Reduced Reactive Oxygen Species Content Research Articles

CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3 regulates salt tolerance through CATALASE 2 in Arabidopsis.

Soil salinization threatens global crop production. Here, we report that a receptor-like cytoplasmic kinase, CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3 (CRCK3), plays an essential role in plant salt tolerance via CATALASE 2 (CAT2), a hydrogen peroxide (H2O2)-scavenging enzyme in Arabidopsis (Arabidopsis thaliana). CRCK3 was induced by salt stress, and its knockout mutant displayed a salt-sensitive phenotype compared with wild-type plants. CRCK3 was activated by salt stress in a calcium-dependent manner, and its kinase activity was required for plant salt tolerance. CRCK3 physically interacted with CAT2, and CRCK3-mediated salt tolerance depended on CAT2. Salt treatment significantly induced CAT2 phosphorylation via the action of CRCK3, and this phosphorylation was required for CAT2-mediated H2O2 scavenging to reduce reactive oxygen species (ROS) content and oxidative damage in plants under saline conditions. CRCK3 phosphorylated CAT2 at the Thr209 residue, resulting in elevated catalase activity to reduce ROS accumulation under saline conditions. Therefore, the CRCK3-CAT2 module mediates plant salt tolerance by maintaining redox homeostasis. This study expands our knowledge of how plants respond to salt stress.

The role of FOXM1 in acetylcysteine improving diabetic periodontitis.

Diabetic periodontitis (DP) stems from hyperglycemia-driven oxidative stress amplification and chronic inflammation, leading to periodontal tissue breakdown. Misregulated forkhead box protein M1 (FOXM1) play key roles in this process, exacerbating both inflammation and oxidative stress. In light of N-Acetylcysteine (NAC)'s potent anti-oxidative capacity and anti-inflammatory potential, understanding how it modulates these central pathways to alleviate DP holds high scientific and clinical importance. An animal model of diabetic mice periodontitis was established, and the model mice were injected with FOXM 1 adenovirus to enrich FOXM 1, and the periodontal pathological histology of each group was evaluated by HE staining. Western blotting and RT-PCR evaluated the expression levels of factors involved in bone destruction. ELISA evaluated the amount of inflammatory factors in mice serum. FOXM 1 over-expression and NAC were treated in murine macrophages, and the intracellular reactive oxygen species(ROS) levels in macrophages were measured using a DCFH-DA probe. Receptor activator of NF-κB ligand (RANKL) and lipopolysaccharide (LPS) were used to establish the macrophage osteoclast differentiation model and test the expression level of osteoclast differentiation factors after giving NAC. Hydrogen peroxide was used to establish a peroxidation environment, the plasmid silenced C-JUN, and the DNA binding activity of activating protein-1(AP1) was detected by EMSA. The effect of peroxidation on the osteoclast differentiation level was determined by WB. Mice with DP model had epithelial damage and inflammatory infiltration in periodontal tissues, and in the FOXM1 enriched group, the periodontal epithelial damage was repaired and inflammation was alleviated. FOXM1 enrichment resulted in DP model lower expression of RANKL (P < 0.01), macrophage colony-stimulating factor (M-CSF) (P < 0.01) and elevated expression of osteoprotegerin (OPG) (P < 0.001). Serum levels of pro-inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNF-α), and inducible nitric oxide synthase (iNOS) were elevated in DP mice (P < 0.001), and anti-inflammatory factor IL-10 was reduced(P < 0.001),, and FOXM1 enrichment significantly reversed inflammatory factor levels (P < 0.01). Overexpression of FOXM1 reduced ROS content in macrophages (P < 0.001), and NAC was performed to further reduce ROS content (P < 0.01). Silencing of FOXM1 elevated the expression of osteoclast-specific genes NFATc1, TRAP and OSCAR (P < 0.01), and the addition of NAC on top of silencing of FOXM1 markedly suppressed the expression level of osteoclast-specific genes (P < 0.01). ROS increased the transcriptional activity of AP1 (P < 0.001), which promoted osteoclast-specific gene expression (P < 0.001), and osteoclast-specific gene expression was decreased after silencing C-JUN (P < 0.01). FOXM1 relieve diabetic periodontitis inflammation and promote bone formation, regulates ROS production and ROS increases the transcriptional activity of AP1 and affects the osteoclastic differentiation of macrophages, which plays a positive role in bone protection in diabetic periodontitis.

