毛酸浆果乙醇提取物抗炎作用及其机制研究

目的 观察毛酸浆果乙醇提取物（AEFPP）的抗炎作用,并探索其作用机制。方法 取二甲苯涂于小鼠耳廓制备耳廓肿胀急性炎症模型,将棉花植入大鼠皮下构建棉球肉芽肿慢性炎症模型,观察AEFPP的在体抗炎作用;采用MTT法检测质量浓度为0.25~20.00 mg/mL的AEFPP对RAW 264.7细胞的细胞毒性;质量浓度为0.625、1.250、2.500 mg/mL的AEFPP预处理3 h后,脂多糖（LPS）作用于RAW 264.7细胞制备炎症模型,ELISA法检测上清中肿瘤坏死因子（TNF-α）和白细胞介素-6（IL-6）的水平;实时定量PCR（qRT-PCR）法检测NF-κB p65 mRNA表达;western blotting法测定NF-κB p65蛋白表达。结果 0.8 g/kg AEFPP可显著抑制小鼠耳廓肿胀率;0.1、0.2、0.4 g/kg剂量组对大鼠棉球肉芽肿具显著的抑制作用,且表现出剂量依赖性;AEFPP在0~3 mg/mL范围内对细胞无明显毒性;另外,0.625、1.250、2.500 mg/mL AEFPP均可显著抑制RAW 264.7细胞上清液中的TNF-α和IL-6的水平;0.625、1.250 mg/mL AEFPP显著抑制NF-κB p65 mRNA及其蛋白的表达。结论 AEFPP具良好的抗炎活性,其作用机制可能与下调p65蛋白的表达和抑制炎症因子TNF-α和IL-6的释放有关。     

二妙散对巨噬细胞分化的调控作用研究

目的 研究二妙散对脂多糖（lipopolysaccharide,LPS）诱导巨噬细胞分化的影响。方法 噻唑蓝（methyl thiazolyltetrazolium,MTT）法测定二妙散水煎液对细胞活力的影响;将LPS （100 ng·mL^-1）及不同浓度（0.1,1.0,10 mg·mL^-1）的二妙散水煎液共同作用RAW264.7细胞后,酶联免疫吸附法（enzyme-linked immune sorbent assay,ELISA）检测上清液中肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、白介素-10（interleukin 10,IL-10）的含量,实时荧光定量PCR （Real-timefluorescent quantitative PCR,qPCR）检测RAW264.7细胞中白介素-1β（Interleukin 1β,IL-1β）,IL-6的mRNA水平,Westernblot检测RAW264.7细胞NOS2的蛋白表达情况。结果 二妙散水煎液在0.01~100 mg·mL^-1内对RAW264.7细胞无明显毒性。与LPS诱导的模型组相比,二妙散水煎液能明显降低细胞上清液中TNF-α的含量（P<0.05）、升高IL-10的含量（P<0.05）,且可显著抑制RAW264.7细胞中M1分化标记物IL-1β、IL-6的mRNA水平（P<0.05）及NOS2的蛋白表达（P<0.05）。结论 二妙散可通过抑制巨噬细胞向M1促炎方向分化,从而发挥抗炎作用。     

秋海棠抗炎作用及其机制研究

摘 要 目的： 探讨秋海棠不同溶剂提取物的抗炎作用及其机制研究。方法: 制备秋海棠醇提取物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物、水提取物,以 脂多糖（LPS）诱导RAW 264.7巨噬细胞活化,不同溶剂提取物作用24 h后,ELISA法检测上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素1β（IL-1β）的含量,Griess法测定一氧化氮（NO）的含量。 结果: 秋海棠乙酸乙酯提取物、正丁醇提取物能极显著抑制TNF-α、IL-1β、NO的含量（P<0.01）,秋海棠醇提取物、乙酸乙酯提取物能极显著抑制NO的含量（P<0.01）。 结论:秋海棠醇提取物、乙酸乙酯提取物、正丁醇提取物抗炎作用显著,其机制可能与减少炎症因子的释放有关。     

雷公藤次碱通过调控TLR4/MyD88/TRAF6信号通路抑制LPS诱导的RAW264.7细胞炎症反应

目的 探讨雷公藤次碱抗炎活性及其作用机制。方法 用细胞计数盒-8 (CCK-8)法考察雷公藤次碱对小鼠单核巨噬细胞白血病细胞RAW 264.7 增殖活性的影响,用酶联免疫吸附法（ELISA）检测雷公藤次碱对脂多糖（LPS）诱导的RAW264.7细胞分泌细胞因子一氧化氮（NO）、白细胞介素-1β（IL-1β）、 肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）的影响,用免疫印迹法考察雷公藤次碱对白介素受体相关激酶（IRAK）、肿瘤坏死因子受体相关蛋白6（TRAF6）、核因子κB抑制因子α（IκBα）、核因子κB（NF-κB p65）、分裂原激活的蛋白激酶p38（p38）、c-Jun氨基末端激酶（JNK）、细胞外调节蛋白激酶（ERK）在LPS刺激的RAW264.7细胞中的表达及其磷酸化的影响。结果 雷公藤次碱在25、50、100 μmol/L浓度下对RAW264.7细胞无显著毒性,并可显著抑制细胞因子NO、IL-1β、TNF-α和IL-6含量。免疫印迹法检测结果显示雷公藤次碱可显著抑制IRAK及TRAF6的表达;显著抑制ERK、P38和JNK的磷酸化;抑制IκBα的降解,降低NF-κB p65的核转运水平。结论 雷公藤次碱具有体外抗炎活性,其作用机制可能介导TLR4/MyD88/TRAF6信号通路。     

