吴茱萸次碱对棕榈酸诱导的脂质损伤肝细胞的影响

目的:研究吴茱萸次碱(Rut)对棕榈酸(PA)诱导的脂质损伤肝细胞(LO2)的细胞活力、活性氧(ROS)以及缝隙连接蛋白32(Cx32)和缝隙连接蛋白26(Cx26)表达水平的影响。方法:加入不同浓度的Rut溶液预处理LO2细胞20 min后,每个孔加入PA(0.2 mmol/L)溶液,共同孵育24 h,分别应用细胞增殖及毒性检测试剂盒(CCK-8)检测细胞活力,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧的表达、划痕负载实验检测缝隙连接细胞间通讯(GJIC)的功能变化及免疫印迹实验(Western blotting)检测缝隙连接蛋白Cx32和Cx26的表达水平。结果:CCK-8实验结果显示,Rut(0.1μmol/L,0.5μmol/L,1μmol/L)能抑制PA诱导的LO2细胞活力的下降;DCFH-DA荧光探针实验显示PA诱导细胞后ROS的表达增强,Rut预处理细胞后能够减少ROS的表达;划痕负载实验结果显示,PA诱导细胞后能够抑制细胞的GJIC功能,预先给予Rut处理能够改善GJIC的功能障碍;Western blotting显示PA诱导细胞后缝隙连接蛋白Cx32和Cx26的表达水平显著降低,给予Rut预处理后能够恢复Cx32和Cx26的表达。结论:PA能够诱导LO2细胞的脂质损伤,导致细胞活性降低、细胞内活性氧增加以及GJIC的功能障碍;Rut预处理LO2细胞后可减轻PA诱导的细胞损伤,并且恢复细胞的缝隙连接蛋白Cx32和Cx26的表达和GJIC的功能。     

大黄素对Cx32组成的细胞缝隙连接通讯功能的影响

目的在转染并稳定表达Cx32的Hela细胞上,观察大黄素对Cx32的GJIC以及Cx32蛋白表达水平的影响。方法采用SRB法检测不同浓度大黄素对Hela细胞的毒性;用细胞接种荧光示踪法("parachute"dye-couplinga ssay)观察不同浓度大黄素对GJIC的影响;用western blot法研究大黄素在影响GJIC功能浓度范围内对Cx32蛋白表达的影响。结果大黄素在0~1μmol/L浓度时对Hela细胞无毒性作用;大黄素(24~600nmol/L)预处理4h,能浓度依赖性地增强GJIC及Cx32蛋白表达量。结论大黄素能够增强Cx32的细胞GJIC;此增强作用可能与其增加Cx32蛋白表达水平有关。     

西替利嗪抑制组胺诱导角质形成细胞IL-8表达

目的:考察西替利嗪对组胺诱导角质形成细胞系HaCaT细胞IL-8表达的影响。方法:采用RT-PCR方法研究组胺H1受体mRNA表达,组胺及西替利嗪对IL-8mRNA表达的调节作用,并用ELISA法对分泌的IL-8进行定量。结果:HaCaT细胞有组胺H1受体mRNA表达,(1×10-5)mol/L组胺刺激细胞5h,显著增强IL-8mRNA表达;(1×10-6)-(1×10-4)mol/L组胺作用细胞24h则显著地诱导了IL-8产生;1×10-6、1×10-5mol/L西替利嗪抑制了组胺的诱导作用。结论:西替利嗪可通过抑制组胺诱导HaCaT细胞IL-8表达而具有H1受体依赖的抗炎作用。     

木犀草素对肝星状细胞迁移和增殖的影响

目的:研究木犀草素对肝星状细胞迁移的影响。方法:体外培养肝星状T6(HSC-T6)细胞,细胞计数Kit8(CCK-8)法检测0、10、20、40μmol/L木犀草素对HSC-T6细胞增殖的影响;通过划痕实验、Transwell实验观察0、10、20、40μmol/L木犀草素对HSC-T6细胞迁移能力的影响;蛋白印迹法检测木犀草素对细胞外信号调节激酶(ERK)5蛋白磷酸化水平的影响。结果:20、40μmol/L木犀草素能明显抑制HSC-T6细胞的增殖(P<0.05);10、20、40μmol/L木犀草素能明显抑制HSC-T6细胞划痕的修复(P<0.05);10、20、40μmol/L木犀草素能明显抑制HSC-T6细胞穿膜(P<0.05);10、20、40μmol/L木犀草素能明显抑制细胞中由血清诱导的去磷酸化(p)-ERK5表达(P<0.05)。结论:木犀草素呈浓度依赖性抑制HSC-T6细胞的增殖和迁移,其机制可能与抑制ERK5蛋白磷酸化水平有关。     

