聚乙二醇对溶菌酶淀粉样纤维形成的作用

目的探索在聚乙二醇(PEG)存在的条件下,溶菌酶淀粉样纤维化及由此生成的聚集体对细胞的毒性作用。方法溶菌酶溶液中分别加入不同相对分子质量的PEG,在pH值为2.0、55℃的条件下孵育。采用硫黄素荧光监测溶菌酶淀粉样纤维化,透射电子显微镜观察聚集体形态,以诱导人红细胞聚集和溶血为指标评估溶菌酶聚集体对细胞膜的损害作用。结果在实验条件下,溶菌酶可形成淀粉样纤维,所有PEG均能够抑制溶菌酶的淀粉样纤维化,改变溶菌酶聚集体的形态,降低聚集体的表面疏水性和聚集体对细胞的损害作用。结论 PEG能够抑制溶菌酶的淀粉样纤维化,并使溶菌酶聚集体的细胞毒性减弱。     

银杏叶提取物对胰岛素淀粉样纤维化的抑制作用

目的探索银杏叶提取物（EGb 761）对胰岛素淀粉样纤维化及纤维细胞毒性的抑制作用。方法在pH 2．0、57℃孵育胰岛素,采用硫黄素荧光检测牛胰岛素形成淀粉样纤维的动力学曲线,透射电镜观察纤维形态;以淀粉样纤维诱导人红细胞的聚集和溶血为指标,评估EGb 761对胰岛素纤维细胞毒性的抑制作用。结果胰岛素在酸性溶液及57℃的条件下孵育可形成淀粉样纤维,EGb 761能够抑制淀粉样纤维的形成,使纤维形态改变,并能够抑制淀粉样纤维诱导的红细胞聚集和溶血。结论 EGb 761能够抑制胰岛素的淀粉样纤维化,并使胰岛素纤维的细胞毒性降低。     

芦丁对β淀粉样肽纤维化及细胞毒性的抑制作用

目的探索芦丁对Aβ25-35肽段淀粉样纤维化及纤维细胞毒性的抑制作用。方法在pH值为7.4、温度37℃孵育Aβ25-35肽,采用硫黄素(thioflavin T,ThT)荧光和透射电子显微镜检测多肽的淀粉样纤维化;以淀粉样纤维处理PC12细胞建立的细胞损伤模型,MTT法检测细胞存活率,以评估芦丁对β淀粉样纤维细胞毒性的抑制作用。结果 Aβ25-35肽段在pH值为7.4、温度37℃条件下,经孵育60h左右形成淀粉样纤维;芦丁抑制Aβ淀粉样纤维的形成,破坏纤维结构,并降低纤维诱导的细胞损害。结论芦丁能够抑制Aβ25-35的淀粉样纤维化和破坏成熟纤维结构,并降低Aβ纤维的细胞毒性。     

谷胱甘肽对胰岛素淀粉样纤维化的抑制作用

目的探索谷胱甘肽抑制胰岛素淀粉样纤维化及纤维细胞毒性的分子机制。方法在pH 2.0、37℃及90r·min-1震荡的条件下孵育胰岛素,采用硫黄素(ThT)荧光检测胰岛素形成淀粉样纤维的动力学曲线,8-苯胺-1-萘磺酸(ANS)荧光检测胰岛素分子聚集体表面疏水性的变化,透射电镜观察纤维形态,以淀粉样纤维诱导人红细胞的聚集为指标,评估谷胱甘肽对胰岛素纤维细胞毒性的抑制作用。结果胰岛素在本文的实验条件下孵育可形成淀粉样纤维,谷胱甘肽能够抑制胰岛素的淀粉样纤维化和降低形成的纤维聚集体的表面疏水性,并降低胰岛素纤维对细胞的损害作用。谷胱甘肽的这种作用与分子中的巯基相关。结论谷胱甘肽能够抑制胰岛素的淀粉样纤维化,改变胰岛素聚集体的表面特性,从而使聚集体的细胞毒性降低。     

丹酚酸B体外抑制淀粉样β蛋白的纤维形成及其细胞毒作用(英文)

