重建线粒体动态平衡对NK细胞NK2型活化影响

目的立于“线粒体功能紊乱-细胞异常活化”视角,探讨NK细胞NK2型活化机制。方法将NK-92MI细胞分为空白组、TSLP组、1、5、10μmol·L^-1 Mdivi-1组。ELISA法测定各组IL-4、IL-5、IFN-γ水平;蛋白印迹法分析各组p-Drp1、MnSOD蛋白表达;DHE染色及流式检测各组ROS水平;激光共聚焦观察各组线粒体形态。结果ELISA显示,与对照组比较,TSLP组IL-4、IL-5水平明显升高,IFN-γ水平下调(P<0.05);与TSLP组比较,5、10μmol·L^-1 Mdivi-1组IL-4、IL-5水平降低,10μmol·L^-1 Mdivi-1组IFN-γ浓度有所回升(P<0.05)。DHE染色及流式显示,TSLP组ROS水平较对照组升高;而5、10μmol·L^-1 Mdivi-1组ROS水平较TSLP组降低(P<0.05)。共聚焦显示,TSLP刺激后,细胞内大量圆球状线粒体生成,5、10μmol·L^-1 Mdivi-1干预后该现象得到改善。蛋白印迹显示,TSLP组p-Drp1水平明显上调,MnSOD表达下降,Mdivi-1干预则有效逆转了上述变化。结论线粒体动态失衡可能是NK细胞异常活化内在机制之一,其或是调控NK细胞NK2型活化介导的变应性炎症反应的重要靶点。     

Endogenous factors regulating neuronal apoptosis]

Apoptosis and necrosis of neurons induced by glutamate and nitric oxide (NO) are associated with various disorders including hypoxic-ischemic brain injury, Alzheimer's disease and Parkinson's disease. In search of endogenous protective factors that inhibit NO-mediated glutamate neurotoxicity, we found that excitotoxicity is suppressed by certain neurotransmitters such as nicotinic acetylcholine and dopamine and growth factors such as NGF and BDNF. We recently purified and isolated a novel neuroprotective substance, which has been named 'serofendic acid', from a lipophilic fraction of fetal calf serum. Mass spectrometry and NMR spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane-type diterpenoid. Serofendic acid exhibited potent protective actions on cortical neurons against neurotoxicity of a NO donor as well as of glutamate, although it did not show appreciable influences on glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on NO radicals but was capable of inhibiting the generation of hydroxyl radicals. These findings suggest that serofendic acid is a low-molecular-weight bioactive factor that promotes survival of CNS neurons, probably through the attenuation of free radical-mediated insults.     

Molecular mechanisms regulating the mitochondrial targeting of microsomal cytochrome P450 enzymes

Cytochrome P450 enzymes (CYPs) are a superfamily of monooxygenases found in almost all living organisms. CYPs are predominantly localized in the endoplasmic reticulum membranes as integral membrane proteins, where they metabolize a variety of endogenous and xenobiotic compounds. CYPs also reside in other subcellular compartments, including the plasma membranes and mitochondria. CYP localization in mitochondria is regulated in one of two ways: (1) direct targeting of inherent CYPs with canonical mitochondrial signals in their protein sequence after synthesis in the cytosol or (2) mitochondrial localization of microsomal CYPs after processing of the NH(2)-terminal region. Microsomal CYPs targeted to mitochondria demonstrate conventional or altered catalytic activities using electrons provided by the mitochondrial electron transport system. Mechanisms of microsomal CYP targeting to mitochondria, regulation of localization, and the implications of these in drug metabolism are described in the present review.     

