Intracellular Nitric Oxide Levels Research Articles

AhGSNOR1 negatively regulates Al-induced programmed cell death by regulating intracellular NO and redox levels

The toxicity of aluminum (Al) in acidic soil inhibits plant development and reduces crop yields. Programmed cell death (PCD) is one of the important mechanisms in the plant response to Al toxicity. However, it is yet unknown if S-nitrosoglutathione reductase (GSNOR) provides Al-PCD. Here, transcription and protein expression of AhGSNOR1 were both induced by Al stress. AhGSNOR1-overexpressing transgenic tobacco plants reduced Al-induced nitric oxide (NO) and S-nitrosothiol accumulation, the inhibitory effect of Al stress on root elongation and the degree of cell death, and enhanced antioxidant enzyme activity to effectively remove hydrogen peroxide. In addition, AhGSNOR1 directly interacted with AhTRXh in vivo. Expression of Trxh3 in AhGSNOR1-overexpressing transgenic plants was significantly upregulated, indicating that AhGSNOR1 positively regulated the transcriptional level of Trxh3. Together, these results suggested that AhGSNOR1 was a negative regulatory factor of Al-induced PCD and improved plant Al-tolerance by modulating intracellular NO and redox homeostasis.

Nitric oxide mediates positive regulation of Nostoc flagelliforme polysaccharide yield via potential S-nitrosylation of G6PDH and UGDH

Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.

Enhancement of lipid synthesis by the transcription factor Asg1 in Saitozyma podzolica zwy-2–3 under dissolved oxygen stress

Microbial oils have been of considerable interest as food additives and biofuel resources due to high lipid contents, but lipid accumulation of oleaginous microorganisms can be induced by environmental stresses, such as dissolved oxygen (DO), which limit large-scale lipid production. Here, DO stress gave rise to the endogenous nitric oxide (NO) level to mediate S-nitrosylation of SpAsg1, regulating the lipid accumulation in Saitozyma podzolica zwy-2–3. Notably, qRT-PCR, yeast one-hybrid, dual-luciferase reporter assays, and metabolomics analysis exhibited that overexpression of SpAsg1 promoted lipid synthesis by directly regulation of glucose metabolism, enhancing glucose uptake, ATP and NADPH contents under DO stress. Meanwhile, SpAsg1 improved the antioxidant capacity to reduce the intracellular reactive oxygen species (ROS) and NO levels. Overall, we systematically investigated the regulation of SpAsg1 on lipid metabolism of S. podzolica zwy-2–3 under DO stress, which sheds light on further studies for alleviating oxygen limitation of lipid production in microbial industry.

Role of Flavohemoglobins in the Development and Aflatoxin Biosynthesis of Aspergillus flavus.

Aspergillus flavus is notorious for contaminating food with its secondary metabolite-highly carcinogenic aflatoxins. In this study, we found that exogenous nitric oxide (NO) donor could influence aflatoxin production in A. flavus. Flavohemoglobins (FHbs) are vital functional units in maintaining nitric oxide (NO) homeostasis and are crucial for normal cell function. To investigate whether endogenous NO changes affect aflatoxin biosynthesis, two FHbs, FHbA and FHbB, were identified in this study. FHbA was confirmed as the main protein to maintain NO homeostasis, as its absence led to a significant increase in intracellular NO levels and heightened sensitivity to SNP stress. Dramatically, FHbA deletion retarded aflatoxin production. In addition, FHbA played important roles in mycelial growth, conidial germination, and sclerotial development, and response to oxidative stress and high-temperature stress. Although FHbB did not significantly impact the cellular NO level, it was also involved in sclerotial development, aflatoxin synthesis, and stress response. Our findings provide a new perspective for studying the regulatory mechanism of the development and secondary mechanism in A. flavus.

Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels

SignificanceNitric oxide (NO) is involved in the regulation of physiological and pathological mechanisms of the cardiovascular, nervous and immune systems; and plays an important role in cancer being involved in tumor growth and suppressing processes depending on its concentration. ApproachThe development of a near-infrared excitable nanoprobe, consisting of gold nanoparticles functionalized with a two-photon excitable NO probe, for the detection of intracellular NO is reported. ResultsThe nanoprobe showed good selectivity towards NO over cellular interferences and excellent stability in aqueous-medium over time. The nanoprobe was able to selectively detect endogenous and exogenous NO in different cell lines and it accumulated in the acidic organelles showing negligible toxicity. Importantly, the nanoprobe showed potential to quantify intracellular NO concentrations in breast cancer cells. ConclusionsThe novel gold-based two-photon nanoprobe showed an excellent performance and versatility and could potentially be applied for the spatiotemporal monitoring of in vivo NO levels.

