NADPH Oxidase Inhibitor Research Articles

Oxidative Stress Inhibition Via Apocynin Prevents Medullary Respiratory Neurodegeneration and Respiratory Pattern Dysfunction in a 6-Hydroxydopamine Animal Model of Parkinson’s Disease

Parkinson’s Disease (PD) is a neurogenerative disorder characterized by the death of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), leading to motor, cognitive, learning, and respiratory dysfunctions. New evidence revealed that breathing impairment in PD mainly results from oxidative stress (OS) that initiates apoptotic signaling in respiratory neurons. Here, we investigated the role of OS inhibition using apocynin (non-specific NADPH oxidase inhibitor) in a 6-OHDA PD animal model in the neural control of breathing. The PD model was confirmed with a 70% reduction in TH-expressing neurons within the SNpc. After 20 and 40 days of PD induction, no differences were observed in superoxide anion levels in any respiratory nuclei. At 30 days after PD induction, 6-OHDA animals presented OS that was prevented in all respiratory nuclei by adding apocynin to the drinking water for 10 days. Forty days after PD animal model induction, impaired motor and breathing function, reduced Phox2b and NK1 receptors-expressing neurons in the medullary respiratory areas; decreased latency to fall in the rotarod motor test; and attenuated respiratory frequency and minute ventilation parameters at rest and under hypercapnia conditions were observed. After 20 days of apocynin treatment, neurodegeneration of respiratory nuclei and breathing dysfunction in 6-OHDA animals were prevented. Thus, OS contributes to respiratory neuron death, consequently leading to breathing dysfunction in the 6-OHDA PD animal model. Furthermore, these results present a new perspective for preventing the onset and progression of PD-related respiratory impairments.

Advanced oxidation protein products induce annulus fibrosus cell senescence through a NOX4-dependent, MAPK-mediated pathway and accelerate intervertebral disc degeneration.

BackgroundIntervertebral disc degeneration (IVDD) is closely associated with senescence. Annulus fibrosus (AF) cell senescence is a crucial driver of AF tissue tearing and fissures, thereby exacerbating IVDD. Increased advanced oxidative protein products (AOPPs) were found in human degenerative discs and aged rat discs and may be involved in IVDD. This study aimed to explore the mechanism of AOPPs-induced senescence in AF cells.MethodsThe pathological effects of AOPPs in vivo were investigated using a rat lumbar disc persistent degeneration model and a rat caudal disc puncture model. Rat primary AF cells were selected as in vitro models, and AOPPs were used as direct stimulation to observe their pathological effects. Setanaxb (NOX1/4 inhibitor), apocynin (NADPH oxidase inhibitor) and adenovirus (ADV) packed NADPH oxidase 4 (NOX4) specific shRNAs were used for pathway inhibition, respectively. Finally, adeno-associated viruses (AAVs) packed with NOX4-specific blocking sequences were used to inhibit the in vivo pathway.ResultsAOPPs accumulated in the rat lumbar and caudal degenerative discs. Intra-discal loading of AOPPs up-regulated the expression of NOX4, p53, p21, p16, IL-1β, and TNF-α, ultimately accelerating IVDD. Exposure of AOPPs to AF primary cells up-regulated NOX4 expression, induced phosphorylation of mitogen-activated protein kinases (MAPK), triggered senescence and increased IL-1β and TNF-α. Apocynin, setanaxib, and ADV pre-cultured AF cells abrogated AOPPs-induced senescence. AAV-mediated inhibition of NOX4 expression in vivo reduced the expression of p53, p21, p16, IL-1β and TNF-α in vivo and delayed IVDD.ConclusionsAOPPs induced AF cell senescence through a NOX4-dependent and MAPK-mediated pathway.

Receptor for Activated C Kinase1B (OsRACK1B) Impairs Fertility in Rice through NADPH-Dependent H2O2 Signaling Pathway.

