NOS Inhibitor Research Articles

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.

Nitric oxide promotes cerebral ischemia/reperfusion injury through upregulating hypoxia-inducible factor1-α-associated inflammation and apoptosis in rats

Nitric oxide (NO) was one of the key factors to sustain hypoxia-inducible factor-1- α (HIF-1α) activation during hypoxia. However, the mechanism by which NO production promotes upregulation of HIF-1α to cause cerebral ischemia/reperfusion (I/R) injury remains unclear. The present study investigated whether eliminating NO would decrease HIF-1α level, and then reduce the subsequent inflammatory actions as well as neuronal apoptotic death in middle cerebral artery occlusion (MCAO) rats. Our results revealed that HIF-1α was correlated with 3-NT, a marker for nitrosative/oxidative stress, in the brain of MCAO rats. Treatment with NOS inhibitor L-NAME suppressed HIF-1α/3-NT double-positive cells, suggesting that HIF-1α was correlated with NO overproduction during cerebral I/R. Furthermore, pro-inflammatory cytokines TNF-α, IL-1β and NF-κB p65 were significantly increased and colocalized with HIF-1α in the brain of MCAO rats, all of which could be attenuated by NO inhibition, suggesting that eliminating NO reduced MCAO-induced HIF-1α upregulation, which in turn exerted anti-inflammatory actions. Accordingly, cleaved caspase-3, as well as HIF-1α and TUNEL double-positive cells in ischemic brain were also decreased by L-NAME treatment. These results suggest that NO accumulation after cerebral ischemia leads to HIF-1α upregulation, which may activate pro-inflammatory cytokines, resulting in neuronal apoptotic death. These findings demonstrate a novel mechanism of NO-induced cerebral I/R injury.

Impact of Growing Location on Kakadu Plum Fruit Composition and In Vitro Bioactivity as Determinants of Its Nutraceutical Potential

Growing location is known to affect the metabolite content and functionality of wild harvested fruits. Terminalia ferdinandiana, commonly known as Kakadu plum (KP), is among the most commercially important native Australian bush foods. Therefore, we evaluated the composition and in vitro bioactivity of aqueous acidified ethanol (AAE) and water extracts prepared from KP fruit wild harvested in the Northern Territory (NT) and Western Australia (WA). Compositional analysis included vitamin C, total ellagic acid (TEA), and total phenolic content (TPC), while in vitro bioactivity was assessed through anti-inflammatory (RAW 264.7 macrophages) activity and cell viability (Hep G2) assay. The IC50 of the extracts ranged from 33.3 to 166.3 µg/mL for NO inhibition and CC50 from 1676 to 7337 µg/mL for Hep G2 cell viability inhibition. The AAE KP fruit extracts from the NT exhibited potent anti-inflammatory activity and impacted Hep G2 cell viability more than other extracts, most likely due to TEA (3189 mg/100 g dry weight (DW)), vitamin C (180.5 mg/g DW) and TPC (196 mg GAE/g DW) being higher than in any other extract. Overall, the findings of the present study are promising for using KP fruit and derived products in functional foods, nutraceuticals, or dietary supplements.

Anti-Inflammatory Activity of Velvet Bean (Mucuna pruriens) Substances in LPS−Stimulated RAW 264.7 Macrophages

In this study, Mucuna pruriens extracts were used to verify their application as a natural-based raw material with anti-inflammatory function. A nitric oxide inhibition activity assay showed that M. pruriens extracted with hot water (MW), M. pruriens extracted with 70% ethanol (ME), and M. pruriens extracted with 70% acetone (MA) presented NO inhibition activity; among them, MW and ME demonstrated the best activity and were selected for Western blot analysis. After identifying the expression patterns of inflammation-related proteins, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB), through Western blots, both MW and ME showed inhibition patterns. As a result of analyzing L-DOPA contained in M. pruriens through ultra-performance liquid chromatography (UPLC), high L-DOPA content was detected in MW, ME, and MA. Therefore, it can be concluded that M. pruriens extracts have the potential for use as an anti-inflammatory material.

