高速逆流色谱法分离制备丹酚酸B

采用高速逆流色谱法分离纯化丹参水溶性成分丹酚酸类物质,制备丹酚酸B化学对照品。分离采用的溶剂系统为正己烷-乙酸乙酯-水-甲醇(1.5:5:5:1.5),上相做固定相,下相做流动相,流速为1.7 mL/min,仪器转速850 rpm,进样量80 mg,纯度用HPLC方法测定。结果表明:一次分离可制备63.4 mg丹酚酸B,其纯度为98.6%。该方法操作简单,可作为高纯度丹酚酸B化学对照品的制备分离方法。     

棉籽糖低钾右旋糖酐液中灌注丹酚酸B对大鼠离体肺的保护作用

目的:研究丹酚酸B对离体供肺的保护作用。方法:将SD大鼠36只,随机分为对照组、低浓度实验组和高浓度实验组,每组各12只。对照组选择棉籽糖低钾右旋糖酐液(R-LPD液),实验组在对照组灌注液的基础上分别加入丹酚酸B注射液800mg/L、1200 mg/L。呼吸机辅助通气下边灌注边取下供肺,离体后再用灌注液行顺灌及逆灌,后放入盛有4℃相应保存液的无菌冰桶内保存。术后6 h、9 h、12 h、24 h分别测两组供肺的湿干重比(W/D)、髓过氧化物酶(MPO)活性、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性。结果:各组内12 h前相邻时间点比较,各参数均无统计学差异(P0.05);24 h后各参数明显高于12 h(P0.01)。各组间同一时间点比较,12 h前组间各参数无统计学差异(P0,05);保存至24 h,W/D、MDA含量、MPO活性比较,对照组低浓度实验组高浓度实验组(P0.05);而SOD活性比较,对照组低浓度实验组高浓度实验组(P0.05)。光镜下各肺组织保存至12 h以前,病理形态学均无明显炎症损伤,仅表现轻度炎症反应。12 h后对照组组光镜病理观察出现肺泡结构破坏、间质水肿、炎症损伤较低浓度实验组组明显,高浓度实验组情况较低浓度实验组肺组织结构更为清晰且炎症损伤较轻。结论:加入丹酚酸B后的灌注液在12 h前未有明显优势,12 h后能更加有效地保证小鼠供肺质量,使供体肺在离体情况下有效保存更长的时间,且高浓度丹酚酸B能更有效的提高对供体肺的保护发挥了重要作用。     

锰,硒对离体大鼠工作心脏心肌收缩功能的影响

我们曾通过对比观察证明,饲喂低硒饲料两个月与注射黄嘌呤-黄嘌呤氧化酶3d对大鼠的影响相似,均能使心肌收缩功能减弱、谷胱甘肽过氧化物酶(GSH-Px)活性降低、脂质过氧化物(LPO)含量增多、电子自旋共振谱线面积增大。本实验沿用相同的低硒饲料与硒、锰添加量,在离体鼠心肌上,观     

丁酸钠对大鼠离体灌流心脏细胞核组蛋白乙酰化的影响

本文用离体心脏灌流技术研究了丁酸钠对~3H-乙酰基参入大鼠心脏细胞核纽蛋白的影响。用蔗糖梯度离心将大鼠离体灌流心脏的细胞核分为心肌的和非心肌的,分别提取组蛋白。尿素-丙烯酰胺凝胶电泳将组蛋白分为五个组分。其比放射性测定的结果表明,~3H-乙酰基只参入核心组蛋白,程度为H_3>H_(2b)>H_4>H_(2a)。Triton-尿素-丙烯酰胺凝胶电泳图放射自显影结果显示,无论心肌细胞核还是非心肌细胞核,在丁酸钠为1m mol/L情况下,组蛋白H_3又可见三个亚组分(H_(3_1)、H_(3_2)及H_(3_3)),H_4可分出四个亚组分(H_(4_1)、H_(4_2)、H_(4_3)及H_(4_4));其总组蛋白乙酰化程度减低至对照组数值的60％。“冷追击”实验的结果提示,丁酸钠引起高乙酰化组蛋白的积蓄,确是通过其对组蛋白脱乙酰基过程的抑制作用而实现的。     

