vascular benefits of high intensity training in PAD remain unexplored. Therefore, the aim of the study was to compare the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on endurance capacity, vascular endothelial function and expression of genes related to nitric oxide (NO) bioavailability in a mouse model of PAD.&#x0D; Methods&#x0D; PAD disease model was generated in male C57BL/6 mice by ligation of the right iliac artery. Mice with PAD were divided into 3 groups: sedentary control group (SED), MICT (40 min running at 70% of maximal aerobic speed) and HIIT (8 intervals of 2.5 minutes running at 90% of maximal aerobic speed combined with 2.5 minutes of running at 50% of maximal aerobic speed). Mice were exercised three times per week for 8 weeks. Maximal running time was determined by an incremental test until exhaustion. Vascular reactivity was assessed by measuring vasoconstrictor responses to phenylephrine as well as endothelium-dependent and -independent vasodilator responses to acetylcholine and DEA-NONOate, respectively, in isolated aortic rings. Gene expression of antioxidant enzymes (catalase and superoxide dismutase isoforms), prooxidant NADPH oxidase subunits (p22phox and p47phox), enzyme involved in NO production (endothelial NO synthase), vascular smooth muscle cell relaxation (soluble guanylate cyclase) and receptors involved in vascular tone regulation (a1 and b2 adrenergic receptors) was determined in aortic tissue by qPCR.&#x0D; Results&#x0D; Maximal running time was improved in both MICT and HIIT as compared to SED at the end of the study, but with no significant difference between the two exercise groups. Phenylephrine-induced vasoconstriction as well as vasodilator responses to acetylcholine and DEA-NONOate were significantly improved in MICT compared to SED while no significant differences were reported between HIIT and SED. There was no significant difference in gene expression among the three groups.&#x0D; Discussion/Conclusion&#x0D; MICT and HIIT were equally effective at improving endurance capacity in mice with PAD. However, only MICT improved vascular reactivity.&#x0D; References&#x0D; Lee, J., Zarezadehmehrizi, A., LaVoy, E., Markofski, M., &amp; Park, Y. (2023). Exercise training improves brachial artery endothelial function, but does not alter inflammatory biomarkers in patients with peripheral artery disease: A systematic review and meta-analysis. Journal of Cardiovascular Translational Research. Advance online publication. https://doi.org/10.1007/s12265-023-10451-0

Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells Promote Muscle Regeneration in a Diabetic Mouse Model of Critical Limb Threatening Ischemia

Critical limb threatening ischemia (CLTI), the end stage of peripheral arterial disease (PAD), is diagnosed in 500,000 patients each year, often results in amputation, and has a ~50% 5-year mortality rate. Diabetic CLTI patients experience especially high morbidity and mortality, and no effective non-surgical therapy exists for this population. Our Phase II MOBILE trial demonstrated that autologous bone marrow nucleated cells were unable to prevent amputations in diabetic patients; however, data from a Phase I trial shows that allogeneic bone marrow-derived mesenchymal stromal cells (BMD-MSC) stimulated angiogenesis in ischemic muscle, including diabetics. While allogeneic MSC may be an effective cell preparation to treat diabetic CLTI, passaging-related cell senescence prevents generation of sufficient cell numbers for therapeutic use. The development of induced pluripotent stem cell (iPSC)-derived MSC overcomes cell senescence issues and offers the possibility of genetic modifications to enhance cell function. The current study was designed to determine potential mechanisms by which iPSC-MSC stimulate muscle regeneration in a rodent CLTI model.&#x0D; The CLTI mouse model was created by ligation/excision of the femoral artery in male polygenic diabetic TallyHo mice. Mice with intramuscular administration of iPSC-MSC displayed positive indicators of muscle regeneration compared to vehicle control mice. Real-time PCR performed with gastrocnemius muscle obtained 7- or 30-days post iPSC-MSC injection showed an increase in mRNA expression for MyH3, MyoD1, Mrc1, FoxP3, and VEGF-A vs. vehicle treated muscle, indicating promotion of muscle regeneration, M2-biased macrophage expression, T regulatory cell (Treg) expansion, and vascular proliferation. Downregulation of the NADPHoxidase subunit p47phox indicated a decrease in oxidative stress in the treated mice. The results are consistent with iPSC-MSC promotion of muscle regeneration via a Treg mediated stimulation of the M1-M2 macrophage phenotypic shift. Thus, human iPSC-MSC could be an effective treatment to stimulate muscle regeneration and ameliorate CLTI in diabetic patients.

