Scavenging of reactive oxygen species induced by hyperthermia in biological fluid

The scavenging activity of rat plasma against hyperthermia-induced reactive oxygen species was tested. The glutathione-dependent reduction of a nitroxyl radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, which was restricted by adding superoxide dismutase or by deoxygenating the reaction mixture, was applied to an index of superoxide (O₂^•−) generation. A reaction mixture containing 0.1 mM 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl and 1 mM glutathione was prepared using 100 mM phosphate buffer containing 0.05 mM diethylenetriaminepentaacetic acid. The reaction mixture was kept in a screw-top vial and incubated in a water bath at 37 or 44°C. The time course of the electron paramagnetic resonance signal of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl in the reaction mixture was measured by an X-band EPR spectrometer (JEOL, Tokyo, Japan). When the same experiment was performed using rat plasma instead of 100 mM PB, the glutathione-dependent reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, i.e., generation of O₂^•−, was not obtained. Only the first-order decay reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, which indicates direct reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, was obtained in rat plasma. Adding 0.5% albumin to the phosphate buffer reaction mixture could almost completely inhibit O₂^•− generation at 37°C. However, addition of 0.5% albumin could not inhibit O₂^•− generation at 44°C, i.e., hyperthermic temperature. Ascorbic acid also showed inhibition of O₂^•− generation by 0.01 mM at 37°C, but 0.02 mM or more could inhibit O₂^•− generation at 44°C. A higher concentration of ascorbic acid showed first-order reduction, i.e., direct one-electron reduction, of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl. Hyperthermia-induced O₂^•− generation in rat plasma can be mostly inhibited by albumin and ascorbic acid in the plasma.     

阿托伐他汀对自发性高血压大鼠血压的影响

目的:观察阿托伐他汀对自发性高血压大鼠(SHR)血压的影响,初步探讨其调节血压的机制。方法:SHR(n=12)随机分为SHR对照组、阿托伐他汀治疗组,同龄WKY大鼠(n=6)作为正常血压对照组,灌胃给药6周。采用尾袖法测量大鼠尾动脉收缩压,硝酸还原酶法测定血清NO浓度,化学比色法检测NOS活性和ROS活性,放免分析法测定心肌AngⅡ含量,透射电镜观察主动脉内皮超微结构改变。结果:阿托伐他汀治疗6周后,SHR治疗组SBP明显下降(P<0.01);心肌AngⅡ浓度明显下降(P<0.05);血清NO浓度和主动脉NOS活性明显增高(P<0.01,P<0.05),血清ROS水平明显降低(P<0.05)并明显改善SHR主动脉内皮的超微结构改变。结论:阿托伐他汀可降低SHR血压,可能是通过增加主动脉NOS活性,升高血清NO含量,减少ROS生成,改善血管内皮功能而发挥降低血压作用。     

远志及其近缘种的ITS序列分析及鉴定

目的:对远志属3种药用植物进行分子鉴定。方法:利用PCR直接测序法对远志ITS序列进行测定,结合Genbank中远志、卵叶远志和瓜子金的ITS序列,经Clustalx比对后,用软件MEGA3.1计算了Kimura 2-Parameter(K2P)距离,并构建了NJ(邻接)树。结果:远志属3种药用植物ITS1长度为269 bp~271 bp,ITS2长度为216 bp~217 bp,变异位点26个,信息位点1个;瓜子金与其他样品的遗传距离最大,卵叶远志与远志2个Genbank样品遗传距离最小。结论:ITS序列无法区别卵叶远志和远志,但可以作为瓜子金的分子鉴定依据。     

A tamoxifen derivative,N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine,selectively targets P-glycoprotein-positive multidrug resistant Chinese hamster cells

DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to DPPE alone correlated with the levels of P-gp expression. Moreover, in MDR cells, DPPE-induced apoptosis was significantly reduced with Bcl2 overexpression and in the presence of P-gp ATPase inhibitor, PSC833. Furthermore, knockdown of P-gp expression in MDR cells with P-gp-siRNA reversed DPPE sensitivity and increased their sensitivity to doxorubicin and taxol but not to cisplatin. The addition of DPPE to membrane fractions led to dose-dependent increase in P-gp ATPase that was inhibited with PSC833. Moreover, incubation of P-gp positive cells with DPPE led to a significant increase in superoxide levels and a drop in cellular ATP and GSH pools that were reversible with inhibitors of P-gp ATPase. The combined presence of DPPE and the mitochondria electron transport complex III inhibitor, antimycin A, synergized in their effects on the growth of MDR cells but had no effect on the growth of parental drug sensitive cells. Collectively, the results of this study provide a possible mechanism that may be relevant to the clinical results of DPPE in breast cancer trial and demonstrates DPPE as P-gp collateral sensitivity drug.     

The relationships among monocyte subsets,miRNAs and inflammatory cytokines in patients with acute myocardial infarction

Background

Acute myocardial infarction (AMI) causes irreversible myocardial damage and release of inflammatory mediators, including cytokines, chemokines and miRNAs. We aimed to investigate changes in the levels of cytokines (IL-6, TNF-α and IL-10), miRNAs profiles (miR-146 and miR-155) and distribution of different monocyte subsets (CD14++CD16-, CD14++CD16+, CD14+CD16++) in the acute and post-healing phases of AMI.

Genetic basis of dyslipidemia in disease precipitation of coronary artery disease (CAD) associated type 2 diabetes mellitus (T2DM)

Type 2 diabetes mellitus (T2DM) and its complications are linked to environmental, clinical, and genetic factors. This review analyses the disorders of lipids and their genetics with respect to coronary artery disease (CAD) associated with T2DM. Cell organelles, hepatitis C‐virus infection, reactive oxygen species produced in mitochondria, and defective insulin signaling due to the arrest of G1 phase to S phase transition of β‐cells have significant roles in the precipitation of the diseases. Adiponectin is anti‐inflammatory and anti‐atherosclerotic and improves insulin resistance. Low‐density lipoprotein (LDL) is atherosclerotic, and LDL‐cholesterol in T2DM is associated with high‐cardiovascular risk. Further, LDL cholesterol reduction significantly reduces cardiovascular morbidity and mortality. High‐density lipoprotein (HDL) is also anti‐atherosclerotic due to HDL associated paraoxonase‐1 serum enzyme, which prevents LDL oxidative modifications and the development of CAD. Moreover, elevated apolipoprotein B and apolipoprotein A‐I (ApoB/ApoA‐I) ratio in plasma is also a risk factor for CAD. LDL receptor, adiponectin, and endocannabinoid receptor‐1 genes are independently associated with CAD and T2DM. Polymorphism of Apo E2 (epsilon2) is a positive factor to increase the T2DM risk and Apo E4 (epsilon4) is a negative factor to reduce the disease risk. Taq 1B polymorphism of cholesterol ester transfer protein (CETP) gene contributes to the development of atherosclerosis, whereas haplotypes of APOA5, APOC3, APOC4, and APOC5 genes are in the same cluster and are independently associated with high plasma triglyceride level, CAD and T2DM. In conclusion, because various genes, LDLR, CETP, APOA5, Apo E, Apo B, and Apo A‐I, are associated with the precipitation of CAD associated with T2DM, a personalized diet–gene intervention therapy may be advocated to reduce the disease precipitation. Copyright © 2014 John Wiley & Sons, Ltd.     

