Increase In Nitric Oxide Concentration Research Articles

Reciprocal modulation of responses to nitrate starvation and hypoxia in roots and leaves of Arabidopsis thaliana

ABSTRACT The flooding of agricultural land leads to hypoxia and nitrate leaching. While understanding the plant’s response to these conditions is essential for crop improvement, the effect of extended nitrate limitation on subsequent hypoxia has not been studied in an organ-specific manner. We cultivated Arabidopsis thaliana without nitrate for 1 week before inducing hypoxia by bubbling the hydroponic solution with nitrogen gas for 16 h. In the roots, the transcripts of two transcription factor genes (HRA1, HRE2) and three genes involved in fermentation (SUS4, PDC1, ADH1) were ~10- to 100-fold upregulated by simultaneous hypoxia and nitrate starvation compared to the control condition (replete nitrate and oxygen). In contrast, this hypoxic upregulation was ~5 to 10 times stronger when nitrate was available. The phytoglobin genes PGB1 and PGB2, involved in nitric oxide (NO) scavenging, were massively downregulated by nitrate starvation (~1000-fold and 105-fold, respectively), but only under ambient oxygen levels; this was reflected in a 2.5-fold increase in NO concentration. In the leaves, HRA1, SUS4, and RAP2.3 were upregulated ~20-fold by hypoxia under nitrate starvation, whereas this upregulation was virtually absent in the presence of nitrate. Our results highlight that the plant’s responses to nitrate starvation and hypoxia can influence each other.

MONITORING IN VITRO AND EX VIVO INFLAMMATION USING SINGLE-WALLED CARBON NANOTUBES (SWCNTS) SENSOR TECHNOLOGY

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation.Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion.The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin.The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo.Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

Exosomes derived from rapamycin-treated 4T1 breast cancer cells induced polarization of macrophages to M1 phenotype.

M2 macrophages are the most prevalent type in the tumor microenvironment and their polarization to M1 type can be used as a potential cancer immunotherapy. Here, we investigated the role of tumor microenvironment and particularly purified exosomes in M2 to M1 macrophage polarization. Rapamycin treatment on triple-negative breast cancer cells (TNBC) was performed. Tumor cells-derived exosomes (called texosomes) were isolated and characterized using scanning electron microscopy, transmission electron microscopy, dynamic light scattering, high-performance liquid chromatography, Fourier transform infrared, and Western blot assays. M2 mouse peritoneal macrophages were treated with rapamycin or rapamycin-texosome. Then, M1/M2 phenotype-specific marker genes and proteins were measured to assess the degree of M2 to M1 polarization. Finally, nitric oxide (NO) production, phagocytosis, and efferocytosis assays were assessed to verify the functionality of the polarized macrophages. Purified rapamycin-texosomes significantly increased the expression of the M1 markers (Irf5, Nos2, and CD86) and decreased M2 markers (Arg, Ym1, and CD206). In addition, the levels of M1-specific cytokines tumor necrosis factor alpha and interleukin 1β (IL-1β) were increased, whereas the levels of M2 specific cytokines IL-10 and transforming growth factor beta were declined. Furthermore, texosome treatment increased NO concentration and phagocytosis and decreased efferocytosis indicating M1 polarization. These findings suggest rapamycin-texosomes can induce M2 to M1 macrophages polarization as a potential immunotherapy for TNBC.

