Effects of vitamin E on oxidative stress and membrane fluidity in brain of streptozotocin-induced diabetic rats

Background: Diabetics and experimental animal models exhibit high oxidative stress due to persistent and chronic hyperglycemia, thereby deplete the activity of the antioxidative defense system and thereby promote the generation of free radicals. The current study examined the effects of vitamin E on oxidative stress and membrane fluidity in the brain of diabetes-induced rats. Methods: Sprague–Dawley male rats were randomly assigned to normal and streptozotocin (STZ)-induced diabetic groups. The diabetic groups were fed a vitamin E-free diet, 40 mg vitamin E/kg diet, or 400 mg vitamin E/kg diet. Diabetes was induced with STZ after 3 weeks of the experimental diet, then the rats were sacrificed 9 days later to determine the oxidative stress and cell membrane fluidity in the brain. Results: Dietary vitamin E strengthened the antioxidative defense system with an increased activity of the antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) and increased vitamin E content, in the brain of the diabetes-induced exeperimental rats. Accordingly, vitamin E was found to reduce the accumulation of reactive oxygen species (ROS), such as superoxide radical decrease the generation of oxidative damage substances, such as the carbonyl value, increase the membrane fluidity lowered by oxidative damage, and significantly improve the lipid composition. Conclusions: Vitamin E was found to be excellent for strengthening the antioxidative defense system, reducing the generation of ROS and damaging oxidative substances, and maintaining membrane fluidity in the brain of diabetes-induced rats.     

培元颗粒的处方与制备工艺考察

目的:优选培元颗粒的处方与成型工艺,为该方的研究与开发提供参考。方法:采用单因素试验法,以颗粒成型性、吸湿性、流动性为评价指标,优选培元颗粒的处方组成和成型工艺参数,并对培元颗粒的水分、流动性、堆密度等参数进行测定。结果:培元颗粒的最佳处方与成型工艺为干膏粉-糊精-可溶性淀粉(5∶3∶3),以90%乙醇为润湿剂,湿法制粒,60℃干燥1 h。收率、水分、休止角、堆密度及临界相对湿度分别为95.78%,5.08%,30.73度,0.526 g·m L-1,70%。结论:优选的培元颗粒处方与成型工艺合理、可行,颗粒剂的水分、流动性、堆密度均符合2010年版《中国药典》的规定。     

三七皂苷Rg1对肝纤维化大鼠线粒体质子跨膜转运的作用机制

目的探讨在三七皂苷(panax notoginseng saponins,Pn S)Rg1干预下,肝纤维化大鼠线粒体质子跨膜转运的变化和肝线粒体膜的流动性的变化,为开发三七单体Rg1在临床抗纤维化的应用提供详尽的试验依据和理论基础。方法 72只Wistar大鼠随机分为对照组、四氯化碳(carbon tetrachloride,CCl4)肝纤维化大鼠模型组、Pn S Rg1组各24只。除对照组外,其余2组用5%CCl4橄榄油按5 m L/kg灌胃制作肝纤维化大鼠模型。Pn S Rg1组在每次CCl4灌胃同时腹腔注射Pn S Rg1(5 mg/kg)用稳态荧光探针标记技术动态观察肝纤维化大鼠线粒体质子跨膜转运的变化,用荧光偏振法测定肝线粒体膜的流动性和膜的微黏度的改变。结果 (1)与对照组相比,肝纤维化模型组大鼠用单因素多组间方差分析,发现模型组大鼠质子跨膜转运中,肝线粒体质子跨膜转运能力显著下降(P0.01)。Pn S Rg1组与对照组相比质子跨膜转运的变化没有显著性意义(P0.05),与模型组相比,有显著性差异(P0.01)。(2)肝纤维化模型组大鼠用单因素多组间方差分析,与对照组相比,证实模型组大鼠线粒体膜的流动性显著下降(P0.01),增加膜的微黏度(P0.01);而Rg1组与模型组相比增加线粒体膜的流动性(P0.01),降低膜的微黏度(P0.01)。结论肝纤维化大鼠肝线粒体质子跨膜转运能力下降和线粒体膜的流动性显著下降是导致肝纤维化的重要原因之一。Pn S Rg1通过增加肝线粒体质子跨膜转运能力和线粒体膜的流动性而防治肝纤维化的发生发展的。     

