Intracellular Superoxide Production Research Articles

Ginsenoside Rc, an Active Component of Panax ginseng, Alleviates Oxidative Stress-Induced Muscle Atrophy via Improvement of Mitochondrial Biogenesis.

Loss of skeletal muscle mass and function has detrimental effects on quality of life, morbidity, and mortality, and is particularly relevant in aging societies. The enhancement of mitochondrial function has shown promise in promoting muscle differentiation and function. Ginsenoside Rc (gRc), a major component of ginseng, has various pharmacological activities; however, its effect on muscle loss remains poorly explored. In this study, we examined the effects of gRc on the hydrogen peroxide (H2O2)-induced reduction of cell viability in C2C12 myoblasts and myotubes and H2O2-induced myotube degradation. In addition, we investigated the effects of gRc on the production of intracellular reactive oxygen species (ROS) and mitochondrial superoxide, ATP generation, and peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) activity in myoblasts and myotubes under H2O2 treatment. Furthermore, to elucidate the mechanism of action of gRc, we conducted a transcriptome analysis of myotubes treated with or without gRc under H2O2 treatment. gRc effectively suppressed H2O2-induced cytotoxicity, intracellular ROS, and mitochondrial superoxide production, restored PGC-1α promoter activity, and increased ATP synthesis. Moreover, gRc significantly affected the expression levels of genes involved in maintaining mitochondrial mass and biogenesis, while downregulating genes associated with muscle degradation in C2C12 myotubes under oxidative stress. We provide compelling evidence supporting the potential of gRc as a promising treatment for muscle loss and weakness. Further investigations of the pharmacological effects of gRc under various pathological conditions of muscle loss will contribute to the clinical development of gRc as a therapeutic intervention.

Scutebarbatine A induces ROS-mediated DNA damage and apoptosis in breast cancer cells by modulating MAPK and EGFR/Akt signaling pathway

Scutebarbatine A (SBT-A), a diterpenoid alkaloid, has exerted cytotoxicity on hepatocellular carcinoma cells in our previous works. Here, the antitumor activity of SBT-A in breast cancer cells and the underlying mechanism were explored. The anti-proliferative effect of SBT-A was measured by trypan blue staining, 5-ethynyl-2′-deoxyuridine (EdU) incorporation and colony formation assay. DNA double-strand breaks (DSBs) were evaluated by observing the nuclear focus formation of γ-H2AX. Cell cycle distribution was assessed by flow cytometry. Apoptosis was determined by a TUNEL assay. Intracellular reactive oxygen species (ROS) generation and superoxide production were measured with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) staining, respectively. The results indicated that SBT-A showed a dose-dependent cytotoxic effect against breast cancer cells while revealing less toxicity toward MCF-10A breast epithelial cells. Moreover, SBT-A remarkably induced DNA damage, cell cycle arrest and apoptosis in both MDA-MB-231 and MCF-7 cells. SBT-A treatment increased the levels of ROS and cytosolic superoxide production. Pretreatment with N-acetyl cysteine (NAC), a ROS scavenger, was sufficient to block viability reduction, DNA damage, apoptosis and endoplasmic reticulum (ER) stress caused by SBT-A. By exposure to SBT-A, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) was upregulated, while the phosphorylation of extracellular signal-regulated kinase (ERK) was downregulated. In addition, SBT-A inhibited the EGFR signaling pathway by decreasing EGFR expression and phosphorylation of Akt and p70S6K. As mentioned above, SBT-A has a potent inhibitory effect on breast cancer cells through induction of DNA damage, apoptosis and ER stress via ROS generation and modulation of MAPK and EGFR/Akt signaling pathway.

3-Nitropropionic Acid Enhances Ferroptotic Cell Death via NOX2-Mediated ROS Generation in STHdhQ111 Striatal Cells Carrying Mutant Huntingtin.