Effect of Exogenous Nitric Oxide on Enterocytozoon hepatopenaei Copy Numbers and Immunity of Exopalaemon carinicauda

To explore the effect of nitric oxide (NO) on Enterocytozoon hepatopenaei (EHP) invading the shrimp, sodium nitroprusside was used as an exogenous NO donor to investigate the effects of different concentrations (0 μg/L (control group), 0.3 μg/L, 0.6 μg/L, and 0.9 μg/L) of sodium nitroprusside on the EHP copy number and the structure in the hepatopancreas of Exopalaemon carinicauda infected by EHP. The results showed that the EHP copy number in the sodium nitroprusside group was significantly lower than that in the control group, and the hepatopancreas in the sodium nitroprusside injection group had less structure damage than the control group. Additionally, superoxide dismutase (SOD) activity in the control group and 0.3 μg/L groups increased and then decreased, while alkaline phosphatase (AKP) activity decreased and then increased, with the same trend, and, the inducible nitric oxide synthase (iNOS) activity in the haemolymph of the 0.3 μg/L group was significantly higher than that of the control group at 3 days ( P < 0.05 ). The NO content the 0.3 μg/L group was also higher from 1 to 4 days than that in the control group and significantly higher than that in the control group at 3 days ( P < 0.05 ). The injection of NO could reduce the EHP copy number in infected E. carinicauda and slow down the pathological changes in the hepatopancreas structure, which might be related to the increase in NO content.

Nitric oxide-releasing gel accelerates healing in a diabetic murine splinted excisional wound model.

According to the American Diabetes Association (ADA), 9-12 million patients suffer from chronic ulceration each year, costing the healthcare system over USD $25 billion annually. There is a significant unmet need for new and efficacious therapies to accelerate closure of non-healing wounds. Nitric Oxide (NO) levels typically increase rapidly after skin injury in the inflammatory phase and gradually diminish as wound healing progresses. The effect of increased NO concentration on promoting re-epithelization and wound closure has yet to be described in the context of diabetic wound healing. In this study, we investigated the effects of local administration of an NO-releasing gel on excisional wound healing in diabetic mice. The excisional wounds of each mouse received either NO-releasing gel or a control phosphate-buffered saline (PBS)-releasing gel treatment twice daily until complete wound closure. Topical administration of NO-gel significantly accelerated the rate of wound healing as compared with PBS-gel-treated mice during the later stages of healing. The treatment also promoted a more regenerative ECM architecture resulting in shorter, less dense, and more randomly aligned collagen fibers within the healed scars, similar to that of unwounded skin. Wound healing promoting factors fibronectin, TGF-β1, CD31, and VEGF were significantly elevated in NO vs. PBS-gel-treated wounds. The results of this work may have important clinical implications for the management of patients with non-healing wounds.

Polyamines and nitric oxide crosstalk in plant development and abiotic stress tolerance.

Polyamines (PAs) and nitric oxide (NO) are crucial signalling molecules that exhibit a promising role in improving stress tolerance in plants, maintaining their growth and development. They act as protecting agents for plants through activation of stress adaptation strategies such as membrane stabilisation, acid neutralisation and suppression of ROS generation. NO interacts with PAs during several developmental processes and stress responses. External supplementation of PAs to plants is also reported to cause an increase in NO content. However, it is unclear whether PAs promote synthesis of NO by either as substrates, cofactors, or signals. Impact of NO on synthesis of PAs has been also reported in some studies, yet the exact governing mechanisms of the interrelation between NO and PAs is currently obscure. Understanding the crosstalk between PAs and NO during growth and stress condition in plants can aid in providing better tolerance to plants against stressful environment.

Oxidation absorption of nitric oxide from flue gas using biochar-activated peroxydisulfate technology

Biochar-activated peroxydisulfate oxidation technology has received more and more attention due to its low metal ion leakage rate and low solid waste post-treatment requirement. In this paper, rice straw biochar-activated peroxydisulfate oxidation technology is used for the first time to oxidize nitric oxide (NO) from flue gas through inducing free radicals. The mechanism of NO oxidation removal is mainly explored. The main factors affecting NO removal, removal products and free radicals are also systematically studied. Results show that the main product of NO oxidation removal is NO3− and no extra by-product is formed. ·OH is identified as the first important free radical for oxidizing NO, while SO4−· is determined to be the second important free radical for oxidizing NO. Peroxydisulfate oxidation only plays a minor supplementary role in NO removal. Increasing rice straw biochar mass concentration and operation temperature promote NO removal. Increasing NO concentration and solution pH value are found to be harmful to NO removal. Increasing peroxydisulfate concentration first increases and then decreases NO removal efficiency. Under optimized process parameters, the maximum NO removal efficiency reaches 95.2%.

