榆干离褶伞溶栓酶对大鼠动脉血栓的抑制作用

目的：研究榆干离褶伞溶栓酶（Lyophyllum ulmarium fibrinolytic enzyme,LUFE）对大鼠动脉血栓形成的抑制作用及其机制。方法：将SD大鼠随机分为假手术组、模型组、阳性对照组和LUFE低、高剂量组,除假手术组以外,其余4 组均以三氯化铁建立颈动脉血栓模型。造模前阳性对照组和LUFE低、高剂量组分别以10 mg/kg mb阿司匹林以及100、400 mg/kg mb LUFE灌胃7 d。观察血栓质量和颈动脉血管组织病理变化;酶联免疫吸附测定法检测肿瘤坏死因子α（tumor necrosis factor alpha,TNF-α）、白细胞介素-6（interleukin-6,IL-6）、血小板活化因子（platelet-activating factor,PAF）、可溶性血栓调节蛋白（soluble thrombomodulin,sTM）、E-选择素和组织因子（tissue factor,TF）水平;流式细胞仪检测血小板CD62P表达水平。为观察LUFE对血小板功能的影响,在体外以胶原诱导血小板活化,观察LUFE对大鼠富血小板血浆中P-选择素、β-血小板球蛋白（β-thromboglobulin,β-TG）和血小板膜糖蛋白IIbIIIa（glycoprotein IIbIIIa,GPIIbIIIa）水平的影响;Western blot方法观察LUFE对大鼠血小板磷脂酰肌醇3激酶（phosphatidylinositol 3 kinase,PI3K）、蛋白激酶B（protein kinase B,Akt）和p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38 MAPK）表达水平的影响。结果：LUFE能明显减轻血栓质量和血管受损程度,抑制血栓模型大鼠血清sTM、E-选择素、TF、TNF-α、IL-6和PAF水平的上调,下调血小板CD62P的表达。同时,在体外LUFE能够下调胶原诱导的富血小板血浆中P-选择素、β-TG和GPIIbIIIa水平,下调血小板PI3K、Akt和p38 MAPK蛋白的磷酸化水平。结论：LUFE具有抗血栓作用,其机制可能与LUFE对血管内皮细胞的保护作用以及对血小板活化的抑制作用有关。     

鱼油对KKAy糖尿病小鼠糖代谢及PI3K/Akt信号通路的影响

目的：探讨鱼油对KKAy糖尿病小鼠糖代谢及磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase,PI3K）/蛋 白激酶B（protein kinase B,Akt）信号通路的影响。方法：40 只KKAy小鼠,分为模型组和鱼油低、中、高剂 量组,每组10 只。另设10 只C57BL/6小鼠为正常对照组。灌胃12 周后,测定血糖、胰岛素、脂联素、瘦素及 PI3K/Akt信号通路中相关基因的表达水平。结果：与模型组相比,鱼油低剂量组血糖水平极显著降低（P＜0.01）, 鱼油低、中剂量组胰岛素水平显著提高（P＜0.05）,鱼油各剂量组的脂联素水平显著提高（P＜0.05）。此外,鱼油 各剂量组可上调胰岛素受体底物-1、PI3K、Akt、葡萄糖转运蛋白-4基因的表达,其中低剂量鱼油对Akt和葡萄糖转 运蛋白-4基因的调控效果更显著。结论：低剂量鱼油可能通过调节PI3K/Akt信号通路改善糖代谢。     

