NOX4 Expression Levels Research Articles

Oxymatrine Ameliorates Memory Impairment in Diabetic Rats by Regulating Oxidative Stress and Apoptosis: Involvement of NOX2/NOX4.

Oxymatrine (OMT) is the major quinolizidine alkaloid extracted from the root of Sophora flavescens Ait and has been shown to exhibit a diverse range of pharmacological properties. The aim of the present study was to investigate the role of OMT in diabetic brain injury in vivo and in vitro. Diabetic rats were induced by intraperitoneal injection of a single dose of 65 mg/kg streptozotocin (STZ) and fed a high-fat and high-cholesterol diet. Memory function was assessed using a Morris water maze test. A SH-SY5Y cell injury model was induced by incubation with glucose (30 mM/l) to simulate damage in vitro. The serum fasting blood glucose, insulin, serum S100B, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were analyzed using commercial kits. Morphological changes were observed using Nissl staining and electron microscopy. Cell apoptosis was assessed using Hoechst staining and TUNEL staining. NADPH oxidase (NOX) and caspase-3 activities were determined. The effects of NOX2 and NOX4 knockdown were assessed using small interfering RNA. The expression levels of NOX1, NOX2, and NOX4 were detected using reverse transcription-quantitative PCR and western blotting, and the levels of caspase-3 were detected using western blotting. The diabetic rats exhibited significantly increased plasma glucose, insulin, reactive oxygen species (ROS), S-100B, and MDA levels and decreased SOD levels. Memory function was determined by assessing the percentage of time spent in the target quadrant, the number of times the platform was crossed, escape latency, and mean path length and was found to be significantly reduced in the diabetic rats. Hyperglycemia resulted in notable brain injury, including histological changes and apoptosis in the cortex and hippocampus. The expression levels of NOX2 and NOX4 were significantly upregulated at the protein and mRNA levels, and NOX1 expression was not altered in the diabetic rats. NOX and caspase-3 activities were increased, and caspase-3 expression was upregulated in the brain tissue of diabetic rats. OMT treatment dose-dependently reversed behavioral, biochemical, and molecular changes in the diabetic rats. In vitro, high glucose resulted in increases in reactive oxygen species (ROS), MDA levels, apoptosis, and the expressions of NOX2, NOX4, and caspase-3. siRNA-mediated knockdown of NOX2 and NOX4 decreased NOX2 and NOX4 expression levels, respectively, and reduced ROS levels and apoptosis. The results of the present study suggest that OMT alleviates diabetes-associated cognitive decline, oxidative stress, and apoptosis via NOX2 and NOX4 inhibition.

Alpha-lipoic acid ameliorates H2O2-induced human vein endothelial cells injury via suppression of inflammation and oxidative stress

The study was aimed to investigate the effect of alpha-lipoic acid (ALA) on human umbilical vein endothelial cells (HUVECs) injury induced by hydrogen peroxide (H2O2) and to explore its possible mechanisms. We established the H2O2-induced HUVECs injury model and the ALA treatment groups in which HUVECs were co-incubated with H2O2 (250μmol/L) and different final concentrations of ALA (100,200,400μmol/L) for 48h. Cell survival rate assay and LDH activity assay were carried out. The levels of related proteins were performed by Western Blot. We observed that H2O2 administration resulted in an increase in the LDH activity and a decrease in cell survival rate. The expression levels of Nox4, Bax, NF-κB p65, Caspase-9, Caspase-3, iNOS, VCAM-1 and ICAM-1 were up-regulated, while the expression level of Bcl-2 was down-regulated. All these factors were significantly improved by ALA treatment. In brief, ALA treatment ameliorates H2O2-induced HUVECs damage by inhibiting inflammation and oxidative stress.

Morroniside Inhibits H2O2-Induced Podocyte Apoptosis by Down-Regulating NOX4 Expression Controlled by Autophagy In Vitro.