Humulus lupulus aqueous extract and hydrolate as a potential ingredient for cosmetics: chemical characterization and in vitro antimicrobial, cytotoxicity, antioxidant and anti-inflammatory assessment

Humulus lupulus extracts have in their composition different molecules, such as polyphenols, α-acids, β-acids, and hydrocarbons, which contribute to the plant's medicinal properties. These molecules are associated with antimicrobial, antioxidant and anti-inflammatory activities. ObjectiveThis work focuses on the evaluation of H. lupulus biological activities, with the aim of evaluating its potential for inclusion in cosmetic formulations. MethodsTwo distinct aqueous extracts and two hydrolates obtained via hydrodistillation were evaluated. These include the flower parts (FE, FH) and the mix of aboveground parts (ME, MH). The chemical profiles for both aqueous extracts and hydrolates were identified by high performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS). Antimicrobial, antioxidant, cytotoxicity, and anti-inflammatory activity were tested in vitro using standard methods. ResultsRutin was the major compound found in FE (40.041 μg mg−1 of extract) and ME (2.909 μg mg−1 of extract), while humulenol II was the most abundant compound in hydrolates (FH: 20.83%; MH: 46.80%). Furthermore, FE was able to inhibit the growth of Staphylococcus aureus and Staphylococcus epidermis with MIC values of 50% and 25% (v/v), respectively. FH showed the same effect in Staphylococcus aureus (50% v/v). FH evidenced poor antioxidant potential in DPPH scavenging test and demonstrated significant antioxidant and anti-inflammatory effects by reducing (***p < 0.001) intracellular reactive oxygen species (ROS), NO (nitric oxide) levels (***p < 0.001) and cyclooxygenase-2 (COX-2) protein expression (***p < 0.001) in lipopolysaccharide (LPS)-stimulated macrophages. Nevertheless, it is important to note that FH exhibited cytotoxicity at high concentrations in 3T3 fibroblasts and RAW 264.7 macrophages. ConclusionThe studied H. lupulus aqueous extracts and hydrolates revealed that FH stands out as the most promising bioactive source for cosmetic formulations. However, future research addressing antimicrobial activity is necessary to confirm its potential incorporation into dermatological and cosmetic formulations.

Engineering hemin-loaded hyaluronan needle-like microparticles with photoprotective properties against UV-induced tissue damage.

This study aimed to develop hyaluronan (HA)-based hydrogel microparticles (MPs) loaded with hemin to address the limitations of traditional macroscale hydrogels. The objective is to design MPs such that they can modulate their physicochemical properties. Given the widespread use of ultraviolet C (UVC) light in various industries and the need for protective measures against accidental exposure, this study evaluated the potential of hemin-loaded MPs to protect human dermal fibroblasts from oxidative stress and cell death caused by UVC exposure. Multiple MP formulations were developed and analysed for size, surface charge, swelling behaviour, degradation rate, and radical scavenging capabilities, both with and without hemin loading. The most promising formulations were tested against UVC-exposed cells to assess cell viability, intracellular nitric oxide (˙NO) and reactive oxygen species levels, and protein carbonylation. The fabricated particles were in the form of microneedles, and the degree of their crosslinking and the role of hemin in the chemical crosslinking reaction were found to influence the surface charge and hydrodynamic diameter of the MPs. Increased crosslinking resulted in reduced swelling, slower degradation, and decreased hemin release rate. MPs with a higher degree of swelling were capable of releasing hemin into the culture medium, leading to enhanced bilirubin generation in dermal fibroblasts following cellular uptake. Pre-treatment with these MPs protected the cells from UVC-induced cell death, nitrosative stress, and protein carbonylation. These findings highlight the potential of the studied MPs to release hemin and to minimise the harmful effects of UVC on dermal fibroblasts.

Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

Mycothiol maintains the homeostasis and signalling of nitric oxide in Streptomyces coelicolor A3(2) M145

BackgroundPrevious studies have revealed a nitric oxide (NO) metabolic cycle in which NO, nitrate (NO3−), and nitrite (NO2−) circulate. The NO produced in this cycle serves as a signalling molecule that regulates actinorhodin (ACT) production via the DevS/DevR NO-dependent two-component system (TCS) in Streptomyces coelicolor A3(2) M145. However, the mechanisms involved in the regulation of NO signalling in S. coelicolor have not yet been elucidated. Mycothiol (MSH), a thiol molecule produced by Actinomyces, is involved in the defence mechanisms against oxidative stress. Therefore, this study focused on the correlation between intracellular NO and MSH levels.ResultsTo investigate the interaction of MSH with endogenously produced NO, we generated an S. coelicolor A3(2) strain deficient in MSH biosynthesis. This mutant strain exhibited a decrease in low-molecular-weight S-nitrosothiols and intracellular NO levels during culture compared to those of the wild-type strain. Moreover, the mutant strain exhibited reduced activity of the DevS/DevR TCS, a regulator of NO homeostasis and ACT production, from the early stage of culture, along with a decrease in ACT production compared to those of the wild-type strain.ConclusionsThis study suggests that MSH maintains intracellular NO homeostasis by forming S-nitrosomycothiol, which induces NO signalling. Finally, we propose a metabolic model in which MSH from endogenously produced NO facilitates the maintenance of both NO homeostasis and signalling in S. coelicolor A3(2) M145.

IL-17A is involved in the hyperplasia of blood vessels in local lesions of psoriasis by inhibiting autophagy.

Increased angiogenesis is a pathological feature of psoriasis, but the pathomechanisms of angiogenesis in psoriasis are not clear. Interleukin-17A (IL-17A) is the major effect factor in the pathogenesis of psoriasis. Our results showed that IL-17A can promote angiogenesis and cause endothelial cell inflammation. Autophagy plays an important role not only in regulating inflammation, but also in regulating angiogenesis. Whether angiogenesis in psoriasis is related to autophagy remains unclear. In this study, we treated human umbilical vein endothelial cells (HUVECs) with IL-17A to simulate increased angiogenesis to study whether increased angiogenesis in psoriasis is related to autophagy. Our results showed that treatment of HUVECs with IL-17A significantly increased angiogenesis and expression levels of mRNA for multiple proinflammatory cytokines (CCL20, IL-8, CCL2, IL-6, and IL-1β) and, while decreasing intracellular levels of nitric oxide (NO) and NO synthase (NOS) activity. Moreover, IL-17A inhibited autophagy as shown that IL-17A significantly increased expression levels of LC3II and p62 proteins. Induction of autophagy ameliorated IL-17A-mediated inflammatory response and inhibited angiogenesis, accompanied by increased p-AMPKα(Thr172) and p-ULK1(Ser555) expression, and decreased p-mTOR(Ser2448) and p-ULK1(Ser757) expression. Furthermore, inhibition of either AMPK or lysosomal acidification completely overrode autophagy-induced changes in angiogenesis and NOS activity. Finally, induction of autophagy decreased apoptosis and caspase-3 activity in IL-17A-treated HUVECs. These results showed that IL-17A is involved in angiogenesis and inflammatory response by inhibiting autophagy through AMPK signaling pathway, suggesting that autophagy may be a new therapeutic target for psoriasis.

Effects of elevated levels of intracellular nitric oxide on Pseudomonas aeruginosa biofilm in chronic skin wound and slow-killing infection models.

Nitric oxide (NO), produced through the denitrification pathway, regulates biofilm dynamics through the quorum sensing system in Pseudomonas aeruginosa. NO stimulates P. aeruginosa biofilm dispersal by enhancing phosphodiesterase activity to decrease cyclic di-GMP levels. In a chronic skin wound model containing a mature biofilm, the gene expression of nirS, encoding nitrite reductase to produce NO, was low, leading to reduced intracellular NO levels. Although low-dose NO induces biofilm dispersion, it is unknown whether it influences the formation of P. aeruginosa biofilms in chronic skin wounds. In this study, a P. aeruginosa PAO1 strain with overexpressed nirS was established to investigate NO effects on P. aeruginosa biofilm formation in an ex vivo chronic skin wound model and unravel the underlying molecular mechanisms. Elevated intracellular NO levels altered the biofilm structure in the wound model by inhibiting the expression of quorum sensing-related genes, which was different from an in vitro model. In Caenorhabditis elegans as a slow-killing infection model, elevated intracellular NO levels increased worms' lifespan by 18%. Worms that fed on the nirS-overexpressed PAO1 strain for 4h had complete tissue, whereas worms that fed on empty plasmid-containing PAO1 had biofilms on their body, causing severe damage to the head and tail. Thus, elevated intracellular NO levels can inhibit P. aeruginosa biofilm growth in chronic skin wounds and reduce pathogenicity to the host. Targeting NO is a potential approach to control biofilm growth in chronic skin wounds wherein P. aeruginosa biofilms are a persistent problem.