Ca2+对水杨酸诱发的丹参幼苗丹酚酸B生物合成的影响

为研究Ca2+在水杨酸诱导丹参幼苗丹酚酸B生物合成过程中的作用,分别用激光共聚焦显微镜和高效液相色谱仪检测胞外Ca2+通道抑制剂Vp和LaCl3,胞内Ca2+通道抑制剂LiCl以及胞内钙调素拮抗剂TFP处理前、后水杨酸诱导丹参叶片保卫细胞内Ca2+荧光强度和丹酚酸B含量的变化。结果表明,水杨酸 (SA) 处理后6 min即可诱发丹参幼苗叶片保卫细胞内Ca2+迸发,持续时间为2~3 min,丹参幼苗丹酚酸B生物合成量亦显著增加,且丹酚酸B合成量的增加发生在Ca2+迸发之后。胞外Ca2+通道抑制剂,胞内Ca2+通道抑制剂以及胞内钙调素拮抗剂均可抑制水杨酸诱导的Ca2+迸发和丹酚酸B的生物合成。结果表明水杨酸诱发的Ca2+对丹参幼苗丹酚酸B生物合成具有重要的调控作用。     

阿司匹林对离体蟾蜍心功能影响的实验研究

目的:观察阿司匹林(aspirin)溶液对离体蟾蜍心脏心率和心肌收缩力的影响。方法:根据斯氏蛙心插管方法,用不同浓度的阿司匹林对离体蟾蜍心脏进行灌流,采用BL-410生物机能实验系统记录心率和心肌收缩力的变化。结果:灌注0.1g/ml的阿司匹林溶液时,离体蟾蜍的心脏收缩力明显下降和心率明显减慢(P0.05),心脏甚至停止跳动,0.05g/ml的阿司匹林溶液时,离体蟾蜍的心脏收缩力降低和心率减慢程度不如0.1g/ml,并且随着阿司匹林浓度的降低离体蟾蜍的心脏收缩力和心率降低程度逐渐较少。结论:阿司匹林溶液降低离体蟾蜍心肌收缩力和减慢心率,并随着阿司匹林浓度升高,其心肌收缩力和心率降低程度更加明显。     

白细胞介素—2对离体大鼠心脏的作用及其机理

The purpose of the present study was to explore the biological effects and mechanism of interleukin-2 (IL-2) on the isolated rat heart. The results showed that hrIL-2 increased the number of premature ventricular contraction, heart rate, left ventricular developed pressure, left ventricular end-diastolic pressure and coronary flow in the isolated perfused rat heart. Heat inactivated hrIL-2 had no effect on the heart. Pretreatment with ryanodine canceled the positive effects of hrIL-2 on left ventricular developed pressure, left ventricular end-diastolic pressure and coronary flow but had no effects on arrhythmogenesis and tachycardia by hrIL-2. Pretreatment with nifedipine or low extracellular calcium abolished the arrhythmogenic effect of hrIL-2 and attenuated partially the augment of heart rate, left ventricular developed pressure, left ventricular end-diastolic pressure and coronary flow. It suggests that the cardiac activity of hrIL-2 depended on the integrity of its spatial structure and transmembrane influx Ca2+ and intracellularly stored Ca2+ were involved in the cardiac activity of hrIL-2.     

丹酚酸B对牙周膜干细胞成骨分化和氧化应激的影响

为了考察丹酚酸B对牙周膜干细胞(PDLSC)成骨分化和氧化应激的影响.本研究使用丹酚酸B(10 μmol/L)处理PDLSC,考察丹酚酸B对PDLSC的成骨或成脂分化的影响.另外,使用Ak-t信号通路抑制剂LY294002 (20 μmol/L)处理PDLSC来考察丹酚酸B对Akt信号通路的调控作用.通过用H2O2 (...     

丹酚酸B对高糖日粮小鼠消化系统自由基水平的影响

本文研究了丹酚酸B(salvianolic acid B,Sal B)对灌注及长期摄食高糖小鼠活性氧自由基(reactive oxygen species,ROS)的影响.结果表明:灌胃高糖使小鼠全血ROS于1.5 h达到最大值,灌胃葡萄糖的ROS释放量显著高于蔗糖(P＜0.05);灌胃高糖使小鼠血糖浓度于0.5 h达到最高,灌胃葡萄糖的小鼠血糖浓度有高于灌胃蔗糖的小鼠血糖浓度的趋势(P＞0.05);添加Sal B后并没有改变灌胃高糖小鼠的全血ROS释放和血糖浓度变化趋势,但有降低血糖趋势(P＞0.05),显著减少了ROS的释放量(P＜0.05);添加Sal B后显著降低长期高糖膳食小鼠的全血、肝脏、胰腺、胃、十二指肠、空肠和回肠的ROS释放量.     

