萸冰制剂对实验性偏头痛大鼠血清NO/NOS含量的影响

萸冰制剂是由华佗再造丸中主药吴茱萸与冰片组成。根据硝酸甘油、NO/NOS、偏头痛的内在联系,文章利用硝酸甘油型实验性偏头痛动物模型,并用硝酸还原酶法测定其血液中的NO/NOS含量。结果表明：硝酸甘油实验性偏头痛大鼠血清中NO/NOS的含量明显增强,萸冰制剂能明显降低偏头痛大鼠血清中NO/NOS的含量,且降低NO/NOS含量的作用明显优于西药阳性组（麦角胺咖啡因）和中药阳性组（正天丸）,与模型组比较有显著或极显著差异。提示：萸冰制剂具有治疗实验性偏头痛动物模型的作用。     

东方田鼠和小鼠感染日本血吸虫后血清NO的变化以及肝、肺病变的比较

目的了解日本血吸虫适宜宿主和非适宜宿主在感染后血清NO含量的变化规律以及肝、肺组织感染后的病变情况，以比较不同宿主对日本血吸虫感染的反应。方法东方田鼠和小鼠分别感染尾蚴1000条和40条，每2d分别剖杀2只，观察两宿主肝、肺变化，同时分别检测各宿主血清中NO的含量。结果小鼠和东方田鼠感染日本血吸虫后，肺部出现程度不同的出血现象。感染10d后出血现象均自行消失。其中，小鼠肝表观正常，但30d后肝中出现卵引起的内芽肿。东方田鼠在感染后6d肝中开始出现童虫所致病变，感染后第12～14d时逐渐减少，20d后病变消失。其间，小鼠和东方田鼠血清NO水平表现出不同的变化规律。东方田鼠感染后血清NO水平由正常的100μmol／L降到16μmol／L，18～20d恢复到原来的水平，完成一个完整和规律的循环。小鼠感染日本血吸虫后NO水平变化较大，第36d后，血清NO水平基本上维持在0～10μmol／L之间较低的、非正常水平。结论日本血吸虫非适宜宿主东方田鼠感染日本血吸虫后肝、肺病变特征以及NO水平变化与日本血吸虫适宜宿主小鼠不同，提示东方田鼠感染后体内存在一个敏感和系统的抗感染过程。     

p38 MAPK信号转导通路参与NO诱导兔关节软骨细胞凋亡

目的探讨p38 MAPK信号转导通路在软骨细胞凋亡中的作用。方法体外培养兔关节软骨细胞,一氧化氮(NO)供体NOC-18和p38 MAPK抑制剂SB203580作用于细胞24 h,用AnnexinV-FITC/PI流式细胞术检测软骨细胞凋亡率,W estern b lot测定p38、磷酸化p38蛋白的表达水平。结果与对照组比较,SB203580显著降低了NOC-18诱导的软骨细胞凋亡率(P<0.05);NOC-18以浓度依赖的方式促进p38 MAPK的磷酸化,而SB203580能抑制其磷酸化(P<0.05)。结论p38 MAPK通路参与了NO诱导的兔关节软骨细胞凋亡的信号转导。     

脑灵颗粒治疗血管性痴呆及对SOD、MDA、NO水平的影响

目的 :探讨脑灵颗粒治疗血管性痴呆及对SOD、MDA、NO水平的影响。方法 :选择血管性痴呆病人 80例 ,随机分为治疗组和对照组各 40例 ,治疗组口服脑灵颗粒 ,每次 1包 ,沸水调服 ,每日 3次 ;对照组用脑通片 10mg ,每日 3次口服 ,疗程 3个月。结果 :治疗组临床疗效、智力、生活能力改善均优于对照组 ,经比较有统计学意义 (P <0 .0 5 ) ,治疗组治疗前后SOD、MDA、NO水平比较均有统计学意义 (P <0 .0 1) ,SOD升高 ,MDA、NO下降。结论 :脑灵颗粒剂治疗血管性痴呆有较好的治疗效果。     

Impact of analysis interval on the multiple exhalation flow technique to partition exhaled nitric oxide