复方苦参酊剂对炎症小鼠和大鼠的抗炎作用研究

摘 要 目的：考察复方苦参酊剂对炎症小鼠和大鼠的抗炎作用,为该药的新药申报奠定基础。 方法: 动物随机分为空白对照组、复方苦参酊剂低、中、高剂量组(33,66,110 mg·ml-1)、消肿止痛酊阳性药物组,分别采用二甲苯致小鼠耳廓肿胀模型、小鼠腹腔通透性模型、大鼠棉球肉芽肿增生模型、角叉菜胶致大鼠足趾肿胀模型,以耳廓肿胀度抑制率、毛细血管通透性、棉球肉芽肿增生、足趾肿胀度为检测指标,比较复方苦参酊剂低、中、高剂量组和空白对照组及阳性药组的差异。 结果: 与空白对照组相比,复方苦参酊剂高剂量组能显著降低小鼠耳肿胀度、降低小鼠腹腔灌洗液吸光度值、减少大鼠棉球肉芽肿的增生、抑制大鼠角叉菜胶引起的足跖肿胀（P＜0.05）。 结论: 复方苦参酊剂对炎症小鼠和大鼠均具有抗炎作用。     

豆豉姜活性成分9,9’-O-二-(E)-阿魏酰基-内消旋-5,5’-二甲氧基开环异落叶松树脂酚(LCA)抗炎作用研究

目的对傣药豆豉姜活性成分LCA在动物及细胞水平上的抗炎作用进行评价。方法采用耳肿胀和棉球肉芽肿模型进行LCA抗炎的体内药效学评价。采用脂多糖(LPS)诱导的小鼠单核巨噬细胞(RAW264.7),通过检测其一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)的分泌量以及诱导型一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)蛋白的表达水平,以评价LCA的体外抗炎活性。结果在小鼠耳肿胀的实验中,LCA组的耳肿胀度明显低于模型组,且高浓度组对小鼠耳肿胀的抑制率高于阳性药组;在大鼠棉球肉芽肿实验中,LCA高剂量组显著减轻了棉球肉芽肿的湿重与干重。此外,LCA显著减少LPS诱导的RAW264.7细胞中NO、TNF-α的分泌,降低iNOS、COX-2蛋白的表达。结论LCA具有显著的体内外抗炎作用,值得进一步深入研究。     

西归含药血清对脂多糖诱导的小鼠巨噬细胞RAW264.7炎症因子的影响研究

目的 研究西归含药血清对脂多糖诱导的巨噬细胞炎症因子的影响。方法 SD大鼠灌胃给予西归乙醇提取物以制备西归含药血清,采用噻唑蓝（MTT）法观察给予不同浓度的西归含药血清对RAW 264.7增殖的影响;采用1 μg·mL^-1的脂多糖（LPS）建立RAW 264.7 细胞体外炎症模型,酶联免疫吸附测定(ELISA)法检测细胞上清液中肿瘤坏死因子α(TNF-α) 、白细胞介素6(IL-6) 、白细胞介素2(IL-2)的含量。结果 脂多糖刺激细胞后,给药组均引起肿瘤坏死因子α、白细胞介素6、白细胞介素2的高表达;而西归含药血清干预后,能显著抑制肿瘤坏死因子α和白细胞介素6的高表达（P<0.05或P<0.01）;但对白细胞介素2没有显著影响。结论 西归具有抗炎的作用,其抗炎作用可能是通过抑制脂多糖诱导的巨噬细胞炎症反应来发挥的。     

