铁螯合剂诱导白血病细胞凋亡中caspase-3的变化

目的探讨铁螯合剂去铁胺（DFO）对诱导白血病细胞HL-60的分子机制。方法 2003年712月用钙黄绿素（calcein）检测HL-60细胞LIP。台盼蓝活细胞拒染实验进行活细胞计数及细胞存活率测定;光镜形态学观察及流式细胞仪（FCM）等方法检测HL-60细胞凋亡;比色法检测caspase-3（基于pNA标记底物的比色法）活性。结果①不同浓度的DFO作用于HL-60细胞后,随培养时间延长及DFO浓度的增加,动态铁池降低,细胞生存率逐渐下降,凋亡率增加,显示一定的时间剂量依赖性。②HL-60细胞在不同浓度的DFO作用下,caspase-3的活性逐渐升高。50、100μmol/LDFO作用于HL-60细胞24h,caspase-3酶活性升高明显,与对照组相比,有统计学意义（P〈0.001）;相关分析结果显示,HL-60细胞LIP的改变与caspase-3活性变化呈负相关系（r=-0.887,P〈0.05）。结论 DFO诱导白血病细胞凋亡的作用可能与螯合细胞内铁,降低细胞LIP,激活caspase-3,最终实施细胞凋亡密切相关。     

去铁胺诱导K562细胞凋亡研究

目的：探讨铁螯合剂去铁胺（DFO）诱导白血病细胞凋亡的分子机制。方法：实验分为DFO组(终浓度分别为10、50、100 μM)、DFO+FeCl3（各10 μmol/L）组及空白对照组。用钙黄绿素检测K562细胞可变铁池。锥虫蓝活细胞拒染实验进行活细胞计数及细胞存活率测定;光镜形态学观察及流式细胞仪方法检测K562细胞凋亡;比色法检测caspase-3活性。结果：（1）DFO作用于K562细胞后,随培养时间延长及DFO浓度的增加,动态铁池降低,细胞生存率逐渐下降,凋亡率增加,显示一定的时间剂量依赖性;而DFO+FeCl3组细胞凋亡率与空白对照组差异无统计学意义。（2）50 μmo/L、100 μmol/L DFO作用于K562细胞24 h时,caspase-3酶活性升高明显,与对照组相比,差异有统计学意义(P<0.01);相关分析结果显示,K562细胞铁池的荧光改变与caspase-3活性变化呈负相关(r=-0.894, P<0.05)。结论：DFO诱导白血病细胞凋亡的作用可能与螯合细胞内铁,降低细胞可变铁池,激活caspase-3有关。     

Effect of N-acetylcysteine and/or deferoxamine on oxidative stress and hyperactivity in an animal model of mania

Studies have consistently reported the participation of free radicals in Bipolar Disorder. Administration of d-amphetamine (d-AMPH) is a relevant animal model of mania and it increases oxidative stress in rat brain. Evidences indicate that the antioxidants N-acetylcysteine (NAC) and Deferoxamine (DFX) exert protective effects in the brain. The present study was designed to evaluate the effects of NAC, DFX or their combination on AMPH-induced hyperactivity. The protein oxidation levels were analyzed in prefrontal cortex and hippocampus. In the first animal model (reversal treatment), adult male Wistar rats received saline or d-AMPH for 14 days, and from the 8th to the 14th day, they were treated with saline, NAC, DFX, or NAC plus DFX. In the second animal model (prevention treatment), rats were pretreated with saline or antioxidant regime, and from the 8th to the 14th day, they also received saline or d-AMPH. In the prefrontal cortex, the protein carbonyls were not affected by the treatment with antioxidants alone but it was increased by treatment with NAC plus DFX. At the same model, NAC plus DFX reversed the protein damage in the hippocampus, but NAC alone increased this damage. In the prevention treatment, it was observed that the protein damage in the prefrontal cortex was prevented by DFX or NAC plus DFX. In the hippocampus, the pretreatment with all antioxidant regime prevented protein damage induced by d-AMPH. At both treatments (reversal or prevention) the antioxidants did not present any effect against d-AMPH-induced hyperactivity. In conclusion, NAC or DFX and the combination of NAC plus DFX reverse and protect against d-AMPH-induced oxidative protein damage. Using these protocols we could not observe affects on locomotion, however this effect varies depending on the brain region and the treatment regime.     

去铁胺减少老年大鼠急性脑出血后神经元凋亡作用及其机制

目的:探讨去铁胺(deferoxamine,DFX)降低急性脑出血(intracerebral hemorrhage,ICH)诱导的神经元凋亡作用及其机制。方法:雄性SD大鼠右侧基底节区尾壳核注射100μL自体全血,随机分为观察组和对照组。观察组ICH后2 h注射DFX,以后每12 h注射一次,直至7 d;对照组注射等量生理盐水(normal saline,NS)。两组均在ICH后1、3、7 d进行行为学检测,并检测血肿大小、神经元凋亡、铁蛋白、血浆铁蛋白水平。结果:观察组ICH后7 d前肢放置明显恢复(P<0.01),ICH后3、7 d右转百分比明显降低(P<0.01);ICH后7 d,观察组血肿分辨率明显降低(P<0.01);ICH后1 d,观察组TUNEL阳性细胞数显著降低(P<0.05),但ICH后3 d,两组TUNEL阳性细胞数差异无统计学意义(P>0.05);观察组铁蛋白重链和轻链表达显著减少(P<0.05);血浆铁蛋白水平在ICH后3、7 d有所降低,但与对照组比较,差异无统计学意义(P>0.05)。结论:DFX缓解老年大鼠急性ICH后神经细胞凋亡及神经功能缺损,可能是通过影响急性ICH的内源性反应而发挥保护作用。     

