力达霉素对人肝癌BEL—7402细胞基因组DNA的影响

目的：深入研究力达霉素对人肝癌BEL－7402细胞基因组DNA的影响。方法：琼脂糖凝胶电泳检测DNA泳带;Suthern杂交检测对基因的损伤;中性凝胶电泳检测DNA断裂修复;超螺旋GEM质粒检测断裂DNA位点。结果：力达霉素1μmol/L处理BEL-7402细胞,15,30,60min,可检测到明显的DNA梯带。低浓度的力达霉素对活跃基因N－ras的外显子和内含子均有明显的损伤作用,但对沉默IL－2基因没有影响。力达霉素断裂DNA后,DNA损伤难以修复。力达霉素断裂GEM质粒DNA的位点是－OH HO－。结论 ：力达霉素明显地损伤人肝癌BEL－7402细胞的基因组DNA。该结果有助于解释其高活性地杀伤肿瘤细胞的分子机制。     

As_2O_3诱导人肝癌细胞凋亡及对Fas表达和钙含量的影响

化疗在原发性肝癌的综合治疗中占重要地位.为寻找肝癌治疗新的有效药物,我们观察了As2O3对人肝癌细胞株BEL7402的生物学效应,以期为As2O3临床治疗肝癌提供实验依据.     

抗癌抗生素力达霉素诱导人肝癌BEL-7402细胞死亡的特征

目的　研究抗癌抗生素力达霉素(LDM)诱导人肝癌BEL-7402细胞死亡的特征。方法用荧光染料Hoechst 33342和PI联染;琼脂糖凝胶电泳检测;流式细胞术检测等。结果　LDM1μmol.L^-1处理该细胞后6h,可观察到一种有别于典型凋亡的染色质凝集方式：核膜一直保持完整,细胞仍贴壁,无凋亡小体形成;琼脂糖凝胶电泳检测到DNA梯带。流式细胞术检测到的G1亚峰,仅在LDM处理BEL-7402细胞后24h出现。LDM处理BEL-7402细胞后6h,caspase-3,6的活性分别增高5,4倍。染色质开始凝集的时间比caspase-6活性达到高峰的时间早。结论　力达霉素诱导人肝癌BEL-7402细胞死亡的特征有别于典型的凋亡,此结果可能有助于解释其极高活性地杀死肿瘤细胞的分子机制。     

酒精诱导肝癌细胞BEL-7402凋亡的作用研究

目的 体外观察不同浓度的酒精对肝癌细胞BEL-7402诱导凋亡的作用,为寻求肝癌的治疗提供新思路.方法 不同浓度的酒精与肝癌细胞BEL-7402通过体外培养,MTT法检测细胞增殖的变化;Hoechst 33258/PI 荧光双染色观察细胞形态学变化;蛋白质免疫印迹(Western blotting)半定量分析Caspase-3和p53和Bax的表达变化.结果 药物浓度在100～300 mmol/L的范围显著抑制肝癌细胞增殖,且抑制率随浓度和时间呈依耐性,Caspase-3,Bax和p53的蛋白表达量明显增加.药物浓度在100～300 mmol/L作用48 h,肝癌细胞皱缩,呈现凋亡各期改变,细胞凋亡率显著高于对照组(P<0.05).结论 不同浓度的酒精对肝癌细胞BEL-7402有诱导凋亡的作用,其抑制作用呈浓度和时间依赖性.     

