Panax Notoginseng Saponins Inhibits Atherosclerotic Plaque Angiogenesis by Down-Regulating Vascular Endothelial Growth Factor and Nicotinamide Adenine Dinucleotide Phosphate Oxidase Subunit 4 Expression

Objective： To investigate the mechanism of Panax notoginseng saponins （PNS）, an effective component extracted from Panax notoginseng, on atherosclerotic plaque angiogenesis in atherosclerosis-prone apolipoprotein E-knockout （ApoE-KO） mice fed with high-fat, high-cholesterol diet. Methods： Twenty ApoE-KO mice were divided into two groups, the model group and the PNS group. Ten normal C57BL/6J mice were used as a control group. PNS （60 mg/kg） was orally administered daily for 12 weeks in the PNS group, The ratio of plaque area to vessel area was examined by histological staining. The tissue sample of aortic root was used to detect the CD34 and vascular endothelial growth factor （VEGF） expression areas by immunohistochemistry. The expression of VEGF and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 （NOX4） were measured by reverse transcription polymerase chain reaction and Westem blotting respectively. Results： After treatment with PNS, the plaque areas were decreased （P〈0.05）. CD34 expressing areas and VEGF expression areas in plaques were significantly decreased （P〈0.05）. Meanwhile, VEGF and NOX4 mRNA expression were decreased after treatment with PNS, VEGF and NOX4 protein expression were also decreased by about 72% and 63%, respectively （P〈0.01）. Conclusion： PNS, which decreases VEGF and NOX4 expression, could alleviate plaque angiogenesis and attenuate atherosclerosis.

Relationship between reduced nicotinamide adenine dinucleotide phosphate oxidase subunit p22phox gene polymorphism and obstructive sleep apnea-hypopnea syndrome in the Chinese Han population

Background Increased production of reactive oxygen species （ROS） is thought to play a major role in the pathogenesis of obstructive sleep apnea-hypopnea syndrome （OSAHS）. The reduced nicotinamide adenine dinucleotide phosphate （NADPH） oxidase complex is an important source of ROS. The p22phox subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site. This study aimed to investigate the relationship between NADPH oxidase subunit p22phox gene polymorphism and OSAHS. Methods The genotypes of p22phox polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphisms （PCR-RFLP） assay in 176 unrelated subjects of the Han population in southern region of China （including 107 OSAHS subjects and 69 non-OSAHS subjects）, while the plasma concentration of superoxide dismutase （SOD） was detected in the two groups, and p22phox mRNA expression in peripheral blood mononuclear cell （PBMC） was determined with reverse transcription polymerase chain reaction （RT-PCR）. Results The phagocyte NADPH oxidase subunit p22phox mRNA expression was significantly increased in the OSAHS group than that in the non-OSAHS group （P 〈0.01）. Compared with the non-OSAHS control group （（85.31±9.23） U/ml）, the levels of SOD were lower in patients with OSAHS （（59.65±11.61） U/ml （P 〈0.01）. There were significant differences in genotypes distribution in p22phox polymorphism between the two groups （P=0.02）. Compared with the non-OSAHS control group, the OSAHS group had a significantly higher T allele frequency in p22phox polymorphism （P=0.03）. There were independent effects of p22phox polymorphism on body mass index （BMI）, neck circumference （NC）, waist-to-hip ratio （WHR） in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, systolic blood pressure （SBP）, diastolic blood pressure （DBP） and apnea-hypopnea index （AHI） （P 〈0.05）, but the carriers of the T allele had lower SOD （P 〈0.01） and lowest SaO2 （P=0.04）. There was no significant difference in p22phox mRNA expression between the OSAHS groups with or without T allele （P=0.45）. Conclusions The NADPH oxidase subunit p22phox gene polymorphism may be associated with susceptibility to OSAHS, and it may be an important candidate gene for OSAHS.

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane （PM） nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content （RWC） and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H2O2 and O2^- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants. Histochemical analyses also revealed increased H2O2 and O2^- production in drought-stressed leaves. Application of diphenylene iodonium （DPI）, an inhibitor of PM NADPH oxidase, did not alleviate drought-induced production of H2O2 and O2^-. Catalysis experiments indicated that the rice PM NADPH oxidase was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H2O2 system, is involved in the response of rice to drought stress.

