泛素-蛋白酶体系统在心肌梗死中的病理生理意义

心肌梗死后心室重塑是慢性心力衰竭的主要原因.因凋亡和坏死而导致的心肌细胞死亡可造成心肌重塑,病理性心肌肥大和左心室扩张是其主要特征.研究表明,泛素蛋白酶体系统(ubiquitin proteasome system,UPS)可能参与这一过程.     

细胞铁死亡发生机制及其敏感性对肿瘤的影响

细胞死亡是机体正常生理和病理过程中均可发生的生物学事件.深入研究细胞死亡对预见或调节(抑制或诱导)细胞死亡、认识不同细胞死亡形式与炎症、肿瘤、免疫的关系有重要意义,进而进一步预测其对机体或疾病可能的影响及可能产生的临床意义.近年对铁死亡的认识和研究逐渐深入,诱导肿瘤细胞铁死亡已成为肿瘤治疗策略,但是诱导癌细胞铁死亡可能...     

白藜芦醇苷通过上调SIRT3和SDF-1延缓大鼠心肌纤维化

目的观察白藜芦醇苷对阿霉素诱导心肌纤维化与免疫细胞因子表达的作用及机制。方法雄性SD大鼠20只,随机分为4组,每组5只。对照组(常规喂养)、阿霉素组(腹腔注射2. 5 mg/kg阿霉素,2次/周)、白藜芦醇苷组(腹腔注射50 mg/kg白藜芦醇苷,1次/日,其余处理同阿霉素组)、尼克酰胺组(腹腔注射500 mg/kg尼克酰胺,2次/周,其余处理同白藜芦醇苷组)。Van Gieson染色法观察心肌纤维化,免疫组织化学染色法检测心肌组织1型胶原蛋白(Col1)、Col3表达,酶标法检测心肌羟脯氨酸(HYP)含量,试剂盒检测超氧化物歧化酶(SOD)、丙二醛(MDA)与还原型谷胱甘肽(GSH)水平,ELISA检测肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1)、IL-10含量,反转录PCR检测沉默信息调节因子1(SIRT1)、SIRT3、基质细胞衍生因子1(SDF-1)与转化生长因子β(TGF-β)的mRNA水平,Western blot法检测心肌SIRT1、SIRT3、SDF-1与TGF-β的蛋白水平。结果阿霉素促进心肌纤维化,降低心肌组织中IL-10的水平并抑制SIRT3、SDF-1 mRNA与蛋白表达,增加TNF-α、IL-1的水平并促进TGF-βmRNA与蛋白表达。白藜芦醇苷明显抑制阿霉素诱导的心肌纤维化效应,降低HYP与Col3水平,同时增加GSH与SOD含量,降低MDA含量。显著增加阿霉素诱导后心肌细胞SIRT3、SDF-1 mRNA与蛋白水平,抑制TGF-β表达,尼克酰胺能抑制白藜芦醇苷上述作用。结论白藜芦醇苷可通过上调SIRT3表达提高抗心肌纤维化能力,降低阿霉素诱导氧化应激水平、抑制炎性细胞因子表达。     

应激所致心肌损伤相关蛋白质的研究进展

应激所致心肌损伤是一个复杂的生物学过程,其中有大量蛋白质家族和成员的参与.蛋白质是生命活动的直接执行者,其功能状况决定着应激性心肌损伤的发生和程度.本文综述了应激诱导心肌损伤发生过程中涉及到的主要蛋白质及其作用.     