Podocyte apoptosis is the common pathological basis for the progression of various kidney diseases. The overexpression of NOX4, a key enzyme involved in oxidative stress, has been proved to participate in the occurrence of podocyte apoptosis. Autophagy is a kind of adaptive response of cells under stress. However, as a “double-edged sword”, the effect of autophagy on apoptosis in different cells and conditions is complex and variable, which has not been fully explained yet. Morroniside, extracted from the traditional medicinal plant Cornus officinalis, has remarkable antioxidant and anti-apoptosis effects, and has been proven to inhibit the overexpression of NOX4 in kidney tissue. Therefore, H2O2 was used in this study to explore the effects of autophagy on podocyte NOX4 overexpression and apoptosis induced by oxidative stress, as well as the protection mechanism of morroniside in podocytes. The results showed that the autophagy activator rapamycin, as well as the autophagy inhibitor chloroquine, could induce podocyte apoptosis cultured in normal condition, and chloroquine could also significantly increase the NOX4 expression. The NOX4 expression and apoptosis rate of podocytes increased after H2O2 treatment, the expression of LC3-II decreased, and the expressions of p62, mTOR, and p-mTOR increased. The intervention of morroniside and rapamycin improved autophagy activity and inhibited NOX4 overexpression and apoptosis induced by H2O2. And chloroquine reversed the inhibitory effect of morroniside on NOX4 overexpression and podocyte apoptosis. Taken together, our results suggest that the expression level of NOX4 in podocytes is regulated by autophagy activity. Morroniside can reduce oxidative stress induced podocyte apoptosis by restoring the damaged autophagy flux and inhibit the overexpression of NOX4.

Isoorientin Attenuates Cisplatin-Induced Nephrotoxicity Through the Inhibition of Oxidative Stress and Apoptosis via Activating the SIRT1/SIRT6/Nrf-2 Pathway.

Cisplatin (CDDP) is a widely used chemotherapeutic agent for various solid tumors, but its severe side effects, particularly nephrotoxicity, limit its clinical application. Isoorientin (Iso) is a flavonoid-like compound known to have antioxidant effects. As oxidative injury plays a vital role in CDDP-induced acute kidney injury (AKI), the effect of Iso on CDDP-induced nephrotoxicity has not yet been researched. We assessed the effects of Iso against CDDP-induced nephrotoxicity in vitro using mTEC cells and further explored the mechanisms underlying CDDP-induced renal dysfunction in vivo in WT and Nrf2–/– mice. The results showed that Iso treatment significantly reduced CDDP-induced nephrotoxicity via attenuating cell damage in vitro and via ameliorating renal injury, as determined by biochemical markers, in mice. The molecular mechanism underlying this protection was also investigated. Iso up-regulated the expression levels of SIRT1 and SIRT6 in vivo and in vitro. In addition, Iso activated Nrf2 translocation and the expression levels of its downstream antioxidant enzymes, such as HO-1 and NQO1, whereas it inhibited the expression level of NOX4, thus decreasing oxidative stress. Notably, the protective effects of Iso observed in WT mice were completely abolished in Nrf2–/– mice. Collectively, these data indicate that the protective effect of Iso on CDDP-induced nephrotoxicity by SIRT1- and SIRT6-mediated Nrf2 activation regulates oxidative stress, inflammation and apoptosis. The absence of Nrf2 exacerbates CDDP-induced renal damage, and the pharmacological activation of Nrf2 may represent a novel therapy to prevent kidney injury.

Enhancer of zeste homolog 2 modulates oxidative stress-mediated pyroptosis in vitro and in a mouse kidney ischemia-reperfusion injury model.

