MCP-1参与缬沙坦对阿霉素心脏毒性的预保护作用

目的　探讨阿霉素诱导SD大鼠心肌损伤模型中单核细胞趋化蛋白-1（MCP-1）蛋白表达及其潜在的干预机制。方法　选择8周龄雄性SPF级SD大鼠30只,按照随机数字表法分为3组：对照组（Con组,生理盐水灌胃＋腹腔注射）、阿霉素组（Dox组,1.25 mL/kg阿霉素腹腔注射+2 mL/kg生理盐水灌胃）及缬沙坦+阿霉素组（Val+Dox组,1.25 mL/kg阿霉素腹腔注射+2 mL/kg缬沙坦灌胃）各10只,连续干预6周。10周时超声心动图检测心功能变化,留取大鼠心肌组织,电镜观察心肌超微结构变化,采用蛋白芯片技术检测各组血清MCP-1、调节激活正常T细胞表达和分泌因子（RANTES）及次级淋巴组织趋化因子（6Ckine）蛋白含量,采用Western blot检测各组心肌组织MCP-1蛋白表达。结果　超声结果显示,与Con组相比,Dox组和Val+Dox组左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）明显降低,左心室收缩末期内径（LVESD）、左心室舒张末期内径（LVEDD）明显升高（P＜0.05）。与Dox组相比,Val+Dox组LVEF、LVFS明显升高,LVESD、LVEDD明显降低（P＜0.05）。电镜下可见Con组心肌细胞结构清晰;Dox组心肌结构模糊,心肌溶解,可见自噬小体;Val+Dox组心肌结构模糊不清,线粒体体积增大、大小不等,但结构完整。与Con组相比,Dox组血清MCP-1、RANTES、6Ckine蛋白分别上调1.87倍、1.40倍和1.26倍（P＜0.05）。Western blot结果显示,Dox组与Con组相比,心肌组织MCP-1蛋白表达显著升高（P＜0.05）;Val+Dox组与Dox组相比,心肌组织MCP-1蛋白表达显著降低（P＜0.05）。结论　MCP-1蛋白可能参与阿霉素诱导的心肌损伤,缬沙坦可能通过下调MCP-1蛋白表达减轻心肌损伤。     

Interspecies prediction of pharmacokinetics and tissue distribution of doxorubicin by physiologically-based pharmacokinetic modeling

The aim of the study was to develop a physiologically-based pharmacokinetic (PBPK) model to describe and predict whole-body disposition of doxorubicin following intravenous administration. The PBPK model was established using previously published data in mice and included 10 tissue compartments: lungs, heart, brain, muscle, kidneys, pancreas, intestine, liver, spleen, adipose tissue, and plasma. Individual tissues were described by either perfusion-limited or permeability-limited models. All parameters were simultaneously estimated and the final model was able to describe murine data with good precision. The model was used for predicting doxorubicin disposition in rats, rabbits, dogs, and humans using interspecies scaling approaches and was qualified using plasma and tissue observed data. Reasonable prediction of the plasma pharmacokinetics and tissue distribution was achieved across all species. In conclusion, the PBPK model developed based on a rich dataset obtained from mice, was able to reasonably predict the disposition of doxorubicin in other preclinical species and humans. Applicability of the model for special populations, such as patients with hepatic impairment, was also demonstrated. The proposed model will be a valuable tool for optimization of exposure profiles of doxorubicin in human patients.     

Glutathione-dependent micelles based on carboxymethyl chitosan for delivery of doxorubicin

Novel glutathione (GSH)-dependent micelles based on carboxymethyl chitosan (CMCS) were developed for triggered intracellular release of doxorubicin (DOX). DOX-33′-Dithiobis (N-hydroxysuccinimidyl propionate)-CMCS (DOX-DSP-CMCS) prodrugs were synthesized. DOX was attached to the amino group on CMCS via disulfide bonds and drug-loaded micelles were formed by self-assembly. The micelles formed core–shell structure with CMCS and DOX as the shell and core, respectively, in aqueous media. The structure of the prodrugs was confirmed by IR and proton nuclear magnetic resonance (¹H NMR) spectroscopy. The drug-loading capacity determined by UV spectrophotometry was 4.96% and the critical micelle concentration of polymer prodrugs determined by pyrene fluorescence was 0.089 mg/mL. Micelles were spherical and the mean size of the nanoparticles was 174 nm, with a narrow polydispersity index of 0.106. Moreover, in vitro drug release experiments showed that the micelles were highly GSH-sensitive owing to the reductively degradable disulfide bonds. Cell counting kit (CCK-8) assays revealed that DOX-DSP-CMCS micelles exhibited effective cytotoxicity against HeLa cells. Moreover, confocal laser scanning microscopy (CLSM) demonstrated that DOX-DSP-CMCS micelles could efficiently deliver and release DOX in the cancer cells. In conclusion, the DOX-DSP-CMCS nanosystem is a promising drug delivery vehicle for cancer therapy.     