细胞内信号通路对心肌肥大调控作用的研究进展

心肌肥大是心肌细胞从成熟的“收缩状态”向“胚胎型合成状态”转化的一种现象,是心力衰竭发展过程中的主要病理生理过程.心肌成纤维细胞约占心肌细胞总数的60％～70％,它们参与细胞外基质的合成和沉积、细胞信号调控、物质代谢等.同时,其能够在受损或衰竭心脏中诱导纤维化性心肌重塑,调节心脏功能.     

circNCX1通过miR-103-3p调节阿霉素诱导的心肌细胞凋亡

目的:探究环状RNA NCX1(circNCX1)对阿霉素(又称多柔比星,DOX)诱导的心肌细胞凋亡的调节作用。方法:通过RT-qPCR检测经DOX处理的H9c2细胞和注射DOX的小鼠心肌组织中circNCX1表达量的变化;TUNEL染色和凋亡相关蛋白caspase-3/-7的活性检测试剂盒检测细胞凋亡水平;台盼蓝染色检测细胞存活率;RNA pull-down实验验证circNCX1与其下游微小RNA-103-3p(miR-103-3p)的直接结合。结果:DOX诱导的H9c2细胞及小鼠心肌组织中circNCX1升高,并且DOX可诱导心肌细胞凋亡,表现为心肌细胞TUNEL阳性率及caspase-3/-7活性升高。抑制circNCX1表达能够显著减少DOX诱导的心肌细胞TUNEL阳性率,并且降低caspase-3/-7的活性。circNCX1通过直接结合miR-103-3p抑制其表达。此外,过表达miR-103-3p同样能够抑制DOX诱导的心肌细胞死亡和caspase-3/-7活性。结论:circNCX1通过靶向miR-103-3p促进DOX诱导的心肌细胞凋亡。     

内皮间质转化参与阿霉素诱导的大鼠心肌纤维化

目的研究阿霉素对心肌纤维化形成的机制。方法构建大鼠阿霉素心肌纤维化模型,HE染色和Masson染色检测心肌病理变化,RT-q PCR和免疫组化检测纤维化相关基因Col1α、Col3α和TGF-β1的表达。免疫荧光技术检测心肌组织内皮细胞标志物v WF和间质细胞标志物α-SMA的表达。结果给大鼠腹腔注射阿霉素(2 mg/kg,每周1次)出现心室腔扩大、心壁变薄、心肌纤维间隙增宽、心肌溶解、心肌纤维化、心肌血管内皮损伤和通透性增加。随着阿霉素作用时间延长,实验组相比于对照组心肌纤维化程度明显(P0.05),纤维化相关因子Col1α、Col3α和TGF-β1的表达增多(P0.05),阿霉素心肌纤维化大鼠心脏血管内皮细胞出现内皮间质转化。结论阿霉素可诱导大鼠不可逆的心肌纤维化,内皮间质转化参与阿霉素心肌纤维化的形成。     

009 死亡受体及线粒体途径与激活诱导T细胞凋亡

免疫系统形成了一系列确保激活的淋巴细胞能被有效清除的分子及信号途径,称之为激活诱导的细胞死亡.激活诱导的细胞死亡主要负责调节免疫细胞稳态及清除自身反应性淋巴细胞.死亡受体途径及线粒体死亡途径分别是细胞凋亡的外在途径和内在途径,也参与了激活诱导的T细胞凋亡,本文就外周成熟T细胞凋亡相关的死亡受体途径、线粒体死亡途径及其相关调控机制研究进展作一综述.     