The Impact of NADPH Oxidase 5 Activity and Reactive Oxygen Species on Capacitated Human Sperm.

Progesterone (P4) activates sperm calcium channels (CatSper), allowing calcium to enter the cell, which activates NADPH Oxidase-5 (NOX5) and produces reactive oxygen species (ROS). While calcium and ROS are essential for sperm capacitation, the role of NOX5 in capacitated sperm is unclear. This study investigated NOX5 activity in capacitated human sperm. Forty semen samples from fertile men were processed, with motile sperm separated and divided into nine groups: control (Ham's F-10), solvent (DMSO), progesterone, diphenyleneiodonium chloride (DPI, NOX5 inhibitor), phorbol-12-myristate 13-acetate (PMA, NOX5 activator), P4+DPI, P4+PMA, Trolox, and P4+ Trolox. Sperm kinematics, membrane integrity, survival rate, and ROS production was evaluated. Data were analyzed using ANOVA and Kruskal-Wallis tests, p≤ 0.05 considered statistically significant. Progressive motility significantly decreased with DPI (56.2±2.1%) and PMA (56.5±2.1%), both alone and combined with progesterone (58.0±2.0% and 57.4±2.2%), compared to the progesterone group (66.0±1.9%). No significant change was observed in the Trolox groups. Progesterone, alone or combined with DPI, PMA, and Trolox, significantly reduced sperm linearity from 0.6±0.01 to 0.5±0.01%. Straight-line velocity decreased in P4+PMA and P4+Trolox groups (88.2±4.4 and 89.7±3.9 μm/s) compared to the control group (105.0±5.5 μm/s). Trolox reduced ROS content, while other treatments had no effect on ROS levels. NOX5 does not play a prominent role in capacitated sperm. The negative effects of PMA and DPI on sperm motility appear independent of their actions on NOX5 and ROS production. Trolox did not affect sperm motility and survival, indicating that capacitated sperm require little or no ROS.

Ginsenoside Rg1 ameliorates cerebral ischemia-reperfusion injury by regulating Pink1/ Parkin-mediated mitochondrial autophagy and inhibiting microglia NLRP3 activation

This study aimed to further elucidate the mechanism of ginsenoside Rg1 in the treatment of cerebral ischemia-reperfusion. In this study, we observed the apoptosis of RM cells (microglia) after oxygen-glucose deprivation/reoxygenation (OGD/R) modeling before and after Rg1 administration, changes in mitochondrial membrane potential, changes in the content of Reactive oxygen species (ROS) and inflammatory vesicles NLR Family Pyrin Domain Containing 3 (NLRP3), and the expression levels of autophagy-related proteins, inflammatory factors, and apoptosis proteins. We further examined the pathomorphological changes in brain tissue, neuronal damage, changes in mitochondrial morphology and mitochondrial structure, and the autophagy-related proteins, inflammatory factors, and apoptosis proteins expression levels in CI/RI rats before and after administration of Rg1 in vivo experiments. In vitro experiments showed that Rg1 induced mitochondrial autophagy, decreased mitochondrial membrane potential, and reduced ROS content thereby inhibiting NLRP3 activation, decreasing secretion of inflammatory factors and RM cell apoptosis by regulating the PTEN induced putative kinase 1(Pink1) /Parkin signaling pathway. In vivo experiments showed that Rg1 induced mitochondrial autophagy, inhibited NLRP3 activation, improved inflammatory response, and reduced apoptosis by regulating the Pink1/Parkin signaling pathway, and Rg1 significantly reduced the area of cerebral infarcts, improved the pathological state of brain tissue, and attenuated the neuronal damage, thus improving cerebral ischemia/reperfusion injury in rats. Our results suggest that ginsenoside Rg1 can ameliorate cerebral ischemia-reperfusion injury by modulating Pink1/ Parkin-mediated mitochondrial autophagy in microglia and inhibiting microglial NLRP3 activation.

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32mg/kg diet.