Seco- and isopimarane diterpenoids from Kaempferia marginata rhizomes and their NO inhibition activities

Three undescribed 9,10-seco-isopimarane diterpenoids, marginols I–K, and an unprecedent isopimara-8(9),15-diene diterpene, 14-epi-boesenberol F, together with a known 9,10-seco-isopimarane diterpenoid, kaemgalangol A, were isolated from the rhizomes of Vietnamese Kaempferia marginata. Marginols I and J contained a naturally very rare 6-oxabicyclo[3.2.1]octane-5-ol ring, while marginol K had a naturally rare oxepan-2-one ring in its structure. The unprecedented structures were elucidated by spectroscopic techniques, including HR-ESI-TOF-MS, UV, IR, and 1D and 2D NMR. The absolute configurations of marginols I–K and 14-epi-boesenberol F were determined by ECD calculations. The NO production inhibitory assay revealed that the isolated compounds, except marginol J, exhibited NO inhibitory activities with IC50 values ranging from 65.06 to 87.70 μM against lipopolysaccharide (LPS)-induced RAW264.7 cells.

Asymmetric dimethylarginine accumulation under hyperglycemia facilitates β-cell apoptosis via inhibiting nitric oxide production

Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) has been shown to suppress apoptosis in pancreatic β-cells. In the present study, the influence of asymmetric dimethylarginine (ADMA), the major endogenous inhibitor of NOS, on the apoptosis-suppressive effect of NO was investigated. The expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2), an ADMA-metabolizing enzyme, in INS-1 β-cells and in mouse pancreatic islets was drastically reduced by in vitro exposure to high-concentration glucose (20 mM) and by in vivo treatment of mice with the insulin receptor blocker S661, which resulted in hyperglycemia, respectively. In line with this, a higher ADMA level was observed in INS-1 cells exposed to 20 mM glucose. The treatment of INS-1 cells with ADMA, similarly to with the NOS inhibitor NG-nitro-L-arginine methyl ester, significantly facilitated 20 mM glucose-induced increase in cleaved caspase-3 protein expression. Furthermore, increased protein expression of cleaved caspase-3 and CHOP was observed in INS-1 cells with knockdown of DDAH2. These results suggest that ADMA accumulation through a decrease in DDAH2 expression in β-cells, which is induced under hyperglycemic conditions, facilitates β-cell apoptosis through suppression of cNOS-mediated NO production.

Abstract 14011: Rare Variants in GUCY1A3 Gene Contribute to Hypertension via NO/cGMP Pathway

Hypertension is a leading risk factor for cardiovascular events and considered as a complex genetic disease. Previous studies have revealed many common variants relevant to hypertension. However, the contributions of rare variants and the underlying mechanisms are still poorly understood. Through a case-control study with whole-exome sequencing, we found that rare nonsynonymous variants in GUCY1A3 gene were enriched in the patients with early-onset hypertension. To assess the effects of rare variants of GUCY1A3 , a knock-in mouse model carrying a variant corresponding to Pro637His in human GUCY1A3 was generated, and the blood pressure (BP) was measured by telemetry.The baseline systolic BP of both homozygous (P637H +/+ ) and heterozygous (P637H + )mice were higher than their wide-type (WT) littermate controls. After administration of eNOS inhibitor, BP elevated in all genotypes, and the P637H +/+ and P637H + mice had profoundly higher BP than WT. The vascular dysfunction in mutated mice was determined as contraction curves of aorta ring elevated earlier and higher, and relaxation curves decreased later than WT rings. Furthermore, compared to WT mice, the protein levels of sGCα1 and sGC in both P637H +/+ and P637H + mice were decreased both at baseline and after NO inhibitor treatment, and the cGMP levels were also significantly decreased. Consistenly, the activity of cGMP-dependent protein kinase1 (cGK-1) was supressed and the phosphorylation of its downstream substrate, myosin light chain, was increased in knock-in mice. Finally, the inhibitory effect on sGC proteins expression and NO/cGMP pathway activity were also observed in vitro on other 5 rare nonsynonymous variants of GUCY1A3 . In conclusion, our results demonstrate that GUCY1A3 defects contribute to hypertension by reducing the activity of NO/cGMP pathway, and support that rare varaints are important components of the genetic architecture of hypertension.

