目的:分离提取心肌缺血预适应(IPC)模型大鼠循环血中的细胞微囊泡(MVs),探究IPC处理对循环血MVs中microRNAs(miRNAs)表达的影响.方法:健康雄性Wistar大鼠8只,随机分为IPC-MVs组和假手术对照(Sham-MVs)组,每组4只.建立大鼠心肌IPC模型,提取循环血IPC-MVs,采用Microarray分析两组循环血MVs中的miRNAs,利用生物信息学进行靶基因预测及功能分析.结果:与Sham-MVs组比较,IPC-MVs组中循环血差异表达显著上调的miRNAs共3个(P<0.01,FER<0.05):miR-1-3p、miR-133a-3p和miR-133b-3p(t=3.194、3.002、3.389,均P<0.05).基因本体研究会数据库(GO)和京都基因与基因组百科全书(KEGG)分析显示,这些miRNAs可能通过调节心肌细胞中丝裂原活化蛋白激酶(MAPK)信号通路和Ras信号通路,发挥抗心肌缺血/再灌注(I/R)损伤的保护作用,可能参与调控的核心靶基因包括Ntrk2、Igf1、Gnai3和Bcl2l1.结论:IPC处理可使大鼠循环血MVs中miR-1-3p、miR-133a-3p和miR-133b-3p表达显著上调.

Tetrathiatriarylmethyl (TAM, trityl) radicals have found wide applications in electron paramagnetic resonance (EPR) oximetry. However, the biomedical applications of TAM radicals were exclusively limited to an extracellular region owing to their negatively charged nature. The intracellular delivery of TAM radicals still remains a challenge. In the present work, we report a liposome-based method to encapsulate the water-soluble Finland trityl radical CT-03 for its intracellular delivery. Using the thin lipid film hydration method, CT-03-loaded liposomes were prepared from DSPC/cholesterol/DOTAP with a mean size of 167.5 ± 2.4 nm and a zeta potential of 27.8 ± 0.8 mV. EPR results showed that CT-03 was entrapped into the liposomes and still exhibited good oxygen (O2) sensitivity. Moreover, CT-03 was successfully delivered into HepG2 cells and HUVECs using the CT-03-loaded liposomes. Importantly, the combination of the liposome-encapsulated radical CT-03 and the other TAM radical CT02-H enabled simultaneous measurements of the intracellular and extracellular O2 concentrations and O2 consumption rates in HepG2 cells. Our present study provides a new approach for intracellular delivery of TAM radicals and could significantly expand their biomedical applications.

Background Microvesicles (MVs) are submicron membrane vesicles as mediators of intercellular communication. The aim of our study was to investigate protective mechanism of circulating MVs derived from ischemic preconditioning (IPC-MVs) on myocardial I/R injury. Results Administration of IPC-MVs reduced infarct size and activity of lactate dehydrogenase (LDH) in myocardial I/R injury in vivo. Meanwhile, IPC-MVs could increase cell viability and reduce LDH activity in hypoxia/reoxygenation (H/R) injured H9c2 cells in vitro. Microarray analysis demonstrated that miR-133a-3p expression in IPC-MVs increased apparently compared with Sham-MVs. We found that miR-133a-3p increased cell viability, decreased LDH activity and apoptosis , as well as suppressed H/R-induced endoplasmic reticulum stress (ERS). MVs induced by hypoxic preconditioning enriched with FAM-miR-133a-3p allowed the transfer of miR-133a-3p to target cells. In addition, miR-133a-3p was significantly increased in H/R injured H9c2 cells by treatment with IPC-MVs. Epidermal growth factor receptor (EGFR) is a target gene of miR-133a-3p. AG1478 (EGFR inhibitor) significantly increased cell viability, decreased LDH activity and ERS-induced apoptosis in H9c2 cells under H/R injury. Conclusions The findings of this study showed that IPC-MVs exerted cardioprotective effects by transferring miR-133a-3p into H/R injured cardiomyocytes targeting EGFR, thus attenuating ERS-induced apoptosis. MiR-133a-3p transferred by IPC-MVs may provide a novel therapy for myocardial I/R injury.