糖尿病足患者血液流变学改变的研究

目的探讨2型糖尿病合并糖尿病足患者血液流变学改变与病情的关系。方法对168例2型糖尿病患者进行分组,糖尿病足组72例,非糖尿病足组96例,测定全血黏度、血浆黏度、血细胞比容、红细胞沉降率、纤维蛋白原。结果年龄大,病程长,合并糖尿病肾脏疾病、神经病变、视网膜病变、周围血管病变的2型糖尿病患者,糖尿病足发生率高;糖尿病足组全血黏度（高切）、血浆黏度、血细胞比容、红细胞沉降率、纤维蛋白原均升高（P〈0.05）;踝肱指数异常组血液流变学参数较踝肱指数正常组均升高（P〈0.05）。结论血液流变学异常能加重2型糖尿病的微循环障碍,促使糖尿病足的发生。有效地控制血液高黏状态,对防治糖尿病足有重要意义。     

左旋卡尼汀对异丙肾上腺素致心肌损害的影响

目的:观察左旋卡尼汀对异丙肾上腺素致心肌损害的保护作用并探讨其机制。方法:采用大鼠皮下注射异丙肾上腺素造成心肌损害模型。心肌切片,HE染色,光镜下观察心肌损害程度;分离心肌线粒体,测定膜脂流动性（LFU）。结果:注射左旋卡尼汀可减轻心肌损害程度;改善线粒体的损伤,改善LFU。结论:左旋卡尼汀能减轻异丙肾对心肌的损害,对线粒体膜脂流动性损伤具有明显的保护作用。     

Mechanisms involved in the antiplatelet activity of Escherichia coli lipopolysaccharide in human platelets

In this study, Escherichia coli lipopolysaccharide (LPS) dose-dependently (100–300 μg/ml) and time-dependently (10–60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LPS also dose-dependently inhibited the phosphoinositide breakdown and the intracellular Ca⁺² mobilization in human platelets stimulated by collagen. LPS (300 μg/ml) also significantly inhibited the thromboxane A₂formation stimulated by collagen in human platelets. Moreover, LPS (100–300 μg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. In addition, LPS (200 and 300 μg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 μg/ml) also significantly increased the production of nitrate within a 30 min incubation period. Rapid phosphorylation of a platelet protein of M _r 47 000, a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 n M ). This phosphorylation was markedly inhibited by LPS (200 μg/ml) within a 30 min incubation period.
These results indicate that the antiplatelet activity of LPS may be involved in two important pathways. (1) LPS may induce conformational changes in the platelet membrane, leading to change in the activity of phospholipase C. (2) LPS also activated the formation of nitric oxide (NO)/cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicaemic and endotoxaemic patients.     

Ceramide transfer protein function is essential for normal oxidative stress response and lifespan

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein. We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display >70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal.     

Effects of type 2 diabetes mellitus on plasma fatty acid composition and cholesterol content of erythrocyte and leukocyte membranes

Abstract Insulin resistance is a major factor in the pathogenesis of type 2 diabetes mellitus (T2DM) and is related to the fatty acid profile of the plasma membranes. The purpose of the present study was to investigate fatty acid composition and cholesterol content of cell membranes in patients with type 2 diabetes and, thus, to evaluate the possible factors leading to the alteration of plasma membrane fluidity. The study was performed in 20 healthy control subjects and 32 patients with type 2 diabetes. The fatty acid profiles and cholesterol content of the erythrocyte (RBC) and leukocyte (WBC) membranes were determined by a gas chromatographic method. When one considers the membrane constituents increasing fluidity and the ones decreasing it, the diabetics had a membrane composition decreasing fluidity compared to controls. On the other hand, when compared to control subjects, type 2 diabetic patients showed a significantly higher proportion of C16:0 components in erythrocyte and leukocyte membranes and plasma samples (25.4±3.1% vs. 31.1±4%; 23.3±2.4% vs. 29.3±5.2%; 27.6±3.9% vs. 34.5±5.7%; p<0.005, p<0.01 and p<0.005, respectively). Our results suggest that the ratio of saturated:unsaturated fatty acids changes in plasma and cell membranes of patients with type 2 diabetes. This situation may cause, at least in part, RBC–WBC function abnormalities and insulin resistance because of inconvenient membrane fluidity.     

