NADPH Oxidase Expression Research Articles

Oleic acid-induced metastasis of KRAS/p53-mutant colorectal cancer relies on concurrent KRAS activation and IL-8 expression bypassing EGFR activation.

Background: Multigene mutations in colorectal cancer (CRC), including KRAS, BRAF, and p53, afford high metastatic ability and resistance to EGFR-targeting therapy. Understanding the molecular mechanisms regulating anti-EGFR-resistant CRC metastasis can improve CRC therapy. This study aimed to investigate the effects of IL-8 and the activation of KRAS on reactive oxygen species (ROS) production and metastasis of hyperlipidemia-associated CRC harboring mutations of KRAS and p53. Methods: The cytokine array analysis determined the up-expression of secreted factors, including IL-8. The clinical relevance of the relationship between IL-8 and angiopoietin-like 4 (ANGPTL4) was examined in CRC patients from National Cheng Kung University Hospital and TCGA dataset. Expressions of IL-8, ANGPTL4, NADPH oxidase 4 (NOX4), and epithelial-mesenchymal transition (EMT) markers in free fatty acids (FFAs)-treated KRAS/p53 mutant CRC cells were determined. The hyperlipidemia-triggered metastatic ability of CRC cells under treatments of antioxidants, statin, and cetuximab or knockdown of IL-8, KRAS, and EGFR was evaluated in vitro and in vivo. In addition, the effects of antioxidants and depletion of IL-8 and KRAS on the correlation between ROS production and hyperlipidemia-promoted CRC metastasis were also clarified. Results: In this study, we found that free fatty acids promoted KRAS/p53-mutant but not single-mutant or non-mutant CRC cell metastasis. IL-8, the most abundant secreted factor in KRAS/p53-mutant cells, was correlated with the upregulation of NOX4 expression and ROS production under oleic acid (OA)-treated conditions. In addition, the metastasis of KRAS/p53-mutant CRC relies on the ANGPTL4/IL-8/NOX4 axis and the activation of KRAS. The antioxidants and inactivation of KRAS also inhibited OA-induced EMT and metastasis. Although KRAS mediated EGF- and OA-promoted CRC cell invasion, the inhibition of EGFR did not affect OA-induced ANGPTL4/IL-8/NOX4 axis and CRC metastasis. The high-fat diet mice fed with vitamin E and statin or in IL-8-depleted cells significantly inhibited tumor extravasation and metastatic lung growth of CRC. Conclusion: The antioxidants, statins, and targeting IL-8 may provide better outcomes for treating metastatic CRC that harbors multigene mutations and anti-EGFR resistance.

Bromelain Ameliorates Atherosclerosis by Activating the TFEB-Mediated Autophagy and Antioxidant Pathways.

Bromelain, a cysteine protease found in pineapple, has beneficial effects in the treatment of inflammatory diseases; however, its effects in cardiovascular pathophysiology are not fully understood. We investigated the effect of bromelain on atherosclerosis and its regulatory mechanisms in hyperlipidemia and atheroprone apolipoprotein E-null (apoe-/-) mice. Bromelain was orally administered to 16-week-old male apoe-/- mice for four weeks. Daily bromelain administration decreased hyperlipidemia and aortic inflammation, leading to atherosclerosis retardation in apoe-/- mice. Moreover, hepatic lipid accumulation was decreased by the promotion of cholesteryl ester hydrolysis and autophagy through the AMP-activated protein kinase (AMPK)/transcription factor EB (TFEB)-mediated upregulation of autophagy- and antioxidant-related proteins. Moreover, bromelain decreased oxidative stress by increasing the antioxidant capacity and protein expression of antioxidant proteins while downregulating the protein expression of NADPH oxidases and decreasing the production of reactive oxygen species. Therefore, AMPK/TFEB signaling may be crucial in bromelain-mediated anti-hyperlipidemia, antioxidant, and anti-inflammatory effects, effecting the amelioration of atherosclerosis.