Nitric oxide and hydrogen sulfide work together to improve tolerance to salinity stress in wheat plants by upraising the AsA-GSH cycle

The participation of nitric oxide (NO) in wheat plant tolerance to salinity stress (SS) brought about by hydrogen sulphide (H2S) via modifying the ascorbate-glutathione (AsA-GSH) cycle was studied. The SS-plants received either 0.2 mM sodium hydrosulfide (NaHS; H2S donor), or NaHS plus 0.1 mM sodium nitroprusside (SNP; a NO donor) through the nutrient solution. Salinity stress decreased plant growth, leaf water status, leaf K+, and glyoxalase II (gly II), while it elevated proline content, leaf Na+ content, oxidative stress, methylglyoxal (MG), glyoxalase I (gly I), the superoxide dismutase, catalase and peroxidase activities, contents of endogenous NO and H2S. The NaHS supplementation elevated plant development, decreased leaf Na+ content and oxidative stress, and altered leaf water status, leaf K+ and involved enzymes in AsA-GSH, H2S and NO levels. The SNP supplementation boosted the positive impact of NaHS on these traits in the SS-plants. Moreover, 0.1 mM cPTIO, scavenger of NO, countered the beneficial effect of NaHS by lowering NO levels. SNP and NaHS + cPTIO together restored the beneficial effects of NaHS by increasing NO content, implying that NO may have been a major factor in SS tolerance in wheat plants induced by H2S via activating enzymes connected to the AsA-GSH cycle.

Nitric oxide mediates melatonin-induced isoflavone accumulation and growth improvement in germinating soybeans under NaCl stress

The involvement of nitric oxide (NO) in exogenous melatonin (MT)-induced isoflavone accumulation and growth improvement in NaCl-stressed soybeans was investigated in this study. The results demonstrated that MT increased the activity of nitrate reductase (NR) and upregulated the relative expression of NR1, NR2, and nitric oxide synthase1, which subsequently led to an increase in NO content. MT and sodium nitroprusside (SNP, as an NO donor) markedly increased isoflavone content by enhancing the activities of cinnamic acid 4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL), and by upregulating gene expression of C4H, Isoflavone synthase, PAL, and Chalcone isomerase 1A, which are involved in isoflavone biosynthesis. Moreover, MT, as well as SNP, improved the growth and biomass of NaCl-treated soybeans by increasing the activities of superoxide dismutase, catalase, and peroxidase, and reducing the accumulation of H2O2 and O2•− in soybeans under NaCl stress. These MT-induced responses were entirely reversed by the supply of 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a specific scavenger of NO), which in turn considerably decreased endogenous NO content. These results suggest that NO acts as an important downstream signal molecule, mediating MT-induced isoflavone accumulation and growth improvement in NaCl-stressed soybeans.

Mitochondrial dysfunction due to in vitro exposure to atrazine and its metabolite in striatum.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) has beendescribed as a potential toxic for dopaminergic metabolism both in vivo and in vitro. Its main metabolite diamino-chloro triazine (DACT) has been shown to achieve higher levels in brain tissue than atrazine. The aim of this study was to evaluate the in vitro effects of atrazine and DACT on striatal mitochondrial function, active oxygen species generation, and nitric oxide (NO) content. Incubation of mitochondria with atrazine (10 µM) was not able to modify oxygen consumption. However, a 50% increase in malate-glutamate state 4 respiratory rates was observed after DACT treatment (100 µM) without changes in respiratory state 3. Atrazine was able to inhibit complex I-III activity by 30% and DACT induced a tendency to decrease by 17% in the striatum. Regardingreactive oxygen species(ROS), DACT increased H2 O2 production by 43%. Also, superoxide anion levels were higher (14%) after atrazine exposure than in control mitochondria. Incubation of striatal mitochondria with atrazine and DACT induced membrane depolarization by 15% and 19%, respectively. Also, atrazine increased NO content by 10% but no significant changes were observed after exposure of mitochondria to DACT. Glutathione peroxidase activity was inhibited (56%) by DACT and atrazine inhibited superoxide dismutase activity by 60%. Also, cardiolipin oxidation (15%) was observed after atrazine treatment. Summing up, the obtained results suggest that in vitro atrazine and DACT induce ROS production affecting striatal mitochondrial function. The atrazine effects would be attributed to a direct effect on the mitochondrialrespiratory chain and superoxide dismutase activity while DACT appears to disturb glutathione-related enzyme system.

