木犀草素对白血病K562/ADR细胞多药耐药的逆转作用及机制研究

目的 探究木犀草素（Lut）对慢性髓系白血病K562/ADR细胞多药耐药性的逆转作用及其机制。方法K562和K562/ADR细胞经不同浓度阿霉素（ADR）处理24 h后，采用CCK-8实验检测K562/ADR细胞耐药倍数。Lut单独或联合ADR作用K562/ADR细胞24 h后，采用CCK-8实验检测Lut的细胞毒性及对ADR的增敏作用。取对数生长期K562/ADR细胞分为0μmol/L Lut组、2μmol/L Lut组、4μmol/L Lut组，采用流式细胞术检测细胞内ADR蓄积量的变化；RT-PCR法和Western blot法分别检测核因子E2相关因子2(Nrf2)、多药耐药相关蛋白1(MRP1)、P-糖蛋白（P-gp）、谷胱甘肽-S-转移酶pi(GST-pi)mRNA和蛋白的表达；谷胱甘肽（GSH）试剂盒检测细胞内GSH的含量。结果 与K562细胞相比，K562/ADR细胞株对ADR具有明显的耐药性，耐药倍数为53.69倍。与0μmol/L Lut相比，不同浓度Lut作用于K562/ADR细胞后，细胞生长均受到不同程度的抑制（P<0.05），其中2、4μmol/L Lu...     

Senkirkine对黑色素瘤细胞B-16的增殖及细胞内谷胱甘肽含量的影响

目的探讨吡咯里西啶生物碱Senkirkine对黑色素瘤细胞B-16生长的抑制作用及细胞内谷胱甘肽含量、谷胱甘肽过氧化物酶(Glutathione Peroxidase,GPx)以及谷胱甘肽还原酶(Glutathione Reductase,GR)活性的影响。方法以黑色素瘤B-16细胞为实验模型,采用MTT法和DTNB法检测Senkirkine与B-16细胞孵育后对B-16细胞存活率的影响,对细胞内还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)含量,GSH/GSSG比率的影响,采用DTNB和NADPH分光光度法检测GPx以及GR的活性。结果Senkirkine可以显著抑制B-16细胞的增殖(P0.001),同时明显降低了细胞内GSH的含量以及GSH/GSSG的比例,并呈时间、剂量依赖性(P0.001);同时GPx活力与对照组相比显著提高(P0.05)而GR活力没有显著性的改变。结论在本试验条件下,Senkirkine能够抑制B-16黑色素瘤细胞的增殖,同时降低了B-16细胞抗氧化应激损伤的能力。     

FAT对K562/A02细胞内GSH含量的影响

目的：探讨FAT对K562/A02细胞内谷胱甘肽（GSH）含量的影响,及其对白血病细胞代谢解毒系统介导的多药耐药（MDR）的逆转效果。方法：采用生化DTNB法测定细胞内谷胱甘肽水平。结果：K562/A02细胞内GSH含量（231.54μmol/106细胞）高于K562细胞内GSH含量（58.03μmol/106细胞）,为K562细胞的3.99倍;VRP（10μmol/L）、FAT（0.01、0.02、0.04、0.08mg/ml）作用48h后,K562/A02细胞内GSH含量从231.54μmol/106细胞分别减少到96.95μmol/106细胞、212.89μmol/106细胞、161.00μmol/106细胞、122.35μmol/106细胞。结论：细胞内GSH含量增加是K562/A02细胞产生MDR的机制之一;FAT降低K562/A02细胞内GSH的含量是FAT逆转MDR的机制之一。     

葛根素对青光眼视网膜氧化应激损伤的保护作用

【摘要】目的研究葛根素对青光眼视网膜氧化应激损伤的保护作用。方法24只健康雄性成年家兔随机分为对照组、模型组及治疗组,每组8只。将2.5%羟丙基甲基纤维素溶液0.2mL注入家兔前房内,制作模型组及治疗组兔青光眼模型,治疗组家兔腹腔注射葛根素10mL/kg,每日1次,共21d。模型建立后第4周,测定视网膜抗氧化酶超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）活性,检测抗氧化物质谷胱甘肽含量及氧化还原状态和丙二醛（MDA）、一氧化氮（NO）含量。结果模型组视网膜抗氧化酶SOD、GSH-Px和CAT活性、还原型谷胱甘肽（GSH）和GSH/氧化型谷胱甘肽（GSSG）均低于对照组,GSSG、MDA和NO含量高于对照组;治疗组视网膜上述抗氧化酶活性、GSH含量、GSH/GSSG均高于模型组,但低于对照组;GSSG、MDA和NO含量均低于模型组,GSSG含量与对照组差异无统计学意义。结论葛根素可以在一定程度上增强抗氧化酶活性及维持谷胱甘肽氧化还原状态,从而减轻青光眼视网膜的氧化应激损伤。      