Exhaled nitric oxide (eNO) is elevated in asthmatics and is a purported marker of airway inflammation. By measuring eNO at multiple flows and applying models of eNO exchange dynamics, the signal can be partitioned into its proximal airway [ (nl/sec)] and distal airway/alveolar contributions [CA_NO (ppb)]. Several studies have demonstrated the potential significance of such an approach in children with asthma. However, techniques to partition eNO are variable, limiting comparisons among studies. The objective of this study is to examine the impact of the analysis interval (time or volume) on eNO plateau concentrations and the estimation of and CA_NO. In 30 children with mild to moderate asthma, spirometry and eNO at multiple flows (50, 100, and 200 ml/sec) were measured. The plateau concentration of eNO at each flow was determined using two different methods of analysis: (1) constant time interval and (2) constant volume interval. For both methods of analysis, a two‐compartment model with axial diffusion was used to characterize and CA_NO. At a flow of 200 ml/sec, the time interval analysis predicts values for eNO that are smaller than the volume interval analysis. As a result, there are significant differences in CA_NO between the methods of analysis (volume > time). When using the multiple flow technique to partition eNO, the method of analysis (constant time vs. constant volume interval) significantly affects the estimation of CA_NO, and thus potentially the assessment and interpretation of distal lung inflammation. Pediatr Pulmonol. 2010; 45:182–191. © 2010 Wiley‐Liss, Inc.     

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg⋅kg⁻¹⋅d⁻¹) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.Cyclic GMP-dependent protein kinase I (cGKI) is expressed in a wide variety of cells including cardiomyocytes (CMs), cardiac myofibroblasts (CMFs), cardiac fibroblasts (CFs), endothelial cells (ECs), and smooth muscle cells (SMCs) (1). Increased cGKI activity has been reported to protect against cardiac hypertrophy induced by pressure overload (2, 3). For example, mice that carry a mutated form of cGKI unable to interact with downstream targets, develop increased pathologic hypertrophy, accelerated mortality, and congestive heart failure when subjected to thoracic aorta constriction (TAC) (4).Hypertrophy induced by angiotensin II (AII) is thought to be attenuated by cGKI, because AII signaling through G_q/11 is abrogated by the regulator of G-protein signaling protein (RGS), a known substrate of cGKI (5). However, selective deletion of the AII receptor 1 (AT-R1) in the kidney ameliorated AII-induced cardiac hypertrophy, suggesting that the cardiac AT-R1 is dispensable for the induction of this response (6), whereas transgenic mice that overexpress the human AT-R1 specifically in CMs develop hypertrophy, fibrosis, dysfunctions, and early death (7). Furthermore, activation of TRPC channels followed by elevated [Ca²⁺]_i may contribute to the induction of the cardiac hypertrophy gene response (8–11). Again, TRPC3 and TRPC6 channels are negatively regulated by cGKI (12–14).It also has been reported that particulate guanylyl cyclase A, the major receptor for atrial natriuretic peptide (ANP) in the heart, couples to and directly activates the TRPC3/C6 channels in chronic cardiac hypertrophy, thus bypassing cGMP and cGKI (15). It was reported that cardiac cGMP can affects cardiac properties by modulating cAMP levels, bypassing again cGKI (16, 17). Thus, the site(s) of action of cGMP/cGKI are not entirely clear (18) and interpretation of cGMP/cGKI effects on cardiac hypertrophy may be quite complicated.Sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, elevates cardiac cGMP at high doses (100 mg⋅kg⁻¹⋅d⁻¹), increases cGKI activity, and has been reported to reverse TAC-induced cardiac hypertrophy (2). This effect is postulated to be caused by an increased activity of RGS2 (19), and in part by the direct regulation of TRPC3/6 conductance by cGKI (11, 14) mentioned above. However, two clinical studies did not report positive therapeutic results after prolonged treatment of diastolic dysfunction with sildenafil (20, 21).We (22) and others (23) have tested the hypothesis that CM-cGKI attenuates cardiac hypertrophy by using the cGKIβ rescue mouse line (βRM). These animals express the cGKIβ isozyme under the SM22α promoter, which is active principally in SMCs and activated CMFs, but do not express cGKIβ in other cell types (24). Chronic infusion of isoproterenol or TAC induced in βRM mice a cardiac hypertrophy that was identical to that of WT littermate controls (22), a result that argues against the hypothesis that CM, EC, or CF cGKI is responsible for the antihypertrophic effects of cGMP in the heart. To examine the role(s) of sildenafil to inhibit cardiac hypertrophy and fibrosis, we now extend these experiments using AII-induced hypertrophy and high concentrations of sildenafil (0.6 mM) in the drinking water. Once again, the lack of cGKI in CM and CF did not cause an increased hypertrophic response as predicted (2). Moreover, little or no antihypertrophic effect of sildenafil was observed in the WT mice and no effect was seen in the βRM mice. However, a large effect of sildenafil was observed on fibrosis in WT but not the βRM mice, suggesting that cGKI present in cardiac SMCs or activated CMFs is an important regulator of cardiac fibrosis.     

