Diphenylene Iodonium Research Articles

FmRbohH Mediates ROS Generation and Enhances Pollen Tube Growth in Fraxinus mandshurica

Flowering plants require normal pollen germination and growth to be fertilized, but studies on the mechanism regulating pollen tube growth in Fraxinus mandshurica are limited. Here, we used transcriptomic data to study the oxidative phosphorylation pathway during pollen tube growth in Fraxinus mandshurica. Our study identified 8,734 differentially expressed genes during the stages S1 to S3 of pollen tube growth. Significant enrichment of the oxidative phosphorylation pathway, amino acid synthesis, protein processing in the ER, carbon metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis were examined using the Kyoto Encyclopedia of Genes and Genomes, and 58 genes linked to ROS synthesis and scavenging during the S1–S3 stages were identified. Also, H2DCFDA staining confirmed ROS formation in the pollen and the pollen tubes, and treatment with copper (II) chloride (CuCl2) and diphenyleneiodonium (DPI) was shown to reduce ROS in the pollen tube. Reduction in ROS content caused decreased pollen germination and pollen tube length. Furthermore, FmRbohH (respiratory burst oxidase homolog H) expression was detected in the pollen and pollen tube, and an antisense oligodeoxynucleotide assay demonstrated reduced ROS and pollen tube growth in Fraxinus mandshurica. This study shed more light on the RbohH gene functions during pollen tube growth.

Hydrogen Peroxide Is Involved in Methane-Alleviated Cadmium Toxicity in Alfalfa (Medicago sativa L.) Seedlings by Enhancing Cadmium Chelation onto Root Cell Walls.

Although previous studies have demonstrated that methane (CH4) can mitigate the toxicity of cadmium (Cd) in alfalfa seedlings, the CH4-rich water used in these studies may create hypoxic conditions, potentially influencing the experimental outcomes. Therefore, this study aimed to investigate whether CH4 can reduce Cd toxicity in alfalfa seedlings without the interference of hypoxia and to analyze its underlying mechanisms. Here, it was observed that supplementing oxygen with saturated CH4-rich water can significantly alleviate the inhibition of 75 μM CdCl2 on the growth of alfalfa (Medicago sativa L.) seedlings. Less Cd accumulation was also observed in both root and shoot parts, which could be explained by the CH4-altered cell wall components in alfalfa seedling roots, including covalent and ionic soluble pectin, and the degree of demethylation in pectin, thus enabling a higher proportion of Cd binding to the cell walls and reducing the entry of Cd into the cells. The above actions of CH4 were accompanied by an increase in hydrogen peroxide (H2O2) content and NADPH oxidase activity, which could be blocked by the addition of the NADPH oxidase inhibitor diphenylene iodonium (DPI). Taken together, these results implied that exogenously applied CH4 could alleviate Cd toxicity in alfalfa seedlings by enhancing Cd chelation onto the root cell walls, which might be closely associated with NADPH oxidase-dependent H2O2 signals. These findings could provide insight into the mechanism through which CH4 alleviates Cd toxicity in alfalfa plants.

CAIP-Induced ROS Production Contributes to Sustaining Atherosclerotic Process Associated with Helicobacter cinaedi Infection through Macrophages and Endothelial Cells Activation.

Several lines of evidence have linked the intestinal bacterium Helicobacter cinaedi with the pathogenesis of atherosclerosis, identifying the Cinaedi Antigen Inflammatory Protein (CAIP) as a key virulence factor. Oxidative stress and inflammation are crucial in sustaining the atherosclerotic process and oxidized LDL (oxLDL) uptake. Primary human macrophages and endothelial cells were pre-incubated with 10 µM diphenyl iodonium salt (DPI) and stimulated with 20 µg/mL CAIP. Lectin-like oxLDL receptor (LOX-1) expression was evaluated by FACS analysis, reactive oxygen species (ROS) production was measured using the fluorescent probe H2DCF-DA, and cytokine release was quantified by ELISA assay. Foam cells formation was assessed by Oil Red-O staining, and phosphorylation of p38 and ERK1/2 MAP kinases and NF-κB pathway activation were determined by Western blot. This study demonstrated that CAIP triggered LOX-1 over-expression and increased ROS production in both macrophages and endothelial cells. Blocking ROS abrogated LOX-1 expression and reduced LDL uptake and foam cells formation. Additionally, CAIP-mediated pro-inflammatory cytokine release was significantly affected by ROS inhibition. The signaling pathway induced by CAIP-induced oxidative stress led to p38 MAP kinase phosphorylation and NF-κB activation. These findings elucidate the mechanism of action of CAIP, which heightens oxidative stress and contributes to the atherosclerotic process in H. cinaedi-infected patients.