A polysaccharide gel made of gellan gum improves oocyte maturation and embryonic development in pigs

Gellan gum (GG) is a soft, tractable, and natural polysaccharide substrate used for cell incubation. In this study, we examined the effects of GG on porcine oocyte maturation. Cumulus cells and oocyte complexes (COCs) were collected from slaughterhouse-derived porcine ovaries and cultured on plastic plates containing 0.05% or 0.1% GG gels. The 0.1% GG gel improved the maturation rate and quality of blastocysts, as determined by the total cell number and the rate of abnormally condensed nuclei. GG gels have antioxidant abilities and oocytes cultured on GG gels (0.05% and 0.1%) have reduced reactive oxygen species (ROS) content. Furthermore, GG gels (0.05% and 0.1%) increased F-actin formation, whereas treatment of oocytes with H2O2 reduced F-actin levels. GG gels increased the ATP content in oocytes but did not affect the mitochondrial DNA copy number or mitochondrial membrane potential. In addition, the medium cultured on 0.05% GG increased the glucose consumption of COCs. In conclusion, GG gel reduced ROS content, increased energy content, and improved subsequent embryonic development in pigs.

Development toxicity and cytotoxicity of pyroxsulam on embryos and adults of zebrafish (Danio rerio)

Pyroxsulam is a typical triazolopyrimidine sulfonamide herbicide, which plays a vital role in weed control for wheat production. Previous studies on pyroxsulam have mainly focused on weed resistance, control effects, and soil microorganisms, however, research on aquatic ecotoxicity is lacking. This study evaluated the toxicity of pyroxsulam in zebrafish embryos (120h) and adults (14 and 28d). Chronic exposure to pyroxsulam (0.2, 2, and 10μgL-1) reduced reactive oxygen species (ROS) content in embryos but increased ROS content in adults. Pyroxsulam caused lipid peroxidation and DNA damage in embryos and adults. The expression of p53, bcl-2/bax, and caspase 3/8/9 indicated that pyroxsulam inhibited apoptosis in embryos but induced apoptosis in adults. By calculating integrated biomarker response, apoptosis was more readily affected than oxidative damage in embryos and adults. The toxicity of pyroxsulam increased with increasing concentration, however, with increasing exposure time, the toxicity of pyroxsulam to adults decreased. Pyroxsulam exerted toxic effects on zebrafish at different life stages (embryos and adults), and different stages had different toxicity. These results indicate that in future studies on the toxicity of pyroxsulam to zebrafish, different life stages (embryos and adults) need to be studied. The present study evaluated the toxicity of environmentally relevant concentrations of pyroxsulam to zebrafish embryos and adults, providing worthy data for assessing its effects on aquatic ecosystems.

Neuroprotective Effect of Ginseng Fibrous Root Enzymatic Hydrolysate against Oxidative Stress.

Oxidative stress is one of the potential causes of nervous system disease. Ginseng extract possesses excellent antioxidant activity; however, little research on the function of the ginseng fibrous root. This study aimed to investigate the neuroprotective effects of ginseng fibrous root to alleviate the pathogenesis of Alzheimer's disease (AD) against oxidative stress. Ginseng fibrous root enzymatic hydrolysate (GFREH) was first prepared by digesting ginseng fibrous roots with alkaline protease. In vitro, the GFREH showed antioxidant activities in free radical scavenging mechanisms. With a cellular model of AD, GFREH inhibited the increase in Ca2+ levels and intracellular ROS content, maintained the balance of mitochondrial membrane potential, and relieved L-glutamic acid-induced neurotoxicity. In vivo, GFREH improved the survival rate of Caenorhabditis elegans (C. elegans) under oxidative stress, upregulated SOD-3 expression, and reduced reactive oxygen species (ROS) content. Therefore, our findings provide evidence for the alleviation effect of GFREH against oxidative stress in neuroprotection, which may accelerate the development of anti-Alzheimer's drugs and treatments in the future.

Photoprotective effects of Sargassum thunbergii on ultraviolet B-induced mouse L929 fibroblasts and zebrafish