缝隙连接蛋白43对人非小细胞肺癌HCC827细胞吉非替尼获得性耐药机制的初步研究

目的探讨缝隙连接蛋白43(connexin 43,Cx43)与非小细胞肺癌(NSCLC)细胞产生吉非替尼(Gefitinib)获得性耐药的关系。方法在Gefitinib敏感细胞株HCC827上,通过逐步递增Gefitinib浓度诱导获得Gefitinib耐药细胞株HCC827 GR;MTT法检测Gefitinib对HCC827/GR细胞的IC_(50);RT-PCR检测Cx43的mRNA水平;Western blot检测Cx43和磷酸化Akt(p-Akt)的蛋白水平;parachute荧光示踪法检测细胞缝隙连接功能(gap junction intercellular communication,GJIC);免疫荧光技术检测Cx43的细胞定位。结果Gefitinib作用于HCC827和HCC827 GR的IC_(50)分别为(0.07±0.019)μmol·L~(-1)、(10.84±0.021)μmol·L~(-1)(P<0.01);HCC827 GR中Cx43的mRNA和蛋白水平较HCC827明显降低(P<0.05),但p-Akt蛋白水平明显升高(P<0.05)。在HCC827 GR细胞上加PI3K的特异性抑制剂LY294002(25μmol·L~(-1),24 h)后,p-Akt蛋白水平明显下降(P<0.01),且Cx43的蛋白水平明显升高(P<0.01)。HCC827及HCC827 GR均未检测到GJIC,用GJIC增强剂RA(retinoic acid)处理(10μmol·L~(-1),24 h)上述细胞,亦未检测到荧光传递,免疫荧光结果显示Cx43表达在细胞胞质。结论胞质中Cx43的下调可能促进NSCLC细胞对Gefitinib的获得性耐药,其机制可能与Cx43非GJIC依赖的PI3K/Akt信号通路激活有关。     

他莫昔芬对肝癌HepG2细胞脂质代谢的影响

目的:研究他莫昔芬对肝癌HepG2细胞脂质代谢的影响。方法:体外培养HepG2细胞,MTT法检测0.5、1、5、15、30μmol/L他莫昔芬对HepG2细胞增殖的影响;油红O染色检测0.5、1、5μmol/L他莫昔芬和200μmol/L棕榈酸(阳性对照)对HepG2细胞内脂质沉积的影响;荧光定量聚合酶链式反应法检测5μmol/L他莫昔芬对HepG2细胞内脂肪酸(FA)合成酶(FAS)、固醇调节元件结合蛋白1c(Srebp-1c)、乙酰辅酶A羧化酶(ACC)的表达和FA氧化相关基因过氧化物酶体增殖物激活受体α(PPAR-α)、解偶联蛋白2(UCP-2)表达的影响;蛋白印迹法检测5μmol/L他莫昔芬对HepG2细胞内ACC蛋白表达及其磷酸化(P-ACC)水平的影响。以上检测结果均与空白对照组进行比较。结果:与空白对照组比较,0.5、1、5μmol/L他莫昔芬对HepG2细胞增殖无明显影响(P>0.05),15、30μmol/L他莫昔芬能明显抑制HepG2细胞的增殖(P<0.05);5μmol/L他莫昔芬能明显增加细胞内脂质沉积,且与阳性对照相似,0.5、1μmol/L他莫昔芬对细胞内脂质沉积无明显影响(P>0.05);细胞内FASmRNA表达明显增强、P-ACC水平明显降低(P<0.05),其余基因和蛋白表达无明显变化(P>0.05)。结论:他莫昔芬能诱导HepG2细胞脂质沉积增加,可能与细胞内P-ACC水平降低、FASmRNA表达上调有关。     

阻断Hedgehog信号通路对HepG2.2.15细胞增殖的影响

目的探讨环耙明阻断Hedgehog信号通路对肝癌细胞HepG2.2.15增殖的影响。方法培养肝癌细胞HepG2.2.15,用5μmol/L、15μmol/L、25μmol/L环耙明处理HepG2.2.15细胞24h、48h、72h,采用MTT检测环耙明对细胞活力的影响;EDU法检测细胞DNA合成情况;Real-timePCR法检测细胞Gli1的表达情况。结果用5μmol/L、15μmol/L、25μmol/L浓度的环耙明处理HepG2.2.15细胞24h、48h、72h后,MTT检测结果显示细胞活力降低,较空白组差异明显(P<0.05);EDU法检测结果显示25μmol/L的环耙明作用于细胞不同时间后,HepG2.2.15细胞的DNA合成率下降,与空白组相比差异有统计学意义(P<0.01);Real-time PCR法实验结果显示5μmol/L、15μmol/L、25μmol/L浓度的环耙明处理组与对照组相比,Gli1表达水平明显降低(P<0.05)。结论不同浓度环耙明能抑制HepG2.2.15细胞的增殖,减少HepG2.2.15细胞的DNA合成率;其作用机制可能与HepG2.2.15细胞中Gli1、mRNA的表达水平下降有关。     