Abstract P397: Preservation Of Renal Microvascular Function In Ischemia-reperfusion Induced Kidney Injury In Rats

Proper renal blood flow and glomerular filtration rate are critical for maintaining normal kidney function by precisely adjusting preglomerular microvascular resistance. Renal ischemia-reperfusion (IR) insult markedly increased mRNA expression of the inflammatory chemokine (C-C motif) Ligand 2 (CCL2) in rat kidney tissues. We hypothesized that depletion of macrophages prevented IR-induced renal autoregulatory impairment via reduction of CCL2 in preglomerular microvessels (PGMV). IR was induced in male rats by 60-minute bilateral renal artery occlusion followed by reperfusion. Some IR rats were treated with the selective macrophage inhibitor, Liposome-encapsulated clodronate (LC) following reperfusion and repeated 72 hours post-IR. Afferent arteriolar autoregulatory reactivity was assessed using the in vitro blood-perfused juxtamedullary nephron preparation in sham, IR, and IR+LC rats on day 7. Afferent arterioles from sham-operated rats exhibited pressure-dependent vasoresponsiveness. The baseline arteriolar diameter averaged 13.4 ± 0.5 μm at renal perfusion pressure (RPP) of 100 mmHg. Diameter increased to 117 ± 4% of baseline at RPP of 65 mmHg and decreased to 69 ± 2% at 170 mmHg, respectively. In contrast, afferent arterioles from IR rats only increased to 104 ± 2% and decreased to 91 ± 5% of baseline at 65 and 170 mmHg, respectively (P &lt; 0.05 vs sham), indicating the impaired autoregulatory capacity in IR rats. LC-treated IR rats, however, markedly improved the pressure-dependent vasoresponsiveness. A decrease in RPP to 65 mmHg resulted in a 124 ± 17% increase in the baseline diameter, whereas increasing RPP to 170 mmHg led to a 74 ± 7% decrease in diameter. Additionally, IR markedly increased CCL2 mRNA expression in isolated PGMV by 10-fold 7 days post-IR. IR also elevated NADPH oxidase subunits (gp91 phox , P67 phox , and P47 phox ). Significantly, LC treatment suppressed mRNA expression of CCL2, gp91 phox , P67 phox , and P47 phox in IR-PGMV. In conclusion, depletion of macrophages preserves afferent arteriolar autoregulatory capacity and reduces PGMV CCL2 mRNA expression in IR rats, suggesting a critical role of macrophages in mediating renal microvascular autoregulatory impairment in IR-induced kidney injury via the increase of CCL2 in PGMV.

Helicobacter pylori infection induces autophagy via ILK regulation of NOXs-ROS-Nrf2/HO-1-ROS loop.

Reactive oxygen species (ROS) can regulate the occurrence of autophagy, and effective control of the balance between ROS and autophagy may be an important strategy for Helicobacter pylori induced gastric-related diseases. In this study, infection with H. pylori led to a lower level of ILK phosphorylation and increased ROS generation. Knockdown of ILK enhanced total ROS generation, and upregulated NADPH oxidase (NOX) subunit p22-phox levels. Inhibition of NOXs affected total ROS generation. The inhibition of NOX and ROS generation reduced Nrf2 and HO-1 levels, and knockdown of ILK significantly enhanced Nrf2 levels in H. pylori-infected GES-1 cells. Activation of Nrf2 by DMF decreased ROS levels. Therefore, NOX-dependent ROS production regulated by ILK was essential for activation of Nrf2/HO-1 signaling pathways in H. pylori-infected GES-1 cells. Beclin1, ATG5 and LC3B-II levels were higher both in H. pylori-infected and ILK-knockdown GES-1 cells. In NAC-pretreated GES-1 cells infected with H. pylori, the LC3B-II level was decreased compared to that in cells after H. pylori infection alone. Stable low expression of ILK with further knockdown of Beclin1 or ATG5 significantly reduced LC3B-II levels in GES-1 cells, while with the addition of the autophagy inhibitor chloroquine (CQ), LC3B-II and p62 protein levels were both remarkably upregulated. H. pylori accelerated the accumulation of ROS and further led to the induction of ROS-mediated autophagy by inhibiting ILK levels. Together, these results indicate that H. pylori infection manipulates the NOX-ROS-Nrf2/HO-1-ROS loop to control intracellular oxygen stress and further induced ROS-mediated autophagy by inhibiting ILK levels.