The scaffold protein receptor for Activated C Kinase1 (RACK1) regulates multiple aspects of plants, including seed germination, growth, environmental stress responses, and flowering. Recent studies have revealed that RACK1 is associated with NADPH-dependent reactive oxygen species (ROS) signaling in plants. ROS, as a double-edged sword, can modulate several developmental pathways in plants. Thus, the resulting physiological consequences of perturbing the RACK1 expression-induced ROS balance remain to be explored. Herein, we combined molecular, pharmacological, and ultrastructure analysis approaches to investigate the hypothesized connection using T-DNA-mediated activation-tagged RACK1B overexpressed (OX) transgenic rice plants. In this study, we find that OsRACK1B-OX plants display reduced pollen viability, defective anther dehiscence, and abnormal spikelet morphology, leading to partial spikelet sterility. Microscopic observation of the mature pollen grains from the OX plants revealed abnormalities in the exine and intine structures and decreased starch granules in the pollen, resulting in a reduced number of grains per locule from the OX rice plants as compared to that of the wild-type (WT). Histochemical staining revealed a global increase in hydrogen peroxide (H2O2) in the leaves and roots of the transgenic lines overexpressing OsRACK1B compared to that of the WT. However, the elevated H2O2 in tissues from the OX plants can be reversed by pre-treatment with diphenylidonium (DPI), an NADPH oxidase inhibitor, indicating that the source of H2O2 could be, in part, NADPH oxidase. Expression analysis showed a differential expression of the NADPH/respiratory burst oxidase homolog D (RbohD) and antioxidant enzyme-related genes, suggesting a homeostatic mechanism of H2O2 production and antioxidant enzyme activity. BiFC analysis demonstrated that OsRACK1B interacts with the N-terminal region of RbohD in vivo. Taken together, these data indicate that elevated OsRACK1B accumulates a threshold level of ROS, in this case H2O2, which negatively regulates pollen development and fertility. In conclusion, we hypothesized that an optimal expression of RACK1 is critical for fertility in rice plants.

BMAL1 modulates ROS generation and insulin secretion in pancreatic β-cells: An effect possibly mediated via NOX2

The pancreatic β cells circadian clock plays a relevant role in glucose metabolism. NADPH oxidase (NOX) family is responsible for producing reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, using NADPH as an electron donor. In pancreatic β-cells, NOX-derived ROS inhibits basal and glucose-stimulated insulin secretion. Thus, we hypothesized that the absence of BMAL1, a core circadian clock component, could trigger an increase of NOX2-derived ROS in pancreatic β cells, inhibiting insulin secretion under basal and stimulated glucose conditions. To test such hypothesis, Bmal1 knockdown (KD) was performed in cultured clonal β-cell line (INS-1E) and knocked out in isolated pancreatic islets, using a tissue-specific β-cells Bmal1 knockout (KO) mice. The insulin secretion was assessed in the presence of NOX inhibitors. The Bmal1 KD within INS-1E cells elicited a rise of intracellular ROS content under both glucose stimuli (2.8 mM and 16.7 mM), associated with an increase in Nox2 expression. Additionally, alterations of glutathione levels, CuZnSOD and catalase activities, reduction of ATP/ADP ratio, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and aconitase activities, followed by glucokinase and Slc2a2 (Glut2) expression were also observed in INS-1E β-cells, reflecting in a diminished insulin secretion pattern. The isolated islets from β-cell Bmal1−/− mice have shown a similar cellular response, where an increased NOX2-derived ROS content and a reduced basal- and glucose-stimulated insulin secretion were observed. Therefore, together with NOX inhibition (Apocynin), polyethene-glycol linked to superoxide dismutase (PEG-SOD), phorbol myristate acetate (PMA), and diethyldithiocarbamate (DDC) data, our findings suggest a possible BMAL1-mediated NOX2-derived ROS generation in pancreatic β cells, leading to the modulation of both basal- and glucose-stimulated insulin secretion.

