铜绿假单胞菌通过MAPK信号传导通路诱导U937细胞表达IL-8

目的探讨铜绿假单胞菌(PA)活菌对不同分化状态的U937细胞表达IL-8的诱导作用及通过MAPK信号传导通路的调控机制。方法应用人单核白血病细胞系-U937细胞,采用ELISA和RT-PCR法对PA诱导不同分化状态的U937细胞IL-8蛋白分泌和其mRNA表达进行研究,并观察MAPKs抑制剂PD98059和SB203580对IL-8表达的影响。结果PA可促进U937细胞及PMA分化的U937细胞IL-8的mRNA及蛋白分泌,而且具有明显的量效和时效关系。分别用SB203580抑制p38MAPK通路、用PD98059抑制ERK通路,均能引起抑制剂浓度依赖的IL-8的表达(P<0.01)。结论PA以浓度和时间依赖的方式感染U937细胞,促进IL-8的分泌和mRNA表达,PA可能通过MAPK信号通路启动IL-8的高效表达和分泌。     

槐定碱对LPS诱导的RAW264.7巨噬细胞p38、iNOS表达的影响

目的探讨槐定碱(sophoridine,SRI)在内毒素导致的炎症反应中的作用。方法采用LPS诱导的RAW264.7巨噬细胞建立细胞炎症反应模型,实验细胞分为5组(n=6):空白对照组、LPS组、槐定碱组、SB203580组、SB203580+槐定碱组,利用反转录聚合酶链反应(RT-PCR)技术检测RAW264.7巨噬细胞p38 mRNA表达量;利用Western blot技术检测RAW264.7巨噬细胞p-p38与iNOS蛋白的表达量。结果槐定碱组与LPS组比较p-p38蛋白表达量和p38 mRNA表达量降低,但高于空白对照组(P<0.01),表明槐定碱可以抑制LPS诱导的RAW264.7巨噬细胞p-p38蛋白表达和p38 mRNA表达量;与LPS组比较,槐定碱组iNOS蛋白表达量降低,但高于空白对照组(P<0.01),SB203580+槐定碱组iNOS蛋白表达量低于槐定碱组和SB203580组(P<0.01),表明槐定碱可以抑制LPS诱导的RAW264.7巨噬细胞P38MAPK信号通路下游iNOS蛋白表达,并且与SB203580有协同作用。结论槐定碱可以通过抑制p38MAPK位点而下调p-p38、iNOS蛋白表达而发挥抗炎作用。     

P38 mAPK信号通路在糖尿病肾病中的作用

目的探讨p38 MAPK信号通路在糖尿病肾病中的作用。方法使用不同浓度葡萄糖及p38 mAPK特异性抑制剂SB203580干预肾小球系膜细胞,采用实时荧光定量PCR法检测各组肾小球系膜细胞p38 MAPK、MMP-9和TIMP-1 mRNA的表达。结果高糖组p38 mAPK、TIMP-1 mRNA的表达明显增加,且呈浓度依赖性;而MMP-9 mRNA表达和MMP-9/TIMP-1比值与正常组相比明显下降,同时p38 MAPK抑制剂SB203580能明显逆转以上变化。结论 p38 MAPK信号通路介导了高糖诱导的肾小球系膜细胞MMP-9和TIMP-1 mRNA表达的改变,进而参与了糖尿病肾病的进展。     

DATS通过p38MAPK通路抑制LPS诱导的小鼠MH-S细胞TNF-α及IL-1β表达

目的探讨p38MAPK在二烯丙基三硫(DATS)抑制脂多糖(LPS)诱导小鼠肺泡巨噬细胞促炎细胞因子表达中的作用。方法体外培养MH-S细胞,用DATS和(或)LPS进行干预,Western blot检测细胞p38及磷酸化p38(p-p38)的表达;用LPS和(或)SB203580孵育细胞,反转录PCR检测细胞中TNF-α、IL-1βmRNA表达,Western blot检测细胞磷酸化(p-IκB)及非磷酸化IκB的表达。结果 LPS刺激MH-S细胞可导致p-p38表达增加,呈时间依赖性;用DATS(0.1、0.5、2.5、5.0 mg.L-1)预处理细胞30 min后再给予LPS刺激,p-p38表达呈剂量依赖性下降;单独DATS对p-p38表达无明显影响。p38特异性抑制剂SB203580可剂量依赖性地抑制LPS诱导的p-IκB蛋白、TNF-α及IL-1βmR-NA表达。结论 DATS可通过抑制p38MAPK通路抑制IκB磷酸化及NF-κB活化,进而下调LPS诱导小鼠肺泡巨噬细胞TNF-α、IL-1βmRNA表达。     

