A Novel Fluorescence Probe 9-(4-(1,2-diamine)benzeneN^1-phenyl)acridine for Nitric Oxide Determination

A novel fluorescent probe 9-（4-（1,2-diamine）benzene-N1-phenyl）acridine（DABPA） was synthesized for the detection of nitric oxide（NO） and characterized by IR, 1H-NMR and EI-MS spectroscopy. Based on a photoelectron transfer mechanism, the fl uorescence intensities of DABPA were investigated with the different concentrations of NO. Under the optimal experimental conditions, the fl uorescence intensity of DABPA had a good linear relationship（R2=0.9977） with NO concentration in the range from 1×10-7 to 1.5×10-6 mol/L with a detection limit of 1×10-8 mol/L. The cytotoxicity induced by DABPA was evaluated by the MTT（3-（4,5-dimethylthiazol-2-yl）-2,5diphenyl tetrazolium bromide） assay for biological application. Furthermore, the probe DABPA had also been successfully applied to real-time image NO produced in PC12 cells in the presence of L-arginine.

The effect of nebivolol on the production of nitric oxide induced by bacterial lipopolysaccharide and peptidoglycan in mice

Nitric oxide (NO) plays a pivotal role in main- taining balance of physiological events in many systems including the autonomic, cardiovas- cular, hematological, and pulmonary systems. Lipopolysaccharide (LPS) and peptidoglycan (PGN), components of the outer cell membranes of Gram-negative bacteria and cell walls of Gram-positive bacteria respectively, are in- criminated in NO-induced septic shock. Ne- bivolol is a third generation β1- adrenoceptor blocker with a vasodilatory property attributed to enhanced availability of nitric oxide and re- duction of cellular oxidative stress through an unknown mechanism. The current study ex- plored the hypothesis that if nebivolol enhances the availability of NO, pretreatment with ne- bivolol may enhance production of NO in re- sponse to subsequent treatment with LPS and PGN, an observation that may have relevance in clinical septic shock. Groups of female BALB/c mice each containing 12 mice (6-8 weeks old) were injected intraperitoneally with LPS (30 μg/mouse), PGN (100 μg/mouse), nebivolol (0.25 μg/g, 0.35 μg/g, 0.7 μg/g), LPS and nebivolol (0.25 μg/g), LPS and nebivolol (0.35 μg/g), LPS and nebivolol (0.7 μg/g), PGN and nebivolol (0.25 μg/g), PGN and nebivolol (0.35μg/g). One group of mice was injected with saline and an- other served as control. Three mice from each group were bled 1, 3, 6 and 9 hours post-injec- tion, the blood was pooled and the nitrite serum levels, reflecting NO concentration, were de- termined using Greiss reagent. The following results were obtained: 1) Treatment with saline did not induce NO production;2) LPS induced NO production to a maximal limit of 545% at 9 hours as compared to treatment with saline;3) PGN did not induce NO production;4) nebivolol at most doses and periods (7 out of 10 deter- minations) increased NO production over a range of 18-110% as compared to treatment with saline;5) Nebivolol enhanced LPS-induced production of NO by 58% at a dose of 0.7 μg/gm at 9 hours. It is concluded that nebivolol in- duces NO production. At low doses nebivolol initially appeared to have a suppressive or no effect on NO production induced by LPS. In- crease in the dose of nebivolol resulted in augmentation of LPS-induced production of NO. PGN, in the dose tested, did not have an effect on NO production.

Programmed Death Ligand-1 on Microglia Regulates Th1 Differentiation via Nitric Oxide in Experimental Autoimmune Encephalomyelitis

Microglia are considered to be potential anti- gen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are lar- gely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis （EAE） induced by myelin oligodendro- cyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 （PD-L1） at the peak phase of EAE. Meanwhile, both the CD4＋ T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Thl cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4＋ T cells and the differentiation of Thl cells, and this was significantly inhibited by PD-L 1 blockade. Further, microglia suppressed Thl cells via nitric oxide （NO）, the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Thl-cell differenti- ation via the PD-L1-NO pathway.