Excessive Copper Induces the Production of Reactive Oxygen Species,which is Mediated by Phospholipase D, Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Antioxidant Systems

Tobacco BY-2 suspension cells were used to study the chemical damage and its associated mechanisms caused by Cu^2＋. Treatment with 100 μmol/L Cu^2＋ generated a large amount of HzOz and thiobarbituric acid-reactive substances （TBARS） in cells. Using phospholipase D （PLD） specific inhibitor （1-butanol） or phosphatidic acid （PA）, we demonstrated that PLD plays an important role in the generation of H2O2 and TBARS. Semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme activity assays with wild type and nicotinamide adenine dinucleotide phosphate （NADPH） oxidaseoverexpressing BY-2 cells revealed that PLD and PA are the key factors leading to NADPH oxidase activation, which is responsible for H2O2 and TBARS production induced by Cu^2＋. Moreover, the content of ascorbic acid （AsA）, an effective antioxidant, was sharply reduced in BY-2 cells exposed to excessive Cu^2＋. Furthermore, a significant downregulation of the enzymes of AsA biosynthesis and the antioxidant system was found. This evidence suggests that excessive Cu^2＋-elevated reactive oxygen species （ROS） production is caused by upregulated PLD that elevates the activity of NADPH oxidase and its collapsed antioxidant systems that scavenges ROS.

Association of nicotinamide adenine dinucleotide phosphate oxidase p22phox gene 549C〉T polymorphism with coronary artery disease

Background The p22phox is a critical component of the superoxide-generating vascular nicotinamide adenine dinucleotide phosphate （NADPH） oxidase. Several polymorphisms in p22phox gene are studied for their association with cardiovascular diseases. However, no publication is available to assess the relation of 549C〉T polymorphism in p22pho~ gene to coronary artery disease （CAD） risk. This study was to investigate the effect of the p22phox gene 549C〉T polymorphism on CAD risk. Methods Hospital-based case-control study was conducted with 297 CAD patients and 343 healthy persons as the control group. Polymerase chain reaction and pyrosequencing using PSQ 96 MA Pyrosequencer （Biotage AB） were used to detect the polymorphisms. Multiple Logistic regression model was used to adjust the potential confounders and to estimate odds ratio （OR） with 95% confidence intervals （CIs）. Results The observed genotype frequencies of this polymorphism obeyed the Hardy-Weinberg equilibrium in both cases （P=0.439） and controls （P=-0.668）. The frequency of mutant genotypes （＇I-I-＋CT） in cases （41.08%） was higher than that in controls （36.73%） with an OR=1.20 （95% C1=0.87-1.65）. After the adjustment of the potential confounders, there was a significant association of the mutant genotypes with increased risk of CAD （OR=1.57, 95% C1=1.01-2.46, P=0.047）. Conclusions The mutant genotypes of the p22phox gene 549C〉T polymorphism had a significant effect on the increased risk of CAD in this studied population.

Pioglitazone inhibits the expression of nicotinamide adenine dinucleotide phosphate oxidase and p38 mitogen-activated protein kinase in rat mesangial cells

Background Oxidative Stress and p38 mitogen-activated protein kinase （p38MAPK） play a vital role in renal fibrosis. Pioglitazone can protect kidney but the underlying mechanisms are less clear. The purpose of this study was to investigate the effect of pioglitazone on oxidative stress and whether the severity of oxidative stress was associated with the phosphorylation level of p38MAPK.