Acute lung injury (ALI) is a major cause of morbidity and mortality globally, and is characterized by widespread inflammation in the lungs. Increased production of reactive oxygen species is hypothesized to be associated with ALI. Matrine and lycopene are active products present in traditional Chinese medicine. Matrine is an effective inhibitor of inflammation, whereas lycopene decreases lipid peroxidation. Therefore, it was hypothesized that combinatorial treatment with matrine and lycopene may provide synergistic protection against ALI. In the present study, mice were treated with dexamethasone (DEX; 5 mg/kg), matrine (25 mg/kg), lycopene (100 mg/kg), and matrine (25 mg/kg) + lycopene (100 mg/kg) for 7 days prior to injury induction using lipopolysaccharide (LPS; 5 mg/kg) for 6 h. Lung tissues were collected following the sacrifice of the mice and hematoxylin and eosin staining was used for histological analysis. Malondialdehyde (MDA), glutathione (GSH) and myeloperoxidas (MPO) levels were examined by respective kits. The expressions of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were evaluated by ELISA. The expressions of I kappa B alpha and NF-kappa B p65 were examined by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. The results indicated that the combined treatment exhibited a similar effect to DEX, both of which attenuated lung structural injuries, downregulated the expressions of IL-6, TNF-alpha, MPO and MDA, and upregulated that of GSH. Furthermore, the combined treatment and DEX inhibited NF-kappa B p65 activation. The present study revealed that combined treatment with matrine and lycopene exhibited protective effects on an LPS-induced mouse model of ALI, suggesting that they may serve as a potential alternative to glucocorticoid therapy for ALI.

目的 研究养正消积胶囊的化学成分.方法 采用气相色谱-质谱(GC-MS)和超高效液相色谱-四极杆串联飞行时间质谱联用技术(UPLC-Q-TOF-MS)对养正消积胶囊中脂溶性成分和水溶性成分进行分析.在GC-MS条件下,选用PH-5毛细管色谱柱(0.25 mm×30 m,0.25 μm),采用程序升温法,EI离子源,质量扫描范围为m/z 40～650;在UPLC-Q-TOF-MS条件下,选用ACQUITY UPLC HSS T3超高效液相色谱柱(2.1 mm×100mm,1.8μm),采用15 mmol·L-1甲酸铵水-乙腈为流动相,梯度洗脱,ESI源,质量扫描范围:m/z 50 ～1 500.结果 采用GC-MS共鉴定出脂溶性成分20种,包括挥发油、倍半萜类、酚类和脂类等成分,主要来源于莪术、徐长卿和茯苓.采用UPLC-Q-TOF-MS共鉴定出水溶性成分38种,包括三萜类及三萜皂苷、黄酮类、环烯醚萜苷类及有机酚酸等,主要来源于人参、黄芪和女贞子.结论 本实验提供了一种快速、有效的方法,全面系统地对养正消积胶囊的化学成分进行定性分析,为药效物质基础研究、药物质量控制、药理作用机制等研究奠定基础.

目的:分离提取缺氧预适应(HPC)诱导人脐静脉内皮细胞(HUVECs)分泌的微囊泡(MVs),探讨HPC-EMVs对正常H9c2心肌细胞的作用和其Caspase 3、Bcl-2和Bax表达的影响.方法:采用HPC方法诱导HUVECs释放MVs,将其和正常培养的大鼠H9c2心肌细胞孵育,通过MTT法检测细胞存活率和比色法测定细胞培养液中乳酸脱氢酶(LDH)活性;通过Hoechst 33258染色观察细胞核的形态变化;比色法测定培养液中Caspase 3活性以及Western blot法检测H9c2细胞中Bcl-2和Bax的表达.结果:HPC模型构建过程中,与缺氧/复氧(H/R)组相比,HPC组细胞存活率显著升高(P＜0.01),即成功构建HUVECs的HPC模型;HPC-EMVs处理H9c2细胞结果发现,与对照组H9c2细胞相比,HPC-EMVs 30、60μg/mL组细胞存活率显著降低(P＜0.001),且LDH活性和Caspase 3活性显著升高(P＜0.01或P＜0.001),凋亡细胞量增加,Bcl-2/Bax比值显著降低(P＜0.001).结论:成功采用HPC的方法诱导HUVECs释放MVs,HPC-EMVs通过影响正常H9c2细胞Caspase 3、Bcl-2和Bax蛋白的表达发挥促凋亡作用.