甲磺酸去铁胺促进大鼠颅骨临界骨缺损血管化骨再生的早期连续观察

目的:连续观察骨缺损愈合早期血管样组织和骨样组织的变化过程,初步了解局部施用甲磺酸去铁胺(deferoxamine mesylate,DFO)对血管化及骨再生的作用,验证DFO维持缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)活性的能力。方法:构建30只6~8周龄雄性SD大鼠颅骨缺损模型,随机分为DFO实验组和生理盐水对照组,于颅骨缺损后第4天分别局部注射200 μmol/L DFO溶液300 μL或生理盐水300 μL,并于缺损后第5、7、10、14和28天,每组每次处死3只大鼠。采用CD31免疫组织化学染色检测血管数量,采用HE染色、Masson染色观察成骨及矿化情况,以HIF-1α免疫组化染色检测HIF-1α蛋白相对表达量。结果:术后第5、7、10、14和28天,实验组血管数量(个)分别为:30.40±12.15、62.00±17.87、73.43±15.63、40.00±7.84和48.71±11.64,对照组血管数量(个)分别为:18.75±6.63、19.13±2.80、51.35±16.21、27.18±7.32和30.88±13.43,各时间点实验组血管数量均显著多于对照组(P<0.05);术后第14和28天,实验组新生骨组织较多,新生骨组织矿化百分比分别为(27.73±5.93)%和(46.53±3.66)%,对照组分别为(11.99±2.02)%和(31.98±4.22)%,这两个时间点实验组矿化百分比显著高于对照组(P<0.001);术后第5、7、10、14和28天,实验组相较对照组的HIF-1α蛋白相对表达量为2.86±0.48、1.32±0.26、1.32±0.32、1.28±0.38、1.05±0.34,仅术后第5天两组间差异有统计学意义(P<0.01)。结论:在骨缺损局部使用DFO可促进血管化及骨再生,可短暂维持HIF-1α蛋白活性。     

Growth,ribonucleotide reductase and metals in murine leukemic lymphocytes

Summary Trace metals are essential for the growth and several other properties of human lymphocytes. We studied the effects of media with variable concentrations of three metals (Fe²⁺, Cu²⁺, Zn²⁺), a metal chelator (deferoxamine, DFX) and a cell-growth inhibitor (hydroxy-urea) on the growth, intracellular metal concentration and activity of the enzyme ribonucleotide reductase in murine leukemic lymphocytes (L1210). Intracellular concentrations of Fe and Cu fluctuated within narrow limits in normal media, but decreased to very low concentrations in metal-poor media. The intracellular Zn concentration did not vary appreciably. Growth in intact cells decreased by 50%–70% when normal media were replaced by metal-poor media, but returned to control values when media were supplemented with gradually increasing concentrations of Fe and Cu. Fe and Cu had synergistic effects, while Zn had no stimulatory action. Hydroxyurea and DFX both inhibited cell growth, but only DFX inhibition was reversed by addition of metals. The addition of the above metals and inhibitors to the cell extracts produced effects on ribonucleotide reductase activity similar to those observed on the growth of whole cell preparations (stimulation by Fe and Cu, inhibition by Zn, DFX and hydroxyurea). These findings show that (a) the intracellular metal concentration is maintained in a narrow range during cell growth; (b) ribonucleotide reductase activity varies with cell growth; (c) ribonucleotide reductase activity and cell growth increase with Fe and Cu and decrease with Zn and DFX. Our data suggest that (a) Fe, Cu and Zn may have some effect on the growth and ribonucleotide reductase activity of L1210 cells, that (b) Fe, Cu and Zn may operate in a related and interdependent way and that (c) DFX inhibits cell growth probably through inhibition of the reductase activity and chelation of the Fe of its Fe-containing subunit. We conclude that any study on one of these metals should always include the other two and that manipulation of intracellular metals should be investigated as a potential therapeutic modulator of growth in leukemic lymphocytes.Abbreviations DFX deferoxamine     

Topical bilirubin-deferoxamine hastens excisional wound healing by modulating inflammation,oxidative stress,angiogenesis, and collagen deposition in diabetic rats