Berry consumption mitigates the hypertensive effects of a high-fat, high-sucrose diet via attenuation of renal and aortic AT1R expression resulting in improved endothelium-derived NO bioavailability

Dysregulation of the renin-angiotensin system (RAS) is a contributor to high-fat diet-related blood pressure (BP) increases. Deleterious effects of dysregulated RAS result in an overproduction of reactive oxygen species and a decrease in endothelial nitric oxide (NO) bioavailability due to increased NADPH oxidase (NOX) expression. Dietary polyphenols have been shown to mitigate the imbalance in the redox state and protect against endothelial dysfunction induced by a high-fat diet. Thus, we aim to determine whether polyphenol-rich blackberry and raspberry, alone and in combination, attenuate the detrimental effects of a high-fat, high-sucrose (HFHS) diet on the vascular endothelium and kidneys of mice. We show that a HFHS diet increased the expression of renal and aortic angiotensin type 1 receptor (AT1R). Further, NOX1 and NOX4 expression were increased in the kidney contributing to fibrotic damage. In human aortic endothelial cells (HAECs), palmitic acid increased the expression of NOX4, potentially driving oxidative damage in the aorta, as evidenced by increased nitrotyrosine expression. Berries reduced the expression of renal and aortic AT1R, leading to a subsequent decrease in renal NOX expression and reduced aortic oxidative stress evidenced by reduced nitrotyrosine expression. Blackberry and raspberry in combination increased the expression of NRF2 and its downstream proteins in HAECs, thereby reducing the oxidative burden to the endothelium. In combination, blackberry and raspberry also increased serum levels of NO metabolites. These findings indicate that blackberry and raspberry unique polyphenols may act synergistically to favorably modulate the abovementioned pathways and attenuate HFHS diet-induced increases in BP.

Insight into the regulation of the Nrf2 pathway in response to ascovirus infection in Spodoptera exigua.

Ascoviruses are a type of entomopathogenic microorganism with high biological pest control potential and are expected to contribute to the natural control of lepidopteran pests. However, knowledge of the molecular mechanism underlying the biocidal activity of ascovirus on its host insects remains limited. In this study, the relative enzyme activity of superoxide dismutase and peroxidase, as well as the expression level of Spodoptera exigua peroxidase (SePOD), were found to be significantly increased at 6h post infection with Heliothis virescens ascovirus 3h (HvAV-3h). H2 O2 accumulation and enhanced expression of NADPH Oxidase (SeNOX) were also observed. In addition, Nuclear Factor erythroid 2-Related Factor 2 (SeNrf2) and muscle aponeurosis fibromatosis (SeMaf) were overexpressed following infection with HvAV-3h. Silencing of SeNrf2 decreased the expression of SePOD, whereas the mortality of SeNrf2-silenced larvae and viral genome copy number also increased. Further RNA interference of SeNOX significantly decreased expression of SeNrf2 and SePOD and therefore increased the mortality and viral genome copy number of the ascovirus-infected host. The HvAV-3h activated Nrf2/ARE pathway of S. exigua and reactive oxygen species were found to respond to ascovirus infection by regulating alterations in antioxidant enzyme genes mediated by the host Nrf2/ARE pathway. These findings enhance our knowledge of ascovirus-host interactions and lay the foundation for the application of ascoviruses in biological pest control. © 2022 Society of Chemical Industry.

High-starchy carbohydrate diet aggravates NAFLD by increasing fatty acids influx mediated by NOX2

Nonalcoholic fatty liver disease (NAFLD) is a high-incidence lipid disorder that affects more than a quarter of the population worldwide, and dietary intervention is the recognized treatment. Starch is the main component of staple foods that are consumed daily, and the effects, metabolic pathway, and molecular mechanism of starch in the context of NAFLD remain unclear. Our study showed that a high-starch carbohydrate diet (HCD) led to the occurrence and exacerbation of NAFLD in mice. Transcriptomics and metabonomic analyses showed that the increased fatty acid influx mediated by NADPH oxidase 2 (NOX2) exacerbated NAFLD. Knocking down NOX2 specifically alleviated HCD-induced NAFLD in vivo and in vitro. Moreover, the large amounts of ROS produced by NOX2 further exacerbated insulin resistance and increased lipolysis in perirenal white adipose tissue (periWAT), thereby providing fatty acids for hepatic lipid synthesis. In addition, the interaction between AMPKα1 and p47phox was the pathway that mediated the high expression of NOX2 induced by a HCD. Our study systematically demonstrated the effect of a HCD on NAFLD. Elevated fatty acid influx is a unique molecular regulatory pathway that mediates HCD-induced NAFLD exacerbation, which is different from the effect of simple sugars. Additionally, NOX2 was suggested to be a specific and effective drug target for NAFLD.