鸢尾素有效降低阿霉素诱导心肌损伤大鼠炎症反应和心肌细胞凋亡

目的研究鸢尾素(Irisin)对阿霉素诱导心肌损伤大鼠的炎症反应和心肌细胞凋亡的影响及其作用机制。方法采用阿霉素诱导方法建立Sprague-Dawley(SD)大鼠心肌损伤模型并随机分成正常对照组(control group)、心肌损伤模型组(model group)和低、中、高浓度鸢尾素处理组。Irisin处理4周后,检测各组大鼠心功能指标(HR、LVSP、LVEDP、+dp/dtmax、-dp/dtmax),HE染色检测心肌组织病理变化,流式细胞术检测心肌组织中活性氧(ROS)水平;ELISA方法检测心肌组织中炎症因子(IL-6、IL-10、TNF-α、IFN-γ)水平;Tunel染色检测心肌组织细胞凋亡情况;免疫印迹法检测心肌组织中p-AKT/AKT水平变化。结果模型组大鼠心功能指标显著下降,且心肌组织病理损伤严重;心肌组织中ROS阳性比例、炎症因子水平和细胞凋亡率均显著上升,而p-AKT/AKT水平显著下降(P0.05)。经低、中、高浓度Irisin作用后,各组大鼠心功能指标逐渐回升,心肌组织病理损伤也逐渐减轻,且心肌组织中ROS阳性比例、炎症因子水平和细胞凋亡率均逐渐降低,而p-AKT/AKT水平显著上升(P0.05)。结论 Irisin有效降低阿霉素诱导的心肌损伤,通过抑制炎症反应、ROS水平和心肌细胞凋亡发挥作用并与AKT磷酸化相关。     

晚期糖基化终产物受体在小鼠慢性阿霉素心脏毒性模型中的表达

目的探讨晚期糖基化终产物受体(RAGE)在小鼠慢性阿霉素心脏毒性模型中的表达及其与化疗药相关心脏毒性的可能内在联系。方法通过多次腹腔注射阿霉素,建立小鼠慢性阿霉素心脏毒性模型,同时设立正常对照组(每组6只)。模型建立后采用超声心动图和组织病理学方法评价小鼠心脏功能及心肌损伤程度;通过免疫印迹及免疫组织化学检测小鼠心肌组织中RAGE表达变化。结果超声心动图检查显示,慢性阿霉素心脏毒性小鼠左室射血分数明显降低;病理学观察显示心肌组织中细胞损伤明显,同时伴有心室肥厚及心肌重构,提示小鼠慢性阿霉素心脏毒性模型建立成功。细胞和分子生物学观察发现,损伤心肌中RAGE表达显著升高。结论RAGE在慢性小鼠阿霉素心脏毒性模型中表达升高,提示其可能参与阿霉素相关心脏毒性的发生发展。     

Influence of copper (I) nicotinate complex and autophagy modulation on doxorubicin-induced cytotoxicity in HCC1806 breast cancer cells

PurposeDoxorubicin is regarded as the most therapeutic active agent available for triple-negative breast cancer (TNBC) treatment. However, the development of drug resistance and toxicity limits its effectiveness. Thus, developing novel strategies for TNBC treatment remains a significant challenge and doxorubicin-based combinations either by metal complexes (Copper I nicotinate complex) or with autophagy modulators could provide novel strategies and alternative strategies contributed to cancer cell death pathways, autophagy and apoptosis.Materials and methodsThe viability of HCC1806 TNBC cells and IC₅₀ values of Doxorubicin (DOX), Torin-1 (TOR), Chloroquine (CQ) and Copper (I) nicotinate complex (CNC) were assessed by MTT assay. ELISA was used for detecting microtubule-associated protein 1 light chain 3 (LC3) level. Real time PCR was used to determine (NBR1) gene expression. Cell cycle analysis and quantitative detection of acid vesicular organelles (AVOs) was performed by flow cytometry. TOR and CQ were used as autophagy modulators for induction and suppression of autophagy, respectively.ResultsThe half-maximal inhibition effect of TOR combination with DOX was revealed to the induction of autophagic cell death and apoptotic cell death. On the other hand, combination of CQ with DOX increased the growth inhibitory effect, induced accumulation of AVOs and suppressed apoptotic cell death. However, combination of CNC with DOX inhibited autophagy and induced cell cycle arrest.ConclusionDoxorubicin drug based combinations either with TOR, CQ or CNC could positively affect DOX effectiveness and reduce DOX doses applied on HCC1806 cells through modulation of autophagy.     

Doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza H3N2 virus: involvement of matrix metalloproteinases

Acute respiratory distress syndrome, a severe form of acute lung injury (ALI), is a major cause of death during influenza pneumonia. We have provided evidence for the involvement of recruited neutrophils, their toxic enzymes such as myeloperoxidase and matrix metalloproteinases (MMPs), and neutrophil extracellular traps in aggravating alveolar-capillary damage. In this study, we investigated the effects of doxycycline (DOX), an inhibitor of MMPs, on influenza-induced ALI. BALB/c mice were infected with a sublethal dose of mouse-adapted virulent influenza A/Aichi/2/68 (H3N2) virus, and administered daily with 20mg/kg or 60 mg/kg DOX orally. The effects of DOX on ALI were determined by measuring inflammation, capillary leakage, and MMP activities. Furthermore, levels of T1-α (a membrane protein of alveolar type I epithelium) and thrombomodulin (an endothelial protein) in the bronchoalveolar lavage fluid were evaluated by Western blot analysis. Our results demonstrate significantly decreased inflammation and protein leakage in the lungs after DOX treatment. Levels of MMP-2 and MMP-9 activity, T1-α and thrombomodulin were also diminished in the DOX-treated group. These findings were corroborated by histopathologic analyses, which demonstrated significant reduction in lung damage. Although DOX treatment reduced ALI, there were no effects on virus titers and body weights. Taken together, these results demonstrate that DOX may be useful in ameliorating ALI during influenza pneumonia. Further studies are warranted to determine whether DOX can be used in combination with anti-viral agents to alleviate severe influenza pneumonia.     

A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex)_1.3(DOX)₂₀. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.     

Clarithromycin effectively enhances doxorubicin-induced cytotoxicity and apoptosis in MCF7 cells through dysregulation of autophagy

PurposeUse of autophagy inhibitors in combination with chemotherapy has become a novel chemotherapeutic strategy. In this study, we aimed to determine whether the effectiveness of doxorubicin (DOX) is augmented by clarithromycin (CAM) in MCF7 cells and the molecular mechanisms involved.Materials and methodsCombined cytotoxicity of CAM and DOX was assessed by MTT assay and was analyzed using the Chou-Talalay's method. To clarify the underlying mechanisms, several factors, including apoptosis (Annexin V/propidium iodide staining), intracellular level of DOX (spectrofluorimetry) and P-glycoprotein activity (Rhodamin 123 efflux assay) were measured. In addition, autophagy was evaluated by intracellular labeling with anti-LC3II and LysoTrackerGreen (LTG) staining and analyzed by flowcytometry.ResultsThe anti-proliferation effect of DOX was synergistically enhanced by CAM in MCF7 cells and was associated with an increase in the apoptotic cell death. However, the intracellular level of DOX remained unchanged in the presence of CAM. Based on the findings, 100 μM of CAM did not exhibit any inhibitory effects on P-glycoprotein activity. Flow cytometric analysis indicated that DOX at IC₂₀ concentration induced the autophagy flux, as confirmed by the increased level of LC3II and LTG signals. Moreover, combined treatment with DOX and CAM resulted in more pronounced LTG signals, but no change in LC3II. These results indicate that CAM blocks the autophagy flux induced by DOX.ConclusionsThese findings suggest that suppression of autophagy by CAM may promote chemotherapeutic outcome in breast cancer. However, further investigations are needed to evaluate the application of CAM in adjuvant breast cancer therapy.     

Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin in vitro

ABSTRACT: BACKGROUND: Doxorubicin (DOX) is an effective antineoplastic drug; however, clinical use of DOX is limited by its dose-dependent cardiotoxicity. It is well known that reactive oxygen species (ROS) play a vital role in the pathological process of DOX-induced cardiotoxicity. For this study, we evaluated the protective effects of guggulsterone (GS), a steroid obtained from myrrh, to determine its preliminary mechanisms in defending against DOX-induced cytotoxicity in H9C2 cells. METHODS: In this study, we used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release measurements, and Hoechst 33258 staining to evaluate the protective effect of GS against DOX-induced cytotoxicity in H9C2 cells. In addition, we observed the immunofluorescence of intracellular ROS and measured lipid peroxidation, caspase-3 activity, and apoptosis-related proteins by using Western blotting. RESULTS: The MTT assay and LDH release showed that treatment using GS (1--30 muM) did not cause cytotoxicity. Furthermore, GS inhibited DOX (1 muM)-induced cytotoxicity in a concentration-dependent manner. Hoechst 33258 staining showed that GS significantly reduced DOX-induced apoptosis and cell death. Using GS at a dose of 10--30 muM significantly reduced intracellular ROS and the formation of MDA in the supernatant of DOX-treated H9C2 cells and suppressed caspase-3 activity to reference levels. In immunoblot analysis, pretreatment using GS significantly reversed DOX-induced decrease of PARP, caspase-3 and bcl-2, and increase of bax, cytochrome C release, cleaved-PARP and cleaved-caspase-3. In addition, the properties of DOX-induced cancer cell (DLD-1 cells) death did not interfere when combined GS and DOX. CONCLUSION: These data provide considerable evidence that GS could serve as a novel cardioprotective agent against DOX-induced cardiotoxicity.     

工程化纳米囊泡结合化疗用于增强肿瘤免疫治疗

目的：结合免疫治疗与化疗,以改善肿瘤的治疗效果,为安全有效的肿瘤免疫治疗策略的发展提供更多见解。方法：使用工程化纳米囊泡,囊泡膜上表达PD-1受体,可以靶向肿瘤细胞表面的PD-L1,通过破坏PD-1/PD-L1免疫抑制通路增强抗肿瘤反应。同时,囊泡包裹的化疗药物阿霉素可以进入肿瘤细胞核,抑制DNA与RNA的合成,诱导肿瘤细胞死亡。结果：实验证实,制备的PD1-阿霉素材料具备良好的稳定性、安全性,能准确靶向肿瘤部位,阿霉素在细胞核部位起作用,能有效地进行肿瘤杀伤。结论：本研究首次将PD-1免疫检查点抑制与化疗药物阿霉素相结合,利用PD-1囊泡安全性高、长循环的特点作为包裹化疗药物阿霉素的载体,这种方法可以进行肿瘤细胞的有效靶向与治疗,实现肿瘤的有效清除。     

Monodisperse core-shell structured up-conversion Yb(OH)CO₃@YbPO₄:Er³+ hollow spheres as drug carriers

In this work, we report a facile solution-phase synthesis of monodisperse core-shell structured Yb(OH)CO?@YbPO? hollow spheres (size around 380 nm) by utilizing the colloidal sphere of Yb(OH)CO? as the sacrificial template via the Kirkendall effect. The Er3+ doped Yb(OH)CO?@YbPO? core-shell hollow spheres can be prepared similarly, which exhibit strong green emission under 980 nm NIR laser excitation even after loading with drug molecules. Most importantly, the sample can be used as an effective drug delivery carrier. The biocompatibility test on L929 fibroblast cells using MTT assay reveals low cytotoxicity of the system. A typical anticancer drug, doxorubicin hydrochloride (DOX), is used for drug loading, and the release properties, cytotoxicity, uptake behavior and therapeutic effects were examined. It is found that DOX is shuttled into cell by core-shell hollow spheres carrier and released inside cells after endocytosis, and the DOX-loaded spheres exhibited greater cytotoxicity than free DOX. These results indicate that the core-shell Er3+ doped Yb(OH)CO?@YbPO? hollow spheres have potential for drug loading and delivery into cancer cells to induce cell death.     

