槲皮素对小鼠体内和体外NIH-3T3细胞的抗氧化作用及其机理

目的 比较槲皮素对小鼠体内和体外H2O2处理前/后的NIH-3T3细胞抗氧化效应的差异并探讨其作用机理。方法 将NIH-3T3细胞分为4组,槲皮素前保护组（Qb）：槲皮素后保护组(Qa)：H2O2组 (H2O2)和对照组(C)。 用试剂盒检测细胞内T-AOC、SOD、GSH-Px、GSH、MDA、NOS和NO2-/NO3-的水平;MTT和TUNEL法检测细胞凋亡率和生存率;免疫印迹和免疫组化技术检测细胞的cyclin D1、PTEN、NF-kB、HSP-70、Bcl-2、Bax、 及caspase-3的表达。另将20只Wistar大鼠等分为对照组和实验组,检测槲皮素灌胃前、后1、2及24h血浆内T-AOC、SOD、GSH-Px、GSH、MDA、NOS和NO2-/NO3-的水平。结果H2O2组细胞凋亡率增高,cyclin D1、PTEN及Bcl-2的表达下调;Bax、HSP-70、NF-kB及caspase-3的表达上调;T-AOC,SOD,GSH-Px及GSH水平降低,NOS、NO2-/NO3-及MDA增高。动物灌胃后1h/2h血浆中T-AOC、SOD、GSH-Px、GSH,NOS及NO2-/NO3-的水平增高,MDA降低;24h后基本恢复。结论 槲皮素在体内外皆可通过调节抗氧化酶体系发挥抗氧化作用,其效应依据H2O2处理与否和处理前/后等细胞的异质性而呈现一定差异。     

丹参酮ⅡA通过AK003290减轻H_2O_2诱导的原代小鼠心肌细胞焦亡

目的:探讨丹参酮ⅡA对过氧化氢(H_2O_2)诱导的原代小鼠心肌细胞焦亡的影响及可能分子机制。方法:分离培养原代小鼠心肌细胞,制备H_2O_2诱导的心肌细胞损伤模型,给予丹参酮ⅡA处理,分为对照(control)组、H_2O_2组(H_2O_2处理12 h)及丹参酮ⅡA组(20、40和60μmolo/L的丹参酮ⅡA预处理12 h+H_2O_2处理12 h);siRNA转染原代小鼠心肌细胞,real-time PCR确认干扰效率,分为对照(control)组、H_2O_2组(H_2O_2处理12 h)、丹参酮ⅡA组(40μmol/L的丹参酮ⅡA预处理12 h+H_2O_2处理12 h)和siAK003290+丹参酮ⅡA组(siAK003290转染+40μmol/L的丹参酮ⅡA预处理12 h+H_2O_2处理12 h)。CKK-8法测定细胞活力,Western blot检测细胞焦亡相关蛋白GSDMD和caspase-1表达,ELISA检测炎症因子白细胞介素1β(IL-1β)和IL-18含量,real-time PCR检测AK003290表达。结果:与control组相比,H_2O_2组细胞活力显著降低(P0.01),焦亡相关蛋白GSDMD和caspase-1表达增高(P0.01),细胞上清IL-1β和IL-18含量显著增加(P0.01),AK003290表达显著降低(P0.01);与H_2O_2组相比,丹参酮ⅡA组细胞活力显著升高(P0.05),焦亡相关蛋白GSDMD和caspase-1表达降低(P0.05),细胞上清IL-1β和IL-18含量显著减少(P0.05),AK003290表达显著增多(P0.05);与丹参酮ⅡA组相比,siAK003290+丹参酮ⅡA组细胞活力显著降低(P0.05),焦亡相关蛋白GSDMD和caspase-1表达增高(P0.05),细胞上清IL-1β和IL-18含量显著增加(P0.05)。结论:丹参酮ⅡA可减轻H_2O_2诱导的原代小鼠心肌细胞焦亡,其机制与上调AK003290的表达有关。     