Myeloperoxidase G-463A and CYBA C242T genetic variants in gestational diabetes mellitus.

Oxidative stress plays an important role in the pathophysiology of gestational diabetes mellitus (GDM). We investigated the relationship between NADPH oxidase p22phox subunit (CYBA) C242T (rs4673) and myeloperoxidase (MPO) G-463A (rs2333227) genetic variants and GDM in 719 patients with GDM and 1205 control women. Clinical, metabolic, and oxidative stress parameters were analyzed. We found that frequencies of the A allele (15.6% vs 12.3%) and GA + AA genotype (28.5% vs 23.2%) of the MPO G-463A variation were significantly higher in patients with GDM than in the control women (OR = 1.318, 95% CI: 1.068-1.625, P = 0.010 for the dominant model; OR = 1.999, 95% CI: 1.040-3.843, P = 0.034 for the recessive model; OR = 1.320, 95% CI: 1.095-1.591, P = 0.004 for the allele model). Genotype GA + AA remained a significant predictor of GDM in a logistic regression model including age and BMI at delivery (OR = 1.282, 95% CI: 1.037‒1.583, P = 0.021). Furthermore, the ‒463A allele was associated with higher TG and the 242T allele was related to higher pre-pregnancy BMI and oxidative stress index in all subjects (P < 0.05). The 242T allele was also associated with higher homeostatic model assessment of insulin resistance but lower serum total antioxidant capacity in patients with GDM (P < 0.05). We conclude that the MPO G-463A, but not the CYBA C242T, genetic variation is associated with an increased risk of GDM in Chinese women. These two genetic polymorphisms may be linked to obesity, dyslipidemia, insulin resistance, and oxidative stress.

Lutein Induces Reactive Oxygen Species-Mediated Apoptosis in Gastric Cancer AGS Cells via NADPH Oxidase Activation.

Disruption of apoptosis leads to cancer cell progression; thus, anticancer agents target apoptosis of cancer cells. Reactive oxygen species (ROS) induce apoptosis by activating caspases and caspase-dependent DNase, leading to DNA fragmentation. ROS increase the expression of apoptotic protein Bax, which is mediated by activation of nuclear factor-κB (NF--κB). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of endogenous ROS, and its activation is involved in apoptosis. Lutein, an oxygenated carotenoid and known antioxidant, is abundant in leafy dark green vegetables, such as spinach and kale, and in yellow-colored foods, such as corn and egg yolk. High amounts of lutein increase ROS levels and exhibit anticancer activity. However, its anticancer mechanism remains unclear. This study aimed to determine whether lutein activates NADPH oxidase to produce ROS and induce apoptosis in gastric cancer AGS cells. Lutein increased ROS levels and promoted the activation of NADPH oxidase by increasing the translocation of NADPH oxidase subunit p47 phox to the cell membrane. It increased NF-κB activation and apoptotic indices, such as Bax, caspase-3 cleavage, and DNA fragmentation, and decreased Bcl-2, cell viability, and colony formation in AGS cells. The specific NADPH oxidase inhibitor ML171, and the known antioxidant N-acetyl cysteine reversed lutein-induced cell death, DNA fragmentation, and NF-κB DNA-binding activity in AGS cells. These results suggest that lutein-induced ROS production is dependent on NADPH oxidase, which mediates NF-κB activation and apoptosis in gastric cancer AGS cells. Therefore, lutein supplementation may be beneficial for increasing ROS-mediated apoptosis in gastric cancer cells.

Novel insights into the mechanism of reactive oxygen species-mediated neurodegeneration.