NOX4 promotes tumor progression through the MAPK-MEK1/2-ERK1/2 axis in colorectal cancer

BACKGROUND Metabolic reprogramming plays a key role in cancer progression and clinical outcomes;however,the patterns and primary regulators of metabolic reprogramming in colorectal cancer(CRC)are not well understood.AIM To explore the role of nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)in promoting progression of CRC.METHODS We evaluated the expression and function of dysregulated and survival-related metabolic genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes.Consensus clustering was used to cluster CRC based on dysregulated metabolic genes.A prediction model was constructed based on survival-related metabolic genes.Sphere formation,migration,invasion,proliferation,apoptosis and clone formation was used to evaluate the biological function of NOX4 in CRC.mRNA sequencing was utilized to explore the alterations of gene expression NOX4 over-expression tumor cells.In vivo subcutaneous and lung metastasis mouse tumor model was used to explore the effect of NOX4 on tumor growth.RESULTS We comprehensively analyzed 3341 metabolic genes in CRC and identified three clusters based on dysregulated metabolic genes.Among these genes,NOX4 was highly expressed in tumor tissues and correlated with worse survival.In vitro,NOX4 overexpression induced clone formation,migration,invasion,and stemness in CRC cells.Furthermore,RNA-sequencing analysis revealed that NOX4 overexpression activated the mitogen-activated protein kinase-MEK1/2-ERK1/2 signaling pathway.Trametinib,a MEK1/2 inhibitor,abolished the NOX4-mediated tumor progression.In vivo,NOX4 overexpression promoted subcutaneous tumor growth and lung metastasis,whereas trametinib treatment can reversed the metastasis.CONCLUSION Our study comprehensively analyzed metabolic gene expression and highlighted the importance of NOX4 in promoting CRC metastasis,suggesting that trametinib could be a potential therapeutic drugs of CRC clinical therapy targeting NOX4.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2） was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco＇s modified Eagle＇s medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-＇reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine （8-OHdG）, 4-hydroxy-2-nonenal （4-HNE） and 3-nitrotyrosin （3-NT） in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

Abdominal paracentesis drainage ameliorates myocardial injury in severe experimental pancreatitis rats through suppressing oxidative stress

BACKGROUND Abdominal paracentesis drainage(APD)is a safe and effective strategy for severe acute pancreatitis(SAP)patients.However,the effects of APD treatment on SAPassociated cardiac injury remain unknown.AIM To investigate the protective effects of APD on SAP-associated cardiac injury and the underlying mechanisms.METHODS SAP was induced by 5%sodium taurocholate retrograde injection in Sprague-Dawley rats.APD was performed by inserting a drainage tube with a vacuum ball into the lower right abdomen of the rats immediately after SAP induction.Morphological staining,serum amylase and inflammatory mediators,serum and ascites high mobility group box(HMGB)1,cardiac-related enzymes indexes and cardiac function,oxidative stress markers and apoptosis and associated proteins were assessed in the myocardium in SAP rats.Nicotinamide adenine dinucleotide phosphate oxidase activity and mRNA and protein expression were also examined.RESULTS APD treatment improved cardiac morphological changes,inhibited cardiac dysfunction,decreased cardiac enzymes and reduced cardiomyocyte apoptosis,proapoptotic Bax and cleaved caspase-3 protein levels.APD significantly decreased serum levels of HMGB1,inhibited nicotinamide adenine dinucleotide phosphate oxidase expression and ultimately alleviated cardiac oxidative injury.Furthermore,the activation of cardiac nicotinamide adenine dinucleotide phosphate oxidase by pancreatitis-associated ascitic fluid intraperitoneal injection was effectively inhibited by adding anti-HMGB1 neutralizing antibody in rats with mild acute pancreatitis.CONCLUSION APD treatment could exert cardioprotective effects on SAP-associated cardiac injury through suppressing HMGB1-mediated oxidative stress,which may be a novel mechanism behind the effectiveness of APD on SAP.

Gene Expressions Underlying Mishandled Calcium Clearance and Elevated Generation of Reactive Oxygen Species in the Coronary Artery Smooth Muscle Cells of Chronic Heart Failure Rats