基于线粒体动力学失衡调控研究锁阳醋酸乙酯提取物对阿尔茨海默症小鼠行为学的改善作用

目的 探究锁阳醋酸乙酯提取物（ECS）对阿尔茨海默症（AD）模型小鼠行为学、线粒体组织形态学及线粒体动力学相关蛋白表达的影响和作用机制。方法 采用8周龄AD模型5xFAD转基因小鼠为模型组,8周龄野生型C57BL/6小鼠为对照组,ECS组为模型鼠ig ECS 47 mg/kg,每天1次,连续14 d。给药6和12周分别进行跳台、水迷宫、旷场行为学检测;给药12周后取海马部位进行电镜观察;Western blotting检测线粒体融合相关蛋白MFN1、OPA1和线粒体分裂相关蛋白DRP1的表达。结果 水迷宫结果显示,给药6周,3组间均无显著性差异;给药12周后,与对照组比较,模型组小鼠潜伏期时间延长（P<0.05）,穿越平台次数显著减少（P<0.05）;与模型组比较,ECS组小鼠潜伏期时间显著缩短（P<0.05）,穿越平台次数显著增加（P<0.05）;跳台结果显示,与对照组比较,给药6周及12周,模型组小鼠跳台潜伏期时间显著缩短（P<0.05）,跳台次数显著增多（P<0.05）;与模型组比较,给药6周,ECS组无显著性差异;给药12周,ECS组跳台潜伏期时间显著延长（P<0.05）,跳台次数显著减少（P<0.05）。旷场结果显示,给药12周,三组均无显著性差异。电镜结果显示,对照组线粒体结构清晰膜完整,嵴排列整齐;模型组线粒体膜破裂、直径变小、嵴扭曲模糊;ECS组线粒体嵴不完全变形,部分基质出现空泡。Western blotting结果显示,与对照组比较,模型组OPA1及MFN1蛋白表达水平显著下调,DRP1蛋白表达水平显著上调（P<0.05）;与模型组比较,ECS组MFN1、OPA1蛋白表达水平显著上调（P<0.05、0.01）,DRP1蛋白表达水平显著下调（P<0.05）。结论 ECS显著改善AD模型小鼠的行为学,机制可能与调节线粒体动力学失衡相关。     

含硅羟基磷灰石调节巨噬细胞极性促进成骨分化机制研究

摘要：目的　探讨无机骨再生修复材料含硅羟基磷灰石（si-HA）通过调节巨噬细胞极性转换促进小鼠前成骨细胞MC3T3-E1（3T3）的增殖及成骨分化的作用机制。方法　制备羟基磷灰石（HA）和si-HA纳米粒子,以10 mg/L的剂量刺激小鼠RAW264.7巨噬细胞（RAW,分别为HA组和si-HA组）,另设Control组（10%FBS的DMEM培养基）。分析RAW细胞的极化状态及炎性因子的表达水平。收集完全培养基、HA和si-HA纳米粒子刺激RAW细胞3 d后获得的上清液,制备RAW条件培养基、HA+RAW条件培养基和si-HA+RAW条件培养基,并培养小鼠3T3前成骨细胞（分别为RAW组、HA＋RAW组和si-HA＋RAW组）,并设空白对照组（完全培养基）,检测各组细胞增殖和成骨相关基因［M1极化标志物诱导型一氧化氮合酶（iNOS）、M2极化标志物Arginase基因及促炎、抗炎因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6、IL-10和IL-1ra］的表达水平。结果　si-HA组巨噬细胞iNOS、TNF-α、IL-1β mRNA表达水平低于Control组和HA组,Arginase、IL-10和IL-1ra mRNA表达水平高于Control组和HA组（P＜0.05）;IL-6 mRNA表达水平低于HA组（P＜0.05）,与Control组差异无统计学意义。si-HA+RAW组3T3细胞增殖能力、碱性磷酸酶（ALP）活性及矿化结节形成明显强于空白对照组、RAW组和HA+RAW组（P＜0.05）。培养14 d时,si-HA+RAW组3T3细胞ALP、OCN、OPN、Col-1、Runx2 mRNA表达水平均高于其他3组（P＜0.05）。结论　si-HA纳米粒子可诱导巨噬细胞产生较有利的骨免疫微环境,增强3T3细胞的增殖和成骨分化。     