Neurite degeneration, a major component of many neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, is not part of the typical apoptosis signaling mechanism, but rather it appears that a self-destructive process is in action. Oxidative stress is a well-known inducer of neurodegenerative pathways: neuronal cell death and neurite degeneration. Although oxidative stress exerts cytotoxic effects leading to neuronal loss, the pathogenic mechanisms and precise signaling pathways by which oxidative stress causes neurite degeneration have remained entirely unknown. We previously reported that reactive oxygen species generated by NADPH oxidases induce activation of the E3 ubiquitin ligase ZNRF1 in neurons, which promotes neurite degeneration. In this process, the phosphorylation of an NADPH oxidase subunit p47-phox at the 345th serine residue serves as an important checkpoint to initiate the ZNRF1-dependent neurite degeneration. Evidence provides new insights into the mechanism of reactive oxygen species-mediated neurodegeneration. In this review, we focus specifically on reactive oxygen species-induced neurite degeneration by highlighting a phosphorylation-dependent regulation of the molecular interaction between ZNRF1 and the NADPH oxidase complex.

Sympathetic nervous system hyperactivity-induced oxidative stress promoting endothelial dysfunction is dependent on the NADPH oxidases/SGLT2 crosstalk: potential role in cardiogenic shock

Abstract Background Hyperactivity of the sympathetic nervous system (SNS) promotes cardiac dysfunction, that can contribute to heart failure and even cardiogenic shock. Endothelial dysfunction associated with increased formation of reactive oxygen species (ROS) has been suggested to promote cardiac dysfunction and heart failure. Our previous results showed that SGLT2 and ROS derived from endothelial NADPH oxidases contribute to cardiac dysfunction. Purpose Therefore, this study examined whether cardiogenic shock and hyperactivation of the SNS involve NADPH oxidases and SGLT2 in the development of endothelial dysfunction using in vitro and in vivo approaches. Methods Peripheral blood from patients with cardiogenic shock and healthy controls was drawn by vein puncture into tubes containing sodium citrate. Cultured human microvascular endothelial cells (HMEC-1) were stimulated with either 100 nM of the β-adrenergic agonist isoproterenol or with heparinized (10 U/ml) patient plasma. Male wild-type mice and mice lacking the NADPH oxidase subunit p22phox in the endothelium (p22phox ecKO, 11-week-old) were treated with isoproterenol (100 mg/kg) for five consecutive days and sacrificed at day 14. ROS levels were assessed by dihydroethidium fluorescence, and the expression levels of target genes and proteins by RT-qPCR and Western blot, respectively. siRNA approaches were used to down-regulate the NADPH oxidase subunit p22phox or SGLT2. Results Exposure of HMEC-1 to either plasma from patients suffering from cardiogenic shock or isoproterenol stimulated the formation of ROS. ROS levels were reduced by N-acetylcysteine (an antioxidant) and silencing of p22phox, by empagliflozin (a selective SGLT2 inhibitor) and silencing of SGLT2 as well as by metoprolol (a beta1 adrenergic receptor antagonist) and ICI-118,551 (a beta2 adrenergic receptor antagonist). Cardiogenic shock patient plasma and isoproterenol upregulated the NADPH oxidase subunits p22phox and Nox4 as well as SGLT2. These responses were prevented by silencing of either p22phox or SGLT2, and by empagliflozin. In addition to improved cardiac function and blunted SGLT2 expression, p22phox ecKO mice were protected against isoproterenol-induced endothelial dysfunction and senescence, characterized by upregulation of eNOS, nitrotyrosine, VCAM-1 and p16 in the left ventricle. Conclusion SNS hyperactivity and cardiogenic shock-derived patient plasma cause a feed forward loop between NADPH oxidases and SGLT2, promoting pro-oxidant responses and endothelial dysfunction via activation of both beta1 and beta2 adrenergic receptors. The NADPH oxidase/SGLT2 crosstalk also contributes to SNS hyperactivity-induced senescence, cardiac and endothelial dysfunction. Thus, the endothelial NADPH oxidases/SGLT2 crosstalk appears to have a major role in cardiac diseases associated with SNS hyperactivity such as cardiogenic shock. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Deutsches Herzzentrum München

Effects of resistance exercise in the cardiac remodeling of myocardial infarction rats