半夏类药材不同提取物对人肝癌细胞Bel-7402的诱导凋亡作用

目的:观察并比较半夏类药材不同提取物诱导体外培养的人肝癌细胞Bel-7402凋亡的作用。方法:应用流式细胞术,检测半夏类药材不同提取物促进人肝癌细胞Bel-7402凋亡的作用,并观察了2种掌叶半夏醋酸乙酯提取物对细胞凋亡的量效关系。结果:3种药材都有诱导Bel-7402肿瘤细胞凋亡的活性,其诱导肿瘤细胞凋亡的有效成分主要集中在总有机酸或醋酸乙酯提取部分。结论:掌叶半夏和半夏诱导肿瘤细胞凋亡的作用较强,水半夏较弱,并且2种掌叶半夏样品的醋酸乙酯部分对促进Bel-7402细胞凋亡呈现一定的剂量依赖性。     

丹酚酸A对过氧化氢诱导的人脐静脉内皮细胞损伤的保护作用

目的研究丹酚酸A对人脐静脉内皮细胞(HUVECs)氧化损伤的保护作用。方法采用过氧化氢(H2O2)诱导的HUVECs氧化损伤模型,观察丹酚酸A(10,20和40μg·mL-1)对血管内皮细胞损伤的保护作用,MTT法检测细胞存活率,试剂盒检测细胞上清液中的超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)、一氧化氮(NO),比色法测定Caspase-3的活性。结果丹酚酸A可抑制H2O2诱导的血管内皮细胞的凋亡,同时增加SOD的活性,降低MDA的水平,减少LDH的漏出量,增加NO的释放,降低Caspase-3的活性。且丹酚酸A的上述作用均呈质量浓度依赖性。结论丹酚酸A可以有效保护内皮细胞免受氧化损伤。     

亚硝酸钠诱导人肝癌SMMC-7721细胞上皮-间质转化

探讨亚硝酸盐诱导人肝癌SMMC-7721细胞发生上皮-间质转化(EMT)的作用。0.25～25 mmol·L-1亚硝酸钠处理细胞48 h后,酶联免疫吸附实验(ELISA)检测细胞培养上清液中的TGF-β1、IL-6和IL-8水平,相差倒置显微镜下观察细胞形态学变化,划痕实验和Transwell侵袭实验检测细胞移动和侵袭能力,同时采用Western blotting方法检测EMT相关标记蛋白和p-AKT及下游信号分子的蛋白表达水平。结果表明,亚硝酸钠处理人肝癌细胞48 h,可以引起TGF-β1的分泌显著升高,并呈现剂量依赖性。亚硝酸钠对IL-8及IL-6的分泌也具有一定的增加作用,但不具有剂量依赖性。0.25 mmol·L-1亚硝酸钠作用48 h,诱导细胞形态向间质转化,增强波形蛋白和p-AKT及其下游信号蛋白的表达,抑制E型钙黏素蛋白的表达,促进细胞迁移和侵袭能力。结果提示,亚硝酸钠可能通过促进细胞分泌TGF-β1和IL-8,诱导人肝癌SMMC-7721细胞上皮-间质转化。     

丁酸钠诱导人肝癌SMMC-7721细胞凋亡作用的评价

通过观察丁酸钠 (Na B)对人肝癌 SMMC-772 1细胞抑制增殖和诱导凋亡作用 ,初步确定丁酸钠对体外培养人肝癌SMMC-772 1细胞的最适作用浓度。将丁酸钠以不同终浓度、不同时间作用于人肝癌 SMMC-772 1细胞 ,用华罗庚优选法确定实验浓度 ,采用 MTT比色法、倒置显微镜观察细胞增殖抑制 ;采用流式细胞术分析细胞凋亡率和细胞周期 ;电镜观察超微结构确定细胞凋亡情况。结果显示 ,丁酸钠对人肝癌 SMMC-772 1细胞生长的抑制呈剂量依赖和时间依赖 ,透射电镜观察到细胞皱缩、核质浓缩、核碎裂以及凋亡小体形成等凋亡特征的形态学改变。高浓度 (≥ 4mmol/L)时 ,细胞在 12 h迅速出现坏死 ,电镜下观察 ,正常细胞膜结构消失 ,线粒体肿胀、破裂。流式细胞术分析细胞阻滞于细胞周期的 G0 /G1期 ,S期比例明显减少 ,细胞增殖指数明显下降     