人参皂苷F2通过PI3K/Akt途径改善3T3-L1脂肪细胞胰岛素抵抗

该研究探讨了人参皂苷F2对3T3-L1脂肪细胞胰岛素抵抗的影响及其机制。将3T3-L1前脂肪细胞诱导分化成熟后,用1 μmol/L地塞米松处理48 h,建立胰岛素抵抗模型。用10 μmol/L的罗格列酮（阳性对照）和25、50、100 μmol/L人参皂苷F2处理胰岛素抵抗细胞12 h,检测细胞对2-NBDG的摄取。实验结束后用RT-qPCR检测各组细胞中葡萄糖转运蛋白4（GLUT-4）和胰岛素底物受体1（IRS-1）mRNA相对表达量,并利用Western blot检测PI3K/Akt信号通路中蛋白表达及其磷酸化水平。结果表明：与模型组相比,25、50、100 μmol/L人参皂苷F2均能促进胰岛素抵抗3T3-L1脂肪细胞对2-NBDG的摄取,分别增加了12.58%、29.07%和34.62%（p<0.05）;人参皂苷F2作用12 h后,GLUT-4和IRS-1 mRNA相对表达量以及PI3K、Akt的磷酸化水平显著提高（p<0.01）。该研究表明人参皂苷F2可通过促进胰岛素抵抗3T3-L1脂肪细胞中GLUT-4和IRS-1mRNA相对表达,增加PI3K和Akt蛋白磷酸化,从而激活PI3K/Akt信号通路,改善其糖代谢和胰岛素抵抗。     

苦荞清蛋白酶解物对高糖诱导的HepG2细胞胰岛素抵抗的保护作用

目的：建立胰岛素抵抗HepG2细胞模型,观察苦荞清蛋白酶解物（tartary buckwheat albumin hydrolysate,TBAH）对HepG2细胞胰岛素抵抗的影响。方法：采用碱性蛋白酶水解苦荞清蛋白制备TBAH,超滤法截留分子质量小于3 kDa的TBAH;利用高糖高胰岛素诱导HepG2细胞36 h建立胰岛素抵抗模型,以2.5、5、10 μg/mL的TBAH培养细胞24 h。观察TBAH对HepG2细胞葡萄糖代谢的影响;检测细胞氧化损伤指标（一氧化氮（nitric oxide,NO）、丙二醛（malondialdehyde,MDA）、乳酸脱氢酶（lactic dehydrogenase,LDH）、超氧化物歧化酶（superoxide dismutase,SOD）和活性氧（reactive oxygen species,ROS））水平;Western blot检测胰岛素受体底物1（insulin receptor substrate 1,IRS-1）/磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase,PI3K）/蛋白激酶B（protein kinase B,Akt）信号通路中相关蛋白表达情况。结果：与IR模型组相比,TBAH组葡萄糖消耗量显著增多（P＜0.05）,且呈剂量依赖性,同时MDA、NO和ROS水平显著降低（P＜0.01,P＜0.05）,SOD活力显著升高（P＜0.05）,LDH活力显著下降（P＜0.05）;p-IRS-1蛋白表达水平显著降低（P＜0.05）,磷酸化Akt、PI3K、葡萄糖转运蛋白4表达水平极显著上升（P＜0.01）。结论：TBAH通过抑制氧化应激改善胰岛素抵抗。     

姜黄素对H2O2诱导血小板凋亡的抑制作用及分子机制

目的：血小板凋亡在心血管疾病发生发展过程中发挥重要作用，姜黄素（curcumin，Cur）是存在于姜科植物根茎中的一种多酚类化合物，具有多种生物学活性，但是Cur对血小板凋亡是否具有调控作用尚鲜见报道，本研究通过体外实验探讨Cur对H2O2诱导血小板凋亡的影响及其潜在的机制。方法：用不同浓度（0、1、10、100 μmol/L）的Cur与健康人纯化血小板在体外共同孵育30 min，然后加入H2O2（100 μmol/L）干预血小板60 min，用流式细胞术检测血小板磷脂酰丝氨酸的暴露水平和线粒体膜电位（ΔΨm）去极化水平；用酶联免疫吸附法测定血小板凋亡蛋白Caspase-3和Caspase-9活化水平、胞内总活性氧以及超氧化物生成水平；用Western blot蛋白免疫印迹法检测血小板促凋亡蛋白Bax、Bak和细胞色素c的表达水平。结果：与对照组相比，10、100 μmol/L Cur显著抑制H2O2诱导的血小板磷脂酰丝氨酸暴露和ΔΨm去极化（P＜0.05）；同时，Western blot结果显示，Cur能显著降低H2O2诱导的血小板促凋亡蛋白Bax、Bak和细胞色素c的表达水平的升高（P＜0.05）；酶联免疫吸附测定结果表明，H2O2诱导的血小板Caspase-3和Caspase-9的活化可被10、100 μmol/L Cur显著抑制（P＜0.05）；此外，H2O2处理时，血小板内总活性氧水平和超氧化物水平显著上调，Cur干预可显著抑制胞内总活性氧和超氧化物生成（P＜0.05）。结论：Cur具有显著抑制H2O2诱导的血小板凋亡的作用。     