Abstract Introduction Exercise is an important therapeutic strategy for heart failure (HF). However, the myocardial effects of resistance exercise during HF are not completely understood. In this study, we investigated the influence of resistance exercise on cardiac remodeling and molecular myocardial changes of rats with myocardial infarction (MI)-induced HF. Methods Three months after MI induction or simulated surgery, Wistar rats were divided into three groups: Sham (n=14); MI (n=9); and MI subjected to resistance exercise (MI-R, n=13). Exercised rats trained 3 times a week during 12 weeks performing four climbs in a ladder with progressive loads. Cardiac structure and left ventricular function were assessed by echocardiogram. Myocyte diameters were measured in histological sections. Energy metabolism, lipid hydroperoxide, antioxidant enzymes activity, malondialdehyde, and protein carbonylation were evaluated by spectrophotometry. NADPH oxidase subunits (Nox2, Nox4, p22phox and p47phox) gene expression was assessed by RT-PCR. Statistical analysis: ANOVA and Tukey test or Dunn's test. Results Mortality did not differ between MI-R and MI groups. MI-R and MI presented dilated left atrium and left ventricle with systolic and diastolic dysfunction. Exercise improved maximum carrying load with no changes in cardiac structure or left ventricle function. Myocyte diameter was lower in MI than Sham and MI-R. Lactate dehydrogenase and creatine kinase activities were lower in MI than Sham. Activity of citrate synthase and catalase was lower in MI and MI-R than Sham. Lipid hydroperoxide concentration was lower in MI-R than MI. Nox2 and p22phox gene expression was higher in MI-R than Sham. Gene expression of Nox4 was higher in both infarcted groups and gene expression of p47phox was lower in MI than Sham. Conclusion Resistance exercise is safe and well tolerated by infarcted rats. Exercise increases maximum carrying load and reduces myocardial oxidative stress with no changes in cardiac structure or left ventricle function of infarcted rats. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): CNPq - National Council for Scientific and Technological Development

Wax apple (Syzygiumsamarangense) fruit extract ameliorates endothelial dysfunction and liver damage in high cholesterol diet-fed rats

Background and aimWax apple fruit (Syzygium samarangense) is one of the most popular tropical fruit in Asia, and contains several essential nutrients. Therefore, this study explored the effects of the wax apple fruit extract on a high-cholesterol diet-induced vascular endothelial dysfunction and fatty liver in rats. Experimental procedureMale Sprague Dawley rats were fed a diet with 1.5% cholesterol (HCD) for 8 weeks, and were given wax apple fruit extract (50 and 100 mg/kg/day) orally for the last 4 weeks. After 8 weeks, blood sample, thoracic aorta, and liver were collected and processed for biochemical and histological analysis. Additionally, vascular endothelial function and the protein expression of oxidative stress markers in aortae were evaluated. Results and conclusionWax apple reduced serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), but increased high-density lipoprotein cholesterol (HDL-C) levels. Furthermore, the liver levels of TG and TC were reduced in wax apple-treated hypercholesterolemic rats. Histological studies revealed that wax apple ameliorated HCD-induced morphologic changes of aortic and liver tissues of rats. In aortic tissues, the impaired endothelium-dependent responses to acetylcholine, the reduced nitric oxide (NO) contents, the elevated endothelin (ET)-1 contents, and the increased expression of NADPH oxidase subunit p47phox and 4-hydroxynonenal in HCD-fed rats were reversed by wax apple treatment. These results suggest that oral administration of wax apple improves vascular dysfunction and damage in hypercholesterolemic rats possibly through increasing NO bioavailability, decreasing ET-1 levels and reducing oxidative stress. Furthermore, wax apple ameliorates the HCD-induced fatty liver in rats.

Saikogenin F From Bupleurum smithii Ameliorates Learning and Memory Impairment via Antiinflammation Effect in an Alzheimer’s Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disease associated with aging. Bupleurum smithii Wolff. is a Chinese folk medicine used to reduce fever and inflammation. Regarding the key role of neuroinflammation in AD pathogenesis, it was speculated that B. smithii may be the source of compounds that treat AD through anti-inflammatory effects. This study aimed to investigate the effects of saikogenin F, a natural active ingredient from B. smithii, on cognition impairment and neuroinflammation in AD mice induced by amyloid β (Aβ). The AD mice model was established by intracerebroventricular (i.c.v.) injection of Aβ, and different doses of saikogenin F (10, 20, and 40 mg/kg) were intragastrically administrated once daily. Results of behavioral experiments, including the novel object recognition (NOR) test, Y-maze test, and Morris water maze (MWZ) test, showed that saikogenin F could ameliorate Aβ-induced cognition impairment in AD mice. Enzyme linked immunosorbent assay (ELISA) results showed that tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and reactive oxygen species (ROS) levels in hippocampal tissue increased after Aβ injection, while saikogenin F could significantly reduce the concentrations of these inflammatory factors. Western blotting results revealed that the Aβ-induced reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits protein expression in mice hippocampus was remarkably downregulated by saikogenin F. Results of Iba-1 immunohistochemical staining showed that saikogenin F could effectively inhibit Aβ-induced activation of microglia in vivo. These results suggested that saikogenin F could relieve Aβ-induced cognitive impairment via inhibiting neuroinflammation and microglial activation. These effects may be achieved by inhibiting the expression of the NADPH oxidase subunits gp91phox and p47phox.