NADPH oxidase inhibitor development for diabetic nephropathy through water tank model.

Oxidative stress can cause generation of uncontrolled reactive oxygen species (ROS) and lead to cytotoxic damage to cells and tissues. Recently, it has been shown that transient ROS generation can serve as a secondary messenger in receptor-mediated cell signaling. Although excessive levels of ROS are harmful, moderated levels of ROS are essential for normal physiological function. Therefore, regulating cellular ROS levels should be an important concept for development of novel therapeutics for treating diseases. The overexpression and hyperactivation of NADPH oxidase (Nox) can induce high levels of ROS, which are strongly associated with diabetic nephropathy. This review discusses the theoretical basis for development of the Nox inhibitor as a regulator of ROS homeostasis to provide emerging therapeutic opportunities for diabetic nephropathy.

Magnetopriming Actuates Nitric Oxide Synthesis to Regulate Phytohormones for Improving Germination of Soybean Seeds under Salt Stress.

In this study, the role of the signalling molecule nitric oxide (NO) in magnetopriming-mediated induction of salinity tolerance in soybean seeds is established. The cross-talk of NO with germination-related hormones gibberellic acid (GA), abscisic acid (ABA) and auxin (IAA) for their ability to reduce the Na+/K+ ratio in the seeds germinating under salinity is highlighted. Salt tolerance index was significantly high for seedlings emerging from magnetoprimed seeds and sodium nitroprusside (SNP, NO-donor) treatment. The NO and superoxide (O2•−) levels were also increased in both of these treatments under non-saline and saline conditions. NO generation through nitrate reductase (NR) and nitric oxide synthase-like (NOS-like) pathways indicated the major contribution of NO from the NR-catalysed reaction. The relative expression of genes involved in the NO biosynthetic pathways reiterated the indulgence of NR in NO in magnetoprimed seeds, as a 3.86-fold increase in expression was observed over unprimed seeds under salinity. A 23.26-fold increase in relative expression of NR genes by the NO donor (SNP) was observed under salinity, while the NR inhibitor (sodium tungstate, ST) caused maximum reduction in expression of NR genes as compared to other inhibitors [L-NAME (N(G)-nitro-L-arginine methyl ester; inhibitor of nitric oxide synthase-like enzyme) and DPI (diphenylene iodonium; NADPH oxidase inhibitor)]. The ratio of ABA/GA and IAA/GA decreased in magnetoprimed and NO donor-treated seeds, suggesting homeostasis amongst hormones during germination under salinity. The magnetoprimed seeds showed low Na+/K+ ratio in all treatments irrespective of NO inhibitors. Altogether, our results indicate that a balance of ABA, GA and IAA is maintained by the signalling molecule NO in magnetoprimed seeds which lowers the Na+/K+ ratio to offset the adverse effects of salinity in soybean seeds.

Identification of Respiratory Burst Oxidase Homolog (Rboh) Family Genes From Pyropia yezoensis and Their Correlation With Archeospore Release.

Reactive oxygen species (ROS) play important regulatory roles in plant growth and development, as well as in cell differentiation and stress responses. Respiratory burst oxidase homolog (RBOH) is the key enzyme in ROS production. So far, the Rboh family genes in Pyropia yezoensis have not been comprehensively characterized, and whether their function was involved in the formation of archeospores is still unknown. In this study, a total of 11 PyRboh genes were identified from the P. yezoensis genome by homology mining. Through phylogenetic analysis, it is suggested that the PyRboh genes were evolutionarily conserved among the lineages of red algae, but a few genes exhibited a species-specific manner. The treatment of P. yezoensis blades with NADPH oxidase inhibitor diphenylene iodonium (DPI) could significantly inhibit the formation of archeospores, suggesting that RBOH may be involved in the formation of archeospores. According to PyRboh gene expression analysis using the P. yezoensis strains with obvious differences in releasing archeospores, it is showed that the expression trends of most genes were consistent, with no significant difference between strains, whereas the expression pattern of the two P. yezoensis-specific genes (PyRbohJ and PyRbohK) was positively correlated with the amount of archeospores. Furthermore, as treatment of blades with allantoin resulted in a significant increase in the release of archeospores, the expression levels of PyRbohJ and PyRbohK were also consistently upregulated, further confirming the relationship between the two genes and archeospore formation. These findings provide insights into the molecular mechanism of P. yezoensis archeospore formation.