二烯丙基二硫活化NADPH氧化酶诱导人白血病K562细胞凋亡

目的研究DADS诱导人白血病K562细胞凋亡的分子机制。方法应用MTT法检测细胞的活性;流式细胞术检测细胞内的活性氧(reactive oxygen species,ROS)水平以及凋亡细胞百分率;Real-time PCR检测NADPH氧化酶各亚基mRNA水平;免疫共沉淀检测蛋白Rac2与蛋白p67phox的结合;Western blot检测Rac2蛋白的表达。结果 DADS能明显抑制K562细胞的增殖,呈时间和剂量依赖性;6 mg·L-1DADS作用人白血病K562细胞6 h后,NADPH氧化酶复合物的6个亚基mRNA水平都明显上调;5.0、10.0 mg·L-1DADS作用人白血病K562细胞24 h后蛋白Rac2的表达水平明显上调;免疫共沉淀结果显示,DADS诱导的K562细胞凋亡过程中有Rac2与p67phox结合;流式细胞术检测凋亡细胞百分率结果显示,PMA能明显提高DADS诱导K562细胞凋亡的作用,而DPI能抑制DADS诱导K562细胞凋亡。PMA能提高DADS诱导K562细胞活性氧的水平,而DPI明显抑制了活性氧的产生。结论 NADPH氧化酶的活化是DADS诱导K562细胞凋亡过程中活性氧的主要来源,DADS通过活化NADPH氧化酶诱导K562细胞凋亡。     

谷胱甘肽合成抑制对人胚肾细胞的毒作用

目的：研究细胞谷胱甘肽（GSH）合成抑制对人胚肾细胞的影响。方法：用GSH合成特异抑制剂（BSO）抑制GSH合成，从而降低胞内GSH的水平。测定BSO处理不同时间人胚肾细胞内GSH的水平、细胞周期的变化、活性氧水平、细胞凋亡率和细胞存活率。结果：细胞内总GSH水平随BSO处理时间的延长而下降。处理24时，总GSH水平下降至对照的10％，分析发现细胞周期G2－M期增多（P＜0．01），S期减少（P＜0．05）；但活性氧水平、细胞凋亡率及细胞存活率没有明显改变。处理48h时，总GSH水平下降至对照的4％，细胞存活率明显降低（P＜0．01），活性氧水平明显升高（P＜0．01）。细胞凋亡率直到处理72h时升高才能显著性（P＜0．01，n=3），此时细胞内总GSH已基本耗竭。结论：由于GSH水平下降而引起的氧化还原状态失衡可能是凋亡发生的普遍诱导因素。     

二氢青蒿素抑制K562细胞血管内皮生长因子的表达

目的通过观察二氢青蒿素抑制K562细胞血管内皮生长因子(VEGF)的表达，探讨青蒿素类药物在抑制血液肿瘤血管新生方面的作用。方法运用MTT法、免疫组化分析和Western blotting分析等探讨了二氢青蒿素对K562细胞增殖以及VEGF表达方面的影响，并进一步对药物预处理后肿瘤细胞的条件培养基在促内皮细胞增殖以及促鸡胚绒毛尿囊膜(CAM)血管新生的作用进行评定。结果二氢青蒿素能有效抑制K562细胞的增殖，并显著下调K562细胞VEGF蛋白和mRNA的表达。同时,药物预处理细胞的条件培养基，其促内皮细胞增殖和促CAM血管新生的能力都有所下降，并呈药物浓度依赖性。结论二氢青蒿素能显著下调K562细胞VEGF的表达，并能抑制由其诱导的血管新生作用。     