Trigonella foenum-graecum L. and Psoralea corylifolia L. Improve Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats through Suppression of Oxidative Stress.

Background Diabetes mellitus-induced erectile dysfunction (DMED) is one of the most common complications of diabetes and is mainly attributed to oxidative stress. Hu-Lu-Ba-Wan (HLBW) is a classic Chinese formulation consisting of Trigonella foenum-graecum L. (TFG) and Psoralea corylifolia L. (PC). HLBW has been used not only for the treatment of diabetes but also for the treatment of erectile dysfunction in clinics. This study aimed to explore the efficacy and underlying mechanism of HLBW in ameliorating erectile function in streptozotocin-induced diabetic rats. Methods The diabetic model was established by tail vein injection of streptozotocin (26 mg/kg), and then DMED rats screened by the apomorphine test were randomly divided into two groups: the model group and the HLBW group. The rats in the HLBW group were administered HLBW granules daily for 12 weeks. Fasting blood glucose and fasting insulin were tested by a commercial kit. Intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured by cavernous nerve electrostimulation before the rats were killed. Erectile function was evaluated with ICP/MAP. The markers of oxidative stress in the corpus cavernosum (CC) were assayed by assay kits. Apoptosis in cavernosal tissue was detected by Western blotting (WB). The expression levels of vascular endothelial marker (vWF), α-smooth muscle actin (α-SMA), endothelial nitric oxide synthase (eNOS), and NADPH oxidase subunit P47phox were determined by WB and PCR. Furthermore, the structure of the CC was further confirmed by Masson's trichrome staining. Results The results showed that HLBW significantly reduced blood glucose and increased insulin sensitivity. HLBW reduced oxidative stress and apoptosis. In addition, we observed that the expression levels of vWF, α-SMA, and eNOS as well as the ratio of smooth muscle to collagen increased in the HLBW group. Conclusions Our results demonstrated that HLBW could reduce oxidative stress damage in CC to improve diabetes mellitus-induced erectile dysfunction in rats by inhibiting NADPH oxidase.

Inhibition of autophagy with expression of NADPH oxidase subunit p22phox in preneoplastic lesions in a high-fat diet and streptozotocin-related hepatocarcinogenesis rat model

To study the effects of autophagy inducer carbamazepine (CBZ) in a high-fat diet (HFD)/streptozotocin (STZ)-related early hepatocarcinogenesis model, we determined autophagic flux by immunohistochemical analysis of autophagy marker expression in preneoplastic liver foci and compared that with the expression of the NADPH oxidase subunit. Male F344 rats were fed a basal diet or HFD and subjected to two-stage hepatocarcinogenesis; diabetes mellitus was induced via STZ administration. Several STZ-treated, HFD-fed rats were administered CBZ (a total of five doses every one or two days) at week 7 and 8. STZ-treated, HFD-fed rats decreased β cells in the islet of Langerhans and increased adipophilin-positive lipid droplets in the liver; moreover, they had a larger area of glutathione S-transferase placental form-immunopositive preneoplastic liver foci, which was associated with inhibition of autophagy and induction of the NADPH oxidase subunit, as demonstrated by increased immunohistochemical expression of an autophagosome receptor marker microtubule-associated protein light chain 3 (LC3)-binding protein p62, and of an NADPH oxidase subunit p22phox in the preneoplastic foci. An increased trend of an autophagy phagophore marker LC3 in preneoplastic foci was also detected. CBZ administration could induce autophagy and impair p22phox expression, as shown by altered expression of autophagy regulators (Atg5, Atg6, Lamp1, Lamp2, and Lc3), NADPH oxidase subunits (P22phox and P67phox), and antioxidant enzymes Gpx1 and Gpx2. These results suggest that inhibition of autophagy and induction of p22phox might contribute to HFD/STZ-related early hepatocarcinogenesis in rats; however, the effects of CBZ administration on the STZ/HFD-increased preneoplastic foci were marginal in this study.