Idiopathic Pulmonary fibrosis is a disease with high morbidity and mortality. Therefore, the development of new drugs is imperative. Gallic acid derivative is a derivative of Gallic acid that can be extracted from Chinese herbal medicine. In previous experiments, we found that Gallic acid derivative played dual roles in inflammatory and antioxidant activities. Meanwhile, Gallic acid derivative could inhibit the proliferation of lung fibroblast. In the present study, we investigated the function of Gallic acid derivative in inhibiting lung fibrosis. 5 mg/kg of bleomycin was administered to mice by a single intratracheal instillation. Three dosages of Gallic acid derivative (75 mg/kg, 150 mg/kg, 300 mg/kg) and Pirfenidone (80 mg/kg) were given to mice for 21 day. Gallic acid derivative treatment significantly reduced lung histological changes and decreased inflammatory cell infiltration. The content of collagen decreased with the decrease of hydroxyproline level. Analogously, the expression of alpha smooth muscle actin was reduced. Gallic acid derivative enhanced the antioxidant status, but reduced the expression of interleukin 6, NADPH oxidase-4. Our study proved that Gallic acid derivative reduced inflammation activation to some extent and could exert its effects through transforming growth factor β1/Smad2 signaling pathway and balancing NOX4/Nrf2.

Microvesicles (MVs) have been shown to be involved in pathophysiology of ischemic heart diseases. However, the underlying mechanisms are still unclear. Here we investigated the effects of MVs derived from ischemic preconditioning (IPC-MVs) on myocardial ischemic/reperfusion (I/R) injury in rats. Myocardial IPC model was elicited by three cycles of ischemia and reperfusion of the left anterior descending (LAD) coronary artery. IPC-MVs from the peripheral blood of the above animal model were isolated by ultracentrifugation and characterized by flow cytometry and transmission electron microscopy. IPC-MVs were administered intravenously (7 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min ischemia and 120-min reperfusion of LAD in rats. We found that total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (< 1 µm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats respectively. Additionally, treatment with IPC-MVs significantly alleviated damage of myocardium, and restored cardiac function of I/R injury rats, as evidenced by increased heart rate, and decreased the elevation of ST-segment. The size of myocardial infarction, lactate dehydrogenase activity, and the number of apoptotic cardiomyocytes were also reduced significantly with IPC-MVs treatment, coincident with the above function amelioration. Moreover, IPC-MVs decreased the activity of caspase 3, and the expression of endoplasmic reticulum stress (ERS) markers, GRP78, CHOP and caspase 12 indicating the involvement of ERS-specific apoptosis in I/R injury, and cardioprotective effects of IPC-MVs. In summary, our study demonstrated a novel mechanism of IPC in which circulating IPC-MVs could protect hearts from I/R injury in rats through attenuation of ERS-induced apoptosis. These findings provide new insight into therapeutic potential of IPC-induced MVs in cardioprotection against I/R injury.

OBJECTIVES:To investigate the effects of Astragaloside IV (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs), and the mechanism of AST.METHODS:H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs in vitro under the condition of hypoxia for 12 hour/Reoxygenation for 4 hour, H/R-EMVs were stored in D-Hank's solution. Male Wistar rats were underwent thoracotomy, the thoracic aorta with intact endothelium were carefully removed and cut into 3~4 mm rings. The experiment was divided into six groups. H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10μg/ml; different doses of AST groups:thoracic aortic rings of rats were treated with 10, 20, 40, 60 mg/L AST co-incubated with 10μg/ml H/R-EMVs respectively; control group were treated with the same volume of D-Hank's solution. Duration of incubation was 4 h, each group was tested in five replicate aortic rings. Effects of AST on endothelium-dependent relaxation were detected. The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinase (Akt), phosphorylated Akt (p-Akt, Ser-473), extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2, Thr202/Tyr204) of rat thoracic aortic rings were detected.RESULTS:Tenμg/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (P<0.01). Compared with H/R-EMVs group, relaxation of rat thoracic aortic rings was increased by 20, 40 and 60 mg/L AST in a concentration-dependent manner (P<0.01), the level of NO production was also enhanced (P<0.05, P<0.01). The level of t-eNOS, t-Akt and ERK1/2 was not changed, but the level of p-eNOS, p-Akt and p-ERK1/2 increased by the treatment with AST (P<0.01).CONCLUSIONS:AST could effectively ameliorate endotheliumdependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner, the mechanism might involve the increase in production of NO, and the protein level of p-eNOS, p-Akt and p-ERK1/2.