Vitamin B1 delays postharvest senescence and enhances antioxidant accumulation by activating NADPH oxidase in Ziziphus jujuba fruit

Thiamine, also known as vitamin B1 (VB1), plays a key role in regulating plant growth and development. However, no studies have addressed its role in regulating postharvest fruit and vegetable senescence. Herein, the effects of VB1 on postharvest senescence and antioxidant-compound accumulation in jujube (Ziziphus jujuba) fruit stored at 25 °C were investigated. Soaking postharvest jujube fruit with 1 mmol/L VB1 drastically decreased its weight loss, decay incidence, and respiration rate and improved firmness during the first seven days of storage. VB1 treatment increased jujube fruit NADPH oxidase, superoxide dismutase, and catalase activities, and changed its H2O2 content. Furthermore, VB1 treatment also dramatically increased the ascorbic acid level, total phenolics content, total flavonoids accumulation, and total antioxidant capacity of the postharvest jujube fruit. However, the increase in antioxidant enzyme activities and antioxidant-compound accumulation were partially reversed by 0.1 mmol/L diphenyleneiodonium chloride, a specific inhibitor of NADPH oxidase. Accordingly, the role of VB1 in delayed fruit senescence and enhanced antioxidant-compound accumulation were discussed in terms of NADPH-oxidase-regulated H2O2 signaling. Overall, this study provides practical and theoretical information regarding a simple new method for the maintenance of jujube fruit quality during storage at ambient temperature.

Induction of reactive oxygen species by mechanical stretch drives endothelin production in neonatal pig renal epithelial cells

Vasoactive endothelin (ET) is generated by ET converting enzyme (ECE)-induced proteolytic processing of pro-molecule big ET to biologically active peptides. H2O2 has been shown to increase the expression of ECE1 via transactivation of its promoter. The present study demonstrates that H2O2 triggered ECE1-dependent ET1-3 production in neonatal pig proximal tubule (PT) epithelial cells. A uniaxial stretch of PT cells decreased catalase, increased NADPH oxidase (NOX)2 and NOX4, and increased H2O2 levels. Stretch also increased cellular ECE1, an effect reversed by EUK-134 (a synthetic superoxide dismutase/catalase mimetic), NOX inhibitor apocynin, and siRNA-mediated knockdown of NOX2 and NOX4. Short-term unilateral ureteral obstruction (UUO), an inducer of renal tubular cell stretch and oxidative stress, increased renal ET1-3 generation and vascular resistance (RVR) in neonatal pigs. Despite removing the obstruction, UUO-induced increase in RVR persisted, resulting in early acute kidney injury (AKI). ET receptor (ETR)-operated Ca2+ entry in renal microvascular smooth muscle (SM) via transient receptor potential channel 3 (TRPC3) channels reduced renal blood flow and increased RVR. Although acute reversible UUO (rUUO) did not change protein expression levels of ETR and TRPC3 in renal microvessels, inhibition of ECE1, ETR, and TRPC3 protected against renal hypoperfusion, RVR increase, and early AKI. These data suggest that mechanical stretch-driven oxyradical generation stimulates ET production in neonatal pig renal epithelial cells. ET activates renal microvascular SM TRPC3, leading to persistent vasoconstriction and reduction in renal blood flow. These mechanisms may underlie rUUO-induced renal insufficiency in infants.