腺病毒介导的TSP1f基因转移对脐静脉内皮细胞株ECV304增殖的影响

目的探讨腺病毒介导的凝血酶敏感蛋白1（TSP1）抗血管新生衍生物（TSP1f）基因转移对人脐静脉内皮细胞株ECV304增殖的影响。方法应用RTPCR方法从正常人外周血单个核细胞扩增TSP1f cDNA序列构建TSP1f重组腺病毒（ADV-TSP1f）;Western blot方法检测ADV-TSP1f介导TSP1f在K562细胞中的表达;台盼蓝拒染检测经ADV-TSP1f感染的K562细胞条件培养基对ECV304细胞增殖的抑制作用。结果K562细胞经ADV-TSP1f感染可高效表达／分泌TSP1f多肽;经ADV-TSP1f感染的K562细胞条件培养基可显著抑制ECV304细胞增殖,以ADV-LacZ及PBS作对照,抑制率分别为51．6％和53．8％。结论ADV-TSP1f可显著抑制人脐静脉内皮细胞株ECV304增殖,可望成为有效的恶性肿瘤血管新生抑制剂。     

木犀草素抑制白血病耐药株K562/A02的GST-π表达

目的 探讨木犀草素(luteolin)对白血病耐药株K562/A02细胞谷胱甘肽S转移酶π(GST-π)的影响.方法 木犀草素30 mmol/L预处理K562/A02细胞第1、3、5d后,MTT法测定阿霉素IC50,采用722分光光度计测定细胞内GSH含量及Western Blot法测定木犀草素处理后GST-π蛋白表达.结果 木犀草素对K562/A02的耐药性具有明显的逆转作用,用木犀草素处理后,对阿霉素药物敏感性的相对逆转效率第1、3、5d分别为10.7％、42.7％和15.8％;木犀草素显著降低K562/A02细胞内GSH含量,GST-π蛋白的表达于用药后第1、3、5d分别下降22％、56％和34％.结论 木犀草素具有较强的逆转K562/A02细胞多药耐药的作用,其逆转机制可能与降低细胞内GSH含量,下调K562/A02细胞GST-π蛋白的表达相关.     

木犀草素抑制白血病耐药株K562/A02的GST-π表达

目的探讨木犀草素（luteolin）对白血病耐药株K562/A02细胞谷胱甘肽S转移酶π（GST-π）的影响。方法木犀草素30 mmol/L预处理K562/A02细胞第1、35、d后,MTT法测定阿霉素IC50,采用722分光光度计测定细胞内GSH含量及Western Blot法测定木犀草素处理后GST-π蛋白表达。结果木犀草素对K562/A02的耐药性具有明显的逆转作用,用木犀草素处理后,对阿霉素药物敏感性的相对逆转效率第1、3、5 d分别为10.7%4、2.7%和15.8%;木犀草素显著降低K562/A02细胞内GSH含量,GST-π蛋白的表达于用药后第1、35、d分别下降22%5、6%和34%。结论木犀草素具有较强的逆转K562/A02细胞多药耐药的作用,其逆转机制可能与降低细胞内GSH含量,下调K562/A02细胞GST-π蛋白的表达相关。     

卡立泊来德对K562细胞诱导的血管生成的影响

目的研究卡立泊来德(cariporide)处理对K562细胞诱导的血管生成能力的影响。方法应用MTT检测K562细胞上清液对脐静脉内皮细胞增殖能力的影响;transwell检测K562细胞上清液对脐静脉内皮细胞迁移能力的影响;基质胶血管形成法检测K562细胞上清液对脐静脉内皮细胞体外血管形成能力的影响;激光扫描共聚焦显微镜测定K562细胞的细胞内的pH;酶联免疫吸附实验(ELISA)检测K562细胞上清中血管内皮生长因子的表达水平。结果cariporide处理可以明显降低K562细胞上清液对脐静脉内皮细胞增殖,迁移和体外成管能力的诱导;cariporide处理后K562细胞的细胞内pH明显下降,分泌VEGF能力也受到抑制。结论 cariporide能抑制K562细胞的血管生成诱导能力,这种抑制是通过细胞内pH下降以及VEGF分泌减少引起的。     

Cyclosporin A sensitises Bcr-Abl positive cells to imatinib mesylate independently of P-glycoprotein expression

The effect of cyclosporin A (CsA) on imatinib treated Bcr-Abl positive K562 cells was studied. Similarly to other authors we found that imatinib induced apoptosis and erythroid differentiation in K562 cells. While its low concentrations induced predominantly erythroid differentiation, higher concentrations induced apoptosis. We found that CsA significantly potentiated cytotoxic effects of imatinib. A detailed analysis revealed that CsA shifted the balance between differentiation and apoptosis in favour of apoptosis. Our findings indicated that the observed effect of CsA was mediated neither through inhibition of ERK1/2 (extracellular signal-regulated kinases 1/2), nor through inhibition of p38 MAPK. We further observed that CsA might sensitise cells to apoptosis due to a changed cellular redox status as combined treatment of cells with imatinib and CsA resulted in a dramatic decrease of the ratio between reduced (GSH) and oxidised (GSSG) glutathione GSH/GSSG and in a significant suppression of thioredoxin reductase enzymatic activity. Our results indicated that K562 cells did not express detectable level of P-glycoprotein (P-gp). In addition, CsA did not affect significantly the intracellular level of imatinib. Therefore we excluded the possibility that CsA increased sensitivity of cells to imatinib by the inhibition of P-gp-mediated drug efflux or by another mechanism involving modulation of intracellular drug concentration.     