Abstract 14718: Using Renal-Specific Cyclo-Oxygenase-2-Deficient Mice to Understand the Pro-Thrombotic Effects of Non-Steroidal Anti-Inflammatory Drugs

Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) work by inhibiting the enzyme cyclooxygenase (COX)2 but their use increases thrombotic risk. We have shown that COX2 inhibition increases plasma methylarginines ADMA and LNMMA in mice and man. Methylarginines are controlled in the kidney but circulate causing inhibition of anti-thrombotic vascular NO by competing with L-arginine for the active site of eNOS. However, the causative links between renal COX2 (which is expressed predominately in fibroblasts), methylarginines and thrombosis remain unproven. Hypothesis: Here we hypothesised that deletion of COX2 from fibroblasts would result in a renal-specific COX2-deficiency and be sufficient to increase methylarginine levels and drive vascular NO dysfunction and a pro-thrombotic phenotype. Methods: Fibroblast COX2-deficient mice were generated from Fsp1-Cre and Ptgs2 flox strains. COX2 levels were measured by RT-qPCR, methylarginines by LC/MS/MS, vascular NO function as acetylcholine-induced dilation by myography and thrombosis in vivo after FeCl 3 -carotid injury. Results: Fibroblast COX2-deficient mice showed reduced COX2 expression in the renal medulla (Fig A) but not renal cortex, aorta, lung, colon, thymus or brain. This was associated with increased plasma ADMA and LNMMA levels (Fig B) and reduced NO-dependent dilatation of carotid arteries (Fig C). Fibroblast COX2-deficiency accelerated thrombosis after carotid artery injury which could be reversed by pre-treating animals with L-arginine (50mg/kg; 5 days) which displaces methylarginines from eNOS (Fig D). Conclusions: These data provide the first direct demonstration that renal COX2 regulates methylarginine levels to control systemic endothelial and thrombotic function. As such, they indicate that inhibition of renal COX2 contributes to the thrombotic risk seen in patients taking NSAIDs and suggest L-arginine supplementation as a potential rescue therapy to mitigate this.

Prediction and minimization of NOx emission in a circulating fluidized bed combustor: Improvement of bed quality by optimizing cyclone performance and coal particle size

• Improvement of cyclone performance contributes to reducing the NO x emission. • Reducing coal particle size can decrease the NO x emission. • NO x emission may be further reduced by reducing the height of the coal inlet. • Effects of cyclone efficiency and coal size on combustion efficiency are discussed. • A comprehensive CFB mathematical model is applied to further analyze the mechanism. The NO x emission of circulating fluidized bed (CFB) boilers can be evidently reduced via optimization of the cyclone performance and the coal particle size, which has been proved in industrial practices, while there are few reasonable interpretations for these phenomena so far. In this paper, a comprehensive 1-dimensional/1.5-dimensional CFB mathematical model was applied to investigate the effect of these two important factors on NO x emission. Simulation results on two commercial CFB boilers indicate that increasing the cyclone efficiency and reducing the coal particle size can significantly decrease the average size of bed materials and increase the solid suspension density in the freeboard zone; namely, the bed quality is improved. The local gas–solid flow behaviors inside the CFB furnace will be accordingly changed, such as the increasing volume fraction of bubble phase in dense bed, shortening of secondary air penetration depth, enrichment of particles in the downward-flow annulus or clusters, etc. Consequently, the reducing atmosphere will be formed or enhanced in local areas, leading to the effective inhibition of NO x formation. Meanwhile, the increase in the bed inventory of fine char particles also facilitates the reduction of NO x . Moreover, if appropriately reducing the height of the coal inlet or feeding fine coal particles from the solid recycle system, the NO x emission may be further reduced due to the adjustment of the volatiles release pattern. In addition, the reduction of coal particle size can contribute to the increase in fuel combustion efficiency, while the unburned carbon content in fly/bottom ash may increase or decrease with the improvement of cyclone efficiency. This study further analyzes the influence of gas–solid flow characteristics on the chemical reaction process and provides a new solution to the low-cost pollutant emission control for CFB boilers.