BackgroundChronic exposure to ultraviolet B (UVB) causes a series of adverse skin reactions, such as erythema, sunburn, photoaging, and cancer, by altering signaling pathways related to inflammation, oxidative stress, and DNA damage. Marine algae have abundant amounts and varieties of bioactive compounds that possess antioxidant and anti-inflammatory properties. Thus, the objective of this study was to investigate the photoprotective effects of an ethanol extract of Sargassum thunbergii.MethodsSargassum thunbergii phenolic-rich extract (STPE) was prepared, and its activity against UVB damage was evaluated using L929 fibroblast cells and zebrafish. STPE was extracted and purified by 40% ethanol and macroporous resin XDA-7. Reactive oxygen species (ROS) and antioxidant markers, such as superoxide dismutase (SOD), catalase (CAT) activities, and malondialdehyde (MDA) content were analyzed. The effect of STPE on UVB-induced inflammation was determined by inflammatory cytokine gene and protein expression. The expression of signaling molecules in the Nuclear Factor KappaB (NF-κB) pathway was determined by western blotting. DNA condensation was analyzed and visualized by Hoechst 33342 staining. In vivo evaluation was performed by tail fin area and ROS measurement using the zebrafish model.ResultsThe total polyphenol content of STPE was 72%. STPE reduced ROS content in L929 cells, improved SOD and CAT activities, and significantly reduced MDA content, thereby effectively alleviating UVB radiation-induced oxidative damage. STPE inhibited the mRNA and protein expression of TNF-α, IL-6, and IL-1α. STPE reversed DNA condensation at concentrations of 20 and 40 μg/mL compared with the UVB control. Moreover, STPE inhibited NF-κB signaling pathway activation and alleviated DNA agglutination in L929 cells after UVB irradiation. Additionally, 1.67 μg/mL STPE significantly increased the tail fin area in zebrafish, and 0.8–1.6 μg/mL STPE effectively eliminated excessive ROS after UVB radiation.ConclusionsSTPE inhibited UVB-induced oxidative stress, inflammatory cytokine expression, and DNA condensation via the downregulation of the NF-κB signaling pathway, suggesting that it prevents UVB-induced photodamage, and has potential for clinical development for skin disease treatment.

Cysteine-Rich Whey Protein Isolate (CR-WPI) Ameliorates Erectile Dysfunction by Diminishing Oxidative Stress via DDAH/ADMA/NOS Pathway.

Nitric oxide synthase- (NOS-) dependent endothelial dysfunction induced by oxidative stress (OS) is assumed to play a pivotal role in the pathogenesis and progression of diabetes mellitus-related erectile dysfunction (DMED). Cysteine-rich whey protein isolate (CR-WPI) is a widely used protein supplement and has been confirmed to reduce reactive oxygen species (ROS) by increasing cellular antioxidant glutathione (GSH). However, it is currently unknown whether CR-WPI elicits therapeutic effects in DMED. Here, we provide diabetic rats with CR-WPI to determine its effect on DMED and the underlying mechanisms. The results suggest that CR-WPI supplementation increased GSH biosynthesis and reduced ROS content and simultaneously upregulated the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetrical dimethylarginine (ADMA)/nitric oxide synthase (NOS) metabolic pathway. Evaluation of intracavernous pressure (ICP) also showed an improvement of penile erectile function in CR-WPI-treated rats. The results of the vitro cell culture showed that glutathione pretreatment protected corpus cavernosum smooth muscle cells (CCSMC) from H2O2-induced apoptosis by decreasing Caspase 9 and Caspase 3 expressions. These results augur well for the potential therapeutic application of dietary CR-WPI supplementation for treating diabetic erectile dysfunction.

Protection and reversion role of a pure stilbene extract from grapevine shoot and its major compounds against an induced oxidative stress

Sulfur dioxide is a controversial preservative used in winemaking. Given its potential toxicity, it is of great interest to find alternatives aimed at replacing or reducing sulfur dioxide. The objective was to assess the antioxidant effects of a grapevine-shoot extract with 99% of stilbenes (ST-99), its major compounds (trans-resveratrol and trans-ε-viniferin) and their mixture (1:3.9) by measuring reactive oxygen species (ROS) and glutathione (GSH) levels in vitro. Their ability to protect against or reverse the effects of H2O2 on cells were also studied. The results showed that ST-99, followed by trans-ε-viniferin and the mixture, were able to reduce ROS levels, increased GSH content and exhibited antioxidant ability against an induced oxidative stress. Trans-resveratrol significantly reduced ROS content only at the highest concentrations. ST-99 at non-cytotoxic concentrations is more effective than the other compounds, which might be attributed to increased levels of GSH. The results suggest a promising use of ST-99.

CircGLI3 Inhibits Oxidative Stress by Regulating the miR-339-5p/VEGFA Axis in IPEC-J2 Cells.