茵陈蒿汤调节胆汁酸代谢并干预阻塞性黄疸大鼠肝细胞线粒体DNA损伤的研究

摘要：目的　探讨茵陈蒿汤调节胆汁酸代谢对阻塞性黄疸大鼠肝细胞线粒体DNA（mtDNA）损伤的影响及其意义。方法　SPF级雄性成年SD大鼠54只,随机分为假手术组、模型组、茵陈蒿汤组,每组18只。梗阻建模第3天后,模型组每日给予生理盐水灌胃,茵陈蒿汤组每日给予茵陈蒿汤灌胃,各组观察时相点为建模后第1、3、8天,每时相点处死大鼠6只。检测血总胆红素（TBIL）、直接胆红素（DBIL）、丙氨酸转氨酶（ALT）、γ-谷氨酰转肽酶（GGT）、总胆汁酸（TBA）,尿TBA及尿量变化情况,实时荧光定量PCR法检测肝细胞mtDNA的相对量。结果　给予茵陈蒿汤治疗的大鼠,循环血中胆汁酸含量降低（P＜0.05）,尿量及尿胆汁酸含量均增加（P＜0.05）。模型组与茵陈蒿汤组mtDNA的相对量均较假手术组减少,茵陈蒿汤组mtDNA的相对量第8天较第3天回升。结论　茵陈蒿汤可能通过肝-肾轴途径调节胆汁酸代谢以减少肝细胞mtDNA损伤,进而改善阻塞性黄疸大鼠肝功能。     

A review on environmental factors regulating arsenic methylation in humans

Subjects exposed to arsenic show significant inter-individual variation in urinary patterns of arsenic metabolites but insignificant day-to-day intra-individual variation. The inter-individual variation in arsenic methylation can be partly responsible for the variation in susceptibility to arsenic toxicity. Wide inter-ethnic variation and family correlation in urinary arsenic profile suggest a genetic effect on arsenic metabolism. In this paper the environmental factors affecting arsenic metabolism are reviewed. Methylation capacity might reduce with increasing dosage of arsenic exposure. Furthermore, women, especially at pregnancy, have better methylation capacity than their men counterparts, probably due to the effect of estrogen. Children might have better methylation capacity than adults and age shows inconsistent relevance in adults. Smoking and alcohol consumption might be associated with a poorer methylation capacity. Nutritional status is important in the methylation capacity and folate may facilitate the methylation and excretion of arsenic. Besides, general health conditions and medications might influence the arsenic methylation capacity; and technical problems can cause biased estimates. The consumption of seafood, seaweed, rice and other food with high arsenic contents and the extent of cooking and arsenic-containing water used in food preparation may also interfere with the presentation of the urinary arsenic profile. Future studies are necessary to clarify the effects of the various arsenic metabolites including the trivalent methylated forms on the development of arsenic-induced human diseases with the consideration of the effects of confounding factors and the interactions with other effect modifiers.     

氯化壳聚糖季铵盐的调血脂活性研究

目的 观察氯化壳聚糖季铵盐(TMCC)对Wistar大鼠的调血脂作用。方法 大鼠随机分为正常(NG)组、高脂模型(HF)组、壳聚糖(CTS)组、TMCC组和考来烯胺(CLST)阳性对照组。除NG组给予普通饲料外,其余4组都给予高脂饲料。4周后,停止给予高脂饲料,NG、HF组给予生理盐水灌胃,其余3组,按166.7mg/(kg·d)分别给予CTS、TMCC、CLST。4周后测定各组血浆总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)。结果　与HF组比较,TMCC组能明显降低TC、TG、LDL-C水平(P<0.01),但对HDL-C作用不明显(P>0.05)。TMCC降低TC、LDL-C的能力比CTS和CLST强。结论CTS结构修饰物TMCC调血脂活性优于CTS。     

表没食子儿茶素没食子酸酯调控食管癌细胞线粒体膜电位诱导细胞凋亡作用研究

目的 探讨表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)调控食管癌细胞Ec9706线粒体膜电位诱导细胞凋亡作用.方法 不同浓度(100、200、300 mg/L) EGCG作用Ec9706细胞24 h后,Annexin V/PI双标流式细胞术方法检测细胞凋亡水平;流式细胞术检测细胞线粒体膜电位;Western Blot方法检测细胞中Caspase-3蛋白表达水平,0.9％氯化钠溶液代替EGCG作为对照组.结果 100、200、300 mg/L EGCG作用Ec9706细胞24 h后,与对照组相比Ec9706细胞凋亡率显著增高(P＜0.01),300 mg/L EGCG组细胞凋亡率显著高于100、200 mg/L EGCG组(P＜0.01).EGCG可显著降低细胞线粒体膜电位(P＜0.01),且具有剂量依赖性.western-blot结果显示,EGCG组与对照组相比Caspase-3蛋白表达水平显著增高(P＜0.05).结论 EGCG具有诱导食管癌Ec9706细胞凋亡作用,其作用机制可能与调控线粒体膜电位引起内源性细胞凋亡有关.     