Enhancer of zeste homolog 2 (EZH2), a well-known methyltransferase, mediates histone H3 lysine 27 trimethylation (H3K27me3) and plays a crucial role in several kidney disease models. However, its role in renal ischemia/reperfusion (I/R) injury still remains unclear. In this study, we found that EZH2 was positively related to renal I/R injury and inhibition of EZH2 with DZNeP alleviated I/R injury and blocked the activation of oxidative stress and pyroptosis in vivo. Similarly, inhibition of EZH2 with either DZNeP or si-RNA also exerted an inhibitory effect on hypoxia/reoxygenation (H/R)-induced oxidative stress and pyroptosis in vitro. Moreover, further study revealed that ablation of reactive oxygen species (ROS) with N-acetyl-cysteine (NAC) suppressed pyroptosis in human renal proximal tubular epithelial cell line cells exposed to H/R stimulation. Furthermore, Nox4, which was positively related to the generation of ROS, was upregulated during H/R process, while it could be reversed by EZH2 inhibition. Consistently, Nox4-mediated ROS generation was attenuated upon inhibition of EZH2 with DZNeP or si-RNA. Additionally, the transcriptional activity of Nox4 was enhanced by the activation of ALK5/Smad2/3 signaling pathway, which was abolished by ALK5 knockdown in vitro. Finally, EZH2 inhibition blocked H/R and I/R-activated ALK5/Smad2/3 pathway and also resulted in an obvious decrease in the transcriptional activity and protein expression levels of Nox4. In conclusion, our results proved that EZH2 inhibition alleviated renal pyroptosis by blocking Nox4-dependent ROS generation through ALK5/Smad2/3 signaling pathway, indicating that EZH2 could be a potential therapeutic target for renal I/R injury.

High nuclear NADPH oxidase 4 expression levels are correlated with cancer development and poor prognosis in hepatocellular carcinoma

NADPH oxidase (NOX) is a key source of reactive oxygen species (ROS). This study aimed to verify NOX2 and NOX4 expression levels in hepatocellular carcinoma (HCC). A total of 134 matched pairs of HCC cells and non-tumour hepatocytes from 134 patients were examined by immunohistochemical staining, and the association of NOX2 and NOX4 expression with clinicopathological parameters was analysed. Western blotting in four HCC cell lines and reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) in 20 pairs of HCC and non-tumour tissue samples were also performed to detect NOX4. Cytoplasmic NOX2 and nuclear NOX4 expression levels were shown by immunohistochemistry to be higher in HCC cells than in non-tumour hepatocytes (p<0.001 each). The western blotting results for NOX4 in four HCC cell lines were consistent with the immunohistochemical results. Increased cytoplasmic expression of NOX2 and NOX4 in HCC cells was significantly correlated with liver cirrhosis (p<0.001 and p<0.031, respectively). However, decreased cytoplasmic expression of NOX2 and NOX4 was significantly correlated with advanced pathological TNM stage (p<0.029 and p<0.007, respectively). Multivariate analysis with clinicopathological parameters showed that high nuclear and low cytoplasmic NOX4 expression levels are correlated with short overall survival (p=0 .021). Our findings imply that cytoplasmic NOX2 and nuclear NOX4 expression is upregulated during HCC development. In particular, NOX4 translocation into the nucleus may affect the development and progression of HCC. NOX2 and NOX4 could be diagnostic markers and have therapeutic implications in HCC.

AVE 0991 Attenuates Pyroptosis and Liver Damage after Heatstroke by Inhibiting the ROS-NLRP3 Inflammatory Signalling Pathway.

We previously demonstrated that angiotensin-(1-7) (Ang-(1-7)), an essential endocrine factor, inhibits the NLRP3 inflammasome by regulating reactive oxygen species (ROS) in fibrotic livers. We also demonstrated that the NLRP3 inflammasome contributes to the liver damage induced by pyroptosis after heatstroke. However, the role of Ang-(1-7) in the hepatocytes under heat stress remains uncertain. We aimed to examine the change in angiotensin peptides in the livers affected by heatstroke and the effect on the ROS-NLRP3 inflammatory signalling pathway. In vivo, increased angiotensin II (Ang II) and decreased Ang-(1-7) in the serum of heatstroke patients suffering from hepatic dysfunction were observed. The change in angiotensin peptides was considered a potential biomarker that could be used to predict hepatic dysfunction. Enhanced Ang II and attenuated Ang-(1-7) levels were also observed in the liver tissue of heatstroke rats, which were consistent with their receptors and converting enzymes. Hepatic damage associated with increased ROS and protein expression levels of NOX4, NLRP3, caspase-1, and IL-1β was attenuated by AVE 0991, an analogue of Ang-(1-7). In vitro, pyroptosis, characterized by activated caspase-1 and IL-1β, was observed in hepatocytes under heat stress, which was enhanced by Ang II and attenuated by antioxidants, NOX4 siRNA, and AVE 0991. In summary, AVE 0991 attenuates pyroptosis and liver damage induced by heat stress by inhibiting the ROS-NLRP3 inflammatory signalling pathway.