伊马替尼对慢性髓系白血病骨髓细胞VEGF表达的影响

目的 研究伊马替尼对慢性髓系白血病(CML)患者骨髓细胞及白血病细胞系K562血管内皮生长因子(VEGF)表达的影响.方法 采用ELISA检测伊马替尼作用前后CML骨髓细胞和K562细胞VEGF蛋白表达水平,实时荧光定量PCR检测伊马替尼对K562细胞VEGF mRNA表达的影响.结果 CML患者骨髓单个核细胞在伊马替尼作用后VEGF蛋白表达量为(130.66±100.58)pg/ml,明显低于未加药组(269.11±176.79)pg/ml(P＜0.01).K562细胞VEGF mRNA和蛋白表达水平于伊马替尼作用后亦呈剂量依赖性下降.结论 伊马替尼对CML患者VEGF的表达有明显抑制作用,提示该药对CML可能具有抗血管新生作用.     

Inhibition of glutathione-related enzymes augments LPS-mediated cytokine biosynthesis: involvement of an IkappaB/NF-kappaB-sensitive pathway in the alveolar epithelium

The regulation of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokine biosynthesis by reduction-oxidation (redox)-sensitive enzymes involved in maintaining intracellular glutathione homeostasis was investigated in fetal alveolar type II epithelial cells (fATII). Inhibition of glutathione-oxidized disulfide reductase, which recycles GSSG --> 2GSH, by the action of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) augmented LPS-dependent secretion of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. BCNU increased [GSSG] concentration at the expense of [GSH], thereby favoring oxidation equilibrium. Inhibition of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of GSH, by the action of L-buthionine-(S,R)-sulfoximine (BSO), potentiated LPS-induced IL-1beta, IL-6 and TNF-alpha production. Similar to BCNU, BSO depleted [GSH] and induced the accumulation of [GSSG]. BCNU and BSO reduced LPS-mediated phosphorylation of inhibitory-kappaB (IkappaB-alpha), allowing its cytosolic accumulation. This effect was associated with the inhibition of the nuclear translocation of selective nuclear factor (NF)-kappaB subunits: NF-kappaB1 (p50), RelA (p65), RelB (p68) and c-Rel (p75), but not NF-kappaB2 (p52). BCNU and BSO reduced LPS-induced NF-kappaB activation as determined by the electrophoretic mobility shift DNA-binding assay. Analytical analysis of the effect of modulating the dynamic redox ratio ([GSH]+[GSSG])/[GSSG] revealed a novel role for GSSG as a disulfhydryl compound which mediates an inhibitory effect on NF-kappaB activation. It is concluded that selective modulation of redox-sensitive enzymes has an immunopharmacological potential in regulating pro-inflammatory cytokines and that the TkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling.     

Thiram-induced cytotoxicity is accompanied by a rapid and drastic oxidation of reduced glutathione with consecutive lipid peroxidation and cell death

The toxic effect of thiram, a widely used dithiocarbamate fungicide, was investigated in cultured human skin fibroblasts. Cell survival assays demonstrated that thiram induced a dose-dependent decrease in the viable cell recovery. Thiram exposure resulted in a rapid depletion of intracellular reduced glutathione (GSH) content with a concomitant increase in oxidized glutathione (GSSG) concentration. Alteration of glutathione levels was accompanied by a dose-dependent decrease in the activity of glutathione reductase (GR), a key enzyme for the regeneration of GSH from GSSG. Thiram-exposed cells exhibited increased lipid peroxidation reflected by enhanced thiobarbituric acid reactive substances (TBARS) production, suggesting that GSH depletion and the lower GR activity gave rise to increased oxidative processes. To investigate the role of decreased GSH content in the toxicity of thiram, GSH levels were modulated prior to exposure. Pretreatment of fibroblasts with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented both lipid peroxidation and cell death induced by thiram exposure. In contrast, thiram cytotoxicity was exacerbated by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO). Taken together, these results strongly suggest that thiram induces GSH depletion, leading to oxidative stress and finally cell death.     