As a new type of noncoding RNA, circular RNA (circRNA) is stable in cells and not easily degraded. This type of RNA can also competitively bind miRNAs to regulate the expression of their target genes. The role of circRNA in the mechanism of intestinal oxidative stress (OS) in weaned piglets is still unclear. In our research, diquat (DQ) was used to induce OS in small intestinal epithelial cells (IPEC-J2) to construct an OS cell model. Mechanistically, dual luciferase reporter assays, fluorescence in situ hybridization (FISH), and western blotting were performed to confirm that circGLI3 directly sponged miR-339-5p and regulated the expression of VEGFA. Overexpression of circGLI3 promoted IPEC-J2 cell proliferation, increased the proportion of S-phase cells (P < 0.01), and reduced reactive oxygen species (ROS) generation when IPEC-J2 cells were subjected to OS. circGLI3 can increase the activity of glutathione peroxidase (GSH-Px) and the total antioxidant capacity (T-AOC) in IPEC-J2 cells and reduce the malondialdehyde (MDA) content and levels of inflammatory factors. Therefore, overexpression of circGLI3 reduced oxidative damage, whereas miR-339-5p mimic counteracted these effects. We identified a regulatory network composed of circGLI3, miR-339-5p, and VEGFA and verified that circGLI3 regulates VEGFA by directly binding miR-339-5p. The expression of VEGFA affects IPEC-J2 cell proliferation, cell cycle progression, and ROS content and changes the levels of antioxidant enzymes and inflammatory factors. This study reveals the molecular mechanism by which circGLI3 inhibits OS in the intestine of piglets and provides a theoretical basis for further research on the effect of OS on intestinal function.

A grape seed extract maternal dietary supplementation improves egg quality and reduces ovarian steroidogenesis without affecting fertility parameters in reproductive hens.

In broiler hens, the genetic selection increased susceptibility to metabolic disorders and reproductive dysfunctions. In human ovarian cells, grape seed extracts (GSE) improved steroid production. Here, we investigated the effects of a GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. For this, we designed two in vivo experiments, the first one included three groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, since week 4), and the second one used two groups of hens: A (control) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23th to 40th weeks and quality at 33th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. The fertility parameters and egg production were unaffected by GSE supplementation whatever the experiment (exp.). However, the rate of double-yolk eggs decreased for all GSE supplemented groups (exp. 1 P <0.01, exp.2, P<0.02). In exp.1, C group eggs were bigger and larger (P<0.0001) and the shell elasticity was higher for both B and C (P<0.0003) as compared to control. In the egg yolk, GSE supplementation in both exp. reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells (P<0.001). Interestingly, in both exp. RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk (P<0.0001). Taken together, maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.

33 Assessment of spindle morphology and reactive oxygen species production after vitrification of bovine oocytes following invitro maturation in the presence of glutathione ethyl ester