云南省16个县(市)黄胸鼠体表恙螨种类调查

目的了解云南省黄胸鼠（Rattus flavipectus）体表恙螨的寄生、种类组成、优势种及其空间分布型.方法选取云南省16个县（市）作为调查点,在现场用鼠笼加食饵诱捕黄胸鼠,收集耳壳及耳窝全部恙螨,用Hoyer＇s液封片后在显微镜下分类、鉴定.恙螨种群的空间分布格局用分布型指数中的聚块指数（m＊/m）测定.结果在所调查的16个县（市）中,从13个县（市）捕获黄胸鼠725只,共采集恙螨2 710只,经鉴定隶属3亚科10属42种.印度囊棒恙螨（Ascoschoengastia indica）、微板无前恙螨（Walchia micropelta）、李氏囊棒恙螨（A.leechi）、蒙大微恙螨（Microtrombicula munda）为优势螨种,其带螨率和螨指数较高,依次分别为6.8%、5.5%、3.9%、4.3%和1.65、0.64、0 43、0 42.结论黄胸鼠体表恙螨种类复杂,数量丰富,呈聚集型分布.     

Reactive oxygen species in rostral ventrolateral medulla modulate cardiac sympathetic afferent reflex in rats

Aim: The aim of the present study was to investigate whether reactive oxygen species (ROS) in rostral ventrolateral medulla (RVLM) modulate cardiac sympathetic afferent reflex (CSAR) and the enhanced CSAR response caused by microinjection of angiotensin II (Ang II) into the paraventricular nucleus (PVN). Methods: Under urethane and α‐chloralose anaesthesia, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic‐denervated and cervical‐vagotomized rats. The CSAR was evaluated by the RSNA response to epicardial application of capsaicin (1.0 nmol). Results: Bilateral RVLM microinjection of tempol (a superoxide anion scavenger) or polyethylene glycol‐superoxide dismutase (PEG‐SOD, an analogue of endogenous superoxide dismutase) attenuated the CSAR, but did not cause significant change in baseline RSNA and MAP. NAD(P)H oxidase inhibitors apocynin or phenylarsine oxide (PAO) also showed similar effects, but SOD inhibitor diethyldithio‐carbamic acid (DETC) enhanced the CSAR and baseline RSNA, and increased the baseline MAP. Bilateral PVN microinjection of Ang II (0.3 nmol) enhanced the CSAR and increased RSNA and MAP, which was inhibited by the pre‐treatment with RVLM administration of tempol, PEG‐SOD, apocynin or PAO. The pre‐treatment with DETC in the RVLM only showed a tendency in potentiating the CSAR response of Ang II in the PVN, but significantly potentiated the RSNA and MAP responses of Ang II. Conclusion: These results suggest that the NAD(P)H oxidase‐derived ROS in the RVLM modulate the CSAR. The ROS in the RVLM is necessary for the enhanced CSAR response caused by Ang II in the PVN.     

NLRP3炎症小体介导血管紧张素Ⅱ诱导的人脐静脉血管内皮细胞炎症因子IL-1β的表达

目的研究血管紧张素Ⅱ（AngⅡ）对人脐静脉血管内皮细胞（HUVECs）NLPR3 炎症小体激活与炎症因子白介素-1β （IL-1β）表达的影响。方法体外培养HUVECs,用AngⅡ的不同浓度和刺激时间刺激HUVECs,找到最适合的AngⅡ刺激浓度 与时间。AngⅡ刺激HUVECs前,预用AngⅡ受体阻断剂氯沙坦阻断AngⅡ作用;NAD（P）H抑制剂DPI或H2O2清除剂CAT降 低胞内活性氧（ROS）含量;用caspase-1抑制剂YVAD阻断caspase-1作用;用NLRP3 siRNA沉默胞内NLRP3表达。运用蛋白 印迹法（western blot）分别检测细胞内NOX4、NLRP3、caspase-1以及IL-1β含量。结果（1）HUVECs在浓度10-9MAngⅡ刺激 12 h后,胞内NOX4、NLRP3、caspase-1以及IL-1β的蛋白表达显著增加;（2）氯沙坦、DPI、CAT、YVAD和NLRP3 siRNA都可减 轻AngⅡ诱导的上述反应。结论AngⅡ通过促进HUVECs活性氧生成,激活NLRP3炎症小体,促进胞内炎症介质IL-1β P17活 性片段生成增加,诱导血管炎症的发生。     

Identification of potential therapeutic target of naringenin in breast cancer stem cells inhibition by bioinformatics and in vitro studies

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.