Plumbagin ameliorates liver fibrosis via a ROS-mediated NF-кB signaling pathway in vitro and in vivo

AimsThe purpose of this study was to investigate plumbagin (PL) on liver fibrosis in vitro and in vivo and to explore the underlying mechanisms. MethodsCarbon tetrachloride (CCl4) was used to establish a rat liver fibrosis model, primary hepatic stellate cells (HSCs) were isolated from the rat liver, and fibrosis-related indicators were detected. ResultsThe results revealed that PL significantly prevented CCl4-induced liver fibrosis, as evidenced by the attenuation of histopathological changes, the decrease of MDA and the increase of SOD and GSH-P X . In addition, PL downregulated the mRNA levels of NOX4 and procollagen I; the protein expression levels of NOX4 and p-IκB; and the transcriptional activity of NF-κB in liver fibrosis rats. Moreover, PL significantly decreased ROS expression, protein expression of α-SMA and collagen III, and activation of NF-κB and inhibited the nuclear translocation of NF-κB p65 in IL-1β-stimulated HSCs in vitro. ConclusionThe results of our study indicate that PL can mitigate liver fibrosis in vitro and in vivo, which may be related to the ROS-mediated NF-кB signaling pathway.

Magnesium isoglycyrrhizinate ameliorates radiation-induced pulmonary fibrosis by inhibiting fibroblast differentiation via the p38MAPK/Akt/Nox4 pathway

Radiation-induced pulmonary fibrosis (RIPF) is a severe complication in patients treated with thoracic irradiation. Until now, there are no effective therapeutic drugs for RIPF. In the present study, we attempted to evaluate the effect of Magnesium isoglycyrrhizinate (MgIG) on RIPF, and to further explore the underlying mechanisms. We found that MgIG treatment markedly improved radiation-induced lung pathological changes, reduced collagen deposition, and decreased the transforming growth factor beta1 (TGF-β1) elevation induced by irradiation. In addition, MgIG treatment significantly relieved oxidative damage of pulmonary fibrosis in mice characterized by increased antioxidant factors expression and reduced oxidative factors expression. And, MgIG treatment also significantly reduced the production of intracellular reactive oxygen species (ROS) in vitro. Interestingly, administration of MgIG achieved lower expression levels of Nox4, and phosphorylation of p38MAPK and Akt in vivo and in vitro. Furthermore, treatment with MgIG notably reduced the expression levels of myofibroblast markers, Nox4, and phosphorylation of p38MAPK and Akt both in vivo and in vitro. More importantly, the inhibitory effects of MgIG on fibroblast differentiation were enhanced when the p38MAPK/Akt/Nox4 pathway was inhibited using their respective antagonists or Nox4 siRNA in vitro. Taken together, these findings suggested that MgIG could attenuate RIPF partly by inhibiting fibroblast differentiation, which was closely related to modulation of the p38MAPK/Akt/Nox4 pathway.

Effect of thermal manipulation of broilers embryos on the response to heat-induced oxidative stress.