BR-Mediated Protein S-Nitrosylation Alleviated Low-Temperature Stress in Mini Chinese Cabbage (Brassica rapa ssp. pekinensis)

Brassinosteroids (BRs), a novel plant hormone, are widely involved in plant growth and stress response processes. Nitric oxide (NO), as an important gas signaling molecule, can regulate target protein activity, subcellular localization and function in response to various stresses through post-translational S-nitrosylation modifications. However, the relationship between BR and NO in alleviating low-temperature stress of mini Chinese cabbage remains unclear. The hydroponic experiment combined with the pharmacological and molecular biological method was conducted to study the alleviating mechanism of BR at low temperature in mini Chinese cabbage. The results showed that low temperature inhibited the growth of mini Chinese cabbage seedlings, as evidenced by dwarf plants and yellow leaves. Treatment with 0.05 mg/L BR and 50 µM NO donor S-nitrosoglutathione (GSNO) significantly increased the leaf area, stem diameter, chlorophyll content, dry and fresh weight and proline content. Meanwhile, the malondialdehyde (MDA) content in 0.05 mg/L BR- and 50 µM GSNO-treated leaves were significantly lower than those in other treated leaves under low-temperature conditions. In addition, BR and GSNO applications induced an increase in NO and S-nitrosothiol (SNO) levels in vivo under low-temperature stress. Similarly, spraying BR after the elimination of NO also increased the level of S-nitrosylation in vivo, while spraying GSNO after inhibiting BR biosynthesis decreased the level of NO and SNO in vivo. In contrast, the S-nitrosoglutathione reductase (BrGSNOR) relative expression level and GSNOR enzyme activity were downregulated and inhibited by BR treatment, GSNO treatment and spraying BR after NO clearance, while the relative expression level of BrGSNOR was upregulated and GSNOR enzyme activity was also increased when spraying GSNO after inhibiting BR synthesis. Meanwhile, the biotin switch assay showed that exogenous BR increased the level of total nitrosylated protein in vivo under low-temperature stress. These results suggested that BR might act as an upstream signal of NO, induced the increase of NO content in vivo and then induced the protein S-nitrosylation modification to alleviate the damage of mini Chinese cabbage seedlings under low-temperature stress.

Nitric Oxide Participates in Aluminum-Stress-Induced Pollen Tube Growth Inhibition in Tea (Camelliasinensis) by Regulating CsALMTs.