Oocyte cryopreservation by vitrification affects spindle integrity and causes oxidative stress induced by reactive oxygen species (ROS). Glutathione (GSH) ensures the correct assembly of microtubules, prevents ROS from attacking tubulin assembly, and ensures normal spindle function during meiosis. We hypothesised that addition of GSH ethyl ester (GSH-OEt), a cell-permeable GSH donor, to IVM medium before vitrification may improve oocyte cryotolerance by maintaining spindle morphology and preventing ROS production. For the IVM, viable cumulus-oocyte complexes were randomly distributed into two groups: 1) control: conventional IVM media with tissue culture medium-199 + 20% fetal calf serum + epidermal growth factor and 2) GSH-OEt: IVM medium supplemented with 5mM GSH-OEt at 38.5°C in a 5% CO2 humidified air atmosphere. After 22h of IVM, half of the oocytes from each group were vitrified/warmed using the Cryotop method (Kitazato Corp.). After 24h of IVM, oocytes were denuded, fixed, and microtubule and chromosome distribution were analysed by immunofluorescence. To measure ROS, oocytes were denuded and incubated in 5μM 2′,7′-dichlorodihydrofluorescein diacetate. Fluorescence was acquired, and resulting images were quantified with ImageJ software. A linear mixed effect followed by a pairwise comparison test (Tukey adjustment) were performed to analyse differences in meiotic spindle configuration and ROS production (P&amp;lt;0.05). No significant differences in percentages of MII or normal spindle configuration were observed among treatments. Treatment with GSH-OEt before vitrification resulted in similar percentages of abnormal spindle configuration to control oocytes, whereas vitrified oocytes showed significantly higher percentages of abnormal spindle configuration relative with control oocytes (Table 1). When the content of ROS was measured, vitrification resulted in significantly higher levels (15.1E+06±1.2E+06; P&amp;lt;0.05) when compared with other treatments. However, oocytes vitrified after IVM with GSH-OEt (11.8E+06±0.6E+06) showed similar ROS levels to control (10.7E+06±0.2E+06) and GSH-OEt (10.0E+06±0.1E+06) groups. In conclusion, addition of GSH-OEt to IVM before vitrification did not have detrimental effects on spindle morphology and was able to reduce ROS content. Further experiments are warranted to assess whether the addition of GSH-OEt to IVM medium can improve oocyte development competence after vitrification. Table 1.Assessment of spindle morphology after oocyte vitrification (VIT) following IVM in the presence of glutathione ethyl ester (GSH-OEt) Item n MII (%) Spindle configuration (%) Chromosome distribution (%) Normal Abnormal Absent Dispersed Decondensed Absent Control 127 79.4±92 74.1±4.4 16.2±5.6a 9.7±1.1 16.2±3.8 9.7±1.1 0 GSH-OEt 113 76.5±6.5 65.6±5.7 26.2±9.0ab 8.1±4.3 19.5±2.6 13.7±3.0 1.1±1.1 VIT 71 60.0±5.6 54.8±7.8 32.7±10.8b 12.4±1.4 34.4±8.7 10.7±6.4 0 GSH-OEt VIT 53 57.4±10.7 58.5±8.8 31.1±9.3ab 10.4±5.8 34.8±5.6 6.7±6.7 0 a,bDifferent superscripts indicate significant differences (P&amp;lt;0.05). *Percentage referred to the total number of oocytes at MII. This work was supported by MCIU, Spain (AGL2016-79802-P) and Generalitat de Catalunya (García-Martínez, 2017_FI_00451).

Melatonin-related mitochondrial respiration responses are associated with growth promotion and cold tolerance in plants

Melatonin has the ability to improve plant growth and strengthened plant tolerance to environmental stresses; however, the effects of melatonin on mitochondrial respiration in plants and the underlying biochemical and molecular mechanisms are still unclear. The objective of the study is to determine possible effects of melatonin on mitochondrial respiration and energy efficiency in maize leaves grown under optimum temperature and cold stress and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance. Melatonin and cold stress, alone and in combination, caused significant increases in activities and gene expressions of pyruvate dehydrogenase, citrate synthase, and malate dehydrogenase, indicating an acceleration in the rate of tricarboxylic acid cycle. Total mitochondrial respiration rate, cytochrome pathway rate, and alternative respiration rate were increased by the application of melatonin and/or cold stress. Similarly, gene expression and protein levels of cytochrome oxidase and alternative oxidase were also enhanced by melatonin and/or cold stress. The highest values for all these parameters were obtained from the seedlings treated with the combined application of melatonin and cold stress. The activity and gene expression of ATP synthase and ATP concentration were augmented by melatonin under control and cold stress. On the other hand, cold stress reduced markedly plant growth parameters, including root length, plant height, leaf surface area, and chlorophyll content and increased the content of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide and oxidative damage, including malondialdehyde content and electrolyte leakage level; however, melatonin significantly promoted the plant growth parameters and reduced ROS content and oxidative damage under control and cold stress. These data revealed that melatonin-induced growth promotion and cold tolerance in maize is associated with its modulating effect on mitochondrial respiration.

Acupuncture improved depressive behavior by regulating expression of hippocampal apoptosis-related factors in psychological stress-induced depression rats