Stereocomplex micelle efficiently transports Doxorubicin for enhanced lymphoma suppression in vivo

The application of Doxorubicin (DOX) in the chemotherapy for lymphoma is seriously hampered by the side effects of DOX, especially the cardiotoxicity and nephrotoxicity. Nanoscale micelle as a promising drug delivery system has gained more and more interest in malignancy chemotherapy. In this study, we successfully fabricated DOX-loaded stereocomplex micelle (SCM/DOX) from the equimolar mixture of the enantiomeric four-armed poly(ethylene glycol)-polylactide (PDM and PLM) copolymers. The SCM/DOX showed proper hydrodynamic size of ~90 nm and slow DOX release in phosphate-buffered saline at pH 7.4. The antitumor activities of DOX, PDM/DOX, PLM/DOX, and SCM/DOX toward lymphoma cells were tested in vitro and in vivo. Our data demonstrated that the SCM/DOX more effectively inhibited the cell proliferation than PDM/DOX, PLM/DOX, and free DOX in vitro. In the in vivo antitumor test, the SCM/DOX more effectively inhibited the growth of EL4 lymphoma, too. In addition, the body weight loss caused by SCM/DOX was alleviated than DOX. More importantly, the cardiotoxicity, nephrotoxicity, and hepatotoxicity caused by DOX in mice were obviously attenuated compared to the free DOX treatment group. Taken together, all the results indicated that the SCM/DOX could inhibit the growth of EL4 lymphoma cells and attenuate the toxicity of DOX more efficiently, which suggested SCM/DOX was promising for the prevention and treatment of lymphoma.     

Wpływ cytotoksyczny R-amfinazy,endorybonukleazy o działaniu przeciwnowotworowym,na komórki chłoniaka rozlanego z dużych komórek B w warunkach in vitro

Onconase (ONC) and R-Amphinase (R-AM) are enzymes with anti-tumor activity, belonging to pancreatic ribonuclease A family, received from eggs collected from frog Rana pipiens. Both proteins can induce death of some types of neoplastic cells by inhibition of protein synthesis, cell growth and proliferation. The aim of this study was to assess the cytotoxicity of R-AM, used alone or in combination with one of the most active anti-leukemic drug, doxorubicin (DOX), on diffuse large B-cell lymphoma (DLBCL)-derived cell line, Toledo. We found high cytotoxic activity of R-AM against DLBCL cells as well as influence on expression of several apoptosis-regulating proteins. Moreover, we observed increase in proapoptotic activity after combination of R-AM and DOX, compared with both drugs used alone. These results may justify further studies on interactions of R-AM with other drugs active in DLBCL.     

Effects of Fullerenol C60(OH)24 Nanoparticles on a Single-dose Doxorubicin-induced Cardiotoxicity in Pigs: An Ultrastructural Study

Abstract

Cardioprotective effects of fullerenol C₆₀(OH)₂₄ nanoparticles (FNP) were investigated in pigs after a single treatment with doxorubicin (DOX). Semithin and ultrathin sections of myocardial tissue routinely prepared for transmission electron microscopy were analyzed. Extensive intracellular damage was confirmed in cardiomyocytes of DOX-treated animals. By means of ultrastructural analysis, a certain degree of parenchymal degeneration was confirmed even in animals treated with FNP alone, including both the oral and the intraperitoneal application of the substance. The cardioprotective effects of FNP in animals previously treated with DOX were recognized to a certain extent, but were not fully confirmed at the ultrastructural level. Nevertheless, the myocardial morphology of DOX-treated animals improved after the admission of FNP. Irregular orientation of myofibrils, myofibrillar disruption, intracellular edema, and vacuolization were reduced, but not completely eliminated. Reduction of these cellular alterations was achieved if FNP was applied orally 6?h prior to DOX treatment in a dose of 18?mg/kg. However, numerous defects, including the inner mitochondrial membrane and the plasma membrane disruption of certain cells persisted. In FNP/DOX-treated animals, the presence of multinuclear cells with mitosis-like figures resembling metaphase or anaphase were observed, indicating that DOX and FNP could have a complex influence on the cell cycle of cardiomyocytes. Based on this experiment, further careful increase in dosage may be advised to enhance FNP-induced cardioprotection. These investigations should, however, always be combined with ultrastructural analysis. The FNP/DOX interaction is an excellent model for the investigation of cardiomyocyte cell death and cell cycle mechanisms.