Activation of endothelial nitric oxide synthase is dependent on its interaction with globular actin in human umbilical vein endothelial cells

Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca²⁺ whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca²⁺ and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3′, 5′-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.     

Anti-asthmatic effects of Angelica dahurica against ovalbumin-induced airway inflammation via upregulation of heme oxygenase-1

Asthma is a chronic immune inflammatory disease characterized by variable airflow obstruction. The present study was undertaken to assess the effects of an Angelica dahurica Bentham et Hooker ethanolic extract (AD) on airway inflammation in an ovalbumin (OVA)-induced airway inflammation model. Mice that received AD displayed significantly lower airway eosinophilia, cytokine levels, including interleukin (IL)-4, IL-5, and tumor necrosis factor (TNF)-alpha levels, mucus production and immunoglobulin (Ig)E, compared with OVA-induced mice. In our experiments, AD treatment reduced airway inflammation and suppressed oxidative stress in the OVA-induced asthma model, partly via induction of heme oxygenase (HO)-1. The effects of AD on OVA-induced HO-1 induction were partially reversed by the HO-1 inhibitor, tin protoporphyrin (SnPP). Our results clearly indicate that AD is a suppressor of airway allergic inflammation, and may thus be effectively used as an anti-inflammatory drug in the treatment of asthma.     

Exposure-driven risk assessment: Applying exposure-based waiving of toxicity tests under REACH

The REACH Regulation 1907/2006/EC aims to improve knowledge of the potential risks to humans and the environment of the large number of chemicals produced and used in the EU. The testing requirements are likely to trigger numerous toxicological studies, potentially involving millions of experimental animals, despite the professed goal of REACH to reduce vertebrate testing. It may be necessary therefore to shift emphasis away from animal studies towards more pragmatic strategies, reserving animal tests for the substances of greatest concern. One approach is to waive certain tests based on levels of exposure to the substance. This review explores application of ‘Exposure-Based Waiving’ (EBW) of toxicity studies, with a particular focus on inhalation where possible, considering the potential qualitative and quantitative supporting arguments that might be made, including the use of thresholds of toxicological concern. Incorporating EBW into intelligent testing strategies for substance registration could advance the goals of REACH and the 3Rs (reduction, replacement and refinement of animals in research) by reducing the usage of animals in toxicity tests, whilst maintaining appropriate protection of human health and the environment. However greater regulatory evaluation, acceptance and guidance are required for EBW to achieve its full impact.     

Effects of red mold dioscorea on oral carcinogenesis in DMBA-induced hamster animal model

Monascus-fermented products offer valuable therapeutic benefits and have been extensively used for centuries in East Asia. Dioscorea has been proved to have anti-cancer effect. The aim of this study is to investigate the anti-tumor ability of the ethanol extract of red mold dioscorea (RMDE) on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. We induced oral squamous cell carcinoma (OSCC) in the buccal pouch of male Syrian golden hamsters by painting with 0.5% DMBA three times a week for 14 weeks. From 9 to 14 weeks, a dose of 50, 100, and 200 mg RMDE per kg body weight were painting with the hamsters for 6 weeks on days alternate to the DMBA application. The results demonstrated that RMDE decreased nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E₂ (PGE₂) overexpression in hamster buccal pouches in the DMBA treatment group and increased p53, serum tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) to significantly stimulate caspase-8 and -3 activities, indicating that RMDE reduced oxidative damage causing by DMBA and induced apoptosis in oral cancer cells. Therefore, RMDE may have therapeutic potentials against OSCC.