卡培他滨联合阿司匹林对SGC-7901细胞增殖的体外抑制作用

目的探讨卡培他滨联合阿司匹林对SGC-7901细胞增殖和凋亡的体外抑制作用及其作用机制。方法体外培养SGC-7901细胞,MTT法检测卡培他滨1.0μmol/L与阿司匹林0.5、1.0、3.0 mmol/L单用和联用对SGC-7901细胞体外增殖的抑制作用;倒置相差显微镜观察卡培他滨1.0μmol/L与阿司匹林3.0 mmol/L单独及联合用药对SGC-7901细胞形态的影响;PI/Annexin V-FITC双染色流式细胞术分析卡培他滨1.0μmol/L与阿司匹林3.0 mmol/L单独及联合用药诱导SGC-7901的细胞凋亡率;Western blotting分析卡培他滨1.0 mmol/L与阿司匹林3.0 mmol/L单独及联合用药对COX-2、VEGF表达的影响。结果卡培他滨与阿司匹林对SGC-7901细胞均有生长抑制作用,且联合组的抑制率高于卡培他滨组和阿司匹林组,两组间比较差异具有统计学意义(P0.05)。用药处理24 h后,在显微镜下可观察到SGC-7901细胞发生细胞凋亡的形态学改变,联合组相对于卡培他滨、阿司匹林单独用药组的变化更明显。PI/Annexin V双染分析表明,卡培他滨1.0μmol/L+阿司匹林3.0 mmol/L联合组的细胞凋亡率明显高于阿司匹林3.0 mmol/L组或卡培他滨1.0μmol/L组。阿司匹林3.0 mmol/L、卡培他滨1μmol/L以及卡培他滨1.0μmol/L+阿司匹林3.0 mmol/L联合组电泳可以检测出环氧合酶-2(COX-2)、血管内皮生长因子(VEGF)的特异蛋白条带。阿司匹林单用时,COX-2表达量受到抑制(P0.01),蛋白表达下调,而VEGF表达无明显抑制;卡培他滨单用时,VEGF表达量受到抑制(P0.01),蛋白表达下调,而COX-2表达无明显抑制;卡培他滨联合阿司匹林处理时,SGC-7901细胞的COX-2、VEGF表达量均受到抑制(P0.01),同时下调COX-2及VEGF蛋白表达。结论卡培他滨联合阿司匹林能够明显抑制胃癌SGC-7901细胞的增殖,并促进其凋亡,其作用机制与COX-2、VEGF蛋白表达的调控有关。     

环氧化酶2介导化学性缺氧诱导的人角质形成细胞的炎症损伤作用

目的探讨环氧化酶2(COX-2)在化学性缺氧模拟剂氯化钴(CoCl2)诱导永生化人皮肤角质形成细胞(HaCaT)毒性及炎症反应中的作用。方法用CoCl2处理HaCaT细胞,建立化学性缺氧诱导人皮肤角质形成细胞损伤的实验模型。CCK-8比色法检测细胞存活率,ELISA试剂盒检测细胞培养基中白介素-6(IL-6)和白介素-8(IL-8)的水平,Western blot法检测COX-2蛋白的表达。结果 2 000μmol.L-1 CoCl2处理HaCaT细胞0～3 h,可上调COX-2的表达,1 h COX-2表达开始升高,2 h表达达到高峰。用0、500、1 000和2 000μmol.L-1 CoCl2处理HaCaT细胞2 h可剂量依赖性地上调COX-2的表达。2 000μmol.L-1 CoCl2处理HaCaT细胞0～8 h可时间依赖性地降低HaCaT细胞存活率,半数致死时间(LT50)在6 h左右。2 000μmol.L-1 CoCl2处理HaCaT细胞6 h可促进HaCaT细胞释放IL-6和IL-8。选择性COX-2抑制剂(NS-398)可明显对抗2 000μmol.L-1 CoCl2处理引起的细胞毒性及CoCl2诱导的IL-6和IL-8的释放增加。结论 COX-2介导化学性缺氧诱导的HaCaT细胞毒性及炎症损伤作用。     