Systemic H2O2 signaling mediates epigallocatechin-3-gallate-induced cadmium tolerance in tomato

Toxic heavy metal cadmium (Cd) reduces crop yield and threatens human health via the food chain. The bioactive flavonoid ‘Epigallocatechin-3-gallate’ (EGCG) affects plant stress response; however, the function of EGCG in Cd tolerance and the molecular pathways remain largely unknown. Here, we revealed that root application of EGCG alleviated Cd stress in tomato plants. While Cd stress decreased Fv/Fm, ФPSII, photosynthetic rate, root growth, root vitality and biomass accumulation by increasing reactive oxygen species (ROS) accumulation and lipid peroxidation, exogenous EGCG minimized excessive ROS accumulation and oxidative stress by promoting the activity of antioxidant enzymes and redox poise in roots and leaves. Moreover, EGCG induced the transcript of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and decreased Cd content and photoinhibition in leaves. Interestingly, similar to EGCG, exogenous H2O2 application also enhanced Cd tolerance; however, the application of an NADPH oxidase inhibitor, diphenyleneiodonium (DPI), aggravated Cd phytotoxicity and attenuated the beneficial effects of EGCG on plant tolerance to Cd stress, suggesting that root applied EGCG-induced expression of RBOH1 and associated H2O2 signaling mediate the EGCG-induced enhanced Cd tolerance. This work elucidates a fundamental mechanism behind EGCG-mediated Cd tolerance and contributes to our existing knowledge of stress resistance properties of EGCG in plants.

Accumulation of advanced oxidation protein products contributes to age-related impairment of gap junction intercellular communication in osteocytes of male mice.

AimsGap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism.MethodsChanges in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks.ResultsThe AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo.ConclusionOur study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing.Cite this article: Bone Joint Res 2022;11(7):413–425.

GC MS Based Comparative Phytochemical Profiling of Rhodiola Imbricata Roots Collected from Different High Mountain Passes of Ladakh India and a First Report of Apocynin from Genus Rhodiola

Rhodiola imbricata is a high value medicinal plant of trans-Himalayan mountain passes in Ladakh. This plant is a highly sought after in national and international herbal product market due to its unique phytochemical composition and resultant medicinal properties. However, compositional variation in the raw material from different geographical locations results in variation in quality as well as efficacy of the final products. The current study was designed to generate the comparative GC-MS profiles of hydro-methanolic extracts of Rhodiola imbricata root samples collected from various locations in Ladakh i.e., Chang La (17605 ft), Khardung La (18,379 ft), and Shashi La (13908 ft) mountain passes. The study highlighted variations in volatile phytochemical composition in root samples collected from different locations, especially with respect to phenols, terpenes and fatty acids. Samples from Chang La had maximum amount of phenolic compounds (96.78 per cent), followed by samples from Khardung La (77.05 per cent) while they were undetected in samples from Shashi La pass. Specifically, comparative GC-MS profiling revealed that peak area percentage of two important bioactive compounds (i.e. piceol and apocynin) varied amongst samples. In the samples collected from Chang La, the piceol covered (94 per cent) and apocynin covered (2.78 per cent) peak area whereas in samples collected from Khardung La, piceol covered (73.8 per cent) and apocynin covered (3.25 per cent) peak areas respectively, however, samples collected from Shashi La showed none of these compounds. Interestingly, in the present study, apocynin (a potent NADPH oxidase inhibitor) is being reported for the first time from Rhodoila genus. On the basis of present findings, samples collected from Chang La and Khardung La passes showed better phytochemical composition of pharmacological active phenolic compounds than Shashi La sample.

Protein phosphatase 1 regulates reactive oxygen species-dependent degradation of histone deacetylase 5 by intermittent hypoxia.