辅酶Q10经蛋白激酶A/胞浆型磷脂酶A2信号通路抑制血小板血栓素A2的生成

目的：血小板来源血栓素A2（thromboxane A2,TxA2）可诱发动脉粥样硬化和血栓形成,辅酶Q10（coenzyme Q10,CoQ10）可以抑制血小板活化、聚集和血栓形成。本实验旨在通过体外实验探讨CoQ10对血小板TxA2生成的影响及其调控机制。方法：用不同浓度（0、1、10、100 μmol/L）CoQ10与健康人纯化血小板在体外共同孵育50 min,在激动剂激活条件下,采用酶联免疫吸附测定法测定血小板分泌TxB2水平;用Western blot蛋白免疫印迹法检测胞浆型磷脂酶A2（cytosolic phospholipase A2,cPLA2）蛋白的磷酸化水平。结果：CoQ10显著抑制多种激动剂（如凝血酶、胶原和Convulxin）诱导的血小板TxA2生成;Western blot结果显示CoQ10下调凝血酶和胶原诱导的血小板cPLA2磷酸化水平;cPLA2特异性抑制剂苯乙酮与CoQ10联合使用时,对血小板TxA2的生成没有协同效果;此外,蛋白激酶A（protein kinase A,PKA）特异性抑制剂H89可完全逆转CoQ10对激动剂诱导的血小板cPLA2 磷酸化和TxA2生成的抑制作用。结论：CoQ10具有显著抑制激动剂诱导的血小板TxA2生成的作用,其机制主要是调控PKA/cPLA2介导的信号通路。     

巴豆醛诱导人支气管上皮细胞(BEAS-2B)自噬及相关通路的时间效应变化

为评估巴豆醛对人支气管上皮细胞(BEAS-2B)的毒性效应,使用细胞计数试剂盒(CCK-8)检测BEAS-2B细胞存活率,免疫荧光法检测自噬小体的聚集和定位,Western Blot实验检测自噬标志物微管相关蛋白1轻链3β(LC3B-Ⅱ)及两个自噬相关通路磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶标(mTOR)和肝脏激酶B1(LKB1)/单磷酸腺苷活化蛋白激酶(AMPK)/Unc-51-类激酶(ULK1)的变化。结果表明:(1)巴豆醛暴露可显著降低BEAS-2B细胞存活率。(2)巴豆醛处理可引起自噬小体的聚集,改变LC3B-Ⅱ蛋白表达量;巴豆醛诱导细胞自噬具有明显的时间效应关系,BEAS-2B细胞自噬强度随巴豆醛暴露时间延长先升高后降低。(3)PI3K/Akt/m TOR通路中磷酸化的PI3K、Akt和4E结合蛋白1(4E-BP1)呈下降趋势,磷酸化的m TOR和核糖体蛋白S6激酶(p70 S6K)先下降后升高;LKB1/AMPK/ULK1通路中LKB1、AMPK和ULK1活性均呈升高趋势。因此,两个通路均参与调控巴豆醛诱导的BEAS-2B细胞自噬水平的升高。     

北五味子乙素对大鼠离体心肌缺血再灌注损伤的保护作用及分子机制

目的：观察北五味子乙素（schisandrin B,SchB）对缺血再灌注（ischemia/reperfusion,I/R）大鼠离体心脏功能、梗死面积、乳酸脱氢酶及相关蛋白表达的影响,探讨其对I/R损伤的保护作用及分子机制。方法：Wistar大鼠50 只,随机分为5 组：空白对照组（CON）、缺血再灌注组（I/R）、SchB预保护组（SchB）、PI3K抑制剂组（I/R＋SchB＋LY）、腺苷酸活化的蛋白激酶（AMP-activated protein kinase,AMPK）抑制剂组（I/R＋SchB＋CC）。采用Langendorff法建立离体大鼠心脏I/R模型,心肌缺血2 h,再灌注5、15、30、60 min。观察I/R期间室内压最大上升速率（＋dp/dtmax）、室内压最大下降速率（－dp/dtmax）、左心室舒张末压、冠脉流量和心率;测定心脏灌流液中乳酸脱氢酶（lactate dehydrogenase,LDH）含量,2,3,5-氯化三苯基四氮唑（2,3,5-triphenyltertrazoliumchloride,TTC）染色测定各组心肌梗死面积;免疫印迹检测心肌中总蛋白激酶B（protein kinase B,PKB）、磷酸化Akt（p-Akt）、总糖原合成酶激酶-3β（glycogen synthease kinase-3β,GSK-3β）、磷酸化GSK-3β（p-GSK-3β）蛋白表达。结果：SchB可显著改善I/R所致的心肌功能损伤,减少梗死面积,提高心肌中p-Akt和p-GSK-3β蛋白表达。结论：SchB对大鼠离体心脏I/R损伤具有一定保护作用,其机制可能与激活AMPK和PI3K/Akt信号通路有关。     

榆干离褶伞溶栓酶对高脂血症大鼠肝损伤的保护作用

目的:探讨榆干离褶伞溶栓酶(LUFE)对高脂血症大鼠肝损伤的保护作用及其机制。方法:以高脂饮食建立高脂血症模型,用不同剂量的LUFE灌胃模型大鼠。HE染色观察肝脏病理形态学变化;比色法检测大鼠血浆生化指标;蛋白免疫印迹法观察炎症相关蛋白的表达水平。结果:LUFE可以缓解高脂血症大鼠的肝组织损伤,降低血浆血脂水平、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、巨噬细胞趋化蛋白-1(MCP-1)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,下调TLR4、MyD88和磷酸化的PI3K、Akt、NF-кB水平。结论:LUFE通过调控PI3K/Akt/NF-кB和TLR4/MyD88/NF-кB信号通路,可以保护高脂血症所致的大鼠肝损伤。     

Inability of probiotic bacterial strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 to induce human platelet aggregation in vitro

Platelet aggregation contributes to the pathogenesis of infective endocarditis, and aggregation of platelets induced by lactobacilli is thought to be an important contributory factor in the development and progression of Lactobacillus endocarditis. The main purpose of this study was to examine the effect of immunity-enhancing probiotic strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 on the activation and aggregation of human blood platelets. Whole blood samples from healthy individuals were incubated in vitro with HN001 or HN019 and subsequently labeled with platelet-specific monoclonal antibodies, fluorescein isothiocyanate-conjugated anti-CD41a (expressed on normal platelets), and phycoerythrin-streptavidin-conjugated anti-CD62p (expressed on activated platelets) before analysis by flow cytometry. Platelet-rich plasma was used to assist the gating of the platelet cluster. ADP and epinephrine were used as the physiological platelet activation agonists. Platelet aggregation-inducing strain Streptococcus sanguis 133-79 was used as a positive control strain. The mean fluorescence intensity of phycoerythrin and the percentage of platelets expressing the CD62p marker were used to assess the degree of platelet activation. The percentage of CD62p-positive platelets and the light scatter profiles of the agonist-activated platelets were used to identify the occurrence and degree of platelet aggregation. HN001 and HN019 had no effect on spontaneous platelet activation and aggregation; they also failed to exacerbate the platelet aggregation activity induced by ADP and epinephrine. Therefore, these test probiotic strains HN001 and HN019 are less likely to participate in the pathogenesis of infective endocarditis or other thrombotic disorders with regard to platelet aggregation factors.     