目的:观察丹酚酸B对淀粉样β蛋白的纤维形成及其细胞毒作用的影响。方法:将不同浓度丹酚酸B与淀粉样β蛋白(1-40)在25℃共同孵育,于不同时间取样品电镜观察纤维形成。用MTT法观察此不同时间点淀粉样β蛋白(1-40)对PC12细胞的毒性作用。另将淀粉样β蛋白(25-35)预先老化7d,用MTT法观察此老化蛋白对PC12细胞的毒性及丹酚酸B的作用。结果:丹酚酸B10—100nmol/L可以完全抑制淀粉样β蛋白(1-40)25℃放置30h的纤维形成,对淀粉样β蛋白(1-40)25℃放置48及100h的纤维形成也有明显抑制作用。MTT法显示,经与丹酚酸B共同孵育的淀粉样β蛋白(1-40)明显较未与丹酚酸B孵育的淀粉样β蛋白对PC12细胞的毒性小。丹酚酸B1μmol/L可明显抑制预先老化的淀粉样β蛋白(25-35)对PC12细胞的毒性作用。结论:丹酚酸B可抑制淀粉样β蛋白的老化及纤维形成,同时可直接抑制老化淀粉样β蛋白对PC12细胞的毒性作用。 (责任编辑 吴民淑)     

丹酚酸B对β淀粉样肽诱导神经毒性的保护作用

目的：应用体内和体外实验观察丹酚酸B(SalB，中药丹参的主要活性成份)对β淀粉样肽(AB)诱导神经毒性的保护作用。方法：用MTT测定和流式细胞仪分析PC-12细胞的存活和凋亡情况。用Aβ1-40或Aβ1-40和SalB孵育PC-12细胞，观察丹酚酸B对抑制Aβ聚集和纤维形成的作用。分离大鼠线粒体，测定钙离     

槲皮素对胃癌MGC-803细胞生长及凋亡作用的研究

目的研究槲皮素对人胃癌MGC-803细胞增殖的影响及诱导凋亡作用。方法用四甲基偶氮唑盐(MTT)比色法检测不同浓度槲皮素对MGC-803细胞的增殖活性的效应;原位末端核苷标记(TUNEL)法检测槲皮素诱导细胞凋亡的凋亡指数(AI)。结果槲皮素浓度为40~100μmol/L时对MGC-803细胞增殖有显著的抑制效应,且呈剂量依赖性;细胞加入槲皮素诱导48h后AI明显高于未加入组(P<0.05)。结论槲皮素在一定浓度范围内能显著抑制MGC-803细胞的增殖并能诱导其凋亡,其作用呈浓度依赖性。     

槲皮素对血小板聚集和胞浆游离钙的影响(英文)

目的:研究槲皮素对凝血酶诱导的血小板聚集和胞浆游离钙浓度的影响及钙对槲皮素的血小板聚集抑制效应的作用。方法:用荧光钙离子指示剂观察槲皮素对血小板胞浆游离钙的影响.结果:槲皮素明显抑制凝血酶诱导的血小板聚集和游离钙的升高.IC_(50)和95％可信区间分别为146.2(92.4～231.3)和78.5(49.5—124.4)μmol·L~(-1).槲皮素对血小板的抑制作用可被钙翻转.槲皮素对凝血酶诱导的钙释放无影响.结论:抑制钙内流是槲皮素抑制血小板聚集和[Ca~(2 )]_i升高的机制.     

槲皮素对肝癌HepG2细胞生长影响的体外研究

目的 观察槲皮素对肝癌HepG2细胞生长及hTERT基因表达的影响.方法 以台盼蓝拒染法计数肝癌细胞的生长抑制率,用透射电镜从形态变化方面了解凋亡的发生,流式细胞术检测细胞周期变化,Western-blot检测hTERT基因表达改变,PCR-TRAP法检测端粒酶活性.结果 槲皮素抑制肝癌HepG2细胞增殖的作用明显,且呈浓度和时间依赖性,槲皮素处理48 h后的半数抑药浓度（IC5o）为25.5μmol/L;形态学检测显示出了细胞凋亡的特征变化,经10-20 μm/L的槲皮素处理,肝癌HepG2细胞周期阻滞于G0/G1期,且HepG2细胞hTERT蛋白降低,端粒酶活性被抑制.结论 槲皮素能呈时间、剂量依赖性地抑制肝癌细胞的生长,能诱导HepG2细胞发生凋亡,其抑制增生与诱导凋亡的机制可能与下调hTERT基因表达、抑制端粒酶活性、破坏端粒稳定性有关.     

Survivin和Bcl-2调节槲皮素诱导的A549细胞凋亡

目的研究槲皮素诱导肺腺癌细胞A549凋亡,探讨survivin和Bcl-2在槲皮素诱导A549细胞凋亡中的调节作用。方法分别采用MTT法、荧光染色、流式细胞仪和免疫细胞化学观察了槲皮素对肺腺癌A549细胞增殖、凋亡、细胞周期和蛋白表达的影响。结果槲皮素抑制肺腺癌A549细胞增殖的作用明显,且呈浓度和时间依赖性。形态学检测显示出细胞凋亡的特征变化,槲皮素能使肺腺癌A549细胞周期阻滞于G0/G1期,且A549细胞survivin和Bcl-2蛋白表达同时下降,而caspase-3活性升高。结论槲皮素能诱导A549细胞凋亡,其机制可能是使肺腺癌A549细胞周期阻滞于G0/G1期,并同时下调survivin和Bcl-2蛋白的表达,直接激活caspase-3而诱导A549细胞凋亡。     