阿司匹林抑制脂多糖诱导RAW264.7细胞MMP-9表达及机制研究

目的:探讨阿司匹林对脂多糖(LPS)诱导RAW264.7细胞中基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的表达及其机制研究.方法:MTT法检测LPS诱导RAW264.7细胞作用下,阿司匹林对细胞毒性的影响.Q-RT-PCR检测MMP-9 mRNA表达,免疫蛋白印迹法(western blot)检测MMP-9、p38 MAPK及磷酸化p38(Phospho-p38,P-p38)蛋白表达.特异性抑制剂SB203580(SB)阻断p38MAPK通路后,分别应用Q-RT-PCR和Western blot检测MMP-9的基因和蛋白表达.结果:阿司匹林呈剂量依赖性抑制MMP-9基因和蛋白表达.与LPS组相比,阿司匹林可明显抑制P-p38MAPK蛋白的表达,而p38MAPK总蛋白无明显变化.抑制p38MAPK通路后,MMP-9 mRNA和蛋白水平均明显下调.结论:阿司匹林抑制LPS诱导RAW264.7细胞中MMP-9表达可能与抑制p38MAPK信号转导通路有关,进而发挥其抗AS药理作用.     

丝裂原活化蛋白激酶抑制剂对急性肺损伤影响

目的探讨p38MAPK抑制剂SB203580对大鼠急性肺损伤影响。方法采用LPS建立SD大鼠急性肺损伤模型，随机分为对照组、LPS组、SB203580组。造模后注射p38MAPK抑制剂SB203580，在1h、3h、6h及12h时，剖杀大鼠，观察肺组织病理改变，ELISA法测血清中的TNF—α及IL-6；免疫组化检测肺组织中的p38MAPK及其磷酸化-p38MAPK。结果注射LPS后，SB203580治疗组较LPS组血清中的TNF—α及IL-6显著减少（P〈0．05 or 0.01）；肺组织中的磷酸化-p38MAPK的表达显著减轻，而p38丝裂原活化蛋白激酶未明显减轻。结论p38MAPK抑制剂SB203580可减轻血清中的TNF-α及IL-6和肺组织中的磷酸化-p38MAPK的表达，从而减轻LPS诱导的急性肺损伤。     

Toll样受体信号途径在RAW264.7细胞川崎病模型炎症反应中的作用及其机制研究

目的探讨Toll样受体(TLR)2信号途径在RAW264.7细胞川崎病模型炎症反应中的作用及其分子机制。方法构建TLR2-干扰小RNA(siRNA),合成2对特异性针对TLR2基因的siRNA序列,并用于转染RAW264.7细胞。实时荧光聚合酶链反应和蛋白质印迹法检测TLR2-siRNA对干酪乳杆菌细胞壁提取物(LCWE)诱导RAW264.7细胞p38促分裂原活化的蛋白激酶(MAPK)、基质多属蛋白酶(MMP)-9表达的影响,乳胶增强免疫比浊定量法检测培养基中高敏C反应蛋白(hsCRP)水平的变化;检测p38MAPK抑制剂SB 203580对LCWE诱导RAW264.7细胞MMP-9表达的影响。结果与对照组相比,LCWE刺激组p-p38MAPK、MMP-9的表达及hs-CRP水平都有明显升高,差异具有统计学意义(P<0.05),与LCWE刺激组相比,TLR2-siRNA+LCWE刺激组的p-p38MAPK、MMP-9表达和hs CRP水平降低,差异具有统计学意义(P<0.05)。与LCWE刺激组相比,SB203580+LCWE刺激组的MMP-9表达减轻,差异具有统计学意义(P<0.05)。结论 TLR2-siRNA可以有效减轻LCWE刺激造成的p-p38MAPK、MMP-9、hs-CRP表达升高。p38MAPK抑制剂SB203580对LCWE诱导RAW264.7细胞MMP-9表达有抑制作用。TLR2参与了RAW264.7细胞川崎病模型中的炎症反应,其机制可能与p38MAPK途径有关。     