菥蓂正丁醇部位抑制TLR-4/NF-κB信号通路抗脂多糖诱导的RAW264.7细胞炎症反应作用研究

目的 研究菥蓂Thlaspi arvense水提醇沉部位、正丁醇部位的抗炎活性以及正丁醇部位的作用机制。方法 将菥蓂水提物经过乙醇、正丁醇萃取后制得菥蓂水提醇沉部位、正丁醇部位。采用MTT法确定水提醇沉部位、正丁醇部位（15.625、31.25、62.5、125、250、500、1 000 μg/mL）对RAW264.7细胞的毒性。采用脂多糖（LPS）1 μg/mL诱导RAW264.7细胞产生炎性反应,建立体外炎症细胞模型。Griess法检测水提醇沉部位、正丁醇部位（7.812 5、15.625、31.25、62.5、125、250、500、1 000 μg/mL）对LPS诱导RAW264.7细胞后NO释放量的影响;ELISA法检测水提醇沉部位、正丁醇部位（125、250、500 μg/mL）对白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）释放量的影响;Western Blotting法检测正丁醇部位（125、250、500 μg/mL）对环氧合酶-2（COX-2）、一氧化氮合酶（iNOS）、白细胞介素1β（IL-1β）、核因子-κB（NF-κB）p-P65、Toll样受体4（TLR-4）、磷酸化NF-κB抑制蛋白（p-IκBα）蛋白表达的影响。结果 菥蓂水提醇沉部位、正丁醇部位质量浓度在1 000 μg/mL以下时对RAW264.7细胞几乎无毒性作用。与模型组比较,菥蓂水提醇沉部位、正丁醇部位62.5、125、250、500、1 000 μg/mL组NO释放量显著下降（P<0.01）;菥蓂水提醇沉部位、正丁醇部位125、250、500 μg/mL组IL-6、TNF-α的分泌量显著降低（P<0.01）,且正丁醇部位作用优于水提醇沉部位;菥蓂正丁醇部位500 μg/mL组的iNOS和250、500 μg/mL组COX-2、IL-1β、NF-κB p-P65、TLR-4、p-IκBα蛋白表达量显著降低（P<0.01）,作用均呈浓度相关性。结论 菥蓂正丁醇部位显著抑制LPS诱导的RAW264.7细胞NO的释放,IL-1β、IL-6、TNF-α的分泌以及COX-2、iNOS蛋白的表达,其抗炎作用机制可能与抑制TLR-4/NF-κB信号通路相关。     

基于Nrf2/HO-1信号通路研究橄榄苦苷的体外抗炎作用

目的 探讨橄榄苦苷对脂多糖（LPS）诱导的小鼠腹腔巨噬细胞（RAW264.7）炎症的保护作用及机制。方法 MTT法检测橄榄苦苷（0、10、20、40 μmol/L）对RAW264.7细胞活性的影响;用橄榄苦苷（10、20、40 μmol/L）预处理细胞1 h后,LPS诱导炎症模型,Griess试剂检测细胞内Nitrite释放;Western blotting方法检测细胞iNOS、COX-2、Nrf2、Keap-1、HO-1、NQO1蛋白表达水平;流式细胞仪检测细胞内NO、ROS、Ca²⁺的水平;荧光显微镜检测线粒体膜电位（MMP）水平;ELISA法检测细胞上清液中肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）的释放。结果 与模型组比较,橄榄苦苷组Nitrite释放水平显著降低（P<0.05、0.01、0.001）,iNOS、COX-2蛋白的表达显著降低（P<0.001）,NO的产生显著减少（P<0.05、0.01）;TNF-α、IL-6的释放受到显著抑制（P<0.001）;Ca²⁺释放受到显著抑制（P<0.01）;ROS生成受到显著抑制（P<0.01）;JC-1单体降低,恢复聚合物状态（红色荧光变多）,MMP稳定性增强;Keap1蛋白表达显著降低, Nrf2、HO-1、NQO1蛋白的表达均显著升高（P<0.05、0.01、0.001）。结论 橄榄苦苷对LPS诱导的RAW264.7细胞炎症具有抑制作用,其作用机制可能与激活Nrf2/HO-1信号通路相关。     

RAW 264.7细胞炎症模型优化及异喹啉类生物碱抗炎活性研究

目的 对RAW 264.7细胞炎症模型进行优化,并考察异喹啉类生物碱的抗炎活性。方法 采用内毒素脂多糖（LPS）诱导RAW264.7细胞炎症反应,以细胞上清液中炎症因子分泌水平为指标,优化LPS诱导浓度、时间及阳性药地塞米松（DEX）孵育时间,并考察异喹啉类生物碱的抗炎活性。结果 RAW 264.7细胞上清液中肿瘤坏死因子-α（TNF-α）分泌水平随LPS诱导浓度、时间增加而升高,但当LPS质量浓度超过100 ng/mL、诱导时间超过12 h后,TNF-α分泌水平趋于平缓;LPS为20 ng/mL、诱导12 h的TNF-α分泌水平明显升高。DEX对TNF-α分泌抑制作用随孵育时间延长而增强,但孵育4 h后,TNF-α分泌抑制作用趋于平缓。异喹啉类生物碱呈现不同程度TNF-α分泌抑制作用,其抑制活性与其季胺型母核结构及R基团烷氧基取代等密切相关。结论 LPS为20 ng/mL、诱导12 h能较好诱导RAW 264.7细胞炎症反应;异喹啉类生物碱的TNF-α分泌抑制作用与其季胺型母核结构、R基团取代等密切相关。     

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.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

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.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.