芒果苷对H_2O_2诱导的BRL细胞氧化损伤的保护作用

目的探讨芒果苷对过氧化氢诱导的正常大鼠肝BRL细胞氧化损伤的保护作用。方法选取正常大鼠肝细胞株BRL,通过过氧化氢诱导法制备细胞氧化损伤模型,实验设对照组、H_2O_2组、芒果苷组。HE染色、台盼蓝染色观察细胞形态变化和存活率变化;比色法检测细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量;Western blot检测细胞中Bcl-2、Bax和Caspase-3蛋白表达变化。结果对照组BRL细胞贴壁生长良好,排列紧密。H_2O_2诱导后细胞形态不规则,胞膜皱缩,贴壁能力下降;细胞存活率降低;抗氧化酶SOD、GSH-Px活性降低,MDA含量升高(P0.05);Bax和Caspase-3蛋白表达升高(P0.05)、Bcl-2蛋白表达降低(P0.05)。与H_2O_2组相比,芒果苷可显著改善H_2O_2引起的细胞形态变化,提高细胞存活率;提高SOD、GSH-Px活性,降低MDA含量(P0.05);抑制Bax和Caspase-3蛋白表达(P0.05),上调Bcl-2蛋白表达(P0.05)。结论芒果苷对BRL细胞氧化损伤具有保护作用,其作用机制可能与其提高细胞清除氧自由基能力和抑制细胞凋亡有关。     

蜕皮甾酮减轻心肌细胞氧化应激损伤

目的:研究蜕皮甾酮对心肌细胞氧化损伤的作用。方法:H9c2细胞常规培养,经过氧化氢(H_2O_2)处理后建立损伤模型,分为:对照组,蜕皮甾酮高剂量(2μmol/L)、中剂量(1.5μmol/L)和低剂量(1μmol/L)组,H_2O_2组。CCK-8法检测蜕皮甾酮对H9c2细胞活力的影响;流式细胞术检测H9c2细胞的凋亡率;用分光光度法检测乳酸脱氢酶、肌酸激酶同工酶的活性以及丙二醛、超氧化物歧化酶含量;激光共聚焦联合流式细胞术观察细胞内活性氧簇(ROS)的产生和线粒体膜电位的变化;用Western blot法检测H9c2细胞内Bax、Bcl-2和cleaved caspase-3的蛋白水平。结果:在所选浓度范围内,蜕皮甾酮对H9c2细胞的活力无明显影响。与对照组比较,H_2O_2组H9c2细胞的LDH、CK-MB、ROS、MDA及凋亡率明显增加,通过不同浓度的蜕皮甾酮作用后,H9c2细胞的LDH、CK-MB、ROS、MDA及凋亡率显著下降,与H_2O_2组比较差异有统计学显著性。与对照组比较,H_2O_2组H9c2细胞的SOD和线粒体膜电位明显下降;与H_2O_2组比较,蜕皮甾酮高、中、低剂量组H9c2细胞的SOD和线粒体膜电位明显提高。对比对照组,H_2O_2组中H9c2细胞Bax和cleaved caspase-3的蛋白水平明显升高,Bcl-2的表达水平明显下降;与H_2O_2组比较,蜕皮甾酮高、中、低剂量组中H9c2心肌细胞表达Bcl-2蛋白上升,Bax和cleaved caspase-3的蛋白水平下降。结论:蜕皮甾酮对氧化应激条件下的H9c2细胞具有保护功能,其机制可能和减少氧化应激产物,增强抗氧化酶功能,调节线粒体功能和抑制细胞凋亡相关。     

枸杞多糖减轻过氧化氢诱导的人内皮样细胞EA.hy926氧化损伤

目的:研究枸杞多糖(LBP)对过氧化氢(H_2O_2)诱导人内皮样细胞EA.hy926氧化损伤的作用及机制。方法:用H_2O_2建立EA.hy926细胞氧化损伤模型,细胞共分为5个组:正常对照(control)组、模型(damage)组(H_2O_2,50 mmol/L)、LBP(100 mg/L)组、抗损伤(anti-damage)组(50 mg/L、100 mg/L和200 mg/L LBP+50 mmol/L H_2O_2)和LY294002(20μmol/L)组。采用CCK-8法检测细胞的活力;用Annexin V/PI双染法流式细胞术检测不同处理后EA.hy926细胞的凋亡;吖啶橙(AO)/溴乙啶(EB)染色法观察细胞凋亡的形态特征;划痕法测定细胞的迁移能力;一氧化氮(NO)试剂盒检测细胞培养上清中NO的水平;用ELISA法测定血管内皮生长因子(VEGF)的水平;Western blot检测cleaved caspase-3、Bax、Bcl-2、内皮型NO合酶(eNOS)、p-eNOS和p-Akt的蛋白水平。结果:量效结果显示浓度为100 mg/L的LBP预处理EA.hy926细胞1 h,然后加入H_2O_2(50 mmol/L)处理24 h对减轻H_2O_2诱导的EA.hy926细胞损伤的作用最显著。与damage组比较,LBP预处理可提高EA.hy926细胞的活力(P0.05),减少细胞凋亡(P0.05),并促进细胞迁移;上清液中VEGF和NO水平升高;Bcl-2/Bax比率明显升高,下调cleaved caspase-3蛋白水平,并上调eNOS和p-eNOS蛋白水平。此外,加入PI3K抑制剂LY294002后,LBP对H_2O_2损伤的EA.hy926细胞的保护作用减弱,表现为NO水平和p-Akt蛋白水平降低。结论:LBP能减轻H_2O_2对EA.hy926细胞的损伤作用,缓解H_2O_2诱导的凋亡,其机制与激活PI3K/Akt/eNOS信号通路密切相关。     