柚皮素对胰腺癌细胞PANC-1增殖和凋亡的影响

目的 探讨柚皮素对胰腺癌细胞PANC-1增殖和凋亡的影响及糖原合成酶激酶-3β(GSK-3β)在其中的作用。方法 PANC-1细胞根据随机数字表分为7组(每组12孔，细胞密度为2 500个/mm²):0μmol/L、50μmol/L、100μmol/L、200μmol/L、400μmol/L、control virus和GSK-3β virus组。0μmol/L组，加入相应体积溶剂；50μmol/L、100μmol/L、200μmol/L和400μmol/L分别在培养液中加入终浓度为50μmol/L、100μmol/L、200μmol/L和400μmol/L的柚皮素进行培养；Control virus组，感染了对照病毒的PANC-1细胞培养液中加入200μmol/L的柚皮素进行培养；GSK-3β组，感染了GSK-3β过表达慢病毒的PANC-1细胞培养液中加入200μmol/L柚皮素。CCK-8检测细胞活力，细胞计数仪测定细胞数量，流式细胞测量凋亡细胞百分比，Western blot方法检测Ki-67、cleaved caspase-3、GSK-3β和p-GSK-3...     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Nachweis einiger Heilmittel in der Kniegelenksynovialflüssigkeit

Zusammenfassung Mittels Gaschromatographie und Dünschichtchromatographie wiesen die Autoren 11 Substanzen nach, welche durch Injektion oder nach Verabreichung per os in die Kniegelenksynovialflüssigkeit eindrangen. In ihrer Aufstellung konnten sie eine direkte Beziehung zwischen Struktur sowie chemischphysikalischen Eigenschaften der Substanz und ihrer Fähigkeit, aus dem Blut in die Kniegelenksynovialflüssigkeit einzudringen, nicht nachweisen, außer der Tatsache, daß Substanzen mit starker Affinität zu Eiweißstoffen erst in höheren Dosen nachweisbar waren.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Gender-related symptoms and correlates of alcohol dependence among men and women with a lifetime diagnosis of alcohol use disorders

This study explored gender-related symptoms and correlates of alcohol dependence in a crosssectional study of 150 men and 150 women with a lifetime diagnosis of alcohol use disorders (AUD). Participants were recruited in equal numbers from treatment settings, correctional centres and the general community. Standardized measures were used to determine participants' use of substances, history of psychiatric disorders and psychosocial stress, their sensation seeking and family history of substance use and mental health disorders. Multivariate analyses were used to detect patterns of variables associated with gender and the lifetime severity of AUD. Men had a longer history of severe AUD than women. Women had similar levels of alcohol dependence and medical and psychological sequelae as men, despite 6 fewer years of AUD. More women than men had a history of severe psychosocial stress, severe dependence on other substances and antecedent mental health problems, especially mood and anxiety disorders. There were differences in family history of alcohol-related problems approximating same-gender aggregation. The severity of a lifetime AUD was predicted by its earlier age at onset and the occurrence of other disorders, especially anxiety, among both men and women. The limitations in the generalizability of these findings due to sample idiosyncrasies are discussed.     

Biochemical and molecular characterisation of cubozoan protein toxins

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as α-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.     

Dose tolerability of chronically inhaled voriconazole solution in rodents

Invasive pulmonary aspergillosis (IPA) is a fungal disease of the lung associated with high mortality rates in immunosuppressed patients despite treatment. Targeted drug delivery of aqueous voriconazole solutions has been shown in previous studies to produce high tissue and plasma drug concentrations as well as improved survival in a murine model of IPA. In the present study, rats were exposed to 20 min nebulizations of normal saline (control group) or aerosolized aqueous solutions of voriconazole at 15.625 mg (low dose group) or 31.25 mg (high dose group). Peak voriconazole concentrations in rat lung tissue and plasma after 3 days of twice daily dosing in the high dose group were 0.85 ± 0.63 μg/g wet lung weight and 0.58 ± 0.30 μg/mL, with low dose group lung and plasma concentrations of 0.38 ± 0.01 μg/g wet lung weight and 0.09 ± 0.06 μg/mL, respectively. Trough plasma concentrations were low but demonstrated some drug accumulation over 21 days of inhaled voriconazole administered twice daily. Following multiple inhaled doses, statistically significant but clinically irrelevant abnormalities in laboratory values were observed. Histopathology also revealed an increase in the number of alveolar macrophages but without inflammation or ulceration of the airway, interstitial changes, or edema. Inhaled voriconazole was well tolerated in a rat model of drug inhalation.