RBOH-dependent signaling is involved in He-Ne laser-induced salt tolerance and production of rosmarinic acid and carnosol in Salvia officinalis

BackgroundIn the past two decades, the impacts of Helium-Neon (He–Ne) laser on stress resistance and secondary metabolism in plants have been studied, but the signaling pathway which by laser regulates this process remains unclear. Therefore, the current study sought to explore the role of RBOH-dependent signaling in He–Ne laser-induced salt tolerance and elicitation of secondary metabolism in Salvia officinalis. Seeds were primed with He–Ne laser (6 J cm− 2) and peroxide hydrogen (H2O2, 5 mM) and 15-old-day plants were exposed to two salinity levels (0, 75 mM NaCl).ResultsSalt stress reduced growth parameters, chlorophyll content and relative water content (RWC) and increased malodialdehyde (MDA) and H2O2 contents in leaves of 45-old-day plants. After 48 h of salt exposure, higher transcription levels of RBOH (encoding NADPH oxidase), PAL (phenylalanine ammonia-lyase), and RAS (rosmarinic acid synthase) were recorded in leaves of plants grown from seeds primed with He–Ne laser and/or H2O2. Despite laser up-regulated RBOH gene in the early hours of exposing to salinity, H2O2 and MDA contents were lower in leaves of these plants after 30 days. Seed pretreatment with He–Ne laser and/or H2O2 augmented the accumulation of anthocyanins, total phenol, carnasol, and rosmarinic acid and increased total antioxidant capacity under non-saline and more extensively at saline conditions. Indeed, these treatments improved RWC, and K+/Na+ ratio, enhanced the activities of superoxide dismutase and ascorbate peroxidase and proline accumulation, and significantly decreased membrane injury and H2O2 content in leaves of 45-old-day plants under salt stress. However, applying diphenylene iodonium (DPI as an inhibitor of NADPH oxidase) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) after laser priming reversed the aforementioned effects which in turn resulted in the loss of laser-induced salt tolerance and secondary metabolism.ConclusionsThese findings for the first time deciphered that laser can induce a transient RBOH-dependent H2O2 burst, which might act as a downstream signal to promote secondary metabolism and salt stress alleviation in S. officinalis plants.

Integrated transcriptomic and metabolomic analysis reveals the effects and potential mechanism of hydrogen peroxide on pigment metabolism in postharvest broccoli.

To understand the effects and related potential mechanism of H2O2 on pigment metabolism in postharvest broccoli, an integrated analysis of transcriptome and metabolome was performed. Results suggested that 65 differentially expressed genes and 26 differentially accumulated metabolites involved in chlorophyll, carotenoid, and flavonoid metabolism were identified. H2O2 treatment delayed the decrease of chlorophyll content by upregulating the expressions of chlorophyll synthetic genes, thylakoid synthetic genes, and 15 light-harvesting complex genes compared with the control and diphenylene iodonium treatments. H2O2 treatment decreased the accumulation of 11 flavonoids and 5 flavonols by downregulating the flavonoid synthetic genes. In addition, H2O2 treatment promoted carotenoid biosynthesis to eliminate reactive oxygen species in thylakoids, thereby protecting chlorophyll molecules from degradation. The inhibition of flavonoids and flavonols accumulation and chlorophyll decrease was the crucial reason for the delayed yellowing in H2O2 treatment. This study provides a new method and theoretical support for delaying the yellowing process in postharvest broccoli.