To investigate the effect of acupuncture on behavior changes and the expression of reactive oxygen species (ROS), cytochrome C, cysteine-containing aspartate-specific proteases-3 (caspase-3), apoptosis inducing factor (AIF) in the hippocampus of rats exposed to chronic restraint stress (CRS), so as to provide experimental evidence for the prevention and treatment of depression. Thirty two male SD rats were equally randomized into control, model, acupuncture and medication (Fluoxetine) groups according to the random number table. The depression model was established by chronic psychological stress stimulation (fasting, water deprivation, chronic restraint combined with solitary raising for 28 days). Manual acupuncture stimulation (2 circles/s) was applied to "Baihui" (GV20), "Yintang" (GV29) and bilateral "Sanyinjiao" (SP6) for 2 min, followed by retaining the needles for 20 min. Rats in the medication group were treated by gavage of Fluoxetine (0.18 mg/mL, 1 mL/100 g body weight). The treatment was conducted once daily for 28 days. The depression-like behavior was evaluated by using open-field test, sucrose preference test, and body weight. The content of ROS of the right hippocampus tissue was detected by using dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay, and the expression levels of cytochrome C, caspase-3 and AIF proteins of hippocampus were determined by using Western blot. After 28 days' stress procedure, the depression rats in the model group showed a significant decrease in the numbers of crossing and rearing of open-field test, sucrose preference index and the body weight compared to the control group (P<0.01). The content of ROS and expression levels of cytochrome C, caspase-3 and AIF in the hippocampus of the model group were significantly increased relevant to the control group (all P<0.01). After the intervention and compared with the model group, the decreased levels of the numbers of crossing and rearing, body weight and sucrose preference index, and the up-regulated levels of content of ROS and expression of cytochrome C, caspase-3 and AIF proteins were all reversed in both acupuncture and medication groups (P<0.01，P<0.05), displaying an anti-depressant effect. The effect of acupuncture was significantly superior to that of medication in up-regulating the numbers of crossing and rearing (P<0.01, P<0.05), and in down-regulating the expression of caspase-3 (P<0.01). No significant differences were found between the medication and acupuncture groups in increasing the body weight and sucrose preference index, and in down-regulating the expression of cytochrome C and AIF proteins (P>0.05). Acupuncture intervention can significantly reduce ROS content and expression level of cytochrome C, caspase-3 and AIF proteins in the hippocampus possibly by inhibiting the chronic psychological stress-hippocampal oxidative stress-mitochondrial apoptotic pathway in psychological stress-induced depression rats, which may contribute to its effect in relieving depression.

Interactive effects of abscisic acid and nitric oxide on chilling resistance and active oxygen metabolism in peach fruit during cold storage.

Cold conditions can accelerate the production of reactive oxygen species (ROS), and excessive ROS may attack biological macromolecules, disrupt related signal pathways, induce oxidative stress and influence plant metabolism. The cross-talk between nitric oxide (NO) and abscisic acid (ABA) and the inhibitions by NO or ABA on oxidative damage have been reported in fruits. However, there are few reports about the roles of NO-ABA interactions in chilling stress and antioxidant defense in fruits during cold storage. This study was conducted to investigate the roles of NO, ABA and interactions between NO and ABA in response to chilling stress on peach fruit (Prunus persica (L.) Batsch, cv. 'Xintaihong'). Treatments with 15 µmol L-1 NO, 100 µmol L-1 ABA and 15 µmol L-1 NO + 5mmol L-1 sodium tungstate solution could reduce ROS content, alleviate lipid peroxidation and enhance antioxidant enzyme activities and antioxidant capacities. However, treatments with 5µmol L-1 potassium 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), 5mmol L-1 sodium tungstate and 100 µmol L-1 ABA + 5µmol L-1 c-PTIO differentially blocked these protective effects and significantly increased ROS content and lipid peroxidation of peaches under low-temperature conditions. Application of exogenous ABA could increase the resistance to cold-induced oxidative stress by enhancing the efficiency of enzymatic and non-enzymatic systems, which were partially mediated by NO. © 2018 Society of Chemical Industry.

Hydroxyurea therapy modulates sickle cell anemia red blood cell physiology: Impact on RBC deformability, oxidative stress, nitrite levels and nitric oxide synthase signalling pathway

Hydroxyurea (HU) has been suggested to act as a nitric oxide (NO) donor in sickle cell anemia (SCA). However, little is known about the HU NO-related effects on red blood cell (RBC) physiology and NO signalling pathway. Thirty-four patients with SCA (22 under HU treatment (HU+) and 12 without (HU-)) and 17 healthy subjects (AA) were included. RBC nitrite content, deformability and reactive oxygen species (ROS) levels were measured. RBC NO-synthase (RBC-NOS) signalling pathway was assessed by the measurement of RBC-NOS serine1177 and RBC-AKT serine473 phosphorylation. We also investigated the in vitro effects of Sodium Nitroprusside (SNP), a NO donor, on the same parameters in SCA RBC. RBC nitrite content was higher in HU+than in HU- and AA. RBC deformability was decreased in SCA patients compared to AA but the decrease was more pronounced in HU-. RBC ROS level was increased in SCA compared to AA but the level was higher in HU- than in HU+. RBC-NOS serine1177 and RBC-AKT serine473 phosphorylation were decreased in HU+compared to HU- and AA. SCA RBC treated with SNP showed increased deformability, reduced ROS content and a decrease in AKT and RBC-NOS phosphorylation. Our study suggests that HU, through its effects on foetal hemoglobin and possibly on NO delivery, would modulate RBC NO signalling pathway, RBC rheology and oxidative stress.