Semisynthesis and Evaluation of Anti-Inflammatory Activity of the Cassane-Type Diterpenoid Taepeenin F and of Some Synthetic Intermediates.

A new strategy for the semisynthesis of the aromatic cassane-type diterpene taepeenin F (6) is reported. The introduction of the methyl group at C-14, characteristic of the target compound, was achieved via dienone 13, easily prepared from abietic acid (10), the major compound in renewable rosin. Biological assays of selected compounds are reported. The antiproliferative activity against HT29, B16-F10, and HepG2 tumor cell lines has been investigated. Salicylaldehyde 21 was the most active compound (IC50 = 7.72 μM). Products 16 and 21 displayed apoptotic effects in B16-F10 cells, with total apoptosis rates of 46 and 38.4%, respectively. This apoptotic process involves a significant arrest of the B16-F10 cell cycle, blocking the G0/G1 phase. Dienone 16 did not cause any loss of the mitochondrial membrane potential (MMP), while salicylaldehyde 21 caused a partial loss of the MMP. The anti-inflammatory activity of the selected compounds was investigated with the LPS-stimulated RAW 264.7 macrophages. All compounds showed potent NO inhibition, with percentages between 80 and 99% at subcytotoxic concentrations. Dienone 16 inhibited LPS-induced differentiation of RAW 264.7 cells, by increasing the proportion of cells in the S phase. In addition, salicylaldehyde 21 had effects on the cell cycle, recovering the cells from the G0/G1 full arrest produced in response to LPS action.

Human epididymis protein 4 indicates progression of cardiac fibrosis in Heart Failure with preserved Ejection Fraction

Abstract Background Cardiac fibrosis is one of the main causes of diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Human epididymis protein 4 (HE4) is a novel pro-fibrotic protein expressed especially in activated fibroblast, so-call myofibroblast [1]. Although our previous study showed that HE4 functioned as a secretory factor promoting cardiac fibrosis in dilated cardiomyopathy [2], little is known about the role of HE4 in HFpEF. Purpose The aim of this study is to evaluate the pathophysiological role of HE4 in HFpEF and the association between serum HE4 levels and clinical course of HFpEF. Methods In basic research, we administered high-fat diet and NOS inhibitor (L-NAME) to mice for 5 weeks to induce HFpEF. We analyzed echocardiographic findings and mRNA expressions of fibrosis-related genes including HE4 by real time PCR. In clinical research, we measured serum HE4 levels of 79 patients with HFpEF and divided these patients into low and high HE4 group using median values of HE4 (102.0 pmol/L). Then, we evaluated their mortality and cardiovascular events retrospectively. Results In vivo, the administration of high fat diet and L-NAME induced significant increase in left ventricular mass on echocardiography compared with control group (p=0.006). The mRNA expressions of aSMA and collagen3a1 in heart tissue was upregulated in HFpEF model mice (p=0.008 and p=0.003, respectively). Furthermore, the mRNA expression of HE4 was also upregulated in HFpEF model mice (n=6) than in control mice (n=5), although these differences did not reach statistical significance (p=0.238). In vitro, recombinant HE4 upregulated mRNA expression of pro-fibrotic genes, such as periostin (p=0.001), collagen3a1 (p=0.037), aSMA (p=0.095), and PAI-1 (p=0.17), indicating HE4 facilitate the activation of fibroblast and cardiac fibrosis although some did not reach statistical significance. In clinical research, Kaplan-Meier curve revealed that the rate of all cause death and rehospitalization for worsening heart failure were significantly higher in the high than the low HE4 group, suggesting HE4 can predicts prognosis in patients with HFpEF. Conclusion HE4 induces the activation of cardiac fibroblasts and is upregulated in the heart of HFpEF model mice. Furthermore, it is a useful biomarker for predicting cardiovascular events in patients with HFpEF. Funding Acknowledgement Type of funding sources: None.