Nitric oxide (NO), as a signal molecule, is involved in the mediation of heavy-metal-stress-induced physiological responses in plants. In this study, we investigated the effect of NO on Camellia sinensis pollen tubes exposed to aluminum (Al) stress. Exogenous application of the NO donor decreased the pollen germination rate and pollen tube length and increased the malondialdehyde (MDA) content and antioxidant enzyme activities under Al stress. Simultaneously, the NO donor effectively increased NO content in pollen tube of C. sinensis under Al stress and could aggravate the damage of Al3+ to C. sinensis pollen tubes by promoting the uptake of Al3+. In addition, application of the NO-specific scavenger significantly alleviated stress damage in C. sinensis pollen tube under Al stress. Moreover, 18 CsALMT members from a key Al-transporting gene family were identified, which could be divided into four subclasses. Pearson correlation analysis showed the expression level of CsALMT8 showed significant positive correlation with the Al3+ concentration gradient and NO levels, but a significant negative correlation with pollen germination rate and pollen tube length. The expression level of CsALMT5 was negatively correlated with the Al3+ concentration gradient and NO level, and positively correlated with pollen germination rate and pollen tube length. The expression level of CsALMT17 showed a significant negative correlation with Al3+ concentration and NO content in pollen tubes, but significant positive correlation with pollen germination rate and pollen tube length. In conclusion, a complex signal network regulated by NO-mediated CsALMTs revealed that CsALMT8 was regulated by environmental Al3+ and NO to assist Al3+ entry into pollen tubes; CsALMT5 might be influenced by the Al3+ signal, stimulate malate efflux in vacuoles and chelate with Al3+ to detoxify Al in C. sinensis pollen tube.

Hydrogen Sulfide Modulates Salinity Stress in Common Bean Plants by Maintaining Osmolytes and Regulating Nitric Oxide Levels and Antioxidant Enzyme Expression

The purpose of the present study is to investigate the role of hydrogen sulfide (H2S), in improving resistance to common bean salt stress. Method shows that common bean seeds were soaked in water and in two concentrations of sodium hydrosulfide (50 and 100 µM) for 8 h. After 25 days from sowing, the pots were irrigated with water and with two concentrations of NaCl (75 and 150 mM) until the end of the experiment. Results revealed that H2S relieved salt stress by decreasing growth inhibition and photosynthetic characteristics, and increasing osmolyte contents (proline and glycine betaine). Furthermore, H2S reduced oxidative damage by lowering lipid peroxidation, electrolyte leakage, and reactive oxygen species production such as hydrogen peroxide, hydroxyl radicals, and superoxide anion by increasing non-enzymatic antioxidants such as ascorbic acid and glutathione, as well as enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), and nitrate reductase (NR). Meanwhile, salt stress and H2S application increased the endogenous level of H2S, which was accompanied by an increase in nitric oxide concentration. H2S, in particular, maintained sodium (Na+) and potassium (K+) homeostasis in the presence of excess NaCl. In general, H2S effectively reduced oxidative stress in common bean plants by increasing relative expression levels of copper-zinc superoxide dismutase (Cu-ZnSOD), CAT, and glutathione S-transferase (GST). Applying H2S to common bean plants could protect them from salinity stress by maintaining the Na+/K+ balance, boosting endogenous H2S and nitric oxide levels, and preventing oxidative damage by increasing antioxidant activity.

Luteolin ameliorates hypoxia-induced pulmonary hypertension via regulating HIF-2α-Arg-NO axis and PI3K-AKT-eNOS-NO signaling pathway