Regulation of intracellular metabolism by biodegradable polyrotaxanes

Cellular response to our designed biodegradable polyrotaxanes was investigated in terms of changes in cytoplasmic calcium levels in platelets. The polyrotaxanes regulated thrombin-induced calcium increase in platelets although constituent molecules of the polyrotaxanes showed fewer effects on the intracellular metabolism. Further, an increase in membrane fluidity of red blood cell ghosts was significantly observed by the addition of the polyrotaxanes. Static light scattering study revealed that the polyrotaxanes formed a supramolecular association state in relation to the molecular weight of PEG: a loosely packed association with a specific molecular shape. From these characteristics, it is suggested that supramolecular level interactions between the polyrotaxanes and cell membranes regulate the intracellular metabolism. It is concluded that these biodegradable polyrotaxanes can be feasible as temporarily-controlled bioactivator.     

Physical mechanisms of amyloid nucleation on fluid membranes

Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein–membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways—protein-rich and lipid-rich—and quantify how the membrane fluidity and protein–membrane affinity control the relative importance of those molecular pathways. We find that the membrane’s susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson’s disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context.

The aggregation of normally soluble proteins into β-sheet-rich amyloid fibrils is a common form of protein assembly that has broad implications across biomedical and biotechnological sciences, in contexts as diverse as the molecular origins of neurodegenerative disorders to the production of functional materials (1, 2). The presence of surfaces and interfaces can strongly influence amyloid aggregation, either catalyzing or inhibiting it, depending on the nature of the surface. This effect has been studied for the cases of amyloid nucleation on nanoparticles (3–5), on flat surfaces (6–10), and on the surface of amyloid fibrils themselves (11, 12).Lipid bilayers are a unique type of surface, which is ubiquitous in biology and is the main contributor to the large surface-to-volume ratio characteristic of biological systems. They are highly dynamic, self-assembled structures that can induce structural changes in the proteins bound to them (13, 14) and markedly affect protein-aggregation propensities (15, 16). While nucleation on the surfaces of lipid membranes can influence fibril formation dramatically, alternative surfactant-driven fibrillation pathways in solution have been proposed at surfactant concentrations where formation of bilayer structures is not observed (17).Increasing experimental evidence supports the principle that the interaction between amyloidogenic proteins and the lipid cell membrane catalyzes in vivo amyloid nucleation, which is involved in debilitating pathologies. Remarkably, through surface-driven catalysis, lipid bilayers can enhance the kinetics of α-synuclein aggregation, the protein involved in Parkinson’s disease, by over three orders of magnitude with respect to nucleation in solution (18).Bilayer membranes can exist in different structural phases and can undergo local and global phase changes. A large body of work has focused on exploring how the membrane’s dynamical properties, such as its fluidity, relate to amyloid aggregation of bound proteins (19–26).For instance, fluid membranes, constituted of short and saturated lipid chains, were found to most effectively catalyze the nucleation of α-synuclein (19), while less-fluid membranes composed of long lipid chains had less catalytic power. Furthermore, the addition of cholesterol to lipid membranes was found to alter its fluidity and govern the nucleation rate of Aβ42 (25), a peptide implicated in Alzheimer’s disease. In these cases, the physical properties of the membrane are controlled through variations in its composition, and decoupling the role of the membrane’s physical properties from its chemical specificity is extremely challenging.The question we focus on here is how the microscopic steps that drive amyloid nucleation at the membrane surface are altered by the inherently dynamic nature of lipid bilayers.Computer simulations can be of great help in this case, enabling us to systematically investigate the role of the physical and chemical properties of lipid membranes independently from one another, thus helping to identify key players behind membrane-driven amyloid nucleation.In this work, we develop a coarse-grained Monte Carlo model for studying the nucleation of amyloidogenic proteins on lipid membranes. We use it to identify the microscopic mechanisms which connect the membrane fluidity, the rate of amyloid nucleation, and the morphology of amyloid aggregates. We find that the membrane most efficiently catalyzes amyloid nucleation by donating its lipids to the nucleating fibril, which depends 1) on the lipid solubility and often correlates with membrane fluidity, and 2) the affinity of proteins to the membrane. This interdependence controls both the morphology of the resulting aggregates, which can range from protein-rich to lipid-rich, and the rate of fibril formation. We then discuss how our results provide a mechanistic explanation for a number of recent experimental observations regarding accelerated nucleation kinetics on fluid membranes (19), lipid–protein coaggregation (27), and altered aggregate morphology (23). Furthermore, the framework developed here offers a platform for studying strategies for bypassing amyloid nucleation in a cellular context.