植物多酚通过PI3K/Akt信号通路抗肿瘤作用研究进展

磷脂酰肌醇3-激酶/丝氨酸/苏氨酸激酶B（phosphatidylinositol 3-kinase/serine/threonine kinase B,PI3K/Akt）信号通路是一条经典的抑制细胞凋亡、促进细胞增殖的信号转导通路,在肿瘤、心血管疾病、神经系统疾病和糖尿病等的防治中发挥重要作用,特别是肿瘤。PI3K/Akt信号通路的异常活化与肿瘤的发生、侵袭和转移等过程密切相关,对该通路的研究已成为当今国内外治疗肿瘤的焦点。植物多酚因具有抗肿瘤作用而成为预防肿瘤的天然药物。本文综述了PI3K/Akt信号通路的结构、活化机制以及与肿瘤的关系,总结植物多酚通过该信号通路对肿瘤细胞的作用机制,以期为研发植物多酚作为预防肿瘤的保健食品或药品提供一定的科学依据。     

EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways

Epidermal growth factor (EGF) is present in the maternal-fetal environment and has an important role in placental development. Matrix metalloproteinase-9 (MMP-9) expression/activation is a pre-requisite in extravillous trophoblast invasion. Whereas EGF up-regulates MMP-9 activity in a variety of cell types, there is no direct evidence for the stimulation of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion by EGF in extravillous trophoblasts. In addition, the signalling pathways involved in this regulation are not clear. In the present study, we have examined the possible involvement of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in the regulation of the MMP-9/TIMP-1 system by EGF in vitro. We used a well-established invasive extravillous trophoblast cell line (HTR8/Svneo) and measured gene and protein expression by semi-quantitative RT-PCR and western analysis respectively. MMP activity was determined by zymography. We showed for the first time that EGF activated both PI3K/Akt and MAPK/extracellular-signal regulated kinase (ERK) signalling in HTR8/SVneo, and increased both MMP-9 and TIMP-1 mRNAs and protein concentrations. Interfering with either signalling pathway via PI3K inhibitor LY294002 or MEK inhibitor U0126 in EGF-stimulated HTR8/SVneo cells blocked the induction of MMP-9 and TIMP-1. LY294002 inhibited Akt phosphorylation, but had no effect on ERK phosphorylation; U0126 suppressed ERK phosphorylation without interfering with the phosphorylation of Akt. In addition, expression of constitutively active Akt (Myr-Akt1, Myr-Akt2, Myr-Akt3) was not sufficient to induce proMMP-9 and TIMP-1 secretion. Our results suggest that the activation of both PI3K and MAPK pathways in extravillous trophoblasts is necessary for the up-regulation of MMP-9 and TIMP-1 expression by EGF.     

In vitro anti-platelet effects of simple plant-derived phenolic compounds are only found at high, non-physiological concentrations

Scope : Bioactive polyphenols from fruits, vegetables, and beverages have anti‐platelet effects and may thus affect the development of cardiovascular disease. We screened the effects of 26 low molecular weight phenolic compounds on two in vitro measures of human platelet function. Methods and results : After platelets had been incubated with one of 26 low molecular weight phenolic compounds in vitro, collagen‐induced human platelet aggregation and in vitro TRAP‐induced P‐selectin expression (as marker of platelet activation) were assessed. Incubation of platelet‐rich plasma from healthy volunteers with 100 μmol/L hippuric acid, pyrogallol, catechol, or resorcinol significantly inhibited collagen‐induced platelet aggregation (all p<0.05; n≥15). Incubation of whole blood with concentrations of 100 μmol/L salicylic acid, p‐coumaric acid, caffeic acid, ferulic acid, 4‐hydroxyphenylpropionyl glycine, 5‐methoxysalicylic acid, and catechol significantly inhibited TRAP‐induced surface P‐selectin expression (all p<0.05; n=10). Incubation with lower concentrations of phenolics affected neither platelet aggregation nor activation. Conclusion : As concentrations of 100 μmol/L are unlikely to be reached in the circulation, it is doubtful whether consumption of dietary phenolics in nutritionally attainable amounts plays a major role in inhibition of platelet activation and aggregation in humans.