沉默FOXO1抑制H2O2诱导的大鼠肺泡2型上皮细胞凋亡

目的探讨沉默叉头蛋白O1(FOXO1)基因表达对H_2O_2诱导的大鼠肺泡Ⅱ型上皮细胞系(RLE-6TN)活力、凋亡的影响及机制。方法随机将RLE-6TN细胞分为对照组、H_2O_2组、si-FOXO1组和NC组。CCK8法及膜联蛋白V-FITC(Annexin V-FITC)/碘化丙啶(PI)细胞凋亡试剂盒分别检测细胞活力和凋亡率;Western blot检测FOXO1、p53、Bcl-2相关X蛋白(Bax)和磷酸化的p38丝裂原活化蛋白激酶(p-p38)蛋白表达。结果 H_2O_2可明显上调RLE-6TN细胞FOXO1、p53、Bax和p-p38蛋白表达,抑制细胞活力,诱导细胞凋亡(P0.05),而转染FOXO1 siRNA后可明显降低H_2O_2对FOXO1、p53、Bax和p-p38蛋白表达上调、细胞活力抑制及凋亡诱导作用(P0.05)。结论沉默FOXO1基因表达可抑制H_2O_2诱导的RLE-6TN凋亡,其机制可能与下调p38MAPK信号通路有关。     

洋葱总黄酮减轻过氧化氢诱导的视网膜色素上皮细胞氧化损伤

目的:探讨洋葱总黄酮(FO)对过氧化氢(H_2O_2)诱导的视网膜色素上皮细胞氧化损伤的影响。方法:将视网膜色素上皮ARPE-19细胞分成对照(control)组、H_2O_2组、低浓度FO(FO-L)+H_2O_2组、中浓度FO(FO-M)+H_2O_2组和高浓度FO(FO-H)+H_2O_2组。MTT法检测细胞活力,流式细胞术检测细胞凋亡。DCFH-DA荧光探针法检测细胞中活性氧簇(ROS)的水平,WST法检测超氧化物歧化酶(SOD)的活性,TBA法检测丙二醛(MDA)的含量,JC-1法检测细胞线粒体膜电位,Westernblot检测胞质中细胞色素C(Cyt-C)及细胞中cleaved caspase-3和cleaved caspase-9的蛋白水平。结果:H_2O_2降低ARPE-19细胞活力,升高细胞凋亡率和ROS水平,降低SOD活性,升高MDA含量,降低线粒体膜电位,升高胞质中Cyt C蛋白水平及细胞中cleaved caspase-3和cleaved caspase-9蛋白水平(P0.05)。与H_2O_2组比较,FO-L+H_2O_2、FO-M+H_2O_2和FO-H+H_2O_2组的细胞活力逐渐升高,凋亡率逐渐降低,细胞中ROS水平逐渐降低,SOD活性逐渐升高,MDA含量逐渐降低,线粒体膜电位逐渐升高,胞质中Cyt C蛋白水平及细胞中cleaved caspase-3和cleaved caspase-9蛋白水平逐渐降低(P0.05)。结论:洋葱总黄酮减轻H_2O_2诱导的视网膜色素上皮细胞氧化损伤。     