L-butyl phthalein improves neural function of vascular dementia mice by regulating the PI3K/AKT signaling pathway.

L-3-n-butylphthalide (L-NBP) is a type of anti-ischemic cranial nerve protective drug that may act on vascular dementia (VD). Phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT/PKB) signaling pathway can up-regulate B-cell lymphoma 2 (Bcl-2) expression, reduce reactive oxygen species (ROS) production, and alleviate cell apoptosis. This study aimed at investigating the role of L-NBP on neurological function and cell apoptosis in VD mouse through regulating PI3K/AKT signaling pathway. The mice were divided into four groups, including Sham, VD, VD + solvent, and VD + L-NBP. HT22 cells were cultured in vitro and treated by ischemia/reperfusion (I/R). HT22 cells were divided into four groups, including I/R, VD + solvent, VD + L-NBP, and VD + L-NBP + LY294002 groups. Phosphorylated AKT (p-AKT) and Bcl-2 expressions were tested. ROS content in hippocampus tissue was detected by flow cytometry. Cell apoptosis was evaluated by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. ROS content and cell apoptosis increased, while p-AKT and Bcl-2 expressions reduced in hippocampus tissue from VD group compared with sham group. L-NBP significantly up-regulated p-AKT and Bcl-2 expressions and decreased ROS content and cell apoptosis in hippocampus tissue. I/R treatment markedly induced HT22 cell apoptosis and ROS production, and reduced p-AKT and Bcl-2 expressions. L-NBP treatment markedly up-regulated p-AKT and Bcl-2 levels, restrained cell apoptosis, and reduced ROS content in TH22 cells intervened by I/R. LY294002 apparently attenuated the protective effect of L-NBP on HT22 cells. L-NBP protects VD by up-regulating PI3K/AKT signaling pathway, elevating Bcl-2 expression, reducing nerve cell apoptosis, and restraining ROS production.

MiR-124 affects the apoptosis of brain vascular endothelial cells and ROS production through regulating PI3K/AKT signaling pathway.

The apoptosis of vascular endothelial cells (VEC) is related to ischemic stroke. Phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT/PKB) signaling pathway can upregulate Bcl-2 expression, reduce reactive oxygen species (ROS) production, and induce apoptosis. The level of miR-124 was significantly increased after cerebral ischemia. This study aimed to investigate the role of miR-124 in regulating PI3K expression, brain VEC apoptosis, and ROS production. The expressions of miR-124, PI3K, p-AKT, and Bcl-2 in brain VEC of rats from the sham group and middle cerebral artery occlusion (MCAO) group were tested. Bioinformatics analysis showed the complementary binding site between miR-124 and PI3K mRNA. ROS content and cell apoptosis were detected by flow cytometry. Rat brain VEC were cultured in vitro and treated by oxygen-glucose deprivation (OGD) for 6 h. VECs were divided into four groups, including miR-NC, miR-124 inhibitor, pIRES2-blank, and pIRES2-PI3K groups, and were further treated by OGD. MiR-124 expression, ROS content, and cell apoptosis were markedly increased, whereas the levels of PI3K, p-AKT, and Bcl-2 were markedly reduced in rat VECs from MCAO group compared with that in the sham group. OGD treatment significantly induced VECs apoptosis, upregulated miR-124 expression and ROS content, and down-regulated the levels of PI3K, p-AKT, and Bcl-2. MiR-124 inhibitor or transfection of pIRES2-PI3K plasmid apparently enhanced PI3K, p-AKT, and Bcl-2 expressions, alleviated cell apoptosis and decreased ROS content in VECs induced by OGD. Our data demonstrated that miR-124 induced the apoptosis of brain vascular endothelial cells via the down-regulation of PI3K/AKT signaling pathway and promotion of ROS production.