BackgroundPulmonary hypertension (PH) is a devastating disease with poor prognosis and high mortality. Hypoxia induced pulmonary hypertension (HPH) is a persistent threat to human health, especially to people who live on high altitude plateau. Pulmonary vascular endothelial cell is involved in numerous pathophysiological processes, including in vasoconstriction, oxidative stress, cell growth and differentiation. Endothelial cells (ECs) are the first layer to be exposed to changed oxygen levels and hypoxia could lead to ECs dysfunction. Endothelial-derived nitric oxide (NO) is the most important bioactive molecule, which could regulate endothelial homeostasis. PH pathophysiology has been linked to the disruption of NO pathways. PurposeLuteolin is a kind of plant active ingredient with multiple pharmacological activities. The purpose of this study is to detect the effect of luteolin on HPH with in vivo, ex vivo and in vitro analyses and to further elucidate luteolin's pharmaceutical mechanism with NO related signaling pathway regulation. MethodsHypobaric chamber was used to establish HPH animal model. Rats were intragastrically administrated luteolin for 28 days. Then hemodynamic indexes, histopathological changes, pulmonary artery endothelial function, NO content and arginase activity in lung tissue, NO related pathway proteins expression were measured to evaluate the effect of luteolin on HPH. PAECs were treated with 1% O2 and incubated with or without luteolin. PAECs vitality, NO content in cells supernatant, and NO related pathway proteins expression were tested to reveal the protective mechanism of luteolin. ResultsLuteolin decreased mean pulmonary hypertension of HPH rats, alleviated right ventricular and pulmonary vascular remodeling. Immunofluorescence staining (vWF), isolated perfused/ventilated rat lung experiment indicated that luteolin protected pulmonary vascular endothelial function of HPH rats. Luteolin increased NO content in PAECs supernatant while decreased NO level in lung tissues of HPH rats. Further, it was demonstrated that luteolin inhibited HIF-2α-Arg axis in PAECs and HPH rats. PI3K-AKT-eNOS signaling pathway was upregulated in PAECs, but which was downregulated in lung tissues of HPH rats. Pharmacological effect of luteolin was equivalent or better than sildenafil. ConclusionLuteolin ameliorated HPH in rats by protecting pulmonary vascular endothelial function via regulating HIF-2α-Arg-NO axis and PI3K-AKT-eNOS-NO signaling pathway. This study may provide a novel perspective and approach to alleviate the devastating disease of HPH.

Nitric Oxide-Induced Calcineurin A Mediates Antimicrobial Peptide Production Through the IMD Pathway.

Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.

The Effect of a Novel Small Molecule Estradiol Guanidine Derivative on the Ovary and Uterus of Female Mice

In this study, a novel small molecule estradiol guanidine derivative (Guanidoestradiol, GDT) for female reproductive system targeting and nitric oxide (NO) release has been constructed by conjugating 17β-estradiol and L-arginine through an esterification reaction. After intraperitoneal administration in female mice, the serum Griess assay and immunohistochemical analysis of ovary and uterus were performed. GDT effectively increased NO concentration in the blood and produced some bio-effects that are beneficial to the reproductive system. The specific advantages are as follows: (1) the introduction of guanidine groups and amino groups improves the water solubility and bioavailability of estradiol and is expected to exert other biofunctions; (2) the estradiol residue of GDT gives the NO donor the ability for ovarian and uterine targeting; (3) combined administration of estradiol and NO to the uterus and ovary promoted endometrial thickening and expression of vascular proliferation markers; (4) the abundance of iNOS in tissues was reduced after treatment with NO donors. We suggest that Guanidoestradiol is a promising small-molecule drug to enhance normal endometrial and ovarian function and may play a role in promoting reproduction.

Ultrasound-Induced Destruction of Nitric Oxide-Loaded Microbubbles in the Treatment of Thrombus and Ischemia-Reperfusion Injury.

Objectives: Early recanalization of large vessels in thromboembolism, such as myocardial infarction and ischemic stroke, is associated with improved clinical outcomes. Nitric oxide (NO), a biological gas signaling molecule, has been proven to protect against ischemia–reperfusion injury (IRI). However, the underlying mechanisms remain to be explored. This study investigated whether NO could mitigate IRI and the role of NO during acoustic cavitation. Methods: In vivo, thrombi in the iliac artery of rats were induced by 5% FeCl3. NO-loaded microbubbles (NO-MBs) and ultrasound (US) were used to treat thrombi. B-mode and Doppler US and histological analyses were utilized to evaluate the thrombolysis effect in rats with thrombi. Immunohistochemistry, immunofluorescence, and western blotting were conducted to investigate the underlying mechanisms of NO during acoustic cavitation. In vitro, hypoxia was used to stimulate cells, and NO-MBs were employed to alleviate oxidative stress and apoptosis. Results: We developed NO-MBs that significantly improve the circulation time of NO in vivo, are visible, and effectively release therapeutic gas under US. US-targeted microbubble destruction (UTMD) and NO-loaded UTMD (NO + UTMD) caused a significant decrease in the thrombus area and an increase in the recanalization rates and blood flow velocities compared to the control and US groups. We discovered that UTMD induced NO generation through activation of endothelial NO synthase (eNOS) in vivo. More importantly, we also observed significantly increased NO content and eNOS expression in the NO + UTMD group compared to the UTMD group. NO + UTMD can mitigate oxidative stress and apoptosis in the hind limb muscle without influencing blood pressure or liver and kidney functions. In vitro, NO-MBs alleviated oxidative stress and apoptosis in cells pretreated with hypoxia. Conclusion: Based on these data, UTMD affects the vascular endothelium by activating eNOS, and NO exerts a protective effect against IRI.