Aim of the studyThe study was performed to understand the detailed mechanism of diabetic wound healing by bilirubin-deferoxamine (DFO) combination on topical application.Materials and methodsThere were two study groups, control, and treatment. The granulation tissues collected on different days (3, 7, 14, and 19) were studied in detail for inflammatory mediators, angiogenesis markers, epithelialization, and oxidative stress parameters.ResultsA significant increase in wound contraction percentage was observed from day 7 in the bilirubin-DFO treatment group. The combinatorial treatment significantly reduced tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), and enhanced IL-10 levels. Upregulated mRNAs of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1 α) along with CD31 immunohistochemistry showed the pro-angiogenesis potential of the combination. Hematoxylin and Eosin (H and E) staining and Masson's trichrome staining showed reduced inflammatory cell infiltration, enhanced fibroblast proliferation, well-organized collagen fibers, and the development of new blood vessels. Collagen deposition is further supported by immunohistochemistry studies and Masson's trichrome staining. Bilirubin-DFO combination also reduced lipid peroxidation and elevated antioxidative enzymes.ConclusionTopical application of bilirubin-DFO showed immense potential in augmenting skin wound regeneration in diabetes by upregulating the antioxidant status as well as increasing angiogenesis, collagen deposition, and modulating cytokines.     

Chelators Controlling Metal Metabolism and Toxicity Pathways: Applications in Cancer Prevention,Diagnosis and Treatment

Chelating drugs and chelator metal complexes are used for the prevention, diagnosis and treatment of cancer. Cancer cells and normal cells require essential metal ions such as iron, copper and zinc for growth and proliferation. Chelators can target the metabolic pathways of cancer cells through the control of proteins involved in the regulation of these metals and also of other molecules involved in cell cycle control, angiogenesis and metastatic suppression. Other targets include the inhibition of specific proteins such as ribonucleotide reductase involved in DNA synthesis, the inhibition of free radical damage on DNA caused by iron and copper catalytic centers, the inhibition of microbial growth in immuno compromised cancer patients and the decorporation of radioactive and other toxic metals causing cancer. Chelating drugs and metal ions can affect the metabolism, efficacy and toxicity of anti-cancer drugs such as doxorubicin, mitozantrone, bleiomycin and hydroxyurea (HU). Although many experimental chelators have been shown to be effective as anti-cancer agents, only a few, e.g., dexrazoxane, deferoxamine (DFO) and triapine, have reached the stage of clinical testing or application. In many experimental models, deferiprone (L1) has been shown to be effective in cancer prevention and treatment, and in the inhibition of doxorubicin-induced cardiotoxicity. New anti-cancer drugs could be developed using chelators and chelator complexes with platinum and other metals, and also new protocols of combinations of chelators with known anti-cancer drugs.     

去铁胺对输血相关性铁过载患者骨密度的影响

目的观察输血相关铁过载患者在使用去铁胺(DFO)降铁治疗后骨密度的变化,探讨去铁胺对铁过载骨质疏松患者辅助治疗的应用前景。方法回顾性研究22例输血相关铁过载患者临床资料,检测患者DFO降铁治疗前后的骨密度和血清生化指标。采用配对样本t检验、Wilcoxon非参数检验分析研究患者DFO降铁治疗前后骨密度以及血清生化指标的变化。结果进行DFO有效的铁螯合治疗后,患者血清铁蛋白(Fer)明显下降,由基线水平的2019.95±630.77 ng/m L降至843.61±91.01 ng/m L(P0.05);股骨颈和腰椎的骨密度明显增加,分别由基线水平的0.73±0.12 g/cm2、0.92±0.14 g/cm2增加至0.77±0.09 g/cm2、0.94±0.14 g/cm2(P0.05);骨量正常、骨量减少和骨质疏松患者分别由治疗前的4、12、6例变为治疗后的10、11、1例(P0.05)。结论 DFO降铁治疗可改善输血相关铁过载患者的低骨量状态。本研究为DFO可能用于伴有铁过载的骨质疏松症的辅助治疗提供了有意义的支撑。     

Protective effect of deferoxamine on experimental spinal cord injury in rat

Objective

To observe the protective effect of deferoxamine on experimental spinal cord injury (SCI) in rats.

Methods

Sprague-Dawley rats were randomly divided into the following four groups. Control group: rats were performed laminectomy only; SCI group: rats were performed laminectomy with SCI; DFO group: rats were injected intraperitoneally a bolus of 100 mg/kg deferoxamine after SCI; vehicle group: rats were injected intraperitoneally 0.9% saline after SCI. The SCI of animal model was made by using a modified Allen's method on T₁₀. Six rats of each group were sacrificed at 4 h after injured, and the levels of free iron and malondialdehyde (MDA) of involved spinal cord segments were measured by bleomycin assay and the thiobarbituric acid (TBA) separately. The recovery of function was assessed by Modified Tarlov's scale and inclined plane method at 7, 14, 21 d after SCI. The histologic changes of the damaged spinal cord were also examined at 7 d after SCI.

Results

Following SCI, the levels of free iron and MDA were increased significantly and the Modified Tarlov's score and inclined plane angles decreased in SCI group and vehicle group. In DFO group, the levels of free iron and MDA were not increased, but the Modified Tarlov's score and inclined plane angles decreased, the histological findings were improved as well.

Conclusion

Deferoxamine can reduce the levels of free iron and lipid peroxidation, and improve the hind limb functional status of rats with spinal cord injury.