Evolution of structural changes leading to haemolysis in human erythrocytes treated with SK&F 95018, an antihypertensive compound with combined vasodilator and β-adrenoceptor antagonist properties

SK&F 95018 is an antihypertensive compound with combined vasodilator and β-adrenoceptor antagonist properties, which, when given to dogs by intravenous infusion, rapidly produced symptoms of intravascular haemolysis. The haemolytic potency of SK&F 95018 was confirmed in vitro using human erythrocytes, was concentration dependent and was associated with dose-specific morphological changes as determined by scanning and transmission electron microscopy. Treatment of washed human erythrocytes with 0.5 mm-SK&F 95018 for up to 30 min resulted in gradual transformation from the biconcave discocyte to stomatocyte forms. Stomatocytes developed more rapidly on exposure to 2 mm-SK&F 95018, exhibited unilateral, multifocal invaginations by 2 min and evolved into spherocytic erythrocytes showing many membrane protuberances and invaginations. At the highest treatment level (10 mm) the crenated erythrocytes seen at time 0 transformed rapidly into spherocytes with many membrane-bound, surface projections that were retained in erythrocyte membrane ‘ghosts’. The membrane-active properties of SK&F 95018 were investigated in a phospholipid-membrane model (an aqueous dispersion of side-chain perdeuterated dipalmitoylphosphatidylcholine) by proton and deuterium nuclear magnetic resonance spectroscopy. The results suggest that SK&F 95018, with its molecular dual polarity, inserts into and effectively disrupts the intergrity of biological membranes by micellar reorganization of the bilayer plasmalemma. The slow change in shape from discocyte to stomatospherocyte at the lowest concentration (without the development of membrane-associated protuberances) suggests a disruptive effect on the erythrocyte osmotic balance by gradual cumulative drug insertion into the membrane. At higher concentrations this initial effect (leading to cell swelling) appears to proceed contemporaneously with micellar membrane reordering, producing membrane protuberances.     

The study of the quercetin action on human erythrocyte membranes

Quercetin is a naturally occurring flavonoid that exerts multiple pharmacological effects. In our previous study, we showed that quercetin greatly affects the lipid membrane. In this report, a study of quercetin on human erythrocyte membrane has been performed to determine the influence of this flavonoid on the fluidity and the conformational changes of membrane proteins. An additional aim of the study was to find how quercetin presence affects the resistance of membrane to haemolytic agents. The results showed that incorporation of quercetin into the erythrocyte membranes caused the changes of the partition coefficient of the Tempo spin label between the water and polar head group phases. In the studies, the W/S ratio has been used as a monitor of changes in protein conformation and in the environment within the membrane. It was observed that quercetin caused an increase in protein-protein interactions in human erythrocyte membranes. Haemolytic action of quercetin in the dark was also investigated. This compound showed protective effect against hypotonic haemolysis. However, in the heat-induced haemolysis quercetin caused acceleration of haemolysis. Dark reaction of erythrocyte with quercetin resulted in a shrinkage of the cells and alteration of their shapes. From the results we have concluded that modification of erythrocyte membrane by quercetin proceeds via reaction with membrane lipids and proteins.     

Possible artefacts in the in vitro determination of antimalarial activity of natural products that incorporate into lipid bilayer: apparent antiplasmodial activity of dehydroabietinol,a constituent of Hyptis suaveolens

Dehydroabietinol isolated from Hyptis suaveolens (L.) Poit. was found to inhibit growth of chloroquine-sensitive as well as chloroquine-resistant strains of Plasmodium falciparum cultivated in erythrocytes in vitro (IC 50 26-27 microM). However, erythrocytes exposed to dehydroabietinol were transformed in a dose-dependent manner towards spherostomatocytic forms with concomitant formation of endovesicles, as disclosed by transmission electron microscopy. The erythrocyte shape alterations caused by dehydroabietinol correlated well with its apparent IC 50 value. Thus, dehydroabietinol incorporates into the erythrocyte membrane, and since invasion and survival of Plasmodium parasites is known to depend on the function of the erythrocyte membrane, the observed antiplasmodial effect of dehydroabietinol is presumably an indirect effect on the host cell. Because of these findings, microscopic investigations should be generally used to support claims of antimalarial effects of apolar natural products.     