Capsular polysaccharide mediates Streptococcus agalactiae to resist Nile tilapia macrophage phagocytosis

Streptococcus agalactiae (S. agalactiae) is a highly pathogenic gram-positive bacterium for farmed tilapia. The surface capsular polysaccharide (cps) containing terminal sialic acid (sia) residues is a crucial virulence factor for S. agalactiae. However, there is limited knowledge regarding the effects of cps and sia on the phagocytosis of S. agalactiae by teleost macrophages (MФ). In this study, we investigated the role of cps and sia in tilapia MФ phagocytosis of S. agalactiae using cps-deficient mutant ∆cps and sia-deficient mutant ∆neuA. Nonopsonic phagocytosis of wild type (WT), ∆cps and ∆neuA was initially analyzed by flow cytometry. The results indicated that MФ phagocytosis of ∆cps and ∆neuA was significantly higher than that of WT. The proportion of MФ with higher intracellular reactive oxygen species (ROS) or nitric oxide (NO) levels, and overall mean fluorescence intensity (MFI) of the ROS or NO in MФ increased higher during nonopsonic phagocytosis in the Δcps and ΔneuA. The expression levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) during S. agalactiae infection revealed that S. agalactiae could dampen the inflammatory response of MФ through the cps and sia. Increased susceptibility of the ∆cps and ∆neuA to phagocytic killing indicated that S. agalactiae could impair the phagocytic clearance of MФ through the cps or sia. Furthermore, opsonic phagocytosis of the ∆cps and ∆neuA by MФ was dramatically enhanced compared to the WT under complement or antibody mediation. Moreover, relative to ∆cps and ∆neuA, nonopsonic and opsonic phagocytic receptors were significantly down-regulated during MФ phagocytosis of WT, indicating that S. agalactiae might resist phagocytosis of MФ by directly or indirectly inhibiting the expression of phagocytic receptors through the cps or sia. Our data suggest that cps plays a crucial role in the interference of S. agalactiae on the phagocytosis of tilapia MФ, and sia may be a significant immune escape site. These results provide valuable insights into the mechanism of S. agalactiae attempting to escape the defenses of teleost innate immune system and offer clues for S. agalactiae vaccine development.

Salicylic Acid and Jasmonic Acid Increase the Polysaccharide Production of Nostoc flagelliforme via the Regulation of the Intracellular NO Level

To significantly improve the polysaccharide production of Nostoc flagelliforme, a total of 12 chemicals were evaluated for their effects on polysaccharide accumulation. The results showed that salicylic acid and jasmonic acid increased the accumulation of the polysaccharides in N. flagelliforme significantly, by more than 20%. Three polysaccharides, namely control-capsule polysaccharide, salicylic acid-capsule polysaccharide, and jasmonic acid-capsule polysaccharide, were extracted and purified from N. flagelliforme under normal, salicylic acid, and jasmonic acid culture conditions, respectively. Their chemical compositions slightly differed regarding the total sugar and uronic acid contents, with average molecular weights of 2.06 × 103, 2.16 × 103 and 2.04 × 103 kDa, respectively. They presented similar Fourier transform infrared spectra and no significant difference in antioxidant activity. It was revealed that the salicylic acid and jasmonic acid significantly increased the level of nitric oxide. By investigating the effects of the exogenous nitric oxide scavenger and nitric oxide donor on the nitric oxide levels and polysaccharide yield of N. flagelliforme, the results showed that the increase in intracellular nitric oxide levels might be an important factor promoting the accumulation of polysaccharides. These findings provide a theoretical foundation for enhancing the yield of secondary metabolites by regulating the intracellular nitric oxide levels.