L-Glutamine mitigates bile acid-induced inhibition of growth factor activity in rat hepatocyte cultures

Bile acid-induced hepatotoxicity is inevitable in Cholestasis pathogenesis and L-Glutamine (L-Gln) has been reported to prevent total parenteral nutrition (TPN)-induced cholestasis in premature neonates. While mechanisms remain unknown, we hypothesize that bile acids impair growth factor (GF) function in hepatocytes which L-glutamine prevents through NAPDH oxidase (NOX) modulation. Glycochenodeoxycholic acid (GCDC, 0–100 µM) when added to primary hepatocyte cultures significantly (p < 0.01) decreased the FBS-induced BrdU incorporation, however inhibition of Fibroblast Growth factor (FGF)- or Hepatocyte growth factor (HGF)-induced DNA synthesis was more pronounced (p < 0.001). L-Gln markedly attenuated GCDC-mediated inhibition of DNA synthesis in both FBS and GF-treated cells. GCDC significantly increased the NADPH oxidase activity and NOX-1 protein expression that were markedly reduced by L-Gln and protein kinase c (PKC) inhibitor, LY-333531. Apocynin (APCN) and diphenyliodonium (DPI) significantly blocked the GCDC-mediated inhibition of GF-induced DNA synthesis. This study demonstrates that bile acid-induced hepatotoxicity involves dysfunction of certain growth factors via protein kinase c (PKC)- mediated NOX modulation which can be corrected, at least partly, by L-glutamine.

The NADPH oxidase inhibitor diphenyleneiodonium suppresses Ca2+ signaling and contraction in rat cardiac myocytes.

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca2+ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca2+ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC50 of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca2+ transient and sarcoplasmic reticulum Ca2+ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca2+ transients by DPI. The L-type Ca2+ current (ICa) was decreased concentration-dependently by DPI (IC50 of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca2+ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca2+ and Na+. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca2+ channel and RyR, thereby attenuating Ca2+-induced Ca2+ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

Induction of the intestinal membrane transporters ABCA1 and ABCG8 by 27-hydroxycholesterol through a redox mechanism

Graphical abstract Redox-induction of expression and synthesis of ATP binding cassette transporters in CaCo-2 cells by externally added 27-hydroxycholesterol. 27OHC: 27-hydroxycholesterol; NOX: NADPH oxidase; ROS: reactive oxygen species; ABCA1: ATP binding cassette A1; ABCG8: ATP binding cassette G8; DPI: diphenylene iodonium. Abstract Objective We tested the effect of 27-hydroxycholesterol (27OHC) on the expression and synthesis of two membrane transporters involved in sterols extrusion from the intestinal epithelium into the gut lumen: ATP-binding cassette A1 (ABCA1) and G8 (ABCG8). Special attention was given to ABCG8, a key player in the intestinal cell discharge of plant sterols. Methods Differentiated CaCo-2 intestinal cells were supplemented with 27OHC, and added to the cell incubation medium at a final concentration of 1 or 5 µM. These 27OHC externally added amounts were proven to reach intracellular oxysterol levels within the range of those normally recovered in the human peripheral blood. Results An up-regulation of the ABCA1 and ABCG8 mRNAs was observed in the CaCo-2 cells supplemented with 27OHC. Moreover, both 1 µM and 5 µM 27OHC induced a net, and steady, statistically significant, increase of both ABCA1 and ABCG8 protein levels. Of interest, the cellular pre-treatment with diphenylene iodonium, a selective inhibitor of NADPH oxidase, i.e. a major intracellular source of reactive oxygen species, fully inhibited the 27OHC enhancement of both ABCA1 and ABCG8 protein synthesis. Conclusion This in vitro study shows for the first time that the addition of 27OHC to intestinal epithelial cells up-regulates ABCG8, the transporter discharging plant sterols into the gut lumen, besides confirming to induce ABCA1 as well. Importantly, the 27OHC-dependent up-regulation of the two transporters appears to involve a redox mechanism rather than the canonical liver-X-receptors-dependent pathway. Significance statement The 27OHC introduced with the diet might modulate the plant sterol extrusion in the gut, in parallel with that of cholesterol.

Diphenylene Iodonium as a Prominent Halogen Bond Donor: The Case of Human Monoamine Oxidase B.

Herein we have investigated the formation and interplay of several noncovalent interactions (NCIs) involved in the inhibition of human monoamine oxidase B (MAO B). Concretely, an inspection of the Protein Data Bank (PDB) revealed the formation of a halogen bond (HlgB) between a diphenylene iodonium (DPI) inhibitor and a water molecule present in the active site, in addition to a noncovalent network of interactions (e. g. lone pair-π, hydrogen bonding, OH-π, CH-π and π-stacking interactions) with surrounding protein residues. Several theoretical models were built to understand the strength and directionality features of the HlgB in addition to the interplay with other NCIs present in the active site of the enzyme. Besides, a computational study was carried out using DPI as HlgB donor and several electron rich molecules (CO, H2O, CH2O, HCN, pyridine, OCN-, SCN-, Cl- and Br-) as HlgB acceptors. The results were analyzed using several state-of-the-art computational tools. We expect that our results will be useful for those scientists working in the fields of rational drug design, chemical biology as well as supramolecular chemistry.