苦参碱诱导人肝癌细胞SMMC-7721凋亡的实验研究

目的研究不同浓度的苦参碱对人肝癌SMMC-7721细胞诱导凋亡的作用。方法将不同浓度的苦参碱作用于培养的SMMC-7721肝癌细胞,通过四氮噻唑蓝(MTT)比色法检测各浓度的药物对细胞的抑制增殖作用;光学显微镜、荧光显微镜下形态学观察;DNA琼脂糖凝胶电泳及流式细胞仪(FCM)研究分析其对肝癌细胞的诱导凋亡作用。结果浓度为2.01,4.02,6.04和8.05mmol·L-1苦参碱对SMMC-7721肝癌细胞都有不同程度的抑制增殖作用,抑制增殖作用随药物浓度的增加及作用时间的延长而加强(P<0.001)。光学显微镜、荧光显微镜下观察,发现细胞凋亡现象;DNA琼脂糖凝胶电泳可见DNA梯形条带;流式细胞仪(FCM)检测分析出现凋亡亚二倍体峰。结论苦参碱对肝癌细胞具有诱导凋亡作用,诱导作用具有明显的量效和时效关系。     

蟾酥注射液诱导人肝癌细胞凋亡的研究

目的观察蟾酥注射液对人肝癌细胞凋亡的诱导作用。方法选择不同浓度蟾酥注射液分别与人肝癌BEL-7404细胞共同孵育24、48h,流式细胞仪检测细胞凋亡率、细胞周期变化：琼脂糖凝胶电泳测定DNA Ladder；均与空白组做对照。结果流式细胞仪检测可见,各实验组细胞_1G、S期下降,G_2/M期升高,产生凋亡峰(sub-G_1期)；孵育48h,可见DNA Ladder出现。结论蟾酥注射液可以诱导人肝癌细胞BEL-7404凋亡。     

卡维地洛对过氧化氢致血管内皮细胞氧化应激损伤的保护作用

目的观察卡维地洛(carved ilol)对过氧化氢(hydrogenperoxide,H2O2)致内皮细胞损伤及表面粘附分子表达的影响。方法采用H2O2作为外源性自由基生成系统,模拟内皮细胞的脂质过氧化损伤,建立离体培养的ECV-304细胞氧化应激损伤模型,观察卡维地洛对H2O2致内皮细胞损伤及表面粘附分子表达的影响。结果卡维地洛各浓度组均明显改善H2O2(1.0×10-6mol.L-1)所致ECV-304细胞形态学损伤,提高细胞生存率,降低LDH释放,并可使细胞内及细胞培养液中MDA含量降低,SOD活性升高,亦可下调ICAM-1蛋白及细胞内ICAM-1mRNA表达水平,上述作用随药物浓度增加呈增强趋势。结论卡维地洛可保护内皮细胞结构和功能的完整性,提高内皮细胞抗氧化能力,并从转录水平抑制脂质过氧化诱导的粘附分子表达增加,降低单核-内皮细胞粘附,有利于减少动脉粥样硬化的始动环节和早期事件的发生。     

Nuclear factor-kappaB mediates cytoprotection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