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels.

Nitric oxide (NO) plays an important role in the regulation of the immune, cardiovascular and nervous systems. Consequently, being able to monitor and quantify intracellular NO levels would provide a greater understanding of the implications of this molecule in the different biological processes, including, for example, in cancer. Here, we report a broadly applicable two-photon excitable fluorescent nanoprobe able to detect and potentially quantify NO levels in an extensive range of cellular environments. The nanoprobe consists of a thiolated photoinduced electron transfer-based two=photon fluorescent probe attached onto the surface of 2.4 ± 0.7 nm gold nanoparticles (DANPY-NO@AuNPs). The nanoprobe, which can be synthesised in a reproducible manner and exhibits great stability when stored at room temperature, is able to selectively detect NO in solution, with a dynamic range up to 150 μM, and at pH values of biological relevance. DANPY-NO@AuNPs were able to selectively detect endogenous NO in RAW264.7γ NO- macrophages and THP-1 human leukemic cells; and endogenous and exogenous NO in endothelial cells. The nanoprobe accumulated in the acidic organelles of the tested cell lines showing negligible toxicity. Importantly, DANPY-NO@AuNPs showed potential to quantify intracellular NO concentrations in MDA-MB-231 breast cancer cells. The biological evaluation of the nanoprobe was undertaken using confocal laser scanning (images and intracellular emission spectra) and multiphoton microscopies, and flow cytometry. Based on their excellent sensitivity and stability, and outstanding versatility, DANPY-NO@AuNPs can be applied for the spatiotemporal monitoring of in vitro and in vivo NO levels.

Berberine hydrochloride inhibits migration ability via increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells

Ethnopharmacological relevanceInadequate trophoblasts migration and invasion is considered as an initial event resulting in preeclampsia, which is closely related to oxidative stress. Berberine hydrochloride (BBR), extracted from the traditional medicinal plant Coptis chinensis Franch., exerts a diversity of pharmacological effects, and the crude drug has been widely taken by most Chinese women to treat nausea and vomit during pregnancy. But there is no research regarding its effects on trophoblast cell function. Aim of the studyThis study aimed to investigate the effect of BBR on human-trophoblast-derived cell line (HTR-8/SVneo) migration ability and its mechanism. Materials and methodsCell viability was detected by CCK-8 assay. The effect of BBR on cells migration function was examined by scratch wound healing assay and transwell migration assay. Intracellular nitric oxide (NO), superoxide (O2−) and peroxynitrite (ONOO−) levels were measured by flow cytometry. The expression levels of inducible NO synthase (iNOS), eNOS, p-eNOS, MnSOD, CuZnSOD, Rac1, NOX1, TLR4, nuclear factor-κB (NF-κB), p-NFκB, pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in cells were analyzed by Western blotting. Uric acid sodium salt (UA), the scavenger of ONOO−, PEG-SOD (a specific superoxide scavenger), L-NAME (a NOS inhibitor) and antioxidants (Vit E and DFO) were further used to characterize the pathway of BBR action. Results5 μM BBR decreased both the migration distance and the number of migrated cells without affecting cells viability in HTR-8/SVneo cells after 24 h treatment. BBR could increase the level of NO in HTR-8/SVneo cells, and the over-production of NO might be attributable to iNOS, but not eNOS. BBR could increase intracellular O2− levels, and the over-production of O2− is closely related with Rac1 in HTR-8/SVneo cells. The excessive production of NO and O2− further react to form ONOO−, and the increased ONOO− level induced by BBR was blunted by UA. Moreover, UA improved the impaired migration function caused by BBR in HTR-8/SVneo cells. The depressed migration function stimulated by BBR in HTR-8/SVneo cells was diminished by PEG-SOD and L-NAME. Furthermore, BBR increased the expression of IL-6 in HTR-8/SVneo cells, and antioxidants (Vit E and DFO) could decrease the expression of IL-6 and iNOS induced by BBR. ConclusionsBBR inhibits the cell migration ability through increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells, indicating that BBR and traditional Chinese medicines containing a high proportion of BBR should be used with caution in pregnant women.

Development of a C6 Glioma Cell Model System to Assess Effects of Cathodic Passively Balanced Electrical Stimulation on Responses to Neurotransmitters: Implications for Modulation of Intracellular Nitric Oxide, Chloride, and Calcium Ions.