Reactive oxygen species derived from NADPH oxidase as signaling molecules regulate fatty acids and astaxanthin accumulation in Chromochloris zofingiensis.

Abiotic stresses can increase the total fatty acid (TFA) and astaxanthin accumulation in microalgae. However, it remains unknown whether a unified signal transduction mechanism exists under different stresses. This study explored the link between nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) and the accumulation of fatty acids and astaxanthin in Chromochloris zofingiensis under three abiotic stresses. Results showed significant increases in fatty acid, astaxanthin, and ROS levels under nitrogen deficiency, phosphorus deficiency, and high-salinity stress. The introduction of the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the content of these components. This underscores the pivotal role of NADPH oxidase-derived ROS in the accumulation of fatty acid and astaxanthin under abiotic stress. Analysis of transcriptomes across three conditions following DPI addition revealed 1,445 shared differentially expressed genes (DEGs). Enrichment analysis revealed that biotin, betalain, thiamine, and glucosinolate may be important in stress responses. The heatmap demonstrated that DPI notably suppressed gene expression in the fatty acid and carotenoid biosynthesis pathways. Our findings underscore the pivotal role of NADPH oxidase-derived ROS in the accumulation of fatty acid and astaxanthin under abiotic stresses.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

GKT137831 and hydrogen peroxide increase the release of 6-nitrodopamine from the human umbilical artery, rat-isolated right atrium, and rat-isolated vas deferens.

Introduction: The human umbilical artery (HUA), rat-isolated right atrium, and rat-isolated vas deferens present a basal release of 6-nitrodopamine (6-ND). The basal release of 6-ND from these tissues was significantly decreased (but not abolished) when the tissues were pre-incubated with Nω-nitro-L-arginine methyl ester (L-NAME). Methods: In this study, the effect of the pharmacological modulation of the redox environment on the basal release of 6-ND was investigated. The basal release of 6-ND was measured using Liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results and Discussion: Pre-incubation (30min) of the tissues with GKT137831 (1μM) caused a significant increase in the basal release of 6-ND from all tissues. In the HUA, pre-incubation with diphenyleneiodonium (DPI) (100μM) also caused significant increases in the basal release of 6-ND. Preincubation of the HUA with hydrogen peroxide (H2O2) (100μM) increased 6-ND basal release, whereas pre-incubation with catalase (1,000U/mL) significantly decreased it. Pre-incubation of the HUA with superoxide dismutase (SOD) (250U/mL; 30min) also significantly increased the basal release of 6-ND. Preincubation of the HUA with either allopurinol (100μM) or uric acid (1mM) had no effect on the basal release of 6-ND. Pre-treatment of the HUA with L-NAME (100μM) prevented the increase in the basal release of 6-ND induced by GKT137831, diphenyleneiodonium, and H2O2. The results obtained indicate a major role of endogenous H2O2 and peroxidases as modulators of 6- ND biosynthesis/release and a lack of peroxynitrite contribution.

Protective Effect of Lavandula officinalis on Pentylenetetrazol-Induced Epilepsy in Mice: Expression of NOS Genes and Caspase-3

Abstract Objective Given the incidence of epilepsy and the adverse effects of unconventional antiepileptic medicines, there is a need for a novel medical treatment strategy for epileptic patients. Materials and Methods The current study involved the selection of 80 male mice, which were then separated into 10 experimental groups: pentylenetetrazol (PTZ), negative control which received normal saline, treatment which received Lavandula officinalis in two doses of 200 and 400 mg/kg, L. officinalis 200 mg/kg and 1400 w, L. officinalis 200 mg/kg and 7-NI, L. officinalis 200 mg/kg and diphenylene iodonium chloride (DPI), L. officinalis 400 mg/kg and 1400 w, L. officinalis 400 mg/kg and 7-nitroindazole (7-NI) and group which received L. officinalis 400 mg/kg and DPI. Each group was stimulated with an 11-day injection cycle (every 48 hours) of PTZ at a dosage of 35 mg/kg. All groups underwent PTZ challenge dosage (75 mg/kg) testing during the 12th injection. Ultimately, the brains of all mice were extracted, and the activity of genes related to neuronal nitric oxide, inducible nitric oxide, and endothelial nitric oxide was assessed. The enzyme-linked immunosorbent assay (ELISA) method was used to assess the quantity of caspase-3 in the groups. Results Lavandula officinalis decreased the severity of seizures. The findings of our study demonstrated that the extract had a suppressive effect on the expression of inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS; p &lt; 0.05), while it had a stimulatory effect on endothelial NOS (eNOS; p &lt; 0.05). In addition, L. officinalis reduced caspase-3 levels in the groups who were administered the extract. Conclusion The hydroalcoholic extract of L. officinalis has been found to be effective. Lavandula officinalis enhanced the expression of endothelial nitric oxide and reduced the levels of neuronal and inducible nitric oxide to a greater extent in brain tissue affected by epilepsy. The groups receiving extract derived from L. officinalis exhibited a reduction in the level of caspase-3.