离体工作心脏模型泵功能稳定性的评定指标

离体灌注的工作心脏模型是在心肌代谢及功能研究领域中广泛采用的实验系统。但离体工作心脏模型本质上是不稳定的,心功能在灌注过程中逐渐下降。本文探讨了对该系统稳定性进行数学描述方法,指出用实验系统平均主动脉流量与灌注时程的回归直线可简明地描述这一基本特征,直线下的面积S可作为判定系统稳定性的简单实用的指标。     

Matrix metalloproteinase-9 induces cardiac fibroblast migration, collagen and cytokine secretion: inhibition by salvianolic acid B from Salvia miltiorrhiza

Cardiac fibroblasts play the key role in cardiac function and matrix metalloproteinases-9 (MMP-9) is a well known contributor to the development of myocardial remodeling. However, the direct regulation of MMP-9 on the function of cardiac fibroblasts and the underlying mechanism are far from elucidation. In the present research, recombinant protein encoding catalytic domain of MMP-9 (MMP-9 CD) was constructed and the function of neonatal cardiac fibroblasts was investigated by cell proliferation assay, migration assay, picrosirius red assay, multiplex cytokine assay and fibroblast phenotype detection. 200 nM MMP-9 CD stimulated cardiac fibroblasts migration (169.4±22.5% versus 100±0%, p<0.01), increased collagen synthesis (1.5±0.2 fold, p<0.05), up-regulated the secretion of ICAM (574.0±40.1 versus 268.5±8.6pg/ml, p<0.01), TNF-α (192.6±11.0 versus 14.4±1.8pg/ml, p<0.001), IL-6 (1500.9±70.2 versus 323.4±40.6pg/ml, p<0.001) and sVCAM-1 (30.3±4.3 versus 7.0±0.1 pg/ml, p<0.05) and down-regulated VEGF (436.5±148.9 versus 1034.3±28.1 pg/ml, p<0.05) significantly with modest effects on proliferation. Accompanying with these regulations, transition of fibroblasts to myofibroblast was confirmed by immunofluorescent stain of α-smooth muscle actin (α-SMA) with MMP-9 CD treatment. Furthermore, salvianolic acid B (SalB) inhibited the effects of MMP-9 CD significantly. In conclusion, our results provide evidence for a direct influence of MMP-9 on cardiac fibroblast migration, collagen and cytokine secretion, which can be attenuated by SalB.     

Dimerumic acid protected oxidative stress-induced cytotoxicity in isolated rat hepatocytes

Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.     

Glycyrrhizin and glycyrrhetinic acid directly modulate rat cardiac performance

Root extract of liquorice is traditionally used to treat several diseases. Liquorice-derived constituents present several biological actions. In particular, glycyrrhizin and its aglycone, glycyrrhetinic acid, exhibit well-known cardiovascular properties. The aim of this research was to explore the direct cardiac activity of glycyrrhizin and glycyrrhetinic acid.The effects of synthetic glycyrrhizin and glycyrrhetinic acid were evaluated on the isolated and Langendorff perfused rat heart. The intracellular signaling involved in the effects of the two substances was analyzed on isolated and perfused heart and by Western blotting on cardiac extracts. Under basal conditions, both glycyrrhizin and glycyrrhetinic acid influenced cardiac contractility and relaxation. Glycyrrhizin induced significant positive inotropic and lusitropic effects starting from very low concentrations, while both inotropism and lusitropism were negatively affected by glycyrrhetinic acid. Both substances significantly increased heart rate. Analysis of the signal transduction mechanisms suggested that glycyrrhizin acts through the endothelin receptor type A/phospholipase C axis while glycyrrhetinic acid acts through endothelin receptor type B/Akt/nitric oxide synthase/nitric oxide axis.To our knowledge, these data reveal, for the first time, that both glycyrrhizin and glycyrrhetinic acid directly affect cardiac performance. Additional information on the physiological significance of these substances and their cardiac molecular targets may provide indication on their biomedical application.     