Direct quantification of mitochondria and mitochondrial DNA dynamics

Mitochondria are known to be one of major organelles within a cell and to play a crucial role in many cellular functions. These organelles show the dynamic behaviors such as fusion, fission and the movement along cytoskeletal tracks. Besides mitochondria, mitochondrial DNA is also highly motile. Molecular analysis revealed that several proteins are involved in mitochondria and mitochondrial DNA dynamics. In addition to the degeneration of specific nerves with high energy requirement, mutation of genes coding these proteins results in metabolic diseases. During the last few years, a significant amount of relevant data has been obtained on molecular basis of these diseases but mitochondrial dynamics in cells derived from the patients is poorly understood. So far time-lapse fluorescence microscopy, fluorescence recovery after photo bleaching and image correlation methods have been used to study organellar motion. Especially, image correlation method has possibility to evaluate diffusion coefficient of mitochondria and mitochondrial DNA simultaneously and directly. When we search candidates for compounds that modulate mitochondrial dynamics by high throughput screening, image correlation method may be useful although the careful interpretation is required for crowded and heterogeneous environment within a cell.     

基于线粒体通透性转换孔调控的脓毒症心肌损伤及普拉克索保护机制的研究

目的: 探究普拉克索能否通过抑制心肌细胞MPTP开放减轻脓毒症大鼠心功能障碍。方法: 将SD大鼠分为假手术组、盲肠结扎穿孔术组(CLP组)、盲肠结扎穿孔术+环孢菌素A组(CsA组)、盲肠结扎穿孔术+普拉克索组(PPX组)。CLP组、CsA组和PPX组统一使用盲肠结扎穿孔术建立脓毒症大鼠模型,其中CsA组静脉注射CsA(2 mg·kg^-1),PPX组予以普拉克索(1 mg·kg^-1)灌胃。观察SD大鼠一般情况、存活率及腹腔解剖情况,超声心动图检测心功能,ELISA法测定血清PCT、NT-proBNP、cTnI水平,通过线粒体肿胀测定MPTP的开放程度,HE染色及电镜观察各组心脏组织病理改变。结果: 术后36 h,假手术组大鼠一般情况、腹腔解剖及心脏病理改变正常,CLP组明显变差,CsA组及PPX组病变程度较CLP组减轻;各组大鼠的存活率:假手术组(100%)＞CsA组(75.0%)＞PPX组(62.5%)＞CLP组(43.8%);CsA组、PPX组心脏左室射血分数均低于假手术组(P＜0.05),均较CLP组升高(P＜0.05);CLP组、CsA组、PPX组血清PCT、NT-proBNP、cTnI水平均高于假手术组(P＜0.05),CsA组、PPX组低于CLP组(P＜0.05);CLP组、CsA组、PPX组心肌细胞MPTP的开放程度大于假手术组(P＜0.05),CsA组、PPX组小于CLP组(P＜0.05)。结论: 心肌细胞MPTP的异常开放是引起大鼠脓毒症心肌损伤的机制之一,普拉克索可以通过抑制心肌细胞MPTP的异常开放减轻脓毒症大鼠的心功能损伤。     

降糖茶调节血糖作用的研究

目的研究降糖茶的调节血糖作用。方法采用四氧嘧啶制备动物模型 ,进行降糖试验和糖耐量试验。结果灌胃 3 0d降糖茶高 ( 3 .75g kg)、中 ( 1.2 5g kg)、低 ( 0 .62 5g kg)剂量组的降糖作用分别为 3 4 .70 %、2 6.2 5 %和2 1.5 4 %。结论降糖茶对四氧嘧啶致糖尿病小鼠具有明显的降血糖作用和增强小鼠糖耐量作用。     