人参皂苷Rg1对H_2O_2诱导的293T细胞NF-κB转录活性影响

目的:观察人参皂苷Rg1对H_2O_2诱导HEK293T细胞损伤过程中NF-κB转录活性的影响,探讨人参皂苷Rg1的抗氧化机制.方法:H_2O_2模拟氧化应激条件,MTT法和台盼蓝染色法检测人参皂苷Rg1对细胞生长的影响;以DCFH-DA为探针流式细胞仪检测细胞内ROS水平;利用双荧光素酶顺式报告系统,检测人参皂苷Rg1对荧光素酶报告基因NF-κB-Luc相对荧光素酶值的影响,以检测人参皂苷Rg1是否具有下调H_2O_2诱导的NF-κB转录激活的作用.结果:H_2O_2诱导损伤的293T细胞存活率随H_2O_2浓度的增高而下降,相同H_2O_2浓度下,人参皂苷Rg1预保护组细胞存活率较损伤组显著提高(P＜0.05);H_2O_2处理后细胞内自由基明显增加,其荧光强度增加了40%～50%,而给予有效浓度的人参皂苷Rg1后,荧光强度降低35%～40%;与正常对照组比较,H_2O_2模拟氧化应激条件下,HEK293T细胞中NF-κB荧光素酶报告活性明显升高(P＜0.05),而在人参皂苷Rg1预保护组则明显受到抑制(P＜0.05).结论:人参皂苷Rg1对H_2O_2所致的细胞氧化应激损伤具有明显的保护作用,其可能机制是有效地清除了细胞内过多的自由基,下调了转录因子NF-κB的转录活性,继而抑制了NF-κB通路的激活.     

乙酰左旋肉碱减轻H_2O_2诱导的PC12细胞氧化损伤

目的:探索乙酰左旋肉碱(ALC)对H_2O_2诱导PC12细胞氧化损伤的作用及其可能的机制。方法:采用H_2O_2诱导PC12细胞造成中度氧化损伤细胞模型,将不同浓度(100、200和400μmol/L)的ALC作用于体外培养的PC12细胞,CCK8法检测细胞的活力。实验分为:正常对照(control)组,模型(H_2O_2)组,低、中、高浓度ALC药物预处理(ALC+H_2O_2)组,共5组。流式细胞术检测细胞凋亡率,Western blot法检测Nrf2和cleaved caspase-3的蛋白水平;免疫荧光染色法检测Nrf2的核转位情况。结果:不同浓度(100、200和400μmol/L)的ALC可显著抑制H_2O_2诱导的PC12细胞凋亡,以中浓度组效果最佳。与模型组相比,低、中、高浓度ALC组均可显著提高H_2O_2诱导的PC12细胞活力,抑制细胞凋亡(P0.05),显著下调cleaved caspase-3的蛋白水平(P0.05),上调Nrf2的表达水平(P0.05),促进胞浆的Nrf2向核内转移。结论:乙酰左旋肉碱对H_2O_2诱导氧化损伤的PC12细胞具有保护作用,抑制细胞凋亡从而提高其活力,其机制可能与激活Nrf2信号通路有关。     

ADAM33基因在过氧化氢诱导的支气管上皮细胞凋亡及氧化损伤中的作用研究

目的:探讨ADAM33基因对过氧化氢诱导的支气管上皮细胞活力、凋亡率、免疫因子表达及氧化损伤的影响及机制。方法:25、50、100、200、400μmol/L的H2O2处理人支气管上皮16HBE细胞2 h,CCK8法检测各浓度H_2O_2对16HBE细胞活力的影响。16HBE细胞分为对照组、NC组和H_2O_2+si-ADAM33组,CCK8检测各组细胞活力;流式细胞仪检测细胞凋亡率及ROS水平; Western blot检测ADAM33、VEGF、COX-2、NF-κB p65和IKKα的蛋白表达。结果:25μmol/L和50μmol/L的H_2O_2不影响16HBE细胞活力(P0. 05),而100～400μmol/L的H_2O_2可明显抑制16HBE细胞活力,与对照组比较差异具有统计学意义(P0. 05)。与NC组比较,H_2O_2组细胞活力显著降低,细胞凋亡率、ROS水平及VEGF、COX-2、NF-κB p65和IKKα的蛋白表达均显著升高(P0. 05),与H_2O_2组比较,H_2O_2+si-ADAM33组细胞活力显著升高,细胞凋亡率、ROS水平及VEGF、COX-2、NF-κB p65和IKKα的蛋白表达均显著降低(P0. 05)。结论:ADAM33基因表达抑制可降低由H_2O_2诱导的支气管上皮细胞凋亡、氧化损伤及免疫抑制,机制可能与下调NF-κB信号有关。     

Development in in vitro toxicology

There are three principal pressures driving the development of in vitro toxicology: (1) the need for more efficient testing systems to cope with the large number of xenobiotics currently being developed; (2) public pressure to reduce animal experimentation; and (3) a need for a better understanding of the mechanisms of toxicity. Within this, in vitro toxicology is focused on local, systemic, and target-organ toxicity. It is becoming increasingly apparent that a step or decision-tree approach using input of a variety of experimental data (physicochemical properties, biokinetics, cytotoxicity) provides the most efficient system for predicting toxicity. Examples of the use of in vitro toxicity systems for prediction of systemic toxicity and target-organ (liver) toxicity are presented.Originally presented at ECCP 93.     