1. Cytoprotection by H(2)O(2) preconditioning against oxidative stress-induced apoptosis of PC12 cells has been demonstrated previously. In the present study, we investigated the effects of H(2)O(2) preconditioning on nuclear factor (NF)-kappaB activation and the role of NF-kappaB in the adaptive cytoprotection of H(2)O(2) preconditioning in PC12 cells. 2. The PC12 cells were preconditioned with 100 micromol/L H(2)O(2) for 90 min, followed by 24 h recovery and subsequent exposure to 300 micromol/L H(2)O(2) for a further 12 h. 3. The results showed that preconditioning with 100 micromol/L H(2)O(2) upregulated NF-kappaB expression and enhanced its nuclear translocation and DNA binding activity. In addition to its own effects on NF-kappaB expression, H(2)O(2) preconditioning also promoted the overexpression of NF-kappaB induced by a lethal concentration of H(2)O(2) (300 micromol/L). 4. N-Tosyl-l-phenylalanine chloromethyl ketone (TPCK; 20 micromol/L), an inhibitor of NF-kappaB, was administered 20 min before preconditioning with 100 micromol/L H(2)O(2). At this concenteration, TPCK blocked the overexpression of NF-kappaB induced by H(2)O(2) preconditioning, accompanied by attenuation of H(2)O(2) preconditioning-induced cytoprotection. The inhibition of NF-kappaB by TPCK enhanced caspase 3 activity induced by 300 micromol/L H(2)O(2). 5. The findings of the present study provide novel evidence for the effects of preconditioning with H(2)O(2) on constitutive activation of NF-kappaB, which contributes to the adaptive cytoprotection of H(2)O(2) preconditioning against PC12 cells apoptosis.     

Protective effect of puerariae radix on oxidative stress induced by hydrogen peroxide and streptozotocin

This study evaluated the protective effect of Puerariae radix against the oxidative stress induced by hydrogen peroxide (H2O2) and streptozotocin in vitro and in vivo, respectively. The ethanol extract scavenged intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and prevented lipid peroxidation. This radical scavenging activity of the ethanol extract protected the cell viability of Chinese hamster lung fibroblast (V79-4) cells exposed to H2O2. Furthermore, this extract reduced the formation of apoptotic cells induced by H2O2, which was demonstrated by the decreased number of sub G(1) hypo-diploid cells and apoptotic cell body formation. The extract increased the activities of the cellular antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Administration of the extract to the streptozotocin induced diabetic rats decreased the blood glucose levels. The diabetic rats showed low activities of superoxide dismutase and catalase in the liver, and the ethanol extract increased the CAT activity. The increased level of lipid peroxidation in the diabetic rats reverted to near normal levels after being treated with the extract. This study showed that Puerariae radix was effective in the amelioration of diabetes, which may be a consequence of its antioxidant potential.     

姜黄素对过氧化氢诱导的血管内皮细胞损伤的双重作用

目的研究姜黄素(Cur)对过氧化氢(H2O2)诱导的血管内皮细胞ECV304的作用及可能机制;方法胎盘兰计数法分组观察在不同的时间点加入姜黄素后对H2O2诱导的ECV304细胞氧化损伤的作用。流式细胞仪分析细胞周期及细胞凋亡率的变化,试剂盒检测一氧化氮(NO)、丙二醛(MDA)含量及超歧化物氧化酶(SOD)、谷胱甘肽还原酶(GR)活性的变化。结果姜黄素0~100μmol·L-1与H2O2500μmol·L-1同时作用5h,随姜黄素浓度升高细胞存活率升高;25~100μmol·L-1姜黄素预先作用1h,再换为H2O2500μmol·L-1作用4h,细胞存活率明显下降;H2O2500μmol·L-1作用4h,再加入25~100μmol·L-1姜黄素作用1h,细胞存活率也升高。姜黄素对H2O2诱导的细胞凋亡没有明显的影响,但同时作用组、H2O2先作用组S期细胞所占比例升高,姜黄素先作用组S期细胞所占比例下降。同时作用组、H2O2先作用组的SOD、NO、GR水平相对升高,MDA水平下降,姜黄素先作用组的SOD、NO、GR水平相对下降,MDA水平升高。结论姜黄素对H2O2诱导的血管内皮细胞ECV304有双重效应,即有保护效应也有细胞毒效应,与其作用时间点有关。     