Association of CYBA C242T and superoxide dismutase 2 A16V genetic variants with preeclampsia.

To investigate the relationship between NADPH oxidase p22phox subunit (CYBA; C242T) and superoxide dismutase 2 (SOD2; A16V) polymorphisms and the risk of preeclampsia (PE) in Chinese women. This case-control study included 325 patients with PE and 1294 controls. The genotypes were determined by polymerase chain reaction-restrictionfragmentslengthpolymorphism method. Clinical, metabolic, and oxidative stress parameters were analyzed. Participants with T allele of the CYBA C242T polymorphism had a decreased risk of PE (OR=0.62, 95% CI: 0.42-0.92, P=0.016) and those with the A allele of the SOD2 A16V polymorphism had increased risk of PE (OR=1.44, 95% CI: 1.09-1.89, P=0.010). The coexistence of the SOD2 AA+AV genotypes and the CYBA CC genotype further increased the risk of PE (OR=2.32, 95% CI: 1.31-4.10, P=0.003) when the CT+TT/VV combined genotype was the reference category. The T allele of the CYBA C242T polymorphism is related to a decreased risk of PE, while the A allele of the SOD2 A16V polymorphism and its combination with the CYBA CC genotype are associated with an increased risk of PE in Chinese women.

Effect and Mechanism of Lidocaine Pretreatment Combined with Dexmedetomidine on Oxidative Stress in Patients with Intracranial Aneurysm Clipping.

This study aimed to explore the effect and mechanism of lidocaine pretreatment combined with dexmedetomidine on oxidative stress in patients with intracranial aneurysm clipping. Many studies have used various drugs such as lidocaine to explore the effect and mechanism of lidocaine pretreatment. A total of 80 patients with intracranial aneurysm clipping surgery were randomly divided into 4 groups: the single lidocaine group, single dexmedetomidine group, lidocaine combined with dexmedetomidine group, and control group. The thread embolism method was used to establish a stable intracranial aneurysm model of Hashimoto rats. Fifty adult rats were randomly divided into a sham operation group, ligation of the left common carotid artery and bilateral posterior branch of renal artery, lidocaine group, dexmedetomidine group, and lidocaine combined with dexmedetomidine group. The colorimetric method was used to determine the oxidative stress indicators in brain tissue: MDA content, SOD activity, and T-AOC content. The western blot method characterized the protein levels related to oxidative stress: nNOS, iNOS, and NADPH oxidase subunits p22phox, gp91phox, and p47phox. The differences in each index between the groups were statistically significant (P < 0.05). Animal experiment results revealed that the content of MDA in the brain tissue of rats in the LD group was significantly lower than that in the single-drug group and sham group. The T-AOC and SOD concentrations in the LD group were significantly higher than those in the single-drug group and sham group, and the differences between the groups were statistically significant (P < 0.05). The protein expression of the LD group was significantly lower than that of the drug-alone group and model group, and the difference between groups was statistically significant (P < 0.05). To sum up, lidocaine pretreatment combined with dexmedetomidine can effectively maintain the hemodynamic stability of patients with intracranial aneurysm clipping and reduce postoperative oxidative stress response. Its mechanism of action may be related to the inhibition of oxidative stress damage mediated by nNOS, iNOS, and p22phox, gp91phox, and p47phox in the hippocampus. Our study has significant and applicable medical aspects in lidocaine pretreatment combined with dexmedetomidine on oxidative stress in patients.

Astragaloside IV Ameliorates Cognitive Impairment and Neuroinflammation in an Oligomeric Aβ Induced Alzheimer's Disease Mouse Model via Inhibition of Microglial Activation and NADPH Oxidase Expression.