OBJECTIVE:To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats.METHODS:I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats.RESULTS:Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs.CONCLUSION:I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

OBJECTIVE L-CNT has certain effects in improving the cardiac toxicity and peripheral nerve toxicity aspects caused by chemotherapy drugs.This study aimed to investigate the proliferation and cell apoptosis effects of combination treatment L-CNT and DTX on NSCLC GLC-82 cells,and to explore its mechanism.METHODS GLC-82 cells were treated with different concentrations of L-CNT(0.625,1.25,2.5,5,10,20,40,80 μg/mL) and DTX(0.01,0.1,1,5,10,20,40,80 μg/mL) in vitro.The inhibitory rates of cells were measured by MTT assay.Morphological changes were observed with inverted microscope.GLC-82 cells were treated with the combination treatment of L-CNT (1.25,20,80 μg/mL) and DTX(9.301 μg/mL) for 24 h,and then the proliferation inhibitory rates were detected by MTT assay.The cell apoptosis was analyzed by Annexin Ⅴ-FITC/PI double staining.Western blot was used to analyzed the expression of Bcl-2,Bax and adenine nucleotide translocator1 (ANT1).RESULTS After 24 h and 48 h,compared with the control group,the proliferation rates of L-CNT (1.25,20,80 μg/mL) were (14.52 ± 0.73)％,(27.03 ±1.21)％,(19.77±0.65)％,F=42.538,P=0.001 and (11.37±0.13)％,(19.56±1.01)％,(14.89±0.51)％,F=52.119,P=0.001,respectively.Compared with DTX(27.319 μg/mL),the cell proliferation rate of L-CNT(1.25,20,80 μg/mL) and DTX combined group increased from(49.49 ± 2.52) ％ to (52.64± 2.43) ％,(59.44 ±-3.20) ％ and (53.15±2.37) ％,F=7.412,P=0.011.Compared with the DTX(9.301 μg/mL)group,cell density of the combination group was increased,Flow cytometry showed that the apoptosis rates were reduced to (21.90±1.98)％,(14.38 ± 2.35) ％ and (22.42± 1.43) ％ from (24.67±1.95) ％,F=15.697,P =0.001.The combination group could increase the expression of Bcl-2 and decrease the expression of Bax,at the same time,the expression of ANT1 was also decreased.CONCLUSIONS Treatment with L-CNT and DTX has impacts on the proliferation of GLC-82 cells.The possible mechanism of action is achieved by regulating the expression of Bcl-2 protein family and regulating the opening and closing of mitochondrial membrane permeability transition pore.

Objective:To evaluate the long-term toxicity of compound glycyrrhetinic acid cream.Methods:One hundred and eightyeight SD rats were randomly divided into six groups:the drug administration groups were divided into small-dose group,abraded skin given 100 mg·kg-1 compound glycyrrhetinic acid cream,medium-dose group (500 mg·kg-1)and high-dose groups (1 000 mg ·kg-1)and normal skin group (given 1 000 mg·kg-1).Solvent control groups were divided into the damaged skin and normal skin groups,which were given the same volume of blank substrate,respectively.The appearance and sign of the rats were observed and body weight of rats were taken to calculate organ coefficient with successive 4-weeks drug administration and 4-weeks withdraw.After drug administration for 4 weeks,the organ coefficients were calculated and hematological as well as blood biochemical indexes were detected.And then the histopathologic examination was carried out.Results:There were no death,weight disorders and toxicological abnormal changes on hematology,biochemics of blood and histopathology for the rats given compound glycyrrhetinic acid cream at 100,500 and 1 000 mg ·kg-1.Also,there were no delayed toxic reactions after drug withdrawal.Conclusion:Compound glycyrrhetinic acid cream has no long-term toxicity in rats by skin administration.