Huntington's disease (HD) is a hereditary neurodegenerative disease that involves an expansion of the CAG repeats of the Huntingtin (HTT) gene, but the disease onset and progression do not necessarily correspond to the extent of CAG repeats. Decreased mitochondrial complex II activity has also been reported to be closely associated with disease pathogenesis. Here, we examined the mechanism of cell death induced by 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor, using striatal cells (STHdhQ111 cells) derived from HD model mice with mutant HTT carrying the CAG repeat extended. Treatment with 3-NP (5 mM) enhanced cell death in STHdhQ111 compared to STHdhQ7 cells with normal HTT. Ferrostatin-1, an inhibitor of ferroptosis, and deferoxamine, an iron chelator, markedly inhibited 3-NP-induced cell death in both the STHdh cell lines. On the other hands, cell death was not abrogated by a broad-spectrum caspase inhibitor, Z-VAD-FMK, indicating that this cell death was caspase-independent. Cell death caused by 3-NP is suggested to be due to ferroptosis. Furthermore, 3-NP-induced cell death was markedly inhibited by GSK2795039, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) inhibitor, suggesting that cell death is mainly mediated by intracellular superoxide anion (O2-) production through NOX2. Furthermore, a mitochondria-targeted superoxide dismutase mimetic (Mito-TEMPO), partially inhibited 3-NP-induced cell death, suggesting that O2- production in the mitochondria is partially responsible for cell death. These results indicate that 3-NP-induced cell death in the STHdhQ111 cells is caspase-independent, non-apoptotic, and that ferroptotic cell death is mainly induced via NOX2 activation.

Nimbin analogs N5 and N7 regulate the expression of lipid metabolic genes and inhibit lipid accumulation in high-fat diet-induced zebrafish larvae: An antihyperlipidemic study

BackgroundExcess accumulation of lipids leads to obesity. Triterpenoids are a group of plant compounds which poses various biological activities. The biological activities of Nimbin analogs N5 and N7 were addressed in this study on inhibiting lipid aggregation and underlying the derivatives molecular mechanisms for a therapeutical approach. AimThis study aims to evaluate the anti-adipogenic activity of semi-natural Nimbin analogs, N5 and N7, on zebrafish larvae induced with oxidative stress due to a high-fat diet (HFD) and adipogenesis using specific fluorescent stains. Materials and methodsZebrafish at 4 days post fertilized (dpf) larvae were divided into groups for the HFD diet along with exposure to various concentrations of N5 and N7. HFD induced accumulation of neutral lipids and triglycerides (Oil Red O and Nile red staining, respectively) with weight gain, which generated intracellular ROS (DCFH-DA staining) and superoxide anion production (DHE staining) with depleted glutathione levels (NDA staining) were assayed. HFD exposure promoted the accumulation of inflammatory macrophages (Neutral red staining) and impaired glucose metabolism (2NBDG staining). The ability of N5 and N7 to reduce total regulating lipogenic specific genes C/EBP-α, SREBP-1 and FAS were evaluated using relative gene expression. Key findingsThe Nimbin analogues N5 and N7 suppressed adipogenesis, forming intracellular ROS and superoxide anion while simultaneously restoring glutathione levels. The analogues significantly lowered total TC and TG levels, prevented inflammatory macrophage build-up and boosted glucose absorption. Also, N5 and N7 down-regulate the lipogenic-specific genes. SignificanceNimbin analogs N5 and N7 enhance lipolysis and inhibit adipogenesis in in-vivo zebrafish larvae model.

Anti-diabetic effects of astaxanthin-rich extract derived from Paracoccus carotinifaciens on pancreatic β cells

Astaxanthin is a natural pigment that is used as a dietary supplement. Many experiments have shown that astaxanthin has positive effects against various diseases including diabetes. Adonixanthin and adonirubin are biosynthetic intermediates of β-carotene to astaxanthin and possess the same radical scavenging activity as astaxanthin. In this study, we evaluated the antidiabetic effect on pancreatic β cells of astaxanthin-rich extract (ARE) derived from Paracoccus carotinifaciens, which contains several carotenoids such as astaxanthin, adonirubin, and adonixanthin. Specifically, we studied the effects on insulin secretion and cytoprotection as well as on intracellular reactive oxygen species (ROS) generation and mitochondrial superoxide production. As a result, pretreatment with ARE derived from P. carotinifaciens increased insulin secretion under the condition of 20 mM glucose stimulation in iGL cells. In addition, ARE derived from P. carotinifaciens significantly decreased streptozotocin (STZ) -induced cell death in INS-1 cells, and isobologram analysis revealed the synergistic effects among astaxanthin, adonixanthin, and adonirubin. We clarified that the production control of mitochondrial superoxide was involved in the mechanism of these effects. These findings suggest that ARE derived from P. carotinifaciens may be used to treat diabetes by increasing glucose-stimulated insulin secretion and protecting pancreatic β cells.