Seasonal variations in acute toxoplasmosis in pregnant women in Slovenia

Between December 1999 and December 2004, 40 081 pregnant women were examined for toxoplasmosis with Toxo-IgG, Toxo-IgM enzyme immunoassay. Women with positive results were then retested with the Toxo-IgG avidity assay for recent toxoplasmosis. Recent acute toxoplasmosis in pregnant women was found to be significantly more frequent (p < 0.01) during winter than summer. The incidence of acute toxoplasmosis during winter-spring was also significantly more frequent (p < 0.025) than summer-autumn. This phenomenon should be taken into account when formulating preventive measures for toxoplasmosis, especially for pregnant women.     

休克过程中肾上腺髓质素变化的研究进展

Adrenomedullin (AM) is a new peptidergic regulator of vascular function. AM serves as a hormone, which has many biological properties, plays an important role in the many pathophysiological processes, especially shock. This review will highlight the structure, biological properties of AM and the relationship between AM and shock.     

Age-related changes in polyamines in memory-associated brain structures in rats

Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines and have important roles in maintaining normal cellular function, regulating neurotransmitter receptors and modulating learning and memory. Recent evidence suggests a role of putrescine in hippocampal neurogenesis, that is significantly impaired during aging. The present study measured the polyamine levels in memory-related brain structures in 24- (aged), 12- (middle-aged) and 4- (young) month-old rats using liquid chromatography/mass spectrometry and high performance liquid chromatography. In the hippocampus, the putrescine levels were significantly decreased in the CA1 and dentate gyrus, and increased in the CA2/3 with age. Significant age-related increases in the spermidine levels were found in the CA1 and CA2/3. There was no difference between groups in spermine in any sub-regions examined. In the parahippocampal region, increased putrescine level with age was observed in the entorhinal cortex, and age did not alter the spermidine levels. The spermine level was significantly decreased in the perirhinal cortex and increased in the postrhinal cortex with age. In the prefrontal cortex, there was age-related decrease in putrescine, and the spermidine and spermine levels were significantly increased with age. This study, for the first time, demonstrates age-related region-specific changes in polyamines in memory-associated structures, suggesting that polyamine system dysfunction may potentially contribute to aged-related impairments in hippocampal neurogenesis and learning and memory.     

Secular change in menarche in women in Madrid

The age at menarche was estimated by recollection in 1617 women between the ages of 18 and 60 in Madrid and a nearby suburb, Pinto. The population of Pinto is working-class and the Madrid group, taken from residential neighbourhoods , belongs to the upper middle class. In both groups we found a diminution in average age at menarche, from 14.04 to 13.02 years in Madrid and from 14.55 to 13.16 years from about 1935 to about 1965 in Pinto. These changes have been more intense in the group which is less well-off economically, where living conditions have varied much more drastically.     

The cyclic-AMP concentration in plasma and in muscle in response to exercise and beta-blockade in man

At rest the cAMP concentration in (muscle samples of) the quadriceps femoris ranged from 1.55 to 3.00 μmol per kg dry muscle and in plasma from 15.3 to 32.3 nmol per 1. Blockade of the beta adrenoreceptors with propranolol resulted in a significant decrease in the concentration in muscle at rest, the magnitude of the fall being related to the inital level. Similarly in plasma there was a trend towards lower levels of cAMP in those with the highest pretreatment levels, but the overall change was not statistically significant. There was no relation between the concentrations in muscle and plasma, before or after beta-blockade. Maximum dynamic exercise for 4–8 min resulted in an approximate doubling in the cAMP concentration in both muscle and blood. The increase in plasma was closely related to that in muscle. Beta-blockade inhibited totally the rise in cAMP in muscle during exercise but was marginally less effective in preventing the increase in blood. No increase in plasma or muscle cAMP levels during 40–70 s isometric contraction were observed.