High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a "danger signal" and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle-associated form (TSE-MV). Inhibition of chromosome region maintenance-1 (CRM1), a nuclear exporter, attenuated TSE-induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE-MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle-associated form. J. Cell. Physiol. 231: 2319-2326, 2016. © 2016 Wiley Periodicals, Inc.

目的:以土槿乙酸结构特征为模板,吲哚为母核合成类土槿乙酸化合物,以期得到有更高免疫抑制活性的吲哚类衍生物.方法:对5-醛基吲哚和5-羧基吲哚的1,3,5位进行改造.1位进行烷基化改造,3位通过维尔斯迈尔-哈克反应与醛胺缩合反应进行延长,5位通过克脑文格尔反应与缩合酰化反应进行延长,得到吲哚类衍生物.经氢谱和高分辨质谱对其进行结构表征.采用噻唑蓝(MYY)法检测目标化合物对小鼠T、B淋巴细胞增殖的抑制作用.结果:合成得到两个系列共7个化合物,所有的化合物都经过HR-ESI-MS和1H-NMR验证,其中化合物5c对T细胞和B细胞有一定的增殖抑制活性,化合物9a,9b对B细胞有一定增殖抑制活性.结论:通过模拟土槿乙酸的两个长链结构合成了新的吲哚的类似物,发现了具有一定的免疫抑制活性的先导化合物.

OBJECTIVE:To investigate the effects of circulating microvesicles (MVs) derived from ischemic preconditioning (IPC) on myocardial ischemia/reperfusion (I/R) injury in rats and explore the underlying mechanism.METHODS:To establish the IPC model, the rats were subjected to brief cycles of left anterior descending (LAD) coronary occlusion and reperfusion. The blood was drawn from abdominal aorta once the operation was finished. IPC-MVs were isolated by ultracentrifugation from the peripheral blood and characterized by flow cytometry. The myocardial I/R model of rats was established in vivo. Rats were injected via the femoral vein with IPC-MVs at 7 mg/kg. Morphological changes of myocardium were observed microscopically after HE staining. Apoptosis of myocardial cells was detected with TUNEL assay. Myocardial infarct size was detected by TTC staining. Moreover, activity of plasma lactate dehydrogenase (LDH) was tested by colorimetry. The activity of caspase 3 in myocardium was assayed with spectrophotometry. Expression levels of Bcl-2 and Bax protein were examined with Western blot.RESULTS:The concentration of IPC-MVs, which was detected by flow cytometry, was 4380±745 cells/μl. Compared with I/R group, IPC-MVs alleviated the damage of tissues in I/R injured rats significantly. The myocardial infarct size and the cardiomyocyte apoptotic index were obviously decreased after IPC-MVs treatment (P<0.01, respectively). The activity of plasma LDH was significantly decreased in IPC-MVs treated rats (P<0.01). Moreover, the activity of caspase 3 was markedly decreased after IPC-MVs treatment (P<0.01). In addition, the expression of Bcl-2 was increased (P<0.01), the expression of Bax was decreased (P<0.01), the ratio of Bcl-2/Bax was significantly increased after IPC-MVs treatment (P<0.01).CONCLUSIONS:IPC-MVs protected myocardial against I/R injury by up-regulating the expression of Bcl-2 protein, down-regulating the expression of Bax protein, increasing the ratio of Bcl-2/Bax and decreasing cleavage of caspase 3.