H2O2 drives the transition from conchocelis to conchosporangia in the red alga Pyropia haitanensis with promotion facilitated by 1-Aminocyclopropane-1-carboxylic acid.

The Bangiales represent an ancient lineage within red algae that are characterized by a life history featuring a special transitional stage from diploid to haploid known as the conchosporangia stage. However, the regulatory mechanisms governing the initiation of this stage by changes in environmental conditions are not well understood. This study analyzed the changes in phytohormones and H2O2 content during the development of conchosporangia. It also compared the gene expression changes in the early development of conchosporangia through transcriptome analysis. The findings revealed that H2O2 was shown to be the key signal initiating the transition from conchocelis to conchosporangia in Pyropia haitanensis. Phytohormone analysis showed a significant increase in 1-aminocylopropane-1-carboxylic acid (ACC) levels during conchosporangia maturation, while changes in environmental conditions were found to promote the rapid release of H2O2. H2O2 induction led to conchosporangia development, and ACC enhanced both H2O2 production and conchosporangia development. This promotive effect was inhibited by the NADPH oxidase inhibitor diphenylene iodonium and the H2O2 scavenger N, N'-dimethylthiourea. The balance of oxidative-antioxidative mechanisms was maintained by regulating the activities and transcriptional levels of enzymes involved in H2O2 production and scavenging. Transcriptome analysis in conjunction with evaluation of enzyme and transcription level changes revealed upregulation of protein and sugar synthesis along with modulation of energy supply under the conditions that induced maturation, and exogenous ACC was found to enhance the entire process. Overall, this study demonstrates that ACC enhances H2O2 promotion of the life cycle switch responsible for the transition from a vegetative conchocelis to a meiosis-preceding conchosporangia stage in Bangiales species.

Helicobacter pylori secretory Proteins-Induced oxidative stress and its role in NLRP3 inflammasome activation

Helicobacter pylori-associated stomach infection is a leading cause of gastric ulcer and related cancer. H. pylori modulates the functions of infiltrated immune cells to survive the killing by reactive oxygen and nitrogen species (ROS and RNS) produced by these cells. Uncontrolled immune responses further produce excess ROS and RNS which lead to mucosal damage. The persistent oxidative stress is a major cause of gastric cancer. H. pylori regulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), nitric oxide synthase 2 (NOS2), and polyamines to control ROS and RNS release through lesser-known mechanisms. ROS and RNS produced by these pathways differentiate macrophages and T cells from protective to inflammatory phenotype. Pathogens-associated molecular patterns (PAMPs) induced ROS activates nuclear oligomerization domain (NOD), leucine rich repeats (LRR) and pyrin domain-containing protein 3 (NLRP3) inflammasome for the release of pro-inflammatory cytokines. This study evaluates the role of H. pylori secreted concentrated proteins (HPSCP) related oxidative stress role in NLRP3 inflammasome activation and macrophage differentiation. To perceive the role of ROS/RNS, THP-1 and AGS cells were treated with 10 μM diphenyleneiodonium (DPI), 50 μM salicyl hydroxamic acid (SHX), 5 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively. Cells were also treated with 10 μM of NOS2 inhibitor l-NMMA and 10 μM of N-acetyl cysteine (NAC), a free radical scavenger·H2O2 (100 μM) treated and untreated cells were used as positive controls and negative control respectively. The expression of gp91phox (NOX2), NOS2, NLRP3, CD86 and CD163 was analyzed through fluorescent microscopy. THP-1 macrophages growth was unaffected whereas the gastric epithelial AGS cells proliferated in response to higher concentration of HPSCP. ROS and myeloperoxidase (MPO) level increased in THP-1 cells and nitric oxide (NO) and lipid peroxidation significantly decreased in AGS cells. gp91phox expression was unchanged, whereas NOS2 and NLRP3 downregulated in response to HPSCP, but increased after inhibition of NO, ROS and MPO in THP-1 cells. HPSCP upregulated the expression of M1 and M2 macrophage markers, CD86 and CD163 respectively, which was decreased after the inhibition of ROS.This study concludes that there are multiple pathways which are generating ROS during H. pylori infection which further regulates other cellular processes. NO is closely associated with MPO and inhibition of NLRP3 inflammasome. The low levels of NO and MPO regulates gastrointestinal tract homeostasis and overcomes the inflammatory response of NLRP3. The ROS also plays crucial role in macrophage polarization hence alter the immune responses duing H. pylori pathogenesis.