Role of the OTUB1/IRF7/NOX4 axis in oxidative stress injury and inflammatory responses in mice with Parkinson's disease.

Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders and is associated with oxidative stress injury (OSI) and inflammatory responses. This study sought to investigate the mechanism of ovarian tumour domain-containing ubiquitin aldehyde binding 1 (OTUB1) in OSI and inflammatory responses in PD, providing a theoretical foundation for PD treatment. The PD mouse model was established by an intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, followed by behavioural tests, observation of brain pathological changes, and quantification of inflammatory (TNF-α, IL-1β, and IL-10) and OS (ROS, SOD, and MDA) factors. Next, the expression levels of OTUB1, interferon regulatory factor 7 (IRF7), and NADPH oxidase 4 (NOX4) levels were determined by real-time quantitative polymerase chain reaction and western blot assay, the binding of OTUB1 to IRF7 was analysed by co-immunoprecipitation, and the ubiquitination level of IRF7 and the enrichment and binding of IRF7 and the NOX4 promoter were measured by chromatin immunoprecipitation and dual-luciferase assays. Afterwards, rescue experiments were performed with IRF7 or NOX4 overexpression in OTUB1 knockout PD mice. OTUB1 was upregulated in brain tissues of PD mice. Inhibition of OTUB1 alleviated PD progression, OSI, and inflammatory responses. OTUB1 stabilized IRF7 through deubiquitination, and IRF7 bound to the NOX4 promoter to promote NOX4 expression. IRF7 or NOX4 overexpression reversed the effects of silencing OTUB1 on OSI and inflammatory responses in PD mice. OTUB1-mediated deubiquitination stabilized IRF7 and upregulated NOX4 expression, thereby promoting OSI and inflammatory responses in PD mice.

Intermittent exposure to chlorpyrifos results in cardiac hypertrophy and oxidative stress in rats

Forbidden in some countries due to its proven toxicity to humans, chlorpyrifos (CPF) still stands as an organophosphate pesticide (OP) highly used worldwide. Cardiotoxicity assessment is an unmet need in pesticide regulation and should be deeply studied through different approaches to better inform and generate an appropriate regulatory response to OP use. In the present study, we used our 4-week intermittent OP exposure model in rats to address the CPF effects on cardiac morphology allied with cardiovascular functional and biomolecular evaluation. Rats were intermittently treated with CPF at doses of 7 mg/kg and 10 mg/kg or saline (i.p.) and assessed for cardiac morphology (cardiomyocyte diameter and collagen content), cardiopulmonary Bezold-Jarisch reflex (BJR) function, cardiac autonomic tone, left ventricle (LV) contractility, cardiac expression of NADPH oxidase (Nox2), catalase (CAT), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2) and cardiac levels of advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). Plasma butyrylcholinesterase (BuChE) and brainstem acetylcholinesterase (AChE) were also measured. Intermittent exposure to CPF induced cardiac hypertrophy, increasing cardiomyocyte diameter and collagen content. An impairment of cardioinhibitory BJR responses and an increase in cardiac vagal tone were also observed in CPF-treated animals without changes in LV contractility. CPF exposure increased cardiac Nox-2, CAT, SOD1, and TBARS levels and inhibited plasma BuChE and brainstem AChE activities. Our data showed that intermittent exposure to CPF induces cardiac hypertrophy together with cardiovascular reflex impairment, imbalance of autonomic tone and oxidative stress, which may bring significant cardiovascular risk to individuals exposed to OP compounds seasonally.