Protection of protein carbonyl formation by quercetin in erythrocytes subjected to oxidative stress

Quercetin, 3,3’,4’,5,7-pentahydroxyflavone, is one of the most abundant naturally occurring polyphenolic compounds. Evidences suggest that quercetin has biological properties that may play an important role in prevention of human diseases, such as cancer, cardiovascular diseases, diabetes, and allergies. Many of the biological actions of this flavonoid have been attributed to its antioxidant properties. In the present study, we have determined the protection of protein carbonyl formation by quercetin in erythrocytes subjected to oxidative stress. In vitro oxidative stress in human erythrocytes was induced by incubating with 10⁻⁵ M tert-butylhydroperoxide. This resulted in a significantly increased level of carbonyl content in erythrocyte membrane. Treatment with quercetin caused a decrease in the carbonyl content. The effect of quercetin was concentration and time-dependent. The protection of carbonyl formation in proteins substantiates the strong biological antioxidant property of quercetin.     

The combined effect of IDA and glutaraldehyde on the erythrocyte membrane proteins

A number of investigators have been focusing their attention on the encapsulation of antineoplastic drugs within erythrocytes to diminish their side-effects. Glutaraldehyde is often used as crosslinking agent to link the drugs (including idarubicin, IDA) to the cells. The previous studies indicated that in glutaraldehyde-treated human erythrocytes the elevated level of drugs was observed but also the various changes in the organization of the red cells were noted. In this study, we continue our investigations on the interaction of IDA and glutaraldehyde on the erythrocytes and now we concentrate on the effect of these compounds with the erythrocyte membrane proteins. For this purpose, SDS-gel electrophoresis of the cell proteins was carried out. Additionally, analysis of the disturbances of erythrocytes shape and size, accompanied by the application of flow cytometry and microscopy examination, were undertaken. The fluorimetric method was used to estimate content of IDA in supernatants, after erythrocyte membranes incubation with different glutaraldehyde concentrations. It was observed that glutaraldehyde caused in gradually dependent manner an increase of percent of IDA linked to the cell membrane proteins. After this incorporation, perturbations in the content of the proteins in the cell membrane were observed. The protein aggregates and changes in the level of spectrin, band 3 protein and small mass proteins were noted. The use of flow cytometry and microscopy technique demonstrated also disturbances in the shape and size of erythrocytes. For all tested concentrations of glutaraldehyde, the changes were statistically significant.     

Syntheses and structure-activity relationships of seven manganese-salen derivatives as anti-amyloidogenic and fibril-destabilizing agents against hen egg-white lysozyme aggregation

Accumulation of intra- and/or extracellular misfolded proteins as amyloid fibrils is a key hallmark in more than 20 amyloid-related diseases. In that respect, blocking or reversing amyloid aggregation via the use of small compounds is considered as two useful approaches in hampering the development of these diseases. In this research, we have studied the ability of different manganese-salen derivatives to inhibit amyloid self-assembly as well as to dissolve amyloid aggregates of hen egg-white lysozyme, as an in vitro model system, with the aim of investigating their structure-activity relationships. By coupling several techniques such as thioflavin T and anilinonaphthalene-8-sulfonic acid fluorescence, congo red absorbance, far-UV circular dichroism, and transmission electron microscopy, we demonstrated that all compounds possessed anti-amyloidogenic activities and were capable of dispersing the fibrillar aggregates. In addition, MTT assay of the treated SK-N-MC cells with the preformed fibrils formed in the presence of compounds at a drug-to-protein molar ratio of 5:1, indicated a significant increase in the viability of cells, compared to the fibrils formed in the absence of each of the compounds. Our spectroscopy, electron microscopy, and cellular studies indicated that EUK-15, with a methoxy group at the para position (group R(5)), had higher activity to either inhibit or disrupt the β-sheet structures relative to other compounds. On the basis of these results, it can be concluded that in addition to aromatic rings of each of the derivatives, the type and position of the side group(s) contribute to lower lysozyme fibril accumulation.     

Effects of the antiepileptic drug carbamazepine on human erythrocytes

The structural effects of the antiepileptic drug carbamazepine (CBZ) on the human erythrocyte membrane and molecular models have been investigated in the present work. This report presents the following evidence that CBZ interacts with red cell membranes: (a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that CBZ perturbed a class of lipids found in the outer moiety of the erythrocyte membrane; (b) in isolated unsealed human erythrocytes (IUM) the drug induced a disordering effect on the polar head groups and acyl chains of the membrane lipid bilayer; (c) in scanning electron microscopy (SEM) studies on human erythrocytes the formation of echinocytes was observed, due to the preferential insertion of CBZ in the outer monolayer of the red cell membrane. The effects of the drug detected in the present work were observed at concentrations of the order of those currently appearing in serum when it is therapeutically administered. This is the first time that toxic effects of carbamazepine on the human erythrocyte membrane have been described.