抗甲胎蛋白单链抗体与阿霉素联合对人肝癌细胞Huh7增殖的抑制作用

探讨抗人甲胎蛋白(AFP)单链抗体和阿霉素(DOX)单用或联用对人肝癌细胞Huh7细胞增殖的抑制作用。采用MTT法检测抗AFP单链抗体、阿霉素单用及联合作用对Huh7细胞增殖的影响;采用Annexin V/PI荧光双染法检测抗AFP单链抗体、阿霉素单用及联合作用对Huh7细胞凋亡的影响;采用PI单染法检测抗AFP单链抗体、阿霉素单用及联合作用对Huh7细胞周期分布的影响。结果表明,抗AFP单链抗体、阿霉素单药组与联用组均能有效抑制Huh7细胞增殖,呈剂量依赖性,且两药联用具有协同效应;抗AFP单链抗体、阿霉素单用及联用均可诱导细胞凋亡且抗AFP单链抗体(40 μg/mL)与阿霉素(0.25 μg/mL)联合组凋亡率明显高于单独给药组,具有统计学意义(P<0.05);抗AFP单链抗体(40 μg/mL)单独作用后,Huh7的细胞周期阻滞于G₀/G₁期,阿霉素(0.25 μg/mL)单独作用Huh7的细胞周期阻滞于S期,而当两种药物联用时,Huh7的细胞周期被阻滞于G₂/M期,抑制细胞增殖。     

阿霉素减轻坐骨神经慢性缩窄性损伤大鼠的神经病理性疼痛及其机制

目的观察阿霉素(DOX)对坐骨神经慢性缩窄性损伤(CCI)模型大鼠的镇痛作用,并从形态学及组织凋亡蛋白的角度对其机制进行分析。方法将SD大鼠随机分为4组:假手术组(Sham)、CCI模型组(Model)、假手术+阿霉素5 mg·kg^-1组(Sham+DOX)、CCI模型+阿霉素5 mg·kg^-1组(Model+DOX)。造模成功后,各组采用尾静脉注射的方式给药,Sham组和Model组给予等量生理盐水,检测各组大鼠机械痛阈值和热痛阈值。在行为学检测结束后,即手术后d 15取大鼠右侧L4-5DRG,观察DRG细胞形态、超微结构及DOX的分布情况,采用Western blot法测定DRG组织中Bax、Bcl-2、PKCɑ、PKCδ及PKCε的蛋白表达。结果静脉注射DOX可在DRG组织检测到其自发荧光表达。与Sham组相比,Sham+DOX组痛阈值在整个观察期未见差别,而Model组在术后d 7痛阈值明显降低。与Model组相比,Model+DOX组的痛阈值在给药后明显回升,并表现出DRG细胞明显损伤,Bax/Bcl-2升高以及PKCδ、PKCε的蛋白表达量降低等现象。结论 DOX静脉注射可以到达并蓄积于DRG组织,明显减轻CCI大鼠的疼痛反应,这一作用与其降低PKCδ和PKCε的蛋白表达,诱导DRG的凋亡有关。     

Sericin nanomicelles with enhanced cellular uptake and pH-triggered release of doxorubicin reverse cancer drug resistance

Drug resistance is the major challenge facing cancer chemotherapy and nanoscale delivery systems based on natural materials, such as sericin, are a promising means of overcoming drug resistance. Yet, no attempt of introducing synthetic poly(γ-benzyl-L-glutamate) (PBLG) onto sericin polypeptide to fabricate a facile biocompatible and biodegradable micelle has been tried. Here, we prepared a polypeptide-based amphiphilic polymer containing hydrophilic sericin polypeptide backbone and PBLG side chains via ring-opening polymerization (ROP) strategy. The introduction of PBLG side chains remarkably enhances the stability of sericin micelles in water. Meanwhile, the micelles exhibited a high loading capacity and pH-responsive release ability for antitumor drug doxorubicin (DOX), called sericin-PBLG-DOX. Owing to the excellent cell membrane penetration of sericin-PBLG, the cellular uptake of DOX when loaded into micelles was improved. Subsequently, sericin-PBLG-DOX was transferred into perinuclear lysosomes, where the release rate of DOX was accelerated. Compared to the same dose of DOX, sericin-PBLG-DOX could induce a more efficient anti-tumor effect both in vitro and in vivo, and these micelles have promise for future clinical applications in overcoming cancer drug resistance with good biosafety, enhanced cellular uptake, pH-triggered drug release, efficient anti-tumor effects, and minimized systemic toxicity.     