We recently reported pheochromocytoma 12 (PC12) cells and rats subjected to intermittent hypoxia (IH), a hallmark manifestation of obstructive sleep apnea (OSA), exhibit reduced histone deacetylase activity and HDAC5 protein. Our study further suggested that posttranslational modifications rather than transcriptional mechanism(s) mediate IH-induced HDAC5 degradation. These observations prompted our current study to investigate the mechanism(s) underlying HDAC5 degradation by IH in PC12 cell cultures. IH-induced HDAC5 degradation was blocked by an antioxidant, and reactive oxygen species (ROS) mimetics decreased HDAC5 protein, suggesting that ROS mediates HDAC5 degradation by IH. NADPH oxidases (NOX) 2 and 4 were identified as sources of ROS that mediate the effects of IH. HDAC5 degradation during IH was associated with dephosphorylation of HDAC5 at serine259, and this response was blocked by a NOX inhibitor, suggesting that ROS-dependent dephosphorylation mediates HDAC5 degradation. IH-induced dephosphorylation of HDCA5 was inhibited by calyculin A, an inhibitor of protein phosphatase (PP)-1 and -2, or by the overexpression of nuclear inhibitor of PP1 (NIPP1). HDAC5 dephosphorylation by IH lead to augmented hypoxia-inducible factor (HIF)-1α protein and an increase in its transcriptional activity. These data suggest that PP1-dependent dephosphorylation of S259 destabilizes HDAC5 protein in response to IH, resulting in HIF-1α stabilization and transcriptional activity. Our findings highlight hither to unexplored role of protein phosphatases, especially PP1 in regulating HDAC5 protein, which is an upstream activator of HIF-1 signaling by IH.

Abstract 3183: Targeting a myofibroblastic prostate cancer-associated fibroblast subtype through pharmacological inhibition of NADPH oxidase 4

Abstract Recent developments in single cell sequencing have revealed significant heterogeneity in the stromal tumor microenvironment. It is now widely accepted that there are different cancer-associated fibroblast (CAF) subtypes with different functions and effects on tumor progression. Since CAFs are generally considered an emerging therapeutic target, it is crucial to identify those subtypes that are tumor-promoting and to characterize their driver pathways. In this study we describe a population of CAFs in prostate cancer (PCa) that express elevated levels of NADPH oxidase 4 (Nox4) and localize adjacent to tumor foci. We previously showed that Nox4 is essential for TGFβ-mediated differentiation to a myofibroblastic CAF phenotype and that its increased expression is associated with biochemical relapse and reduced survival. Thus, this study aims to investigate whether pharmacological Nox4 inhibition can be used as an adjuvant therapeutic approach in PCa and which molecular pathways are regulated by Nox4 in the tumor microenvironment. Experiments using GKT831, a small molecule Nox1/Nox4 inhibitor with promising effects in clinical trials for fibrotic diseases, so far support its use as a therapeutic target. Most importantly, Nox4 inhibition reduced CAF marker expression in primary prostate CAFs and attenuated the expression of PSA, the key clinical biomarker of PCa progression, in ex vivo cultured human PCa tissue. The translational application of Nox4 inhibition is currently under investigation in vivo using CAF-PCa cell xenografts. To elucidate the molecular mechanisms regulated by Nox4 we performed integrative bioinformatics and functional assays, which revealed that Nox4 regulates CAF adhesion and migration. In addition, we found that Nox4 regulated genes were associated with a YAP signature and the transcription factor TEAD1, a major cofactor of YAP. Ongoing experiments suggest that during adhesion Nox4 signals via oxidation of SHP2, a redox-sensitive phosphatase that has previously been implicated in regulating YAP transcriptional activity. Previous studies have shown that adhesion and mechanotransduction pathways are commonly altered in myofibroblastic CAFs and that they support the formation of a tumor-promoting microenvironment. Our data suggest that Nox4 acts as a central regulator of these key oncogenic pathways and thus represents a promising therapeutic target. Citation Format: Elena Brunner, Lucy Neumann, Elisabeth Damisch, Martin Puhr, Georg Schäfer, Cédric Szyndralewiez, Helmut Klocker, Natalie Sampson. Targeting a myofibroblastic prostate cancer-associated fibroblast subtype through pharmacological inhibition of NADPH oxidase 4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3183.