Background： The calcium clearance and reactive oxygen species （ROS） generations in the coronary artery smooth muscle cells in chronic heart failure （HF） have not been fully investigated. Therefore, we attempted to understand the gene expressions underlying the mishandling of calcium clearance and the accumulations of ROS. Methods： We initially established an animal model of chronic HF by making the left anterior descending coronary artery ligation （CAL） in rats, and then isolated the coronary artery vascular smooth muscle cells from the ischemic and the nonischemic parts of the coronary artery vessels in 12 weeks after CAL operation. The intracellular calcium concentration and ROS level were measured using flow cytometry, and the gene expressions of sarco/endoplasmic reticulum Ca2＋-ATPase （SERCA2a）, encoding sarcoplasmic reticulum Ca2＋-ATPase 2a, encoding sodium-calcium exchanger （NCX）, andp47phox encoding a subunit of the nicotinamide adenine dinucleotide phosphate （NADPH） oxidase were examined using real-time quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Results： We found that the calcium accumulation and ROS generation in the coronary artery smooth muscle cells isolated from either the ischemic or the nonischemic part of the CAL coronary artery vessel were significantly increased irrespective of blood supply （all P 〈 0.01 ）. Moreover, these were accompanied by the increased expressions of NCX and p47phox, the decreased expression of S ERCA2a, and the increased amount of phosphorylated forms of p47phox in NADPH oxidase （all P 〈 0.05）. Conclusions： Our results demonstrated that the disordered calcium clearance and the increased ROS generation occurred in the coronary artery smooth muscle cells in rats with chronic HF produced by ligation of the left anterior descending coronary artery （CAL）, and which was found to be disassociated from blood supply, and the increased generation of ROS in the ceils was found to make concomitancy to the increased activity of NADPH oxidase in cytoplasm.

Role of reactive oxygen species in the renal fibrosis

Renal fibrosis is a common pathway of progressive renal diseases leading to end-stage renal disease regardless of the etiology. Accumulating evidence indicates that oxidative stress, resulting in generation of reactive oxygen species （ROS）, plays a critical role in the initiation and progression of fibrotic diseases. Nicotinamide adenine dinucleotide phosphate （NADPH） oxidase is the predominant enzyme source for ROS generation and is now recognized as a key mediator of cell proliferation and matrix accumulation in renal disease. Multiple stimuli and agonists, such as transforming growth factor , tumor necrosis factor, platelet derived growth factor, angiotensin II, hyperglycemia, oxidized low-density lipoprotein and albumin have been shown to alter the activity or expression of the NADPH oxidase and ultimately increase ROS production. ROS directly incites damage to biologically important macromolecules and leads to generation of the so-called advanced oxidation protein products （AOPPs） and advanced glycation end products, which are not only markers of oxidative stress but also cause renal injury. Targeting NADPH oxidase and/or reducing AOPPs production miaht be a novel strateav for the theraoeutic intervention of varietv of fibrotic kidney disorders.

The Notch pathway attenuates burn-induced acute lung injury in rats by repressing reactive oxygen species

Background:Acute lung injury(ALI)is a common complication following severe burns.The underlying mechanisms of ALI are incompletely understood;thus,available treatments are not sufficient to repair the lung tissue after ALI.Methods:To investigate the relationship between the Notch pathway and burn-induced lung injury,we established a rat burn injury model by scalding and verified lung injury via lung injury evaluations,including hematoxylin and eosin(H&E)staining,lung injury scoring,bronchoalveolar lavage fluid and wet/dry ratio analyses,myeloperoxidase immunohistochemical staining and reac-tive oxygen species(ROS)accumulation analysis.To explore whether burn injury affects Notch1 expression,we detected the expression of Notch1 and Hes1 after burn injury.Then,we extracted pulmonary microvascular endothelial cells(PMVECs)and conducted Notch pathway inhibition and activation experiments,via aγ-secretase inhibitor(GSI)and OP9-DLL1 coculture,respectively,to verify the regulatory effect of the Notch pathway on ROS accumulation and apoptosis in burn-serum-stimulated PMVECs.To investigate the regulatory effect of the Notch pathway on ROS accumulation,we detected the expression of oxidative-stress-related molecules such as superoxide dismutase,nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(NOX)2,NOX4 and cleaved caspase-3.NOX4-specific small interfering RNA(siRNA)and the inhibitor GKT137831 were used to verify the regulatory effect of the Notch pathway on ROS via NOX4.Results:We successfully established a burn model and revealed that lung injury,excessive ROS accumulation and an inflammatory response occurred.Notch1 detection showed that the expression of Notch1 was significantly increased after burn injury.In PMVECs challenged with burn serum,ROS and cell death were elevated.Moreover,when the Notch pathway was suppressed by GSI,ROS and cell apoptosis levels were significantly increased.Conversely,these parameters were reduced when the Notch pathway was activated by OP9-DLL1.Mechanistically,the inhibition of NOX4 by siRNA and GKT137831 showed that the Notch pathway reduced ROS production and cell apoptosis by downregulating the expression of NOX4 in PMVECs.Conclusions:The Notch pathway reduced ROS production and apoptosis by downregulating the expression of NOX4 in burn-stimulated PMVECs.The Notch-NOX4 pathway may be a novel therapeutic target to treat burn-induced ALI.