Doxorubicin induces ZAKα overexpression with a subsequent enhancement of apoptosis and attenuation of survivability in human osteosarcoma cells

Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX‐treated human OS cells.     

Characterization of the interaction forces in a drug carrier complex of doxorubicin with a drug‐binding peptide

Polypeptide‐based materials are used as building blocks for drug delivery systems aimed at toxicity decrease in chemotherapeutics. A molecular‐level approach is adopted for investigating the non‐covalent interactions between doxorubicin and a recently synthesized drug‐binging peptide as a key part of a system for delivery to neoplastic cells. Molecular dynamics simulations in aqueous solution at room and body temperature are applied to investigate the structure and the binding modes within the drug–peptide complex. The tryptophans are outlined as the main chemotherapeutic adsorption sites, and the importance of their placement in the peptide sequence is highlighted. The drug–peptide binging energy is evaluated by density functional theory calculations. Principal component analysis reveals comparable importance of several types of interaction for the binding strength. π‐Stacking is dominant, but other factors are also significant: intercalation, peptide backbone stacking, electrostatics, dispersion, and solvation. Intra‐ and intermolecular H‐bonding also stabilizes the complexes. The influence of solvent molecules on the binding energy is mild. The obtained data characterize the drug‐to‐peptide attachment as a mainly attractive collective process with interactions spanning a broad range of values. These results explain with atomistic detail the experimentally registered doxorubicin‐binging ability of the peptide and outline the complex as a prospective carrying unit that can be employed in design of drug delivery systems.     

P167肽修饰的多柔比星长循环脂质体在大鼠体内的药动学和药效学

目的研究P167肽修饰对多柔比星脂质体大鼠静脉给药的体内药动学和药效学的影响。方法通过pH梯度法分别制备多柔比星长循环脂质体（DOX—SSL）和P167肽修饰的多柔比星长循环脂质体（DOX—P167一SSL）。12只sD大鼠随机分为2组,分另q尾静脉注射DOX．SSL和DOX—P167-SSL,给药剂量为2．5mg·kg-1。采用HPLC内标法测定不同给药时间的血清多柔比星浓度,用WinNonlin软件拟合并计算药动学参数。制备大鼠脑胶质瘤模型,经核磁扫描测定肿瘤体积,均匀分组,分别尾静脉注射生理盐水、DOX—SSL和DOX．P167．SSL,每组6只动物,给药剂量为2．5mg·kg-1,3d给药1次,共给药5次。以相对肿瘤增殖率（T／C％）为指标评价药效。结果DOX—SSL和DOX—P167-SSL大鼠尾静脉给药后,均符合二室模型。DOX—P167-SSL组的tl／,t1/2及AUC分别为4．89min、837．57rain、610．35min·mg·L-1,均低于DOX—SSL组的16．23、2940．48和1301．16min·mg·L-1。DOX—P167-SSL组T／C％为7．32％,而DOX—SSL组为37．07％。结论P167肽的修饰使多柔比星长循环脂质体的长循环效果降低,但药效得到明显提高,这可能与P167肽的穿透作用有关。     

泊洛沙姆188-PLGA纳米给药系统对抗耐药肿瘤的研究

目的 利用泊洛沙姆188对PLGA进行化学修饰,制备包载阿霉素的纳米粒,并评价纳米粒在人耐药乳腺癌细胞中的摄取能力及毒性。方法 通过EDC/NHS法合成泊洛沙姆188-PLGA,通过核磁共振对其结构进行表征并测定临界胶束浓度;通过纳米沉淀法制备包载阿霉素的纳米粒,通过粒度仪对纳米粒的粒径及分布进行分析,通过细胞摄取实验及细胞毒性实验对纳米粒的摄取效果及毒性进行评价。结果 成功合成了泊洛沙姆188-PLGA,并制备了粒径在140 nm左右的纳米粒,该纳米粒在人耐药乳腺癌细胞中有较好的摄取效果及较强的毒性。结论 泊洛沙姆188能够逆转耐药,增强耐药细胞对化疗药物的敏感程度。