Effects of ROS and caspase-3-like protein on the growth and aerenchyma formation of Potamogeton perfoliatus stem.

Aerenchyma formation plays an important role in the survival of Potamogeton perfoliatus in submerged environment. To understand the regulatory role of reactive oxygen species (ROS) and caspase 3-like protein signaling molecules in aerenchyma formation, we investigated the effects of exogenous NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), catalase inhibitor (3-amino-1,2,4-triazole, AT), and caspase-3-like protein inhibitor (AC-DEVD-CHO, DEVD) on morphological and physiological characteristics and aerenchyma formation in P. perfoliatus. The results showed that after DPI treatment, caspase-3-like protein activity decreased, ROS-related enzyme activities increased, and H2O2 content decreased, thereby inhibiting aerenchyma formation. When the concentration of DPI was approximately 1μmol/L, the inhibitory effect was the most obvious. On the contrary, after the AT treatment, caspase-3-like protein activity increased, ROS-related enzyme activities decreased, and the H2O2 content increased, ultimately promoting aerenchyma formation, and the promotion was the most obvious under treatment with approximately 500μmol/L AT. After DEVD treatment, the inhibition of vegetative growth caused by DPI or AT treatment was alleviated, significantly reducing caspase-3-like activity and inhibiting aerenchyma development. The results of this study show that ROS has a positive regulatory effect on aerenchyma formation, and caspase-3-like protein is activated to promote ROS-mediated aerenchyma formation. This experiment provides a new theoretical basis for further exploration of the signal transduction effects of ROS and caspase-3-like protein in plant cells and their roles in plant development.

Trigonella foenum-graecum L. and Psoralea corylifolia L. Improve Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats through Suppression of Oxidative Stress.

Background Diabetes mellitus-induced erectile dysfunction (DMED) is one of the most common complications of diabetes and is mainly attributed to oxidative stress. Hu-Lu-Ba-Wan (HLBW) is a classic Chinese formulation consisting of Trigonella foenum-graecum L. (TFG) and Psoralea corylifolia L. (PC). HLBW has been used not only for the treatment of diabetes but also for the treatment of erectile dysfunction in clinics. This study aimed to explore the efficacy and underlying mechanism of HLBW in ameliorating erectile function in streptozotocin-induced diabetic rats. Methods The diabetic model was established by tail vein injection of streptozotocin (26 mg/kg), and then DMED rats screened by the apomorphine test were randomly divided into two groups: the model group and the HLBW group. The rats in the HLBW group were administered HLBW granules daily for 12 weeks. Fasting blood glucose and fasting insulin were tested by a commercial kit. Intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured by cavernous nerve electrostimulation before the rats were killed. Erectile function was evaluated with ICP/MAP. The markers of oxidative stress in the corpus cavernosum (CC) were assayed by assay kits. Apoptosis in cavernosal tissue was detected by Western blotting (WB). The expression levels of vascular endothelial marker (vWF), α-smooth muscle actin (α-SMA), endothelial nitric oxide synthase (eNOS), and NADPH oxidase subunit P47phox were determined by WB and PCR. Furthermore, the structure of the CC was further confirmed by Masson's trichrome staining. Results The results showed that HLBW significantly reduced blood glucose and increased insulin sensitivity. HLBW reduced oxidative stress and apoptosis. In addition, we observed that the expression levels of vWF, α-SMA, and eNOS as well as the ratio of smooth muscle to collagen increased in the HLBW group. Conclusions Our results demonstrated that HLBW could reduce oxidative stress damage in CC to improve diabetes mellitus-induced erectile dysfunction in rats by inhibiting NADPH oxidase.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK-dependent Cyclooxygenase-2.

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG2 receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.