Synthesis and biological evaluation of nitric oxide(NO)-hydrogen sulfide(H2S) releasing derivatives of(S)-3-n-butylphthalide as potential antiplatelet agents

In the present study, a series of novel nitric oxide-hydrogen sulfide releasing derivatives of(S)-3-n-butylphthalide((S)-NBP) were designed, synthesized, and evaluated as potential antiplatelet agents. Compound NOSH-NBP-5 displayed the strongest activity in inhibiting the arachidonic acid(AA)- and adenosine diphosphate(ADP)-induced platelet aggregation in vitro, with 3.8- and 7.0-fold more effectiveness than(S)-NBP, respectively. Furthermore, NOSH-NBP-5 could release moderate levels of NO and H2 S, which would be beneficial in improving cardiovascular and cerebral circulation. Moreover, NOSH-NBP-5 could release(S)-NBP when incubated with rat brain homogenate. In conclusion, these findings may provide new insights into the development of novel antiplatelet agents for the treatment of thrombosis-related ischemic stroke.

Luteolin prevents uric acid-induced pancreatic β-cell dysfunction

Elevated uric acid causes direct injury to pancreatic β-cells. In this study, we examined the effects of luteolin, an important antioxidant, on uric acid-induced β-cell dysfunction. We first evaluated the effect of luteolin on nitric oxide （NO） formation in uric acid-stimulated Min6 cells using the Griess method. Next, we performed transient transfection and reporter assays to measure transcriptional activity of nuclear factor （NF）-κB. Western blotting assays were also performed to assess the effect of luteolin on the expression of MafA and inducible NO synthase （iNOS） in uric acid-treated cells. Finally, we evaluated the effect of luteolin on uric acidinduced inhibition of glucose-stimulated insulin secretion （GSIS） in Min6 cells and freshly isolated mouse pancreatic islets. We found that luteolin significantly inhibited uric acid-induced NO production, which was well correlated with reduced expression of iNOS mRNA and protein. Furthermore, decreased activity of NF-κB was implicated in inhibition by luteolin of increased iNOS expression induced by uric acid. Besides, luteolin significantly increased MafA expression in Min6 cells exposed to uric acid, which was reversed by overexpression of iNOS. Moreover, luteolin prevented uric acidinduced inhibition of GSIS in both Min6 cells and mouse islets. In conclusion, luteolin protects pancreatic β-cells from uric acid-induced dysfunction and may confer benefit on the protection of pancreatic β-cells in hyperuricemiaassociated diabetes.

(5R)-5-hydroxytriptolide inhibits the inflammatory cascade reaction in astrocytes

Many studies have shown that(5R)-5-hydroxytriptolide is the optimal modified analogue of triptolide, possessing comparable immunosuppressive activity but much lower cytotoxicity than triptolide. Whether(5R)-5-hydroxytriptolide has preventive effects on neuroinflammation is unclear. This study was designed to pretreat primary astrocytes from the brains of neonatal Sprague-Dawley rats with 20, 100 and 500 nM(5R)-5-hydroxytriptolide for 1 hour before establishing an in vitro neuroinflammation model with 1.0 μg/mL lipopolysaccharide for 24 hours. The generation of nitric oxide was detected by Griess reagents. Astrocyte marker glial fibrillary acidic protein was measured by immunohistochemical staining. The levels of tumor necrosis factor-α and interleukin-1β in the culture supernatant were assayed by enzyme linked immunosorbent assay. Nuclear factor-κB/p65 expression was examined by immunofluorescence staining. The phosphorylation of inhibitor of nuclear factor IκB-α and the location of nuclear factor-κB/P65 were determined using western blot assay. Our data revealed that(5R)-5-hydroxytriptolide inhibited the generation of nitric oxide, tumor necrosis factor-α and interleukin-1β from primary astrocytes activated by lipopolysaccharide, decreased the positive reaction intensity of glial fibrillary acidic protein, reduced the expression of tumor necrosis factor alpha and interleukin-1β in culture supernatant, inhibited the phosphorylation of IκB-α and the translocation of nuclear factor-κB/P65 to the nucleus. These results have confirmed that(5R)-5-hydroxytriptolide inhibits lipopolysaccharide-induced glial inflammatory response and provides cytological experimental data for(5R)-5-hydroxytriptolide in the treatment of neurodegenerative diseases.