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.

Neuroprotective effect of Biochanin a against Bisphenol A-induced prenatal neurotoxicity in zebrafish by modulating oxidative stress and locomotory defects

Exogenous toxicants cause oxidative stress and damage to brain cells, resulting in inflammation. Neuroinflammation is important in the pathobiology of various neurological illnesses, including Alzheimer's disease (AD). In this context, Bisphenol A (BPA), a common toxin, causes oxidative damage and has been linked to neurological problems. An O-methylated isoflavone known as Biochanin A (5,7-dihydroxy-4′-methoxy-isoflavone, BCA) is considered to be a phytoestrogen, which is abundant in some legume plants and soy which have preventive effects against cancer, osteoporosis, menopausal symptoms and oxidative stress. However, the mechanism by which BCA protected the prenatal neurological stress are not known. So that, in this study we investigated the BCA neuroprotective effect against BPA-induced neuroinflammation in zebrafish embryo models. For this study, fertilized zebrafish embryos are exposed to BPA (1 µM) with or without BCA. Our finding suggested that BCA co-exposure prevented the depletion of antioxidant defense enzymes by BPA and reduced the production of intracellular ROS production, superoxide anion (O2–), lipid peroxidation (LPO), lactate dehydrogenase (LDH) and nitric oxide (NO) levels in the head that aided in safeguarding neuronal development. Baseline locomotion was rendered and a total distance was calculated to assess the motor function. Exposure to BCA increased acetylcholinestrase (AChE) and improved motor neuron functions. It also reduced the pro-inflammatory response expression and prevented neuroinflammation. Our study suggests that BCA has a positive role in the attenuation or amelioration of neuronal oxidative damage and locomotory behaviour induced by BPA.

Curcumin Blocks High Glucose-Induced Podocyte Injury via RIPK3-Dependent Pathway.

Podocyte loss is well known to play a critical role in the early progression of diabetic nephropathy. A growing number of studies are paying attention to necroptosis, a programmed form of cell necrosis as a mechanism of podocyte loss. Although necroptosis is a recently established concept, the significance of receptor interacting serine/threonine kinase 3 (RIPK3), a gene that encodes for the homonymous enzyme RIPK3 responsible for the progression of necroptosis, is well studied. Curcumin, a natural hydrophobic polyphenol compound responsible for the yellow color of Curcuma longa, has drawn attention due to its antioxidant and anti-inflammatory effects on cells prone to necroptosis. Nonetheless, effects of curcumin on high glucose-induced podocyte necroptosis have not been reported yet. Therefore, this study investigated RIPK3 expression in high glucose-treated podocytes to identify the involvement of necroptosis via the RIPK3 pathway and the effects of curcumin treatment on RIPK3-dependent podocytopathy in a hyperglycemic environment. The study discovered that increased reactive oxygen species (ROS) in renal podocytes induced by high glucose was improved after curcumin treatment. Curcumin treatment also significantly restored the upregulated levels of VEGF, TGF-β, and CCL2 mRNAs and the downregulated level of nephrin mRNA in cultured podocytes exposed to a high glucose environment. High glucose-induced changes in protein expression of TGF-β, nephrin, and CCL2 were considerably reverted to their original levels after curcumin treatment. Increased expression of RIPK3 in high glucose-stimulated podocytes was alleviated by curcumin treatment as well as N-acetyl cysteine (NAC, an antioxidant) or GSK′872 (a RIPK3 inhibitor). Consistent with this, the increased necroptosis-associated molecules, such as RIPK3, pRIPK3, and pMLKL, were also restored by curcumin in high glucose-treated mesangial cells. DCF-DA assay confirmed that such a result was attributed to the reduction of RIPK3 through the antioxidant effect of curcumin. Further observations of DCF-DA-sensitive intracellular ROS in NAC-treated and GSK′872-treated podocyte groups showed a reciprocal regulatory relationship between ROS and RIPK3. The treatment of curcumin and GSK′872 in podocytes incubated with high glucose protected from excessive intracellular superoxide anion production. Taken together, these results indicate that curcumin treatment can protect against high glucose-induced podocyte injuries by suppressing the abnormal expression of ROS and RIPK3. Thus, curcumin might be a potential therapeutic agent for diabetic nephropathy as an inhibitor of RIPK3.