目的 探讨左卡尼汀对氟尿嘧啶(5-fluorouracil,5-Fu)抑制人胃腺癌MGC803细胞增殖作用的影响.方法 将MGC803细胞分为阴性对照组、5-Fu组、左卡尼汀和5-Fu联合用药组(左卡尼汀+/→5-Fu)进行实验.四甲基偶氮唑蓝(MTT)法检测细胞的增殖,流式细胞术检测细胞凋亡和细胞周期,蛋白质印迹法检测Bcl-2、Bax、ANT1和cleaved-PARP蛋白的表达.结果 与5-Fu-单药组相比,左卡尼汀和5-Fu联合用药组抑制MGC803细胞增殖的作用增强,细胞凋亡百分率增大,细胞周期S期阻滞,上调Bax、ANT1和cleaved-PARP蛋白的表达,下调Bcl-2蛋白的表达.实验结果还显示,左卡尼汀和5-Fu联合应用时,给药方法可以影响5-Fu对MGC803细胞的抑制作用.与左卡尼汀+5-Fu组相比,左卡尼汀→.5-Fu组细胞凋亡百分率由(19.60±1.06)％增加至(24.17±3.12)％(P＜0.05),Go/G1期细胞比例由(72.95±0.91)％降低至(62.62±1.04)％,S期细胞比例由(27.05±0.91)％增加至(37.35±1.03)％(P＜0.001).结论 左卡尼汀可能通过影响Bcl-2家族蛋白的表达和细胞周期,增强5-Fu对MGC803细胞增殖的抑制作用.

OBJECTIVE:To investigate the effect of Iptakalim (Ipt) preventing injury of endothelial microvesicles (EMVs) derived from hypoxia/reoxygenation (H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism.METHODS:H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope (TEM). Thoracic aortic rings of rats were incubated with 10-7-10-3 mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) was recorded in vitro. The nitric oxide (NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinas (Akt) and phosphorylated Akt (p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting.RESULTS:H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles (100-1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner (P<0.05, P<0.01). The expression of total eNOS (t-eNOS) and total Akt (t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-eNOS and p-Akt increased after treated with Ipt (P<0.01).CONCLUSIONS:Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-eNOS and p-Akt expression.

Vascular endothelial dysfunction is the closely related determinant of ischemic heart disease (IHD). Endothelial dysfunction and ischemia/reperfusion injury (IRI) have been associated with an increase in microvesicles (MVs) in vivo. However, the potential contribution of endothelial microvesicles (EMVs) to myocardial damage is unclear. Here we aimed to investigate the role of EMVs derived from hypoxia/reoxygenation (H/R) -treated human umbilical vein endothelial cells (HUVECs) on cultured H9c2 cardiomyocytes.

OBJECTIVE:To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats.METHODS:Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining.RESULTS:Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01).CONCLUSION:The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.

目的 探讨吸入长效β2 受体激动剂联合糖皮质激素制剂对血管内皮功能的影响.方法 募集呼吸内科诊治的中-重度慢性阻塞性肺疾病(COPD)稳定期患者50例,随机分为两组,观察组规律吸入沙美特罗氟替卡松联合制剂,对照组仅按需吸入短效支气管舒张剂.所有受试者常规行血液分析、C反应蛋白(CRP)、肺功能、心脏彩超等检查,收集两组患者外周血上清液采用双抗体夹心-ELISA法测定内皮素-1 (ET-1)水平,12周后复查上述指标.结果 观察组22 例患者治疗12 周后,血浆ET-1 含量为(60.47 ±12) ng/mL,较基线水平(56.23 ±10)ng/mL无明显上升(P=0.13),17例对照组在基线访视12周后复查血清ET-1含量增加[治疗前后分别为(55.5.±19)ng/mL、(72 ±18)ng/mL],差异有统计学意义(P<0.05).观察组治疗后复查肺功能FEV1 由(1.71 ±0.22)L上升至(2.04 ±0.07)L,差异有统计学意义(P=0.002),呼气峰值流速治疗前(4.29 ±1.7) L/s,12周后上升至(5.13 ±0.6) L/s(P<0.001),但FEV1/FVC比值无明显变化(治疗前后分别为59 ±5、60 ±3,P=0.29),对照组肺功能则无明显改善,部分患者甚至有下降,两组差值比较差异有统计学意义(P<0.05).观察组FEV1 变化与血浆ET-1水平呈负相关(r=-0.1572).两组肺动脉平均收缩压、外周血白细胞总数、中性粒细胞百分比及CRP水平在试验前后差异无统计学意义.结论 中重度COPD患者规律使用沙美特罗氟替卡松吸入剂后血浆ET-1水平不再进行性升高,肺功能改善,内皮素水平与肺功能两者之间存在负相关.但肺动脉平均压力与CRP无明显变化.COPD稳定期规律吸入沙美特罗氟替卡松吸入剂可能有助于延缓内皮功能损伤进程.