Astragaloside IV ameliorates sepsis-induced myocardial dysfunction by regulating NOX4/JNK/BAX pathway

AimsSepsis can induce multiple organ dysfunction, and sepsis-induced myocardial dysfunction (SIMD) is relatively common. The current dilemma might ascribe partly to SIMD's lack of unified molecular mechanisms. Our study aims to assess the function of Astragaloside IV (ASI) in cecal ligation and puncture (CLP)-induced cardiac dysfunction and explore its underlying mechanisms. Main methodsIn vivo, ASI (30 mg/kg/day), NADPH oxidase 4 (NOX4) inhibitor 4-hydroxy-3-methoxyacetophenone (APO, 30 mg/kg/day), reactive oxygen species (ROS) inhibitor N-Acetylcysteine (NAC, 150 mg/kg/day) and c-Jun NH2-terminal kinase (JNK) inhibitor (SP600125, 15 mg/kg/day) were severally administered to Sprague Dawley rats following the CLP surgery. The cardiac function, cardiac enzyme markers, proinflammatory cytokine, and cell apoptosis-associated proteins were detected. In vitro, cardiomyocyte H9C2 cells were treated with lipopolysaccharide (LPS, 40 μg/ml) after the presence of ASI (100 μmol/ml), SP600125 (10 μmol/ml), APO (10 μmol/ml). A series of experiments verified the relationship among NOX4, JNK, and BAX. Key findingsThe results indicated that CLP-induced sepsis increased the secretion of creatine kinase isoenzymes (CKMB), brain natriuretic peptide (BNP), cardiac troponin T (c-TnI), interleukin-1β (IL-1β) and interleukin-18 (IL-18), as well as the protein expression of NOX4 and Caspase-3 in vivo. LPS increased the protein level of NOX4 and Caspase-3, upregulated the rate of p-JNK/JNK, and downregulated the rate of Bcl2/BAX in vitro. ASI can reverse these changes in vivo and has a synergistic effect with APO and SP600125 in vitro. SignificanceThis study suggested that ASI may ameliorate SIMD, through regulating NOX4/JNK/BAX signaling pathway, which may be a feasible therapeutic strategy.

Telomere Shortening in Hypertensive Heart Disease Depends on Oxidative DNA Damage and Predicts Impaired Recovery of Cardiac Function in Heart Failure.

Heart failure (HF) coincides with cardiomyocyte telomere shortening. Arterial hypertension is the most prominent risk factor for HF. Both HF and arterial hypertension are associated with dysregulation of the neurohormonal axis. How neurohormonal activation is linked to telomere shortening in the pathogenesis of HF is incompletely understood. Cardiomyocyte telomere length was assessed in a mouse model of hypertensive HF induced by excess neurohormonal activation (AngII [angiotensin II] infusion, high salt diet, and uninephrectomy), in AngII-stimulated cardiomyocytes and in endomyocardial biopsies from patients with HF. Superoxide production, expression of NOX2 (NADPH oxidase 2) and PRDX1 (peroxiredoxin 1) and HDAC6 (histone deacetylase 6) activity were assessed. Telomere shortening occurred in vitro and in vivo, correlating with both left ventricular (LV) dilatation and LV systolic function impairment. Telomere shortening coincided with increased superoxide production, increased NOX2 expression, increased HDAC6 activity, loss of the telomere-specific antioxidant PRDX1, and increased oxidative DNA-damage. NOX2 knockout prevented PRDX1 depletion, DNA-damage and telomere shortening confirming this enzyme as a critical source of reactive oxygen species. Cotreatment with the NOX inhibitor apocynin ameliorated hypertensive HF and telomere shortening. Similarly, treatment with the HDAC6 inhibitor tubastatin A, which increases PRDX1 bioavailability, prevented telomere shortening in adult cardiomyocytes. To explore the clinical relevance of our findings, we examined endomyocardial biopsies from an all-comer population of patients with HF with reduced ejection fraction. Here, cardiomyocyte telomere length predicted the recovery of cardiac function. Cardiomyocyte telomere shortening and oxidative damage in heart failure with reduced ejection fraction induced by excess neurohormonal activation depends on NOX2-derived superoxide and may help to stratify HF therapy.

KCNH6 Enhanced Hepatic Glucose Metabolism through Mitochondrial Ca2+ Regulation and Oxidative Stress Inhibition.