(E)-5-hydroxy-7-methoxy-3-(2′-hydroxybenzyl)-4-chromanone isolated from Portulaca oleracea L. suppresses LPS-induced inflammation in RAW 264.7 macrophages by downregulating inflammatory factors

Context Portulaca oleracea L. is herbaceous succulent annual plant, which belongs to the Portulacaceae family. Many studies have shown its wide spectrum of pharmacological activities such as anti-cancer and anti-diabetic effects. Objectives The objective of this study was to identify the anti-inflammatory effects of HM-chromanone isolated from Portulaca oleracea L. in LPS-stimulated RAW 264.7 macrophages. Materials and methods LPS (1 μg/ml)-stimulated mouse RAW 264.7 macrophages were used to assess the anti-inflammatory effect of HM-chromanone (10–50 μM). Cell viability was evaluated by MTT assay. In addition, the production of intracellular ROS, superoxide anion, lipid peroxide, NO, and PGE2, and activity of antioxidant enzymes were analyzed. The expressions of iNOS, COX-2, IκB, NF-κB, TNF-α, IL-1β and IL-6 were evaluated by western blot analysis. Results HM-chromanone has demonstrated that there is no significant cytotoxic effect on the viability of RAW 264.7 macrophages. In LPS-stimulated RAW 264.7 cells, HM-chromanone treatment was found to significantly inhibit the production of intracellular ROS, superoxide anion and lipid peroxide, while enhancing the activity of antioxidant enzymes such as SOD, catalase, and GSH-px. Additionally, HM-chromanone treatment was observed to inhibit NO and PGE2 production by inhibiting the expression of iNOS and COX-2. Subsequently, HM-chromanone was observed to significantly suppress LPS-induced expression of IκB, NF-κB, TNF-α, IL-1β and IL-6. Discussion and conclusion Overall, our results suggested that HM-chromanone suppresses LPS-induced inflammation in RAW 264.7 macrophages by downregulating the expression of inflammatory factors.

Metformin effect on driving cell survival pathway through inhibition of UVB-induced ROS formation in human keratinocytes

Human skin functions go beyond serving only as a mechanical barrier. As a complex organ, the skin is capable to cope with external stressors cutaneous by neuroendocrine systems to control homeostasis. However, constant skin exposure to ultraviolet (UV) radiation causes progressive damage to cellular skin constituents, mainly due excessive reactive oxygen species (ROS) production. The present study shows new approaches of metformin (MET) as an antioxidant agent. Currently, MET is the first line treatment of type 2 diabetes and has attracted attention, based on its broad mechanism of action. Therefore, we evaluated MET antioxidant potential in cell-free systems and in UVB irradiated human keratinocyte HaCaT cells. In cell-free system assays MET did not show intrinsic scavenging activity on DPPH radicals or superoxide (O2−) xanthine/luminol/xanthine oxidase-generated. Cell-based results demonstrated that MET was able to reduce UVB-induced intracellular ROS and NADPH oxidase-dependent superoxide (O2−) production. MET posttreatment of HaCaT cells reduced ERK 1/2 phosphorylation, NADPH oxidase activity, and cell death by apoptosis. These findings suggest that the protection mechanism of MET may be through the inhibition of ROS formation enzyme. These results showed that MET might be a promising antioxidant agent against UV radiation induced skin damage.

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.