Intracellular redox status modulates monocyte chemoattractant protein-1 expression stimulated by homocysteine in endothelial cells

Homocystinemia has been identified as an independent risk factor for atherosclerosis. Monocyte chemoattractant protein-l (MCP-l) is a potent chemokine that stimulates the migration of monocytes into the intima of the arterial wall. The authors investigated the role of intracellular redox status in the expression of MCP-l stimulated by homocysteine in endothelial cells. Homocysteine stimulated MCP-1 mRNA expression and protein production in a time-dependent and dose-dependent manner in endothelial cells, decreased intracellular glutathione (GSH) and protein thiol levels, as well as G6PDH activity and NADPH levels. Thiol reduced reagents, GSH, and dithiothreitol levels, and reversed the MCP-l mRNA expression and protein production in endothelial cells; in addition, thiol oxidized reagent, diamide, and BSO levels, and markedly potentiated homocysteine-mediated up-regulation of MCP-l mRNA expression and protein production in endothelial cells. These results demonstrate that homocysteine can trigger overexpression of the MCP-1 gene by altering the intracellular redox status, suggesting that the homocysteine-induced changes in the intracellular redox status play an important role in modulating the expression of MCP-l in endothelial cells.     

In vivo effects of fenthion on oxidative processes by the modulation of glutathione metabolism in the brain of Oreochromis niloticus

The present study was designed to understand the oxidative stress potential of fenthion, an organophosphate (OP) pesticide and its involvement in glutathione metabolism modulated buthionine sulfoximine (BSO, 50 mg/kg) and N-acetylcysteine (NAC, 100 mg/kg) in the brain of fish, Oreochromis niloticus. A sublethal fenthion concentration (0.45 mg/L) was applied for 24, 48, and 96 h together with injection with BSO or NAC; following treatment, recovery periods for 24, 48, and 96 h were allowed. Total glutathione (tGSH), oxidized glutathione (GSSG), lipid peroxidation, protein level, and GSH-related enzyme activities were analyzed by using spectrophotometric methods. Fenthion in applied concentration did not change GSH levels, but increased GSSG levels. BSO application in fenthion exposure caused a depletion in GSH, while increasing the GSSG levels. Glutathione peroxidase (GPx; EC 1.11.1.9) specific activity increased in fenthion-applied groups at 24-h treatment. gamma-Glutamylcysteinyl synthetase (gamma-GCS; EC 6.3.2.2) was not detected in the brain. NAC injection in fenthion treatment decreased GSH and increased GSSG levels and GST activity. In conclusion, fenthion in sublethal concentration induced an oxidative stress processes in brain. BSO application provided an evidence for the involvement of fenthion in GSH metabolism. NAC elevated the fenthion-induced effects in spite of its antioxidant properties. Recovery period for 96 h was not adequate to eliminate the fenthion-induced changes.     

The relationship between the anti-inflammatory effects of curcumin and cellular glutathione content in myelomonocytic cells

Oxidative stress plays an important role during inflammatory diseases and recent therapies have focused on antioxidant administration to diminish oxidative stress and to arrest inflammatory processes. In this study, we investigated the impact of the GSH modulating effects of curcumin, a naturally derived polyphenol, on inflammatory processes in myelomonocytic U937 cells. One hour after administration of 10 micromol/l curcumin reactive oxygen species (ROS) production was significantly increased in undifferentiated U937 cells (+43%). Twenty-four hour after addition of curcumin, a significantly decreased ROS concentration was found (-32%), whereas GSH (+110%) and GSSG (+88%) content increased. A higher concentration of curcumin (25 micromol/l) caused an even stronger increase of GSH (+145%) and GSSG (+101%), but significantly decreased percentage of living cells to 84%. The increased GSH content of differentiated U937 cells after pre-incubation with curcumin was associated with lowered ROS production, nuclear factor kappa B (NFkappaB) activation (-34%) and tumor necrosis factor alpha (TNF-alpha) secretion (-51%) after LPS exposure. Curcumin inhibited TNF-alpha formation was also seen after GSH depletion by buthionine sulfoximine (BSO). This study shows that the antioxidative effects of curcumin are preceded by an oxidative stimulus, which is time and dose-dependent. Excessive concentrations of curcumin may even harm cells, as cell viability was decreased, in spite of elevated GSH contents. There was no clear relationship between intracellular GSH concentrations and the anti-inflammatory effects of curcumin.