Association between eNOS gene promoter polymorphism (-786T>C) and idiopathic recurrent pregnancy loss in Iranian women

Objective:To investigate the frequency of-786T>C variant in endothelial nitric oxide synthase(eNOS)gene promoter in Iranian women with recurrent pregnancy loss.Methods:Blood samples were obtained from 100 unrelated women affected by recurrent pregnancy loss and 100 unaffected women as the controls.Genomic DNA was extracted and-786T>C polymorphism in eNOS gene promoter was investigated by PCR-RFLP method.Statistical analyses and Hardy-Weinberg equilibrium in the groups of patients and controls were performed by Chi-square test and SPSS standard software(Version 21).Results:The frequency of homozygous TT was 40%in cases and 46%in the control group;the frequency of CC was 7%in cases and 5%in the control group;frequency heterozygote TC was 53%in cases and 49%in the control group.Genotype frequencies between the two groups showed no significant differences(P>0.05).Conclusions:The-786T>C polymorphism is not more frequent in recurrent pregnancy loss in this population.

Regulation of adenosine triphosphate-sensitive potassium channels suppresses the toxic effects of amyloid-beta peptide(25-35)

In this study, we treated PC12 cells with 0-20 μM amyloid-β peptide （25-35） for 24 hours to induce cytotoxicity, and found that 5-20 μM amyloid-β peptide （25-35） decreased PC12 cell viability, but adenosine triphosphate-sensitive potassium channel activator diazoxide suppressed the decrease in PC12 cell viability induced by amyloid-β peptide （25-35）. Diazoxide protected PC12 cells against amyloid-β peptide （25-35）-induced increases in mitochondrial membrane potential and intracellular reactive oxygen species levels. These protective effects were reversed by the selective mitochondrial adenosine triphosphate-sensitive potassium channel blocker 5-hydroxydecanoate. An inducible nitric oxide synthase inhibitor, Nw-nitro-L-arginine, also protected PC12 cells from amyloid-β peptide （25-35）-induced increases in both mitochondrial membrane potential and intracellular reactive oxygen species levels. However, the H202-degrading enzyme catalase could not reverse the amyloid-β peptide （25-35）-induced increase in intracellular reactive oxygen species. A 24-hour exposure to amyloid-13 peptide （25-35） did not result in apoptosis or necrosis, suggesting that the increases in both mitochondrial membrane potential and reactive oxygen species levels preceded cell death. The data suggest that amyloid-β peptide （25-35） cytotoxicity is associated with adenosine triphosphate-sensitive potassium channels and nitric oxide. Regulation of adenosine triphosphate-sensitive potassium channels suppresses PC12 cell cytotoxicity induced by amyloid-β peptide （25-35）.

The Releasing Effects of Captopril and Sodiun Nitroprusside on Endothelin-1 in Vascular Smooth Muscle Cell

Objective To investigate the influence of captoprial and SNP on the release of ET -1 in cultured VSMC of rats. Methods Measurement of endothelin - 1 levels by radioimmljnoassay in various concentrations of captopril and/or sodium nitroprusside in cultured vascular smooth muscle cell (VSMC) of rats. Results Both captopril and SNP could reduce the high ET - 1 levels of VSMC which were caused by Ang Ⅱ. There was a linear relationship between Ang Ⅱlevels and ET -1 production ( r = 0. 760, P <0. 001 ) . Conclusion Endothelin - 1 may accelerate the formation and development of atherosclerosis through inhibiting endogenous NO production by VSMC. ACEI or NO inhibition of ET -1 release could reduce atherosclerosis formation.