KCNH6 has been proven to affect glucose metabolism and insulin secretion both in humans and mice. Further study revealed that Kcnh6 knockout (KO) mice showed impaired glucose tolerance. However, the precise function of KCNH6 in the liver remains unknown. Mitochondria have been suggested to maintain intracellular Ca2+ homeostasis; ROS generation and defective mitochondria can cause glucose metabolism disorders, including type 2 diabetes (T2D). Here, we found that Kcnh6 attenuated glucose metabolism disorders by decreasing PEPCK and G6pase abundance and induced Glut2 and IRS2 expression. Overexpression of Kcnh6 increased hepatic glucose uptake and glycogen synthesis. Kcnh6 attenuated intracellular and mitochondrial calcium levels in primary hepatocytes and reduced intracellular ROS and mitochondrial superoxide production. Kcnh6 suppressed oxidative stress by inhibiting mitochondrial pathway activation and NADPH oxidase expression. Experiments demonstrated that Kcnh6 expression improved hepatic glucose metabolism disorder through the c-Jun N-terminal kinase and p38MAPK signaling pathways. These results were confirmed by experiments evaluating the extent to which forced Kcnh6 expression rescued metabolic disorder in KO mice. In conclusion, KCNH6 enhanced hepatic glucose metabolism by regulating mitochondrial Ca2+ levels and inhibiting oxidative stress. As liver glucose metabolism is key to T2D, understanding KCNH6 functions may provide new insights into the causes of diabetes.

Effect of Artemisiae Argyi Folium Extract on Alcohol-Induced Gastritis

Objective: Alcohol is known to cause inflammation in the stomach by decreasing the protective substances of the gastric mucosa and increasing oxidative stress. The purpose of this study was to investigate the anti-inflammatory effect of Artemisiae Argyi Folium extract (AF) on alcohol-induced gastritis.Methods: The total polyphenol and flavonoid contents of AF were confirmed through an in vitro experiment. Radical scavenging activities were confirmed using DPPH and ABTS assays. In vivo experiments were conducted on mice divided into 5 groups (n=8): a normal group (Nor), an alcohol-induced gastritis group (Con), an alcohol-induced gastritis group administered 10 mg/kg sucralfate (SC), an alcohol-induced gastritis group administered 100 mg/kg AF (AFL), and an alcohol-induced gastritis group administered 200 mg/kg AF(AFH). The serum levels of reactive oxygen species (ROS) were determined, and protein expressions were confirmed in gastric tissues.Results: In alcohol-induced gastritis, AF alleviated damage to the gastric mucosa caused by alcohol. AF also decreased the serum ROS levels. Western blots showed that AF decreased the expression of NADPH oxidase and decreased the expression of the NF-κB pathway associated with inflammation. AF also decreased the expression of adhesion molecules and chemokine proteins, and increased the expression of anti-inflammatory proteins.Conclusions: AF not only reduced oxidative stress in alcohol-induced gastritis, but it also relieved gastric mucosal inflammation by regulating the expression of the NF-κB pathway.

Natural variation in macrophage polarization and function impact pneumocyte senescence and susceptibility to fibrosis.

Radiation-induced pulmonary fibrosis (RIPF), a late adverse event of radiation therapy, is characterized by infiltration of inflammatory cells, progressive loss of alveolar structure, secondary to the loss of pneumocytes and accumulation of collagenous extracellular matrix, and senescence of alveolar stem cells. Differential susceptibility to lung injury from radiation and other toxic insults across mouse strains is well described but poorly understood. The accumulation of alternatively activated macrophages (M2) has previously been implicated in the progression of lung fibrosis. Using fibrosis prone strain (C57L), a fibrosis-resistant strain (C3H/HeN), and a strain with intermediate susceptibility (C57BL6/J), we demonstrate that the accumulation of M2 macrophages correlates with the manifestation of fibrosis. A comparison of primary macrophages derived from each strain identified phenotypic and functional differences, including differential expression of NADPH Oxidase 2 and production of superoxide in response to M2 polarization and activation. Further, the sensitivity of primary AECII to senescence after coculture with M2 macrophages was strain dependent and correlated to observations of sensitivity to fibrosis and senescence in vivo. Taken together, these data support that the relative susceptibility of different strains to RIPF is closely related to distinct senescence responses induced through pulmonary M2 macrophages after thoracic irradiation.

The impacts of vorinostat on NADPH oxidase and mitochondrial biogenesis gene expression in the heart of mice model of depression.

The comorbidity of depression and high risk of cardiovascular diseases (CVD) have been reported as major health problems. Our previous study confirmed that fluoxetine (FLX) therapy had a significant influence on brain function but not on the heart in depression. In the present study, suberoyanilide hydroxamic acid (SAHA) was proposed as another therapeutic candidate for treatment of depression comorbid CVD in maternal separation model, following behavioral analyses and gene expression level in the heart. Our data demonstrated that SAHA significantly attenuates the NOX-4 gene expression level in treated mice with SAHA and FLX without significant change in NOX-2 expression level. SAHA decreased the gene expression level of peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α) and nuclear respiratory factors (Nrf2) in heart tissues of maternally separated mice. It supposed that non-effectiveness of FLX on mitochondrial biogenesis and NOX gene expression level in the heart of depressed patient can be related to recurrence of depression. It revealed that SAHA not only reversed the depressive-like behavior similar to our previous data but also recovered the heart mitochondrial function via effect on NOX-2, NOX-4, and mitochondrial biogenesis genes' (PGC-1α, Nrf-2, and peroxisome proliferator-activated receptor-α (PPAR-α)) expression levels. We suggest performing more studies to confirm SAHA as a therapeutic candidate in depression comorbid CVD.

Decreased Polymorphonuclear Myeloid-Derived Suppressor Cells and ROS Production Correlated Closely with Bronchopulmonary Dysplasia in Preterm Infants.

Background Bronchopulmonary dysplasia (BPD) is one of the most serious complications in premature infants. Myeloid-derived suppressor cells (MDSCs) have been indicated to promote immune tolerance and induce anti-inflammatory responses during the neonatal stage. However, the role of MDSCs in BPD has not been completely expounded. Methods 130 cases of newborns were collected from six tertiary hospitals in Guangzhou from August 2019 to June 2022. They were divided into BPD group, non-BPD preterm infants group, and term infants group according to gestational age and presence of BPD. The peripheral blood was collected and used to analyze the proportion, phenotypic, and function of MDSCs at 3 to 7 days and 8 to 14 days after birth, respectively. Results We indicated that the number of both MDSCs in premature infants is reduced, and the number of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in peripheral blood of BPD infants was significantly lower than that of non-BPD infants under 34 weeks of gestational age (P < 0.05). Furthermore, PMN-MDSCs from peripheral blood of patients presented inhibitory effect on proliferation of CD4+T and CD8+T cells in each group. However, PMN-MDSCs from BPD group had obviously weaker inhibitory effect on proliferation of CD4+T and CD8+T cells than that from non-BPD preterm infants group. In addition, we demonstrated that the expression of NADPH oxidase (Nox2) and reactive oxygen species (ROS) in PMN-MDSCs of BPD children was significantly lower than that in non-BPD preterm infants, suggesting that ROS pathway was affected in BPD in premature infants. Conclusion This study preliminarily revealed the role of PMN-MDSCs in the pathogenesis of BPD in premature infants. The specific immune regulation mechanism of PMN-MDSCs in BPD will provide new ideas and strategies for clinical prevention and treatment of BPD in premature infants.

Chitooligosaccharide Maintained Cell Membrane Integrity by Regulating Reactive Oxygen Species Homeostasis at Wounds of Potato Tubers during Healing.

Chitooligosaccharide (COS) is a degradation product of chitosan. Although COS increased fruit resistance by regulating the metabolism of reactive oxygen species (ROS), few reports are available on whether COS regulates ROS homeostasis at wounds of potato tubers during healing. In this study, COS increased gene expression and activities of NADPH oxidase and superoxide dismutase, and promoted the generation of O2●− and H2O2. Moreover, COS increased gene expression and activities of catalase, peroxidase, and AsA–GSH cycle-related enzymes, as well as the levels of ascorbic acid and glutathione levels. In addition, COS elevated the scavenging ability of DPPH, ABTS+, and FRAP, and reduced cell membrane permeability and malondialdehyde content. Taken together, COS could maintain cell membrane integrity by eliminating excessive H2O2 and improving the antioxidant capacity in vitro, which contributes to the maintainance of cell membrane integrity at wounds of potato tubers during healing.