Redox-related Molecular Mechanism of Sensitizing Colon Cancer Cells to Camptothecin Analog SN38.

The aim of this study was to elucidate the possibility of sensitizing colon cancer cells to the chemotherapeutic drug SN38 and investigate its mechanism of action after combined treatment with electroporation (EP). Cells were treated with SN38, EP and their combination for 24/48 h. The cell viability, actin cytoskeleton integrity, mitochondrial superoxide, hydroperoxides, total glutathione, phosphatidyl serine expression, DNA damages and expression of membrane ABC transporters were analyzed using conventional analytical tests. The combination of EP and SN38 affected cell viability and cytoskeleton integrity. This effect was accompanied by: (i) high production of intracellular superoxide and hydroperoxides and depletion of glutathione; (ii) increased DNA damage and apoptotic/ferroptotic cell death; (iii) changes in the expression of membrane ABC transporters - up-regulation of SLCO1B1 and retention of SN38 in the cells. The anticancer effect of the combined treatment of SN38 and EP is related to changes in the redox-homeostasis of cancer cells, leading to cell death via apoptosis and/or ferroptosis. Thus, electroporation has a potential to increase the sensitivity of cancer cells to conventional anticancer therapy with SN38.

An approach to rescue the fertility of stallions with a high level of hemospermia

A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control-pure raw semen, (b) WB50-50% (v/v) whole blood added into semen, (c) E1-WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2-WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O2 ) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p<.05) in E1 than in CT and E2 samples. Progressive motility decreased (p<.05) with an increase in the percentage of blood in the samples. The PMI and PER did not differ between groups (p>.05); however, O2 production was higher (p<.05) in WB50 than in E2 samples, while the values were intermediate (p>.05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p<.05) fertility than the others (WB50-0% and E1-25%). It was concluded that sperm motility and fertility of semen with a large amount of blood can be rescued by dilution with a 2:1 extender:semen ratio using a milk-based extender.

Vascular Protection by Exercise in Obesity: Inflammasome-associated Mechanisms.

The nodlike receptor family pyrin domain containing 3 (NLRP3) inflammasome is a critical player in vascular pathology as it regulates caspase-1-mediated interleukin (IL)-1β processing. Physical activity ameliorates obesity-induced inflammation and vascular dysfunction, but the mechanisms responsible for these positive changes are incompletely understood. Here, the protective effect of physical activity on the inflammasome-associated vascular dysfunction in obesity and its putative mechanisms were investigated. Mice were fed a control low-fat diet (LFD) or a high-fat diet (HFD; 45% of calories from fat) and provided with running wheel access (LF-RUN or HF-RUN) or denied wheel access for our sedentary condition (LF-SED or HF-SED). The NLRP3 inflammasome-associated pathway, including NLRP3, caspase-1, and IL-1β, in mice aorta was examined by RT-qPCR and FLICA and DAB staining. The protein expression of zonula occluden-1 (ZO-1), ZO-2, adiponectin (APN), and adiponectin receptor 1 (AdipoR1) in aortic endothelial cells was determined by immunofluorescence double staining. Intracellular reactive oxidative stress and nitric oxide (NO) production were monitored with fluorescence probes, dihydroethidium, and diaminofluorecein. HFD increased caspase-1 and IL-1β at mRNA and protein levels in endothelial cells of the aorta, and this was attenuated by voluntary running. HFD decreased ZO-1 and ZO-2 expression and reduced APN and AdipoR1 signaling; these were restored by running. The elevated intracellular superoxide (O2) production observed in HF-SED was ameliorated in HF-RUN. Finally, HF-RUN improved NO production in the aorta compared with HF-SED. Our findings suggest that voluntary running ameliorates mechanisms associated with vascular dysfunction by suppressing NLRP3 inflammasome, improving NO production, and reducing oxidative stress. Such benefits of physical activity may be, at least in part, associated with APN-AdipoR1 signaling and tight junction protein expression.

Proanthocyanidin-Rich Grape Seed Extract Reduces Inflammation and Oxidative Stress and Restores Tight Junction Barrier Function in Caco-2 Colon Cells.