Effects of embryonic thermal manipulation (TM) on mRNA expressional levels and total antioxidant capacity of genes associated with heat-induced oxidative stress (NOX4, GpX2, SOD2, catalase, and AvUCP) in 2 breeds of broiler chicken were investigated. Fertile Cobb and Hubbard eggs (n = 1,200) were divided into 4 treatment groups: Cobb control, Cobb TM, Hubbard control, and Hubbard TM. Control groups were maintained under standard conditions (37.8°C; 56% relative humidity), whereas TM groups were incubated at 39°C and 65% relative humidity for 18 h a day from embryonic days (ED) 10 to 18. On post-hatch day 28, the broilers were subject to acute heat stress (AHS) at 40°C for 7 h. At certain intervals (0, 1, 3, 5, and 7h), 12 chickens from each of the 4 groups were humanely euthanized, and liver samples were immediately isolated. During AHS, in both breeds, the mRNA expression levels of NOX4, GPx2, SOD2, and catalase in TM chickens were significantly lower than in controls, but AvUCP mRNA expression in the TM group was higher. The total antioxidant capacity and activity of superoxidase dismutase and catalase were significantly lower in the TM than in the control group in both breeds. The results of this study suggest that TM has a long-lasting effect on the acquisition of thermotolerance in 2 broiler chicken breeds as indicated by the reduction of system genes associated with heat-induced oxidative stress.

Effect of Ambrisentan Therapy on the Expression of Endothelin Receptor, Endothelial Nitric Oxide Synthase and NADPH Oxidase 4 in Monocrotaline-induced Pulmonary Arterial Hypertension Rat Model.

Background and ObjectivesElevated endothelin (ET)-1 level is strongly correlated with the pathogenesis of pulmonary arterial hypertension (PAH). Expression level of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4 is increased in the PAH patients. Ambrisentan, a selective endothelin receptor A (ERA) antagonist, is widely used in PAH therapy. The current study was undertaken to evaluate the effects of ambrisentan treatment in the monocrotaline (MCT)-induced PAH rat model.MethodsRats were categorized into control group (C), monocrotaline group (M) and ambrisentan group (Am). The M and Am were subcutaneously injected 60 mg/kg MCT at day 0, and in Am, ambrisentan was orally administered the day after MCT injection for 4 weeks. The right ventricle (RV) pressure was measured and pathological changes of the lung tissues were observed by Victoria blue staining. Protein expressions of ET-1, ERA, endothelial nitric oxide synthase (eNOS) and NOX4 were confirmed by western blot analysis.ResultsAmbrisentan treatment resulted in a recovery of the body weight and RV/left ventricle+septum at week 4. The RV pressure was lowered at weeks 2 and 4 after ambrisentan administration. Medial wall thickening of pulmonary arterioles and the number of intra-acinar arteries were also attenuated by ambrisentan at week 4. Protein expression levels of ET-1 and eNOS were recovered at weeks 2 and 4, and ERA levels recovered at week 4.ConclusionsAmbrisentan administration resulted in the recovery of ET-1, ERA and eNOS protein expression levels in the PAH model. However, the expression level of NOX4 remained unaffected after ambrisentan treatment.

Metformin Regulates the Expression of SK2 and SK3 in the Atria of Rats With Type 2 Diabetes Mellitus Through the NOX4/p38MAPK Signaling Pathway

We previously found that metformin regulates the ion current conducted by the small conductance calcium-activated potassium channels (SK channels) in the atria of rats with type 2 diabetes mellitus (T2DM) as well as the mRNA and protein expression of the SK2 and SK3 subtypes of SK channels. In this study, we hypothesized that the nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)/p38 mitogen-activated protein kinase (p38MAPK) signaling pathway was involved in the metformin-mediated regulation of SK2 and SK3 expression in the atria of rats with T2DM. We randomly divided Wistar rats into the control group, the untreated T2DM group, the metformin-treated group, the group receiving subcutaneous injections of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor diphenyleneiodonium (DPI), and the group receiving tail vein injections of the p38MAPK agonist anisomycin. Real-time polymerase chain reaction, Western blot, and immunohistochemistry were applied to examine the expression levels of SK2, SK3, NOX4, and phospho-p38MAPK (p-p38MAPK) mRNAs and proteins in the atrial tissue of relevant groups. We observed that the expression levels of NOX4 mRNA and protein and p-p38MAPK protein were significantly elevated in the atria of rats with T2DM compared with the control group. In addition, SK2 protein expression was reduced, whereas SK3 protein expression was increased. The 8-week treatment with metformin markedly reduced the expression levels of NOX4 mRNA and protein and p-p38MAPK protein, upregulated the SK2 expression, and downregulated the SK3 expression. Tail vein injection with anisomycin significantly increased the p-p38MAPK expression while further inhibiting the expression of SK2 and enhancing the expression of SK3. Subcutaneous injection with DPI considerably inhibited the expression of NOX4, further enhanced the expression of SK2 and suppressed the expression of SK3. In addition, subcutaneous injection with DPI significantly suppressed the phosphorylation of p38MAPK. In conclusion, the NOX4/p38MAPK signaling pathway mediates the downregulation of SK2 and the upregulation of SK3 in the atria of rats with T2DM. Long-term metformin treatment upregulates SK2 protein expression and downregulates SK3 protein expression by inhibiting the NOX4/p38MAPK signaling pathway.

Inhibition of NADPH oxidase activities ameliorates DSS-induced colitis

NADPH oxidases (NOX) act to generate reactive oxygen species (ROS) and exhibit microbicidal bioactivity, whereas their roles in mediating immune responses of inflammation in intestine remain to be further elucidated. The study was performed to explore the effects of NOX activity on regulation of macrophage functions. Macrophage responses were induced by lipopolysaccharides (LPS) in RAW 264.7 cells (in vitro) or dextran sulfate sodium (DSS) in BALB/c mice (in vivo) respectively. LPS induced NOX2 expression and initiated NOX activities in RAW 264.7 cells. Conversely, inhibition of NOX activity by DPI and VAS2870 diminished LPS induced NOX activities and the downstream signaling in RAW 264.7 cells. Murine colitis was characterized by macrophage accumulation and elevation of NOX activities in colon tissues. DPI and VAS2870 administration overrode NOX activities and ROS productions in colon tissues, and ameliorated DSS-induced colitis evidenced with the reduced disease activities and the decreased cytokine levels. Intriguingly, NOX2 expression levels were elevated in colon tissues of patients with active inflammatory bowel disease. Together, our data show a crucial role of NOX activity in regulation of macrophage functions and responses, and suggest that NOX represents a novel therapeutic approach for the management of immune diseases.

The Expression and Prognostic Value of NOX4 in Gastric Cancer

Background: Gastric cancer (GC) is one of the most common malignant tumors worldwide which threaten the health of human. A lot of work has been done in tumor pathogenesis in recent years, while modest progress in diagnosis and treatment of gastric cancer have been made. Methods: Multiple databases including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were explored to identify the expression pattern of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 (NOX4), the main source of reactive oxygen species, in gastric cancer. We also analyzed the correlation between the expression level of NOX4 and gastric cancer patients’ clinical features. The prognostic value of NOX4 was explored in TCGA and K-M Plotter. Last, we utilized TCGA and DAVID databases to uncover the underlying molecular mechanisms of NOX4 with TCGA and DAVID databases. Results: We found that NOX4 was upregulated in tumors compared with adjacent non-tumor tissues and overexpression was correlated with tumor invasion and TNM stage in gastric cancer. Furthermore, NOX4 level could be an independent prognostic marker for GC, unacted on the choice of therapy or HER2 expression. Besides, we investigated the potential mechanisms of NOX4 in gastric cancer. Conclusions: Our findings proved that NOX4 may be a new prognostic factor or therapeutic marker for gastric cancer.

Abstract 036: Interference With PPARγ in the Endothelium Produces Endothelial Dysfunction in the Cerebral Circulation in Response to Activation of the Endogenous Renin-Angiotensin System