Potential cardioprotective effects of Amentoflavone in doxorubicin-induced cardiotoxicity in mice

Doxorubicin (DOX) is an available chemotherapeutic drug for treating various tumors. However, its effectiveness is limited by cardiotoxicity. Amentoflavone (AMF), a natural biflavonoid separated from Cycas thouarsii ethyl acetate fraction, displays promising anticancer, anti-inflammatory, and antioxidant effects. Thus, our research aims to explore whether AMF could boost cardioprotective effects against DOX cardiotoxicity and reveal the potential underlying mechanisms of cardioprotection. Mice were classified into four groups; Normal control, Untreated DOX group, and DOX groups treated with AMF (40 and 80 mg/kg, respectively) intraperitoneal injection daily for four days before doxorubicin administration and for additional three days following DOX administration to assess cardiotoxicity. Echocardiography showed that AMF 80 treated group was protected from DOX cardiotoxicity. Additionally, it alleviated histopathological structural alterations and effectively restored heart weight and body weight ratio. These effects were confirmed biochemically by a substantially reduced serum creatine kinase-MB (CK-MB) and aspartate aminotransferase (AST) levels. AMF effectively restored nuclear respiratory factor-1(NRF-1), mitochondrial transcription factor A (TFAM), and normalized heat shock protein − 27(HSP-27) expression levels compared to the DOX group. Moreover, AMF mitigated oxidative stress conditions and significantly suppressed NADPH oxidase (NOX) expression levels. It also showed significant anti-inflammatory effects via suppressing interleukin-6 (IL-6) expression and decreasing nuclear factor Kabba B (NF-κb) immune-staining. In addition, AMF markedly reduced FAS ligand (FASL) expression and p53 immune staining in cardiac tissue. This study is the first for the in vivo potential beneficial effects of AMF against acute DOX cardiotoxicity, possibly via exerting antioxidant, anti-inflammatory, and anti-apoptotic effects and restoring mitochondrial function.

Exercise Training Ameliorates Renal Oxidative Stress in Rats with Chronic Renal Failure.

In patients with chronic kidney disease, exercise training with moderate intensity protects renal function and improves mortality. However, the mechanisms of the renal protective effects of exercise training in chronic kidney disease have not been clarified. This study investigated the effects of exercise training on renal NADPH oxidative and xanthine oxidase, which are major sources of reactive oxygen species, in rats with chronic renal failure. Six-week-old, male Sprague–Dawley rats were divided into the sham operation, 5/6 nephrectomy (Nx)+ sedentary, and Nx+ exercise training groups. The Nx+ exercise training group underwent treadmill running. After 12 weeks, systolic blood pressure, renal function, malondialdehyde, renal NADPH oxidase, and xanthine oxidase were examined. Nx induced hypertension, proteinuria, and renal dysfunction, and exercise training attenuated these disorders. Although the plasma levels of malondialdehyde were not different among the group, urinary levels were increased by Nx and decreased by exercise training. Renal activity and expression of NADPH oxidase and xanthine oxidase were increased by Nx and decreased by exercise training. These results indicate that exercise training attenuates hypertension and renal dysfunction and ameliorates NADPH oxidase and xanthine oxidase in rats with chronic renal failure, suggesting that the reduction of reactive oxygen species generation may be involved in the renal protective effects of exercise training.

Long Noncoding RNA-5657 Inhibits the Proliferation of Pancreatic Cells and Insulin Secretion in Gestational Diabetes by Inhibiting the NADPH Oxidase 4 (NOX4)

Multiple comorbidities are observed during pregnancy and there are many challenges for pregnant women with a previous history of diabetes. Pancreatic β-cell dysfunction is associated with gestational diabetes mellitus (GDM). This study intends to explore LncRNA-5657’s effect on proapoptotic cell proliferation and insulin secretion in gestational diabetes. Real-time quantitative fluorescence PCR measured LncRNA-5657 expression in GDM patient placental tissues along with analysis of cell activity, proliferation and insulin secretion by MTT assay, BrdU staining and ELISA. The relation between LncRNA-5657 and NOX4 was assessed by dual luciferase reporter assay. LncRNA-5657 was overexpressed in placental tissue of GDM patients compared to normal pregnant women. LncRNA-5657 overexpression promoted cell proliferation and insulin secretion, which was reversed by LncRNA-5657 downregulation. In addition, LncRNA-5657 negatively regulated NOX4 expression. Overexpression of NOX4 abolished the effect of LncRNA-5657 mimics on pancreatic β-cells, and knockdown of LncRNA-5657 promoted insulin secretion by upregulating NOX4 expression. In conclusion, LncRNA-5657 inhibits insulin secretion and β-cell proliferation by targeting NOX4, indicating that LncRNA-5657 might be a novel target for the treatment of GDM.

Abstract P134: Tnfα Impairs Endothelial Calcium Signaling And Elevates Blood Pressure In Obesity.

Endothelial dysfunction in small arteries is a hallmark of obesity-induced hypertension. Recent studies show that endothelial upregulation of inducible nitric oxide synthase (iNOS) and NADPH oxidase 1 (NOX1) plays a critical role in obesity-induced endothelial dysfunction. Endothelial upregulation of iNOS and NOX1 results in excessive formation of peroxynitrite, a reactive nitrogen species, which impairs endothelium-dependent vasodilation. It is well-known that inflammatory mediators elevate cellular iNOS, NOX1, and peroxynitrite levels. In this regard, tumor necrosis factor-alpha (TNFα) signaling has been proposed as a critical contributor to endothelial dysfunction in obesity. Notably, vascular SMCs TNFα levels were increased in heart failure and diabetes. Therefore, we hypothesized that vascular wall-derived TNFα acts locally to impair endothelial function in obesity. Flow cytometry studies showed an increase in TNFα levels in freshly isolated SMCs from small mesenteric arteries (MAs) of high-fat diet-fed obese mice compared to normal chow-fed control mice. However, TNFα levels in ECs from obese mice were not different from those in ECs from control mice. The pro-inflammatory effects of TNFα are mediated mainly through TNFα receptor I activation. Treating the obese mice with a selective inhibitor of TNFα receptor I signaling reduced the blood pressure to normal levels in obese mice but did not affect blood pressure in control mice. Treatment with R7050 also abolished the obesity-induced increase in iNOS and NOX1 expression in endothelial cells. Further, R7050 administration restored acetylcholine-induced, endothelium-dependent vasodilation of MAs in pressure myography experiments. Previous studies showed a decrease in Ca 2+ influx through endothelial TRPV4 channels contributes to blood pressure elevation in obese mice. Notably, R7050 administration restored the Ca 2+ influx signals through endothelial TRPV4 channels in MAs, as studied using high-speed confocal Ca 2+ imaging. Our findings collectively indicate that TNFα signaling contributes to the impairment of endothelial calcium signaling and blood pressure elevation in obesity and highlights the role of SMCs as a source of TNFα in obesity

Ganoderma Lucidum Polysaccharide Peptide attenuates post myocardial infarction fibrosis via down-regulating TGF-β1/SMAD and relieving oxidative stress

The Ganoderma lucidum, a well-known Chinese traditional medicine, has been used to prevent and treat cardiovascular diseases. The purpose of this study was to determine whether Ganoderma lucidum polysaccharide peptide (GLPP) has therapeutic effect on cardiac fibrosis. Using both mouse myocardial infarction (MI) model and primarily cultured cardiac fibroblast model, we found that GLPP (150 mg/kg/day) ameliorated cardiac fibrosis and improved cardiac function following MI. We further revealed that GLPP down regulated the expression of fibronectin, collagen I, collagen III and α-smooth muscle actin in the cardiac tissue, mainly through hindering the over activation of TGF-β/SMAD signaling. On the other hand, GLPP significantly decreased the expression of NADPH oxidase 4 (NOX4), and reduced the MDA, SOD and 8-OHdG in myocardial tissue, suggesting GLPP alleviated cardiac fibrosis partially via relieving oxidative stress. The inhibitory effect of GLPP on TGF-β/SMAD and NOX4-derived ROS signaling pathways was further confirmed in TGF-β or H2O2 stimulated neonatal rat cardiac fibroblast model. Our experimental results indicate that GLPP is a promising agent for the prevention and treatment of cardiac fibrosis.