Preclinical studies of NOS inhibitor T1059 vasopressor activity on the models of acute hemorrhagic shock in rats and dogs.

The development of new effective and safe vasopressors is one of the ways to increase the effectiveness of the treatment of hypotensive disorders, the severe forms of which remain a common cause of death in all countries of the world. Previously, we synthesized the original compound T1059, a selective inhibitor of eNOS/iNOS which has a pronounced vasoconstrictive effect. Here we show its vasopressor activity in models of the early stage of acute hemorrhagic shock in rats and dogs, as part of preclinical studies. The results indicate NOS inhibitor T1059 as a potent long-acting vasopressor. Its single parenteral administration in sufficiently safe doses (1/50–1/9 LD10), caused in rats and dogs a rapid increase in vascular tone, accompanied by a prolonged hypertensive effect (within 90–120 min in rats, and within 115 min in dogs). The repeated administration of T1059 at low doses (1/3 of the first dose) made it possible to considerably (by at least 60 min) prolong a significant vasopressor effect. In all schemes, T1059 administration considerably inhibited the development of threatening cardiorespiratory disorders and significantly (p = 0.0026–0.0098) increased the short-term survival of experimental animals, formally extending the duration of the “golden hour” by 2 times. These data indicate that NOS inhibitors and, in particular, compound T1059, are able to create new opportunities in the treatment of hypotensive disorders, including the provision of assistance at the prehospital stage of treatment of such pathologies.

The effect of montelukast, a leukotriene receptor antagonist, on the acetic acid-induced model of colitis in rats: Involvement of NO-cGMP-KATP channels pathway.

Inflammatory bowel disease is a chronic autoimmune disorder that may involve entire gastrointestinal tract. The leukotrienes have a role as mediators in the pathophysiology of colitis. Here, we investigated the effect of a leukotriene receptor antagonist, montelukast, and also the role of the NO-cGMP-KATP channel pathway in acetic acid-induced colitis. Rectal administration of acetic acid (4%) was used for induction of colitis in rats. To investigate our hypothesis, the rats were intraperitoneally pre-treated with L-NAME (NOS inhibitor), L-arginine, sildenafil, methylene blue, glibenclamide, or diazoxide 15 min before treatment with montelukast (5–20 mg/kg, i. p.), for three consecutive days. Then, microscopic, macroscopic, and inflammatory parameters were evaluated. Montelukast reduced the microscopic and macroscopic damage induced by acetic acid. Montelukast also reduced the level of IL-1β and TNF-α. We also showed that the effects of montelukast were significantly attenuated by L-NAME, methylene blue (guanylate cyclase inhibitor), and an ATP-sensitive potassium channel blocker (glibenclamide). Also, the administration of L-arginine, sildenafil, and diazoxide before montelukast produced protective effect. In conclusion, the pathway of the NO-cGMP-KATP channel is involved in the protective effect of montelukast in acetic acid-induced colonic tissue damage.

Dibutyl phthalate promotes angiogenesis in EA.hy926 cells through estrogen receptor-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways

Dibutyl phthalate (DBP) is an endocrine disruptor that has been widely used in various products of human use. DBP exposure has been associated with reproductive and cardiovascular diseases and metabolic disorders. Although dysfunction of the vascular endothelium is responsible for many cardiovascular and metabolic diseases, little is known about the effects of DBP on human endothelium. In this study, we investigated the effect of three concentrations of DBP (10−6, 10−5, and 10−4 M) on angiogenesis in human endothelial cell (EC) line EA.hy926 after acute exposure. Tube formation assay was used to investigate in vitro angiogenesis, whereas qRT-PCR was employed to measure mRNA expression. The effect of DBP on extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt), and endothelial nitric oxide (NO) synthase (eNOS) activation was examined using Western blotting, whereas the Griess method was used to assess NO production. Results show that the 24-h-long exposure to 10−4 M DBP increased endothelial tube formation, which was prevented by addition of U0126 (ERK1/2 inhibitor), wortmannin (PI3K-Akt inhibitor), and l-NAME (NOS inhibitor). Short exposure to 10−4 M DBP (from 15 to 120 min) phosphorylated ERK1/2, Akt, and eNOS in different time points and increased NO production after 24 and 48 h of exposure. Application of nuclear estrogen receptor (ER) and G protein-coupled ER (GPER) inhibitors ICI 182,780 and G-15, respectively, abolished the DBP-mediated ERK1/2, Akt, and eNOS phosphorylation and increase in NO production. In this study, we report for the first time that DBP exerts a pro-angiogenic effect on human vascular ECs and describe the molecular mechanism involving ER- and GPER-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways.

Diverse ent-kaurane diterpenoids from Isodon henryi

Phytochemical investigation of Isodon henryi led to the isolation of two new diterpenoids, isohenolides A-B (1–2), one new natural diterpenoid (3) and thirteen known ones (4–16). These diterpenoids showed various ent-kaurane skeletons, in which isohenolide A (1) was a rare 6,8‑cyclo ent-kaurane containing an unprecedented 11,15-lactone group, and possessed a 6/5/6/6/6/6 ring system. Their structures were confirmed by comprehensive UV, IR, HRESIMS, NMR analyses and ECD calculations. Most compounds were also screened for anti-inflammatory actions on LPS-induced RAW 264.7 cells, and compounds 8 and 16 exhibited stronger NO inhibition.

Excessive Zinc Ion Caused PC12 Cell Death Correlating with Inhibition of NOS and Increase of RAGE in Cells.

Zinc ion (Zn2+) is an important functional factor; however, excessive Zn2+ can be toxic. To understand the neurotoxicity of excessive Zn2+ and the underlying mechanism, PC12 cells were treated with excessive Zn2+ and Zn2+ plus N, N, N', N'-Tetrakisethylenediamine (TPEN), a zinc ion chelator agent. Trypan blue and 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, thiazolyl blue tetrazolium bromide (MTT) assays were used to test cell viability; the relative kits were used to detect the activity of NOS synthase and the content of the receptor for advanced glycation end product (RAGE) in cells. We observed that excessive zinc caused PC12 cell damage and that TPEN partially reversed cell damage caused by excessive zinc. In addition, excessive zinc decreased total nitric oxide synthase (TNOS) activity in cells, in which constitutive nitric oxide synthase (cNOS) activity was significantly reduced; however, inducible nitric oxide synthase (iNOS) activity was extremely promoted. Moreover, excessive zinc upregulated the expression of RAGE, and TPEN effectively reversed the increase in RAGE induced by excessive zinc ions. Therefore, we concluded that excessive zinc caused PC12 cell damage, correlating with the inhibition of NOS and increase of RAGE induced in cells.

Sesquiterpene lactones from Artemisia vulgaris L. as potential NO inhibitors in LPS-induced RAW264.7 macrophage cells.

Twelve new guaianolide sesquiterpene lactones (1–12), along with ten known analogs (13–22) were isolated from an EtOH extract of the dried aerial parts of Artemisia vulgaris L. The new structures were elucidated via abundant spectroscopic data analyses (HRESIMS, IR, 1D, and 2D NMR), and the absolute configurations of these compounds were determined by X-ray crystallography and ECD calculations. The compounds (1−22) were identified as guaiane-type sesquiterpenes with characteristic α-methylene-γ-lactone and α,β-unsaturated carbonyl moieties. All compounds were tested for their inhibitory activity against NO production in lipopolysaccharide-stimulated RAW264.7 macrophages. The isolated sesquiterpenoids dose-dependently exhibited an NO production inhibitory activity by inhibiting the expression of inducible NO oxidase (iNOS) and cyclooxygenase-2 (COX-2) with IC50 values ranging from 1.0 to 3.6 μM. The inhibitory effect on the NO production of the compounds (1–4 and 6–22) is better than that of the positive control (dexamethasone). The different substitutions of compounds on C-8 influence anti-inflammatory effects, as evidenced by the in silico analysis of related binding interactions of new compounds (1–12) with iNOS.