尼莫地平对过氧化氢诱导猪脑基底动脉氧化应激损伤的保护作用

目的：探讨尼莫地平对过氧化氢（H2O2）诱导猪脑基底动脉损伤的保护作用。方法：采用去内皮离体血管环灌流的方法，建立H2O2损伤模型。比较正常对照组，H2O2（2×10^-4mol／L）损伤组，维生素C（10^-4mol／L）组，尼莫地平高、中、低剂量（5×10^-6、5×10^-7、5×10^-8mol／L）组血管环对KCl、苯肾上腺素的张力；检测各组血管组织匀浆中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH—PX）活性及丙二醛（MDA）含量。结果：（1）H2O2损伤组与正常对照组比较，血管环对KCl、苯肾上腺素的收缩反应明显增加；尼莫地平高、中、低剂量组呈剂量依赖性抑制KCl、苯肾上腺素对血管环的收缩反应。（2）H2O2损伤组MDA含量升高，SOD、CAT、GSH—PX活性降低，与正常对照组比较，差异有统计学意义（P〈0．05）。尼莫地平高、中、低剂量均能降低组织中MDA含量，增强SOD、CAT、GSH-PX，的活性，与H2O2损伤组比较，差异有统计学意义（P〈0．05）。结论：钙通道阻断剂尼莫地平具有抗氧化应激作用，能预防H2O2对脑基底动脉血管的氧化损伤。     

Response of rat alveolar type II cells and human lung tumor cells towards oxidative stress induced by hydrogen peroxide and paraquat

The expression of MDR1b coding mRNA is increased in alveolar type II cells from juvenile rat lung in culture. Hydrogen peroxide and paraquat-induced further upregulation supporting that oxidative stress mediated mechanisms are involved in the regulation of MDR1b in rat lung. The expression rates of mRNA for catalase, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Mn-superoxide dismutase (Mn-SOD) remains constant during culture and were not modulated by hydrogen peroxide or paraquat. Thus, antioxidative enzymes in primary A II cells from rat lung are not regulated by reactive oxygen species dependent mechanisms. Primary A II cells were substantially more sensitive towards paraquat-induced cytotoxicity and lipid peroxidation than the permanent human lung tumor cell lines H322 and H358. A 100 microM hydrogen peroxide for 2h induces substantial DNA damage which is not paralleled by an increased rate of lipid peroxidation. The expression rate of mRNA coding for catalase and Mn-SOD was not changed and almost the same is true for the activity of catalase and Cu/Zn-SOD. Only 50 microM paraquat induced a significant decrease in catalase activity and an increase in Cu/Zn-SOD activity.     

Effect of standardized fruit extract of Luffa cylindrica on oxidative stress markers in hydrogen peroxide induced cataract

Objective:

The ability of Luffa cylindrica Roem fruit extract (LCE) to modulate biochemical parameters was investigated by in vitro studies for its role in hydrogen peroxide induced cataract on isolated goat lenses which were incubated for 72 h at 37°C.

Materials and Methods:

Test groups contained 5, 10, 15, 20, 25, and 30 µg/ml of LCE along with 1 ml of H₂O₂ (0.5 mM) as cataract inducer. Lenses were examined for morphological variation and transparency periodically during the incubation. Biochemical parameters such as superoxide dismutase (SOD), reduced glutathione (GSH), total protein content (TPC), and malondialdehyde (MDA) were estimated.

Results:

SOD, GSH, and TPC levels were found to increase proportionally with the concentration of LCE. However, MDA levels were found to be inversely proportional to the concentration of LCE. Opacity was graded as per “lens opacities classification system III.” Morphological examination suggested that LCE (25 µg/ml) maintained a vision for 44 h. No lens in LCE dose groups developed dense nuclear opacity after 24 h as opposed to 80% in negative control.