Nitric oxide alleviates salt-induced stress damage by regulating the ascorbate–glutathione cycle and Na+/K+ homeostasis in Nitraria tangutorum Bobr

Nitric oxide (NO) is an important signaling molecule involved in mediation of salt stress induced physiological responses in plants. In this study, we investigated the effect of NO on Nitraria tangutorum seedlings exposed to salt stress. Exogenous application of NO donor, sodium nitroprusside (SNP) increased fresh weight, shoot and root elongation and decreased electrolyte leakage and malondialdehyde (MDA) content in N. tangutorum seedlings under salt stress. Simultaneously, leaf senescence and root damage induced by salt stress were alleviated. SNP effectively increased NO content both in leaves and roots of plants under salt stress. Meanwhile, SNP activated the ascorbate-glutathione (AsA-GSH) cycle by increasing antioxidants contents, antioxidant enzymes activities, and related genes expression, thereby scavenging reactive oxygen species (ROS) and alleviating oxidative damage caused by salt stress. SNP alleviated salt stress induced ion toxicity by promoting Na+ efflux and ion transporter gene expression and reducing Na+ content and the Na+/K+ ratio. In addition, application of NO specific scavenger cPTIO and mammalian NO synthase inhibitor L-NAME sifnificantly aggravated stress damage in plant under salt stress. These results show the beneficial impacts of NO as a stress-signaling molecule that positively regulates defense response in N. tangutorum to salt stress.

Antioxidant and anti-inflammatory protective effects of rutin and kolaviron against busulfan-induced testicular injuries in rats

ABSTRACT There are few treatment options, including the use of natural phenolics-based combination therapy for mitigating male infertility conditions associated with chemotherapy. Busulfan is an anti-cancer drug that leads to testicular problems in humans. Here, we studied the effect of co-treatment of rutin and kolaviron against busulfan-induced testis damage. Young adult male Wistar rats were intraperitoneally injected busulfan (4 mg/kg b.w), and then orally administered rutin (30 mg/kg b.w), and kolaviron (50 mg/kg b.w) alone and combined for 60 days. Results revealed that rutin and kolaviron alone or in combination reversed busulfan-induced increase in oxidative stress along with sperm quality of treated animals. However, kolaviron and rutin separately improved the concentrations of MDA and GSH and sperm quality more than when they were combined. Similarly, rutin and kolaviron separately or in combination preserved spermatogenesis and relieved busulfan-induced increase in nitric oxide concentration, myeloperoxidase and 3β-hydroxysteroid dehydrogenase activities. Co-supplementation with kolaviron but not rutin nor when rutin was combined with kolaviron also improved the testicular level of tumor necrosis-alpha. Finally, the histological features in the testes caused by busulfan were reversed by rutin, whereas treatment with kolaviron alone or in combination with rutin partially protected the testis from busulfan-induced injury as demonstrated by the appearance of few germ cells, damaged tubules, loss of round spermatids and defoliation of the seminiferous epithelium. Thus, the combined treatment regimen of rutin and kolaviron sparingly prevented busulfan-induced testicular injuries in rats. Abbreviations: CAT: Catalase; GSH: Glutathione; 3β-HSD: 3β- hydroxysteroid Dehydrogenase; MDA: Malondialdehyde; TNF-α: Tumor necrosis-alpha; BUS: Busulfan; RUT: Rutin; KV: Kolaviron; TBARS: Thiobarbituric Acid Reactive Substances; MPO: Myeloperoxidase; ELISA: Enzyme-Linked Immunoassay; NAD: Nicotinamide Adenine Dinucleotide (oxidized); ROS: Reactive Oxygen Species.