Low salt diet (LSD) is beneficial in salt-sensitive hypertension but may provoke cardiovascular risk in patients with heart failure, diabetes, or other cardiovascular abnormalities because of renin-angiotensin system (RAS) activation. PPARγ is a transcription factor which promotes an anti-oxidant pathway in the endothelium. We studied transgenic mice expressing a dominant-negative mutation in PPARγ selectively in the endothelium (E-DN) to test the hypothesis that endothelial PPARγ plays a protective role in response to LSD-mediated RAS activation. Plasma renin and angiotensin were significantly and equally increased in all mice fed LSD for 6-weeks (Renin - NT: 39±7 vs 20±1 ng/ml; E-DN: 34±1 vs 16±4 ng/ml; Ang - NT: 257±54 vs 47±6 pg/ml; E-DN: 294±69 vs 63±14 pg/ml p&lt;0.05, n=5). Vasorelaxation to acetylcholine was not affected in basilar artery from E-DN at baseline, but was significantly and selectively impaired in E-DN after LSD (33±5 vs 69±2%, p&lt;0.05, n=6). Unlike basilar artery, LSD was not sufficient to induce vascular dysfunction in carotid artery (carotid artery: 86±4 vs 92±3%, n=5). Endothelial dysfunction in the basilar artery from E-DN mice fed LSD was attenuated by scavengers of superoxide (improved from 29±5% to 55±7%, n=6), inhibitors of NADPH oxidase (improved from 23±3% to 54±7%, p&lt;0.05, n=6), or blockade of the angiotensin-II AT1 receptor (improved from 31±5% to 64±9%, p&lt;0.05, n=5). Gene expression levels of Nox2 was elevated (2.1±0.3 vs 0.4±0.1, p&lt;0.05, n=7) while those of antioxidant enzymes catalase and SOD3 were blunted in cerebral vessels of E-DN mice on a LSD (catalase: 0.5±0.1 vs 2.5±0.2; SOD3: 0.2±0.1 vs 1.1±0.1, p&lt;0.05, n=7). Simultaneous AT1 and AT2 receptor blockade revealed the restoration of endothelial function after AT1 receptor blockade was not a consequence of AT2 receptor activation (59±10 vs 48±2, p&lt;0.05, n=4). We conclude that interference with PPARγ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to LSD-mediated activation of the endogenous RAS, mediated at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the cerebral circulation may be particularly sensitive to inhibition of PPARγ activity and RAS activation.

Inhibition of NADPH Oxidase Activities Ameliorates DSS-Induced Colitis

NADPH oxidases (NOX) act to generate reactive oxygen species (ROS) and exhibit microbicidal bioactivity, whereas their roles in mediating immune responses of inflammation in intestine remain to be further elucidated. The study was performed to explore the effects of NOX activity on regulation of macrophage functions. Macrophage responses were induced by lipopolysaccharides (LPS) in RAW 264.7 cells (in vitro) or dextran sulfate sodium (DSS) in BALB/c mice (in vivo) respectively. LPS induced NOX2 expression and initiated NOX activities in RAW 264.7 cells. Conversely, inhibition of NOX activity by DPI and VAS2870 diminished LPS induced NOX activities and the downstream signaling in RAW 264.7 cells. Murine colitis was characterized by macrophage accumulation and elevation of NOX activities in colon tissues. DPI and VAS2870 administration overrode NOX activities and ROS productions in colon tissues, and ameliorated DSS-induced colitis evidenced with the reduced disease activities and the decreased cytokine levels. Intriguingly, NOX2 expression levels were elevated in colon tissues of patients with active inflammatory bowel disease. Together, our data show a crucial role of NOX activity in regulation of macrophage functions and responses, and suggest that NOX represents a novel therapeutic approach for the management of immune diseases. Funding Statement: This work was supported by National Natural Science Foundation of China, No. 81770555, 81470828, 81270472(AB); Principle Investigator Program of Jiangxi Province, No. 20153BCB22010 (AB); and Natural Science Foundation of Jiangxi Province, No. 20142BAB205048 (AB). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was reviewed and approved by the Ethics Committee of Nanchang University.

Methyl ferulic acid exerts anti-apoptotic effects on L-02 cells via the ROS-mediated signaling pathway.