Conclusion:

The results suggest that LCE can delay the onset and/or prevent the progression of cataract which can be attributed to the presence of adequate phenolics, flavonoids, and Vitamin A and its high nutritional value. This preliminary study can be further synergized by testing LCE against other in vivo and in vitro models of cataract.KEY WORDS: Anticataract, antioxidant, hydrogen peroxide, Luffa cylindrica, sponge gourd     

Hexose monophosphate shunt activities in human erythrocytes during oxidative damage induced by hydrogen peroxide

Human red blood cells (HRBCs) were exposed to H(2)O(2) either as bolus or as a flux generated by a glucose-glucose oxidase system. H(2)O(2) concentrations were in the range 10(-5)-10(-3) M and exposure times to the oxidative stress were 10 min and 60 min. The production of NADPH by the hexose monophosphate shunt (HMPS) was accurately measured by gas chromatography-isotope ratio mass spectrometry as the production of (13)CO(2) from [1-(13)C]glucose. Depending on the duration of exposure and H(2)O(2) concentration, the production of (13)CO(2) by HRBCs under a flux of H(2)O(2) was increased two- to eight-fold in comparison with that obtained under a bolus of H(2)O(2). Under flux stimulation, spectral data show the formation of compound I, and a red shift caused by the presence of compounds II and III, whereas under a bolus stress no obvious spectra changes were observed. Inhibition of catalase by 3-amino-1,2,4-triazole (3-AT) or by sodium azide, followed by a bolus of H(2)O(2) led to a two- to five-fold increases in (13)CO(2) production compared with controls, depending on H(2)O(2) concentration. In contrast, 3-AT-inhibited HRBCs exposed to a flux of H(2)O(2) did not present an increase in (13)CO(2) production. The present paper emphasizes the importance and role of NADPH production following a bolus or a flux stimulation of H(2)O(2). The difference between responses in HMPS activities under the two types of stress could be related to a different balance of activity between 'catalatic' and 'peroxidatic' modes of catalase following H(2)O(2) exposure.     

Tri-n-butyltin delays the cell death induced by hydrogen peroxide in rat thymocytes

Tri-n-butyltin (TBT), one of environmental pollutants accumulated in mollusks, at nanomolar concentrations decreases cellular content of glutathione (GSH), suggesting that TBT increases cell vulnerability to oxidative stress because GSH has a role in catabolizing hydrogen peroxide (H(2)O(2)). In order to examine this possibility, the effect of tri-n-butyltin chloride (TBTCl) on rat thymocytes suffering from oxidative stress induced by H(2)O(2) was examined using a flow cytometer with four fluorescent probes; ethidium bromide, 2',7'-dichlorofluorescin diacetate, 5-chloromethylfluorescein diacetate and annexin-V-FITC. TBTCl at concentrations ranging from 100 nM to 1 μM attenuated H(2)O(2)-induced decrease in cell viability in a dose-dependent manner. It was unlikely that TBTCl reduced H(2)O(2)-induced oxidative stress because TBTCl failed to affect H(2)O(2)-induced oxidation of intracellular molecule (2',7'-dichlorofluorescin) and H(2)O(2)-induced decrease in cellular content of GSH. Results suggest that TBTCl may inhibit the pathway of cell death induced by H(2)O(2) or that TBTCl may induce a protective substance against the oxidative stress produced by H(2)O(2).     

Protective effects of Castanopsis cuspidate through activation of ERK and NF-kappaB on oxidative cell death induced by hydrogen peroxide

The protective effect of Castanopsis cuspidate var. sieboldii was examined on H2O2-induced cell damage. The ethanol extract of Castanopsis cuspidate was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and reduce intracellular reactive oxygen species (ROS) generation, and thus prevent lipid peroxidation and cellular DNA damage induced by H2O2. As a result, Castanopsis extract reduced H2O2-induced cell death of V79-4 cells via inhibition of apoptosis. Castanopsis extract was also found to increase catalase activity and its protein expression. Further molecular mechanistic studies revealed that Castanopsis extract enhanced phosphorylation of extracellular signal regulated kinase (ERK) and activity of nuclear factor kappa B (NF-kappaB). Taken together, the results suggest that Castanopsis extract protects V79-4 cells against oxidative damage by enhancing catalase activity and modulating the ERK and NF-kappaB signal pathway.