Endogenous factors regulating neuronal death induced by radical stress

Neurons in the central nervous system (CNS) are vulnerable to radical stress caused by reactive oxygen species, including nitric oxide (NO). Those radicals play crucial roles in glutamate neurotoxicity associated with ischemic brain injury and a wide range of neurodegenerative disorders. In our previous studies, we have shown evidence suggesting that glutamate neurotoxicity is regulated by certain endogenous substances such as neurotrophins, nicotinic acetylcholine, prostanoids and vitamins. Based on those findings, we have used the term 'neuroprotective factor' for endogenous substances possessing protective activity against glutamate neurotoxicity, and have further searched for a candidate with unique structure. We isolated a novel neuroprotective substance named 'serofendic acid' derived from fetal calf serum. The compound exhibited potent protective action against neurotoxicity induced by glutamate and by an NO donor without inhibiting glutamate receptors. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on NO, but was capable of inhibiting the generation of a hydroxyl radical, a presumed 'executor' radical in the nitric oxide-mediated neurotoxic cascade. The chemical structure was determined by mass spectrometry and nuclear magnetic resonance spectroscopy, and was confirmed by synthesis. The structure was unique among known endogenous substances because the compound was a sulfur-containing atisane type diterpenoid. The discovery of serofendic acid may provide a new scope for the investigation of low-molecular weight bioactive factors promoting the survival of CNS neurons.     

A novel pathway regulating the mammalian target of rapamycin (mTOR) signaling

Originally discovered as an anti-fungal agent, the bacterial macrolide rapamycin is a potent immunosuppressant and a promising anti-cancer drug. In complex with its cellular receptor, the FK506-binding protein (FKBP12), rapamycin binds and inhibits the function of the mammalian target of rapamycin (mTOR). By mediating amino acid sufficiency, mTOR governs signaling to translational regulation and other cellular functions by converging with the phosphatidylinositol 3-kinase (PI3K) pathway on downstream effectors. Whether mTOR receives mitogenic signals in addition to nutrient-sensing has been an unresolved issue, and the mechanism of action of rapamycin remained unknown. Our recent findings have revealed a novel link between mitogenic signals and mTOR via the lipid second messenger phosphatidic acid (PA), and suggested a role for mTOR in the integration of nutrient and mitogen signals. A molecular mechanism for rapamycin inhibition of mTOR signaling is proposed, in which a putative interaction between PA and mTOR is abolished by rapamycin binding. Collective evidence further implicates the regulation of the rapamycin-sensitive signaling circuitry by phospholipase D, and potentially by other upstream regulators such as the conventional protein kinase C, the Rho and ARF families of small G proteins, and calcium ions. As the mTOR pathway has been demonstrated to be an important anti-cancer target, the identification of new components and novel regulatory modes in mTOR signaling will facilitate the future development of diagnostic and therapeutic strategies.     

新型前体脂质体载药及影响因素考察

Aim A new proliposomal technology was used to trap several drugs, such as tegafur, silymarin, cistanosides, oleanolic acid. And then these proliposomal characters were studied. Methods These proliposomes formed liposomes after mixing with water. And then the liposomal morphology was determined by electron microscope, and the liposomal particle size determined by particle sizes instrument. The trap efficiency was determined by the column chromatography, and then the influence factors on the trap efficiency were investigated. Results The liposomes looked round, some with multiply layers, the particle was small, and the ξ potential was about -30 mV. The trap efficiency changed with the partition coefficient and pH. When the partition coefficient and pH increased, the trap efficiency increased. Furthermore, the trap efficiency was not influenced by the molecular weight. Conclusion This kind of liposomal technology trapped the drugs efficiently, and the lipophilic drugs were trapped more easily. Some Chinese traditional drugs could be trapped too.     

Membrane potentials regulating GPCRs: insights from experiments and molecular dynamics simulations

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Targeting protein interaction networks in mitochondrial dynamics for cancer therapy

Mitochondria are crucial organelles that provide energy via oxidative phosphorylation in eukaryotic cells and also have critical roles in growth, division, and the cell cycle, as well as the rapid adaptation required to meet the metabolic needs of the cell. Mitochondrial processes are highly dynamic; fusion and fission can vary with cell type, cellular context, and stress levels. Accumulating evidence demonstrates that an imbalance in mitochondrial dynamics leads to death in numerous types of human cancer cells. Therefore, modulating mitochondrial dynamics could be a therapeutic target. In this review, we provide an overview of the protein interaction networks involved in mitochondrial dynamics as effective and feasible drug targets and discuss the related potential therapeutic strategies for cancer.