AQP3 Promotes the Invasion and Metastasis in Cervical Cancer by Regulating NOX4-derived H2O2 Activation of Syk/PI3K/Akt Signaling Axis.

Unrestrained chronic inflammation leads to the abnormal activity of NOX4 and the subsequent production of excessive hydrogen peroxide (H2O2). Excessive H2O2 signaling triggered by prolonged inflammation is thought to be one of the important reasons for the progression of some types of cancer including cervical cancer. Aquaporin 3 (AQP3) is a member of the water channel protein family, and it remains unknown whether AQP3 can regulate the transmembrane transport of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4)-derived H2O2 induced by the stimulation of inflammatory factors to facilitate the malignant progression in cervical cancer. In this study, cervical cancer HeLa cell line was respectively treated with diphenyleneiodonium (DPI), N-Acetylcysteine (NAC) or lentivirus-shRNA- AQP3. Plate cloning, cell migration or transwell invasion assays, etc. were performed to detect the invasive and migration ability of the cells. Western blot and CO-IP were used to analyze the mechanism of AQP3 regulating H2O2 conduction. Finally, in vivo assays were performed for validation in nude mice. AQP3 Knockdown, DPI or NAC treatments all reduced intracellular H2O2 influx, and the activation of Syk/PI3K/Akt signal axis was inhibited, the migration and invasive ability of the cells was attenuated. In vivo assays confirmed that the excessive H2O2 transport through AQP3 enhanced the infiltration and metastasis of cervical cancer. These results suggest that AQP3 activates H2O2/Syk/PI3K/Akt signaling axis through regulating NOX4-derived H2O2 transport to contribute to the progression of cervical cancer, and AQP3 may be a potential target for the clinical treatment of advanced cervical cancer.

Regulation of respiratory complex I assembly by FMN cofactor targeting

Respiratory complex I plays a crucial role in the mitochondrial electron transport chain and shows promise as a therapeutic target for various human diseases. While most studies focus on inhibiting complex I at the Q-site, little is known about inhibitors targeting other sites within the complex. In this study, we demonstrate that diphenyleneiodonium (DPI), a N-site inhibitor, uniquely affects the stability of complex I by reacting with its flavin cofactor FMN. Treatment with DPI blocks the final stage of complex I assembly, leading to the complete and reversible degradation of complex I in different cellular models. Growing cells in medium lacking the FMN precursor riboflavin or knocking out the mitochondrial flavin carrier gene SLC25A32 results in a similar complex I degradation. Overall, our findings establish a direct connection between mitochondrial flavin homeostasis and complex I stability and assembly, paving the way for novel pharmacological strategies to regulate respiratory complex I.

The Role of Reactive Oxygen Species in the In Vitro Germination and Growth of the Petunia (Petunia hybrida E. Vilm.) Male Gametophyte

The in vitro growth of the pollen tube (PT), an object of comprehensive and intensive research, is a model for studying the mechanisms of sexual reproduction in higher plants. We have studied the potential role of reactive oxygen species (ROS) in the in vitro germination and growth maintenance of the petunia (Petunia hybrida E. Vilm.) male gametophyte. The exogenous treatment with H2O2 influences the PT germination and polar growth in vitro. The addition of H2O2 to culture medium increases both the percentage of pollen grain germination and the PT length in the case of long cultivation, but inhibits both processes during the first hour of cultivation. This suggests that endogenous ROS play a decisive role in the early stages of pollen germination, with the sensitivity to endogenous ROS emerging later over the course of their growth. The addition of diphenylene iodonium chloride (DPI), a NADPH oxidase inhibitor, considerably decreases both the germination and the growth of the petunia male gametophyte at low concentrations (0.1 μM), and completely arrests the growth at high concentrations (1 μM). ROS are necessary for polar growth of the petunia male gametophyte; they are secreted in the early stages of pollen grain activation and are further localized to the initiation of the PT, mainly in the PT apical part, during polar growth, as confirmed with the help of intravital fluorescence microscopy.