丹参活性成分的现代中药药理研究进展

对传统中药丹参中丹参酮、丹酚酸、丹参单体IH764-3三种主要活性成分及其近年来的中药药理学研究进展进行综述,为进一步开发利用这一传统中药提供理论依据。     

Production of lithospermic acid B and rosmarinic acid in hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium rhizogenes ATCC 15834, and the transformation was proved by direct detection of the inserted T-DNA by the polymerase chain reaction. As determined by HPLC, these hairy root cultures had the ability to produce lithospermic acid B (LAB), rosmarinic acid (RA) and other related phenolic compounds, the water-soluble active components of the plant. The effect of five different basal media, MS, MS-NH<INF>4</INF> (MS without ammonium nitrate), B5, WPM and 6,7-V on the root growth and phenolic compound production was studied. It was found that MS-NH<INF>4</INF> and 6,7-V media were superior to MS, B5 and WPM media in terms of both root growth and phenolic compound production. The time course of biomass accumulation and phenolic compound formation was also examined in the culture using MS-NH<INF>4</INF>medium. During cultivation, the content of RA in the roots was stable being approximately 0.48% of dry weight while the content of LAB fluctuated between 0.73% and 1.61% of dry weight, and decreased gradually at the stationary phase of growth. The highest production of LAB and RA was about 64 mg L⁻¹ and 23 mg L⁻¹, respectively. Received 05 November 1998/ Accepted in revised form 06 February 1999     

注射用丹酚酸A防治肝纤维化的实验研究

目的：探讨注射用丹酚酸A抗肝纤维化的作用,为丹酚酸A的临床应用提供理论依据。方法采用CCl4体外诱导肝细胞损伤,观察丹酚酸A对肝细胞活性及其细胞培养上清液ALT、AST、LDH水平和细胞裂解液中SOD活性和MDA含量的变化;另采用皮下注射CCl4诱导大鼠肝纤维化模型,观察丹酚酸A对肝纤维化大鼠血清LN、HA、SOD和MDA含量的影响以及肝脏组织病理改变情况。结果与模型对照组比,丹酚酸A高、低剂量组和Vit E组的细胞存活率显著提高（P ＜0.01）,丹酚酸A高剂量组ALT、AST和LDH活性显著降低（P ＜0.01）,丹酚酸A高剂量组和Vit E组SOD活性明显升高（P ＜0.05）,MDA含量显著降低（P ＜0.05）;体内试验发现,与模型对照组比,丹酚酸A高剂量组纤维化大鼠的血清LN和HA水平显著降低（P ＜0.05）,高、低剂量组SOD活性显著升高（P ＜0.05, P ＜0.01）,MDA含量显著降低（P ＜0.01, P ＜0.05）,并能改善肝脏病理形态。结论注射用丹酚酸A可通过抗脂质过氧化作用,起到保护肝细胞,减轻肝纤维化的作用。     

水杨酸诱发的NO介导了丹参悬浮培养细胞中丹酚酸B的生物合成

水杨酸(SA)可诱导丹参悬浮培养细胞中一氧化氮(NO)产生、苯丙氨酸解氨酶(PAL)活化及丹酚酸B(Sal B)的生物合成。为了阐明NO对丹参悬浮培养细胞中Sal B生物合成的影响及作用机理,本实验利用NO供体硝普钠(SNP)、NO合成酶抑制剂L-NNA(Nω-nitro-L-arginine)、NO淬灭剂c PITO(carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)以及PAL抑制剂L-AOPP(L-2-aminooxygen-3-phenyl acrylic acid)分别处理丹参悬浮培养细胞,并对其胞内NO水平、PAL活性和Sal B积累量进行了检测。结果表明,硝普钠(SNP)处理显著促进了NO产生、PAL活性和Sal B的积累,而L-NNA和c PITO抑制上述过程,说明NO诱发PAL活性提高并参与了SA诱导的Sal B生物合成;L-AOPP显著抑制了PAL活性及Sal B积累,却对NO产生没有显著影响,揭示NO位于PAL的上游。这说明SA诱发的NO产生、PAL活化及Sal B合成之间存在因果关系,即NO通过激活PAL触发Sal B生物合成。     

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.     

Performance of the isolated and perfused working heart of the teleostConger conger: study of the inotropic effect of prostacyclin

Summary An in vitro preparation of the heart of the teleostConger conger, isolated without the pericardium, was set up. The procedure allowed subambient pressures to develop in the perfusion chamber during contraction, mimicking the in vivo situation with the pericardium intact. The ventricle produced a cardiac output of about 15 ml·min^-1·kg wet body weight^-1 at subambient input pressure, and was able to double the stroke work with an increase of preload up to about 0.2 kPa. Using this preparation it was found that prostacyclin has a positive inotropic effect on the atrium and ventricle, but it does not affect the heart rate. Semilogarithmic doseresponse curves of prostacyclin on the atrium are reported, showing a threshold concentration of about 10^-9 M. The isolated and perfusedConger conger heart provides a useful model for a detailed analysis of the action of prostacyclin on myocardial contractility.