The present study aimed to investigate the anti-apoptotic effects of methyl ferulic acid (MFA) on L-02 cell apoptosis induced by ethanol, and to elucidate the possible underlying mechanisms. L-02 cells were examined after being soaked in ethanol (400mM) to allow the ethanol to permeate into the cells for 24h. Cell survival was measured by MTT assay. Cell apoptosis was assessed by both flow cytometry and single-stranded DNA assays. Intracellular reactive oxygen species(ROS) production was determined using the 2',7'-dichlorofluorescein-diacetate dye. The protein expression levels of p38, p-p38, JNK, p-JNK, NADPH oxidase4(NOX4), p22, Bax and Bcl-2 were measured by western blot analysis. The mRNA expression levels of NOX4 and p22 were measured by RT-PCR. It was identified that MFA markedly suppressed the ethanol-induced apoptosis and necrosis of L-02 cells. In addition, MFA decreased the expression levels of superoxide dismutase, catalase and phospholipid hydroperoxide gluthione peroxidase, and downregulated the levels of Bax/Bcl-2 and the cleaved forms of caspase-3 in a dose- and time-dependent manner. This indicated that MFA attenuated the apoptosis of L-02 cells. MFA also decreased the elevated mRNA and protein expression levels of Nox4 and p22phox, and the production of intracellular ROS triggered by ethanol. Further analysis demonstrated that MFA significantly attenuated the phosphorylation of JNK and p38, which are major components of the mitogen-activated protein kinase(MAPK) pathways. On the whole, the findings of this study demonstrated that MFA attenuated the apoptotic cell death of L-02 cells by reducing the generation of ROS and inactivating the MAPK pathways.

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-oxidative 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 improved 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.

NADPH oxidase 4 and endothelial nitric oxide synthase contribute to endothelial dysfunction mediated by histone methylations in metabolic memory.

"Metabolic memory" is identified as a phenomenon that transient hyperglycemia can be remembered by vasculature for quite a long term even after reestablishment of normoglycemia. NADPH oxidases (Noxs) and endothelial nitric oxide synthase (eNOS) are important enzymatic sources of reactive oxygen species (ROS) in diabetic vasculature. The aim of this study is to explore the roles of epigenetics and ROS derived from Noxs and eNOS in the metabolic memory. In this study, we demonstrated that vascular ROS was continuously activated in endothelium induced by transient high glucose, as well as sustained vascular endothelial dysfunction. The Nox4 and uncoupled eNOS are the major sources of ROS, while inhibition of Nox4 and eNOS significantly attenuated oxidative stress and almost recovered the endothelial function in metabolic memory. Furthermore, the aberrant histone methylation (H3K4me1, H3K9me2, and H3K9me3) at promoters of Nox4 and eNOS are the main causes for the persistent up-regulation of these two genes. Modifying the histone methylation could reduce the expression levels of Nox4 and eNOS, thus obviously attenuating endothelial dysfunction. These results indicate that histone methylation of Nox4 and eNOS play a key role in metabolic memory and may be the potential intervention targets for metabolic memory.

Involvement of NADPH oxidase in high-dose phenolic acid-induced pro-oxidant activity on rat mesenteric venules.

In the present study, we investigated the potential role of phenolic acids in initiating oxidative damage to microvascular endothelial cells and the underlying mechanism mediating the pro-oxidant action. Male Wistar rats received high doses of phenolic acid [caffeic acid (CA), salvianolic acid B (SAB), chlorogenic acid (ChA) or ferulic acid (FA)]. The creation of reactive oxygen species in mesenteric microcirculation endothelial cells and adherent leukocytes along with venules were assessed using intravital microscopy. The expression levels of NADPH oxidase subunits (Nox4 and p22phox) in terminal ileum tissues were determined by western blot analysis. Intravenous injection of high-dose ChA or CA (7 mg/kg) markedly increased the peroxide production in the venular walls and upregulated the protein expression levels of Nox4 and p22phox in the ileum tissues, while the same dose of CA and SAB made no difference within the observation period. No changes were observed in the number of leukocytes adhering to the venular walls. High-dose ChA and FA led to an imbalance between the oxidant and antioxidant mechanism by boosting the expression levels of NADPH oxidase. Thus, we clarified the rationale behind the adverse effects of a herbal injection containing high levels of phenolic acid compounds.