Grape polyphenols have previously been shown to improve gut health and attenuate the symptoms of metabolic syndrome; however, the mechanism of these beneficial effects is still debated. In this study, we investigated the protective effect of proanthocyanidin-rich grape seed extract (GSE) on bacterial lipopolysaccharide (LPS)-induced oxidative stress, inflammation, and barrier integrity of human Caco-2 colon cells. GSE significantly reduced the LPS-induced intracellular reactive oxygen species (ROS) production and mitochondrial superoxide production, and upregulated the expression of antioxidant enzyme genes. GSE also restored the LPS-damaged mitochondrial function by increasing mitochondrial membrane potential. In addition, GSE increased the expression of tight junction proteins in the LPS-treated Caco-2 cells, increased the expression of anti-inflammatory cytokines, and decreased pro-inflammatory cytokine gene expression. Our findings suggest that GSE exerts its beneficial effects on metabolic syndrome by scavenging intestinal ROS, thus reducing oxidative stress, increasing epithelial barrier integrity, and decreasing intestinal inflammation.

Synergistic effects of the β-1,3 glucan paramylon and vitamin C on immunological responses of hybrid striped bass (Morone chrysops × M. saxatilis) were pronounced in vitro but more moderate in vivo.

The objective of this study was to evaluate if immunostimulation by the β-1,3-glucan paramylon (PML) could be further enhanced with vitamin C (vit. C) for hybrid striped bass (HSB). Two separate trials were conducted in vitro and in vivo, using a 2 × 2 factorial design, where the main effects tested were PML and vit. C, or their combination (PML + vit. C), to measure possible synergistic effects on various immunological responses. Supplementation of PML in vitro to whole-blood significantly (P < .001) increased the plasma lysozyme activity, and vit. C increased the concentration of total protein (P < .01) and immunoglobulins (P < .001) in the plasma. Synergistic effects (P < .0001) were observed for the production of reactive oxygen species in whole-blood and isolated phagocytes. After corroborating the interaction of PML and vit. C in vitro, a feeding trial was conducted using HSB juveniles (~9.30 g initial weight). Fish were fed diets with 100 mg of PML kg−1, 500 mg of vit. C kg−1 or their combination (100 mg of PML + 500 mg of vit. C kg−1). A basal diet with no supplementation served as the control. Fish were fed for 8 weeks after which production parameters were recorded, and proximate composition of whole-body tissues, immunological components of the blood and plasma, phagocytes isolated from the head kidney, and enzyme activities in intestinal sections were analyzed. Supplementation of PML slightly impaired HSB growth performance and feed efficiency, and decreased the moisture content of whole-body tissues when compared to the non-supplemented group. However, fish fed diets supplemented with PML presented higher concentrations of plasma immunoglobulins, intracellular superoxide anion production, acid phosphatase activity in the posterior part of the intestine, and lysozyme activity in the anterior part of the intestine. A synergistic effect between PML and vit. C for acid phosphatase activity was observed in the anterior part of the intestine, and for whole-blood respiratory burst. Therefore, based on the results from both trials, synergism between PML and vit. C was most pronounced in vitro in terms of reactive oxygen species production, and limited enhancement of immunological responses were observed in vivo after the feeding trial.

Enhanced Antioxidative Defense by Vitamins C and E Consumption Prevents 7-Day High-Salt Diet-Induced Microvascular Endothelial Function Impairment in Young Healthy Individuals.