The Changes of Baroreflex Sensitivity During Head-up Tilt Test and Its Clinical Significance in the Patients with Vasovagal Syncope

Objective To study thechanges of baroreflex sensitivity (BRS) during head - up tilt test (HUT) in patients with vasovagal syncope (VS), and to examine the relationship between baroreflex sensitivity and neurohormonal factors. Furthermore, to investigate the effects of the changes of BRS on VS. Methods Forty - two patients with unexplained syncope (Among the 42 patients, there were 22 patients with positive HUT and 20 patients with negative HUT respectively) and 20 healthy volunteers (with negative HUT) underwent passive head - up tilt testing, Ante-cubital vein blood samples were taken before and after HUT, or at syncope. The fasting plasma endothelin , serum nitric oxide (NO), serum NE were measured, the BRS was assessed on the basis of the linear regression slope the RR interval versus systolic arterial blood pressure during the increment in blood pressure after intravenous administration of phenylephrine. Results (1) During the syncope, the BRS significantly reduced in HUT(+) group than baseline. At the end of tilt, the level of plasma ET, serum NO in patients with positive HUT significantly increased compared with baseline or normal controls, and the plasma concentration of NE also had the trend of increase. (2) By multiple regression analysis, a significant negative correlation was found between baroreceptor sensitivity and the plasma ET, NO at the end of HUT in patients with positive HUT, but there was no relationship between BRS and NE. Conclusions During the syncope occure, the BRS in patients with VS decreased significantly compared with normal controls. The abnormal plasma ET, NO concen-tration might contribute to the mechanism of VS.

依达拉奉对实验性自身免疫性脑脊髓炎大鼠的保护作用

目的观察依达拉奉对实验性自身免疫性脑脊髓炎大鼠的保护作用。方法将40只健康成年Wistar大鼠随机分成正常组、EAE组、LDE组、HDE组各10只,EAE组、LDE组、HDE组建立EAE模型,LDE组、HDE组分别腹腔注射相应剂量的药物。正常组、EAE组仅腹腔注射生理盐水0.5ml/d,所有大鼠24h后处死。结果EAE组神经功能缺损评分>LDE组>HDE组,组间比较有显著性差异(P<0.05);EAE组iNOS、OPN阳性细胞表达>LDE组>HDE组,组间比较有显著性差异(P<0.05)。结论依达拉奉对EAE的保护作用可能与其清除自由基、减轻炎症反应、减少i-NOS和OPN表达有关。

Arsenic exposure decreases rhythmic contractions of vascular tone through sodium transporters and K^+ channels

Arsenic-contaminated drinking water is a public health problem in countries such as Taiwan, Bangladesh, United States, Mexico, Argentina, and Chile. The chronic ingestion of arsenic-contaminated drinking water increases the risk for ischemic heart disease, cerebrovascular disease, and prevalence of hypertension. Although toxic arsenic effects are controversial, there is evidence that a high concentration of arsenic may induce hypertension through increase in vascular tone and resistance. Vascular tone is regulated by the rhythmic contractions of the blood vessels, generated by calcium oscillations in the cytosol of vascular smooth muscle cells. To regulate the cytosolic calcium oscillations, the membrane oscillator model involves the participation of Ca2+ channels, calcium-activated K+ channels, Na+/Ca2+exchange, plasma membrane Ca2+-ATPase, and the Na+/K+-ATPase. However, little is known about the role of K+ uptake by sodium transporters [Na+/K+-ATPase or Na+-K+-2Cl-(NKCC1)] on the rhythmic contractions.Vascular rhythmic contractions, or vasomotion are a local mechanism to regulate vascular resistance andblood flow. Since vascular rhythmic contractions of blood vessels are involved in modulating the vascular resistance, the blood flow, and the systemic pressure,we suggest a model explaining the participation of the sodium pump and NKCC1 co-transporter in low dose arsenic exposure effects on vasomotion and vascular dysfunction.

诱导型一氧化氮合酶与环氧合酶-2在鼻咽癌中表达的初步研究

诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）与环氧合酶-2（cyclooxygenase-2，COX-2）的表达在许多肿瘤中一致上调，两者间呈正相关。在鼻咽癌（NPC）中两种酶的表达及作用，国内未见报道。国外仅有的1篇报道中提到COX-2的表达与iNOS无关。为此，笔者应用免疫组化法对这两种酶在NPC组织中的表达情况进行了初步研究，现报道如下。