EFFECTS OF APOCYNIN TREATMENT AND HBO PRECONDITIONING ON IMMUNOHISTOCHEMICAL 4-HNE, HIF1 ALPHA, HIF2 ALPHA AND NGAL TISSUE EXPRESSION IN POSTISCEMIC ACUTE KIDNEY INJURY IN SHR RATS

Objective: Oxidative stress has been considered as a central aggravating factor in the development of postischemic acute kidney injury (AKI). 4-hydroxynonenal (4-HNE) is a product of lipid peroxidation and may serve as non-invasive biomarker of oxidative stress while neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker of AKI. Hypoxia-inducible factors (HIFs) are the “master’’ transcription factors responsible for gene expression in hypoxia, which play an important role in kidney injury and repair by regulating HIF target genes, including microRNAs. Design and method: The study was conducted on male SHR rats, randomly divided into following groups: sham-operated group (SHAM) AKI control group, AKI group with apocynin treatment (AKI+APO), group with HBO preconditioning before AKI induction (AKI+HBO) and group with HBO preconditioning before and apocynin treatment after AKI induction (AKI+APO+HBO). HBO preconditioning was performed by exposing to pure oxygen (2.026 bar) twice a day for two consecutive days for 60 minutes, and 24 h before AKI induction. Apocynin, NADPH oxidase inhibitor was injected 5 minutes before reperfusion. Results: AKI induction significantly decreased creatinine (CCr), urea (CU) and phosphate (CPhos) clearances when compared to SHAM group. Remarkable increase in CCr, CU, CPhos levels were observed in all treated groups (AKI+APO, AKI+HBO, AKI+APO+HBO) in comparison to AKI group. Sham – operated rats did not express 4-HNE and NGAL in any parenchymal structure. AKI induced abundant and strong glomerular expression of 4-HNE along with expression in interstitial compartment. All treatments (AKI+APO, AKI+HBO, AKI+APO+HBO) significantly decreased 4-HNE expression both in glomeruli and interstitium. Also, AKI stimulated widespread NGAL expression in renal epithelial tubular cells with fine granular appearance on the apical surface of the cells, affecting entire circumference of tubular cross-sectioning, which was decreased in all treated groups. In SHAM group, the expression of HIF1 alpha and HIF2 alpha was focal with weak intensity in glomeruli and interstitium. In AKI group, the expression was diffuse and strong, with remarkable improvement in all treated groups. Conclusions: Considering our results, we can conclude that apocynin treatment, as well as HBO preconditioning may have protective effects in postischemic acute kidney injury induced in spontaneously hypertensive rats.

Effects of polystyrene nanoplastics on endothelium senescence and its underlying mechanism.

Global plastic use has increased rapidly, and environmental pollution associated with nanoplastics (NPs) has been a growing concern recently. However, the impact and biological mechanism of NPs on the cardiovascular system are not well characterized. This study aimed to assess the possibility that NPs exposure promotes premature endothelial cell (EC) senescence in porcine coronary artery ECs and, if so, to elucidate the underlying mechanism. Treatment of ECs with NPs promoted the acquisition of senescence markers, senescence-associated β-galactosidase activity, and p53, p21, and p16 protein expression, resulting in the inhibition of proliferation. In addition, NPs impaired endothelium-dependent vasorelaxation associated with decreased endothelial nitric oxide synthase (eNOS) expression. NPs enhanced reactive oxygen species formation in ECs, and increased oxidative stress levels were associated with the induction of NADPH oxidases expression, followed by the subsequent downregulation of Sirt1 expression. The characteristics of EC senescence and dysfunction caused by NPs are prevented by an antioxidant (N-acetylcysteine), an NADPH oxidase inhibitor (apocynin), and a Sirt1 activator (resveratrol). These findings indicate that NPs induced premature EC senescence, at least in part, through the redox-sensitive eNOS/Sirt1 signaling pathway. This study suggested the effects and underlying mechanism of NPs on the cardiovascular system, which may provide pharmacological targets to prevent NPs-associated cardiovascular diseases.