PeAP1-mediated oxidative stress response plays an important role in the growth and pathogenicity of Penicillium expansum.

Penicillium expansum is the causal agent of post-harvest blue mold in various fruits and serves as a model for understanding fungal pathogenicity and mycotoxin production. The relevance of oxidative stress response in the growth and virulence of P. expansum has been largely unexplored. Here, we identify the transcriptional factor PeAP1 as a regulator of oxidative stress response in P. expansum. Gene expression and protein abundance of PeAP1, as well as its nuclear localization, are specifically induced by H2O2. Deletion of PeAP1 results in increased sensitivity to H2O2, and PeAP1 mutants exhibit a variety of defects in hyphal growth and virulence. PeAP1 prevents the accumulation of both intracellular H2O2 during vegetative growth and host-derived H2O2 during biotrophic growth. Application of an antioxidant glutathione and a NADPH oxidase inhibitor, diphenylene iodonium, to the PeAP1 mutant partially restored fungal growth and virulence. RNA sequencing analysis revealed 144 H2O2-induced PeAP1 target genes, including four antioxidant-related genes, PeGST1, PePrx1, PePrx2, and PeTRX2, that were also demonstrated to be involved in oxidative stress response and/or virulence. Collectively, our results demonstrate the global regulatory role of PeAP1 in response to oxidative stress and provide insights into the critical role of the PeAP1-mediated oxidative stress response to regulate growth and virulence of P. expansum. IMPORTANCE Reactive oxygen species are the core of host plant defense and also play a vital role in the successful invasion of host plants by pathogenic fungi. Despite its importance, the relevance of oxidative stress response in fungal growth and virulence is poorly understood in P. expansum. In this study, we reveal that the transcription factor PeAP1 acts as a central regulator of oxidative stress response in P. expansum and that there is a major link between PeAP1-mediated oxidative stress response and fungal growth and virulence. To explore the underlying mechanisms, we performed comparative transcriptomic studies and identified a number of H2O2-induced PeAP1 target genes, including four novel ones, PePrx1, PePrx2, PeGST1, and PeTRX2, whose functions were linked to PeAP1 and pathogenicity. These findings provide novel insights into the regulation mechanism of PeAP1 on growth and virulence, which might offer promising targets for control of blue mold and patulin contamination.

Red light activates H2O2 signal involved in powdery mildew resistance in oriental melon seedlings

Melon is one of the most important horticultural crops in Asia. Powdery mildew (PM) is frequently occurred in facility cultivation, which seriously affects its yield and fruit quality of melon. Light signal is the basis of plant growth and development, and appropriate red light (RL) plays an important role in regulating the resistance of plant pathogens. Reactive oxygen species (ROS) also have been shown to be participated in plant defence against pathogen infection. However, whether light is involved in PM resistance through ROS remains unclear. Here, we found that RL+PM treatment significantly enhanced the resistance of PM in melon seedlings, improved NADPH oxidase activity, upregulated respiratory burst oxidase homologue 1 (RBOHD) expression, and increased hydrogen peroxide (H2O2) level. Compared with the PM treatment alone, diphenylene iodonium (DPI)+PM treatment significantly enhanced the sensitivity to PM in melon seedlings and weakened the effect of RL-induced PM resistance. Further studies showed that silencing CmRBOHD significantly inhibited the RL-induced PM resistance by inhibiting H2O2 synthesis. In addition, CmWRKY50 could activate CmRBOHD expression by binding to the promoter of CmRBOHD. Taken together, the results suggest that RL can increase endogenous H2O2 levels and induce the initiation of defence through activation of CmWRKY50, and therefore, when plants are subjected to pathogen stress, higher H2O2 levels in the plants are beneficial for organizing the invasion of pathogens and enhancing powdery mildew resistance.