阻断p38丝裂原活化蛋白激酶信号通路对高糖培养肾小球系膜NF-κB信号通路调控影响

目的:探讨阻断p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38MAPK)信号通路对高糖培养肾小球系膜NF-κB信号通路调控影响。方法:大鼠系膜细胞株分别培养在正常糖浓度(5.5 mmol/L,对照组),高糖浓度(25 mmol/L,高糖组)及25 mmol/L葡萄糖+10μmol/L p38 MAPK特异性抑制剂SB203580(SB组)。CCK-8测定系膜细胞增殖;Phospho-ELISA法分别检测胞浆及胞核内p38MAPK、磷酸化p38 MAPK蛋白和总NF-κBp65、活性NF-κB p65、磷酸化NF-κB p65(S276)表达。结果:与正常对照组比较,高糖组系膜细胞出现增殖增加;胞浆及胞核内磷酸化p38MAPK蛋白表达上调;胞核内NLS-NF-κB、Ser276-NF-κB的表达增加;SB203580干预则能逆转这一变化。结论:阻断p38 MAPK信号通路能下调高糖培养的系膜细胞NF-κB信号通路的活化,进而抑制系膜细胞的异常增殖。     

阿霉素诱导胰腺癌BxPC-3细胞凋亡过程中p38MAPK的表达

目的研究阿霉素诱导胰腺癌细胞凋亡过程中p38MAPK表达的变化,探讨p38MAPK在其中的作用。方法用膜联蛋白V-PI(annexinV-PI)染色及流式细胞技术分析阿霉素及应用p38MAPK抑制剂SB203580对胰腺癌细胞凋亡的影响,同时利用免疫细胞化学法观察经阿霉素及SB203580处理人胰腺癌BxPC-3细胞后,p38MAPK的表达水平。结果SB203580(20μmol/L)干预组胰腺癌BxPC-3细胞凋亡率为(20.1±1.4)%,阿霉素作用24 h后诱导胰腺癌BxPC-3细胞凋亡,凋亡率为(31.1±2.7)%,加用SB203580抑制p38MAPK通路后可增强阿霉素诱导的凋亡作用,凋亡率达(40.4±2.6)%。采用单因素方差分析F=136.79,组间比较组与组之间差异有统计学意义。以20μmol/L阿霉素作用BxPC-3胰腺癌细胞24 h后可见p38MAPK在细胞染色后呈现深褐色颗粒散在分布于部分或整个细胞核及细胞质内,联合应用SB203580后可见p38MAPK表达颗粒密度减低,数量减少。结论阿霉素可以活化p38MAPK通路,p38MAPK可能起到保护胰腺癌BxPC-3细胞逃避阿霉素诱导的凋亡,阻断该通路可增强阿霉素诱导胰腺癌细胞凋亡的作用。     

广州城区大气细颗粒物对PC-12细胞IL-8表达的影响

目的 研究广州城区大气细颗粒物(PM2.5)对PC-12细胞IL-8表达的影响,并从p38 MAPK途径探索其作用机制,促进对PM2.5毒性的认识.方法 采集广州城区大气中的PM2.5,对PC-12细胞染毒,设对照组、不同浓度PM2.5组和SB203580+PM2.5组(用20 μ mol/L的SB230580预处理1h后再给予100μg/ml的PM2.5),Trizol法提取RNA用定量PCR法检测细胞IL-8表达情况,RIRP法提取细胞总蛋白,用Western blot法检测p38 MAPK的磷酸化改变.结果 定量PCR检测显示用25、50和100μg/ml的PM2.5染毒后PC-12细胞IL-8表达明显增高;Western blot结果显示PM2.5可磷酸化激活p38 MAPK信号分子,加入p38 MAPK抑制剂SB230580可以抑制PM2.5诱导的IL-8表达.结论 PM2.5可通过p38 MAPK途径诱导PC-12细胞表达细胞因子IL-8,这可能是其导致神经细胞毒性的机制之一.     

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.     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

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.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.