High salt-induced weakness of anti-oxidative function of natriuretic peptide receptor-C and podocyte damage in the kidneys of Dahl rats

Background:Atrial natriuretic peptide(ANP)and its natriuretic peptide receptors A(NPR-A)and C(NPR-C)are involved in the regulation of physiological and pathophysiological process of blood pressure.The present study aimed to determine the role of NPR-C in the development of salt-sensitive hypertension.Methods::The Dahl salt-sensitive(DS)and salt-resistant(DR)rats were used in this study.Animals were matched according to their age and weight,and then placed on either a high-salt(HS,8%)or a normal-salt(NS,0.4%)diet for 6 weeks randomly using random number table.The systolic blood pressure(SBP),plasmatic sodium concentration(PL Na),urinary sodium excretion(UV Na),and serum creatinine concentration(Scr)were measured.The concentration of ANP in blood and tissues(heart and kidney)was detected by enzyme-linked immunosorbent assay.The expression of ANP,NPR-A,and NPR-C in kidney was evaluated with western blot analysis.Regarding renal redox state,the concentration changes in malondialdehyde(MDA),lipofuscin,nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(Nox),and nitric oxide synthase(NOS)in kidney were detected by a spectrophotometric method.The kidney damage was evaluated using pathological techniques and the succinodehydrogenase(SDHase)examination.Furthermore,after an intra-peritoneal injection of C-atrial natriuretic peptide(ANP)4-23(C-ANP 4-23),an NPR-C receptor agonist,the SBP,biochemical values in blood and urine,and renal redox state were evaluated.The paired Student’s t test and analysis of variance followed by the Bonferroni test were performed for statistical analyses of the comparisons between two groups and multiple groups,respectively.Results::The baseline SBP in all groups was within the normal range.At the end of the 6-week experiment,HS diet significantly increased the SBP in DS rats from 116.63±2.90 mmHg to 162.25±2.15 mmHg(t=-10.213,P<0.001).The changes of SBP were not significant in DS rats on an NS diet and DR rats on an NS diet or on an HS diet(all P>0.05).The significant increase of PL Na,UV Na,and Scr related to an HS diet was found in both DS and DR rats(all P<0.05).However,significant changes in the concentration(t=-21.915,P<0.001)and expression of renal ANP(t=-3.566,P=0.016)and the expression of renal NPR-C(t=5.864,P=0.002)were only observed in DS hypertensive rats.The significantly higher desmin immunochemical staining score(t=-5.715,P=0.005)and mitochondrial injury score(t=-6.325,P=0.003)accompanied by the lower SDHase concentration(t=3.972,P=0.017)revealed mitochondrial pathologic abnormalities in podocytes in DS rats with an HS diet.The distinct increases of MDA(t=-4.685,P=0.009),lipofuscin(t=-8.195,P=0.001),and Nox(t=-12.733,P<0.001)but not NOS(t=-0.328,P=0.764)in kidneys were also found in DS hypertensive rats.C-ANP 4-23 treatment significantly decreased the SBP induced by HS in DS rats(P<0.05),which was still higher than NS groups with the vehicle or C-ANP 4-23 treatment(P<0.05).Moreover,the HS-induced increase of MDA,lipofuscin,Nox concentrations,and Nox4 expression in DS rats was significantly attenuated by C-ANP 4-23 treatment as compared with those with HS diet and vehicle injection(all P<0.05).Conclusions::The results indicated that the renal NPR-C might be involved in the salt-sensitive hypertension through the damage of mitochondria in podocytes and the reduction of the anti-oxidative function.Hence,C-ANP 4-23 might serve as a therapeutic agent in treating salt-sensitive hypertension.