Decreased nitric oxide content mediated by asymmetrical dimethylarginine and protein l-arginine methyltransferase 3 in macrophages induces trophoblast apoptosis: a potential cause of recurrent miscarriage.

Is the protein l-arginine methyltransferase 3 (PRMT3)/asymmetrical dimethylarginine (ADMA)/nitric oxide (NO) pathway involved in the development of recurrent miscarriage (RM), and what is the potential mechanism? Elevated levels of PRMT3 and ADMA inhibit NO formation in the decidua, thereby impairing the functions of trophoblast cells at the maternal-foetal interface. Decreased NO bioavailability is associated with RM. ADMA, an endogenous inhibitor of nitric oxide synthase (NOS), is derived from the methylation of protein arginine residues by PRMTs and serves as a predictor of mortality in critical illness. A total of 145 women with RM and 149 healthy women undergoing elective termination of an early normal pregnancy were enrolled. Ninety-six female CBA/J, 24 male DBA/2 and 24 male BALB/c mice were included. CBA/J × DBA/2 matings represent the abortion group, while CBA/J × BALB/c matings represent the normal control group. The CBA/J pregnant mice were then categorised into four groups: (i) normal + vehicle group (n = 28), (ii) abortion + vehicle group (n = 28), (iii) normal + SGC707 (a PRMT3 inhibitor) group (n = 20) and (iv) abortion + SGC707 group (n = 20). All injections were made intraperitoneally on Days 0.5, 3.5 and 6.5 of pregnancy. Decidual tissues were collected on Days 8.5, 9.5 and 10.5 of gestation. The embryo resorption rates were calculated on Day 9.5 and Day 10.5 of gestation. NO concentration, ADMA content, NOS activity, expression levels of NOS and PRMTs in decidual tissues were determined using conventional assay kits or western blotting. PRMT3 expression was further analysed in decidual stromal cells, macrophages and natural killer cells. A co-culture system between decidual macrophages (DMs) and HTR-8/SVneo trophoblasts was constructed to study the roles of the PRMT3/ADMA/NO signalling pathway. Trophoblast apoptosis was analysed via Annexin V-fluorescein isothiocyanate/propidium iodide staining. CBA/J × DBA/2 mouse models were used to investigate the effects of SGC707 on embryo resorption rates. Our results show that NO concentration and NOS activity were decreased, but ADMA content and PRMT3 expression were increased in the decidua of RM patients. Moreover, compared with the normal control subjects, PRMT3 expression was significantly up-regulated in the macrophages but not in the natural killer cells or stromal cells of the decidua from RM patients. The inhibition of PRMT3 results in a significant decrease in ADMA accumulation and an increase in NO concentration in macrophages. When co-cultured with DMs, which were treated with SGC707 and ADMA, trophoblast apoptosis was suppressed and induced, respectively. In vivo experiments revealed that the administration of SGC707 reduced the embryo resorption rate of CBA/J × DBA/2 mice. All sets of experiments were not performed with the same samples. The main reason is that each tissue needs to be reserved for clinical diagnosis and only a small piece of each tissue can be cut and collected for this study. Our results indicate that the PRMT3/ADMA/NO pathway is a potential marker and target for the clinical diagnosis and therapy of RM. This study was supported by the National Key Research and Development Program of China (2017YFC1001401), National Natural Science Foundation of China (81730039, 82071653, 81671460, 81971384 and 82171657) and Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015). The authors have declared no conflict of interest. N/A.