This research focused on the development of an astrocyte cell model system (C6 glioma) for the assessment of molecular changes in response to cathodic passively balanced pulsed electrical stimulation at a rate of 50 Hz (60 µs duration, 0.15 mA intensity). Cells treated with selected neurotransmitters (glutamate, adenosine, D-serine, and γ-aminobutyric acid) were monitored (using specific fluorescent probes) for changes in levels of intracellular nitric oxide, calcium ions, and/or chloride. ES exerted an inhibitory effect on NO, increased calcium and had no effect on chloride. Using this model, cells can be assessed qualitatively and quantitatively for changes and these changes can be correlated with the putative molecular effects that electrical stimulation has on astrocytes and their role in glia-mediated diseases. This model system allows for faster and cheaper experiments than those involving animal models due to the potential to easily vary the conditions, reduce the number of variables (especially problematic in animal models), and closely monitor the cellular effects.

Markers of Endothelial Dysfunction Are Attenuated by Resveratrol in Preeclampsia.

Preeclampsia (PE) is characterized by great endothelial dysfunction, decreased nitric oxide (NO) bioavailability, and higher levels of arginase activity. In the present study, we investigated the potential modulatory effects of trans-resveratrol (RSV) on arginase and endothelial dysfunction biomarkers in endothelial cells exposed to plasma from patients with PE and healthy pregnant (HP) women, and umbilical arteries from patients with PE. Human umbilical vein endothelial cells (HUVECs) were incubated with pooled plasma from 10 HP or 10 PE pregnant women and RSV; umbilical arteries from patients with PE were incubated with RSV; intracellular NO and total reactive oxygen species (ROS) levels were assessed using a probe that interacted with these radicals; total arginase activity was evaluated measuring the urea produced; total antioxidant capacity was measured using the ferric reduction ability power (FRAP) assay; and endothelial dysfunction biomarkers were assessed using qPCR in endothelial cells and umbilical arteries. RSV increased NO levels and decreased total arginase activity in endothelial cells incubated with plasma from patients with PE. In addition, RSV increased total antioxidant capacity and downregulated endothelial dysfunction biomarkers, such as intercellular adhesion molecule-1 (ICAM-1), von Willebrand factor (vWF), and Caspase-3, (CASP-3), in endothelial cells and umbilical arteries from PE patients. RSV treatment positively modulated the L-arginine-NO pathway, decreased arginase activity, and increased antioxidant capacity, in addition to downregulating endothelial dysfunction biomarkers.

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

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

N-isopropylbenzylamine, a methamphetamine mimics, produces toxicity via increasing nitric oxide in vitro

N-isopropylbenzylamine, an isomer of methamphetamine, has been used to adulterate methamphetamine, and distributed as fake “Ice” methamphetamine by illicit manufacturers, leading to a world problem of N-isopropylbenzylamine exposure. Though it is unclear whether N-isopropylbenzylamine has addictive potential like methamphetamine, N-isopropylbenzylamine users reported side effects such as headaches and confusion. However, the pharmacological targets and cytotoxicity of this chemical remained unknown. In this study, in vitro toxicity of N-isopropylbenzylamine and its toxicity-related targets were investigated in SN4741, SH-SY5Y or PC12 cell lines that model neurons. The cell viability was analyzed by using MTT assay after incubation with N-isopropylbenzylamine for 24 h in cells. N-isopropylbenzylamine caused cell death with IC50 values at around 1–3 mM in these cell lines. N-isopropylbenzylamine time- and concentration-dependently facilitated the expression of neuronal nitric oxide synthase (nNOS), and increased intracellular nitric oxide (NO) in SN4741 cells. Furthermore, 7-nitroindazole, a specific inhibitor of nNOS, significantly prevented N-isopropylbenzylamine-induced toxicity in vitro. These results suggested that N-isopropylbenzylamine-induced toxicity is at least partially related to the increased intracellular NO levels and the activated nNOS. Considering the circumstances that N-isopropylbenzylamine was used to adulterate and mimic methamphetamine, and the side effects associated with N-isopropylbenzylamine in abusers, our findings sounded an alarm for abuser and warn the dangerousness of N-isopropylbenzylamine for public health.