This study aimed to examine whether the oral supplementation of vitamins C and E during a seven-day high salt diet (HS; ~14 g salt/day) prevents microvascular endothelial function impairment and changes oxidative status caused by HS diet in 51 (26 women and 25 men) young healthy individuals. Laser Doppler flowmetry measurements demonstrated that skin post-occlusive reactive hyperemia (PORH), and acetylcholine-induced dilation (AChID) were significantly impaired in the HS group, but not in HS+C+E group, while sodium nitroprusside-induced dilation remained unaffected by treatments. Serum oxidative stress markers: Thiobarbituric acid reactive substances (TBARS), 8-iso prostaglandin-F2α, and leukocytes’ intracellular hydrogen peroxide (H2O2) production were significantly increased, while ferric-reducing ability of plasma (FRAP) and catalase concentrations were decreased in the HS group. All these parameters remained unaffected by vitamins supplementation. Matrix metalloproteinase 9, antioxidant enzymes Cu/Zn SOD and glutathione peroxidase 1, and leukocytes’ intracellular superoxide production remained unchanged after the protocols in both HS and HS+C+E groups. Importantly, multiple regression analysis revealed that FRAP was the most powerful predictor of AChID, while PORH was strongly predicted by both FRAP and renin-angiotensin system activity. Hereby, we demonstrated that oxidative dis-balance has the pivotal role in HS diet-induced impairment of endothelial and microvascular function in healthy individuals which could be prevented by antioxidative vitamins consumption.

P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK

Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression.

Pueraria lobata Root Constituents as Xanthine Oxidase Inhibitors and Protective Agents against Oxidative Stress Induced in GES-1 Cells

Ultrafiltration coupled with liquid chromatography-mass spectrometry (LC-MS) was established to screen xanthine oxidase (XO) inhibitors from Pueraria lobata root extract. Four compounds were screened out and identified as puerarin, daidzin, daidzein and genistein with half-maximal inhibitory concentration (IC50) values of 30.8, 5.31, 14.5 and 3.02 µg mL−1 on XO, respectively. The interactions between these compounds and XO were investigated by fluorescence spectroscopic method. The hydrogen peroxide induced oxidative stress model of human normal gastric epithelial cell lines (GES-1) was used to investigate the protections on injured cell. As a result, four XO inhibitors exhibited protective effects without cytotoxicity. With the increased concentrations of four inhibitors, cell viability was higher with decreased mortality rate, the decrease of superoxide dismutase activity, leakage of lactate dehydrogenase and increase of intracellular superoxide anion production induced by hydrogen peroxide were restrained. It showed that these four XO inhibitors could effectively enhance cell viability and protect injury of GES-1 cells from oxidative stress.

Identification of in vitro effect of 4-octylphenol on the basal and human chorionic gonadotropin (hCG) stimulated secretion of androgens and superoxide radicals in mouse Leydig cells

The aim of our in vitro study was to assess the potential effect of 4-octylphenol (4-OP) on the basal and human chorionic gonadotropin (hCG)-stimulated cholesterol levels and biosynthesis of steroid hormones in cultured mouse Leydig cells. In addition, we evaluated the intracellular superoxide production following 4-OP treatment. Isolated mouse Leydig cells were cultured in the presence or absence of 1 IU/mL (hCG) with the addition of 0.04; 0.2; 1.0; 2.5 and 5.0 µg/mL 4-OP during 44 h. The level of cholesterol was determined from the culture medium using photometry. Quantification of steroid hormones was performed by the enzyme linked immunosorbent assay and intracellular generation of superoxide radicals was assessed by the nitroblue-tetrazolium assay. Administered concentrations of 4-OP (0.04-5.0 µg/mL) did not affect basal and hCG-stimulated cholesterol level significantly. However, basal DHEA secretion was increased significantly (P < 0.001) in the highest experimental dose (5 µg/mL) of 4-OP. By hCG-stimulated DHEA secretion, a significant (P < 0.001) decrease was recorded at 5.0 µg/mL 4-OP in comparison to the control group. The stimulatory effect of 4-OP was also confirmed in androstenedione secretion, when 2.5 and 5.0 µg/mL increased hormone secretion significantly (P˂0.05; P˂0.001). Exposure to experimental concentrations (0.04 to 5.0 µg/mL) of tested chemical reduced hCG-stimulated androstenedione formation, but not significantly. Measurements of basal testosterone production have shown significant (P˂0.01; P˂0.001) increase at 1.0; 2.5 and 5.0 µg/mL of 4-OP. Furthermore, 44 h treatment by 4-OP (1.0-5.0 µg/mL) caused significant (P˂0.01; P˂0.001) intracellular accumulation of superoxide radicals in exposed cells. A considerably more detailed and systematic research is required for a better understanding of risks associated with male reproductive system in humans and wildlife.