Microglial activation and neuroinflammation induced by amyloid β (Aβ) play pivotal roles in Alzheimer's disease (AD) pathogenesis. Astragaloside IV (AS-IV) is one of the major active compounds of the traditional Chinese medicine Astmgali Radix. It has been reported that AS-IV could protect against Aβ-induced neuroinflammation and cognitive impairment, but the underlying mechanisms need to be further clarified. In this study, the therapeutic effects of AS-IV were investigated in an oligomeric Aβ (oAβ) induced AD mice model. The effects of AS-IV on microglial activation, neuronal damage and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were further studied. Different doses of AS-IV were administered intragastrically once a day after intracerebroventricularly oAβ injection. Results of behavioral experiments including novel object recognition (NOR) test and Morris water maze (MWM) test revealed that AS-IV administration could significantly ameliorate oAβ-induced cognitive impairment in a dose dependent manner. Enzyme linked immunosorbent assay (ELISA) results showed that increased levels of reactive oxygen species (ROS), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 in hippocampal tissues induced by oAβ injection were remarkably inhibited after AS-IV treatment. OAβ induced microglial activation and neuronal damage was significantly suppressed in AS-IV-treated mice brain, observed in immunohistochemistry results. Furthermore, oAβ upregulated protein expression of NADPH oxidase subunits gp91phox, p47phox, p22phox and p67phox were remarkably reduced by AS-IV in Western blotting assay. These results revealed that AS-IV could ameliorate oAβ-induced cognitive impairment, neuroinflammation and neuronal damage, which were possibly mediated by inhibition of microglial activation and down-regulation of NADPH oxidase protein expression. Our findings provide new insights of AS-IV for the treatment of neuroinflammation related diseases such as AD.

Endothelial NADPH oxidases protect against sympathetic nervous system hyperactivity-induced cardiac dysfunction and remodeling in mice: contribution of SGLT2

Abstract Background NADPH oxidase-derived reactive oxygen species (ROS) contribute to cardiac dysfunction, often characterized by coronary microvascular dysfunction, an inflammatory response and cardiomyocyte hypertrophy. Hyperactivity of the sympathetic nervous system (SNS) induces oxidative stress, promoting cardiac dysfunction and the development of heart failure. Selective inhibitors of sodium-glucose co-transporter 2 (SGLT2), have shown remarkable cardioprotective effects in clinical studies. Recently, SGLT2 inhibitors have been reported to prevent endothelial dysfunction and pro-inflammatory responses in endothelial cells in response to angiotensin II involving NADPH oxidases. Purpose Therefore, the aim of the study was to determine whether endothelial NADPH oxidases promote SNS-induced cardiac dysfunction and to clarify the role of SGLT2. Methods Male wild-type mice and mice lacking the NADPH oxidase subunit p22phox in the endothelium (p22phox ecKO, 11-week-old) were treated with isoproterenol (100 mg/kg) for five consecutive days and sacrificed at day 14. Hemodynamic measurements of left (LV) and right (RV) ventricles were performed by a transthoracic approach. Heart tissue sections were stained with Sirius red to evaluate fibrosis and wheat germ agglutinin to assess cardiomyocyte size. Cultured human microvascular endothelial cells (HMEC-1) were stimulated with 100 nM isoproterenol and ROS levels were assessed by dihydroethidium fluorescence. The expression level of target genes and proteins was assessed by quantitative real-time PCR and Western blot, respectively. siRNA approaches were used to down-regulate either the NADPH oxidase subunit p22phox or SGLT2. Results The isoproterenol treatment increased LV and RV systolic pressures in wild-type mice but not in p22phox ecKO mice. p22phox ecKO mice were protected against isoproterenol-induced fibrosis, cardiac remodeling characterized by upregulation of mRNA levels of ANP, BNP and β-MHC, and pulmonary congestion. LV remodeling was associated with upregulation of the NADPH oxidase subunits p22phox, Nox2, and Nox4 as well as of SGLT2 in wild-type mice, however no such effects were observed in p22phox ecKO mice. Exposure of HMEC-1 to isoproterenol stimulated the formation of ROS and caused an upregulation of p22phox and SGLT2 protein levels in a time- and concentration-dependent manner. No such effects were observed following silencing of either p22phox or SGLT2, or use of a selective SGLT2 inhibitor. Conclusion Deletion of the NADPH oxidase subunit p22phox in the endothelium protected against SNS hyperactivity induced LV cardiac dysfunction and remodeling, and prevented upregulation of SGLT2. Since depletion of SGLT2 prevented the pro-oxidant response to isoproterenol in endothelial cells, the endothelial NADPH oxidase/SGLT2 pathway seems to have a prominent role in promoting cardiac remodeling and dysfunction in SNS hyperactivity. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Deutsches Herzzentrum München