Design and construction of an O-Au-O coordination environment in Au single atom-doped Ti4+ defected TiO2 for an enhanced oxidative ability of lattice oxygen for Hg0 oxidation

Transition metal oxide (TMO)-decorated TiO2 catalysts excel at low-temperature Hg0 oxidation. However, TMOs are typically found in large nanoparticles (NPs), resulting in a lower atom utilization efficiency. The current paper presents the design of an Au single atom (SA)-doped Ti4+ defected TiO2 (Au SAC-Ti1-xO2) and investigations concerning the influence of an O-Au-O coordination environment on Hg0 oxidation. Au SAC-Ti1-xO2 was designed using an O-Au-O coordination environment and the O-Au-O orbital interaction was elucidated using density functional theory (DFT). The enhanced oxidative capability of lattice oxygen atoms and the interaction between Hg0 and oxygen atoms were investigated. The O-Au-O coordination environment containing Au SAC Ti1-xO2 was then synthesized and characterized using X-ray absorption fine structure (XAFS), X-ray photoelectron spectroscopy (XPS), etc. Finally, Hg0 oxidation catalytic activity was determined. Due to the increasing population of Au-O antibonding orbital interactions in the valence band, the bond order of O-Au-O in Au SAC Ti1-xO2 decreased significantly, thereby enhancing the oxidation ability of its adjacent oxygen atoms. Au SA and ultra-small Au clusters coexisted on a Ti1-xO2 catalyst, and the Hg0-oxidation efficiency at 150–200 °C was 14 times and 1.5 times greater than those of TiO2 and 0.1 % Au/TiO2, respectively. Furthermore, Au SAC Ti1-xO2 was more resistant to the inhibition of SO2 and NO. It provides an efficient method for developing highly active SA catalysts for Hg0 oxidation.

Dopaminergic and nitric oxide systems interact to regulate the electrical activity of neurons in the medial septal nucleus in rats.

Research characterizing the neuronal substrate of anxiety has implicated different brain areas, including the medial septal nucleus (m-SEPT). Previous reports indicated a role of dopamine and nitric oxide (NO) in anxiety-related behaviors. In this study, the extracellular single-unit recording was performed from the m-SEPT in adult male albino Wistar rats. Baseline activity was recorded for 5min, and the post-injection recording was performed for another 5min after the microinjection of each drug. The results showed that (1) both D1- and D2-like receptor agonists (SKF-38393 and quinpirole) enhanced the firing rate of m-SEPT neurons; (2) both D1- and D2-like antagonists (SCH-23390 and sulpiride) attenuated the firing rate of m-SEPT neurons; (3) L-arginine (NO precursor) increased the firing rate of m-SEPT neurons, but a non-specific NOS inhibitor, L-NAME, elicited no significant alterations; (4) the non-specific NOS inhibitor reversed the enhanced firing rate produced by SKF-38393 and quinpirole; (5) neither of the dopaminergic antagonists changed the enhanced activity resulted from the application of the NO precursor. These results contribute to our understanding of the complex neurotransmitter interactions in the m-SEPT and showed that both dopaminergic and NO neurotransmission are involved in the modulation of the firing rate of neurons in the m-SEPT.

L-Arginine increases AMPK phosphorylation and the oxidation of energy substrates in hepatocytes, skeletal muscle cells, and adipocytes.

Previous work has shown that dietary L-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400µM) or 400µM Arg + 0.5mM NG-nitro-L-arginine methyl ester (L-NAME; an NOS inhibitor) for 48h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P < 0.05) the oxidation of glucose and oleic acid to CO2 in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400µM also stimulated (P < 0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P < 0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P < 0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P > 0.05) on these variables inC2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms.