美洲大蠊提取物抗肝损伤的实验研究

目的:观察美洲大蠊活虫冷浸药渣水提物对小鼠急性肝损伤模型及CCl4所致大鼠慢性肝损伤模型的影响,为美洲大蠊抗肝损伤的临床应用及物质基础研究提供一定实验依据。方法:美洲大蠊活虫冷浸药渣水提物连续灌胃给予小鼠10天,分别采用灌胃醋氨酚、60%乙醇及腹腔注射CCl4进行小鼠急性肝损伤造模,测定模型小鼠血清ALT与AST水平、肝组织SOD与MDA水平及肝脏、脾脏指数以评价受试药物的抗急性肝损伤作用;采用每周两次灌胃CCl4,连续8周的方式制备慢性肝损伤大鼠模型,测定模型动物血清ALT、AST水平及肝、脾、胸腺指数以评价药物的抗慢性肝损伤作用。结果:美洲大蠊活虫冷浸药渣水提物49.13 mg﹒kg~(-1)显著降低酒精性与CCl4性急性肝损伤模型小鼠血清AST、ALT水平(P0.05),对醋氨酚模型小鼠血清AST、ALT水平有降低趋势(P0.05),能显著性降低醋氨酚模型小鼠脾脏指数(P0.05);65.51 mg﹒kg~(-1)显著降低酒精性急性肝损伤模型小鼠血清ALT、AST水平(P0.05),32.75 mg﹒kg~(-1)显著降低血清ALT与肝组织MDA水平(P0.05)。美洲大蠊活虫冷浸药渣水提物75.78 mg﹒kg~(-1)显著降低CCl4所致慢性肝损伤模型大鼠血清AST、ALT水平(P0.05),45.76、75.78 mg﹒kg~(-1)显著提高模型大鼠胸腺指数(P0.05),45.76 mg﹒kg~(-1)明显降低肝脏指数(P0.05)。结论:美洲大蠊活虫冷浸药渣水提物具有抗急性和慢性肝损伤的作用,该部位可能是其保肝作用的物质基础之一。     

水飞蓟素对伴刀豆球蛋白A致免疫性肝损伤小鼠的保护作用

目的研究水飞蓟素对伴刀豆球蛋白A(concanavalin A,Con A)诱导的免疫性肝损伤小鼠的保护作用,探讨其可能的肝保护机制。方法将实验小鼠30只随机分为对照组、模型组、水飞蓟素组(200 mg·kg~(-1)),各10只。对照组和模型组小鼠按10 m L·kg~(-1)灌胃0.5%羧甲基纤维素钠(CMC-Na)水溶液,水飞蓟素组小鼠灌胃同等容量的水飞蓟素(200 mg·kg~(-1),0.5%CMC-Na混悬),每日1次,共10 d。实验末期,模型组和水飞蓟素组小鼠尾静脉注射Con A(15 mg·kg~(-1))建立小鼠免疫性肝损伤模型,检测小鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)活性及肝组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)、丙二醛(MDA)含量;苏木精-伊红(HE)染色法观察肝组织病理学变化。ELISA法检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)和白细胞介素-6(IL-6)含量。结果水飞蓟素明显降低Con A所致免疫性肝损伤小鼠血清ALT、AST和ALP水平(P<0.01),还可增加肝组织SOD、CAT、GSH的活性,降低肝组织MDA水平(P<0.05或P<0.01),明显改善肝脏病理组织状况;显著降低小鼠血清TNF-α、IL-4和IL-6含量(P<0.01)。结论水飞蓟素能明显减轻Con A诱导的小鼠免疫性肝损伤,该作用与其增加肝组织中抗氧化酶的活性、降低脂质过氧化水平、降低TNF-α、IL-4和IL-6等促炎因子水平有关。     

刀豆蛋白A对小鼠肝线粒体结构和功能的影响

目的研究刀豆蛋白A(Con A)致小鼠免疫性肝损伤时,肝线粒体结构和功能的改变。方法C57BL/6J小鼠随机分为正常对照组、Con A 10和20 mg·kg~(-1)组,分别尾静脉注射灭菌生理盐水、Con A 10和20 mg·kg~(-1),给药8 h后,测定血清谷丙转氨酶(GPT)活性和肝组织病理变化。通过检测血清谷氨酸脱氢酶(GDH)活性评价线粒体膜通透性的改变,用透射电镜观察肝细胞线粒体结构,用高效液相色谱(HPLC)测定肝组织匀浆液中ATP,ADP和AMP含量,计算肝总腺苷酸库(TAN)及能荷水平(AEC),评价线粒体功能状态。结果与正常对照组比,Con A 10和20 mg·kg~(-1)组小鼠血清GPT和GDH活性均显著升高(P<0.05,P<0.01)。用透射电镜观察发现,肝细胞线粒体膜结构破坏,线粒体肿胀,形状高度不规则。功能学研究表明,小鼠肝组织中ATP含量分别降低了25.6%和38.3%(P<0.01),ADP含量分别降低了36.0%和47.1%(P<0.01),TAN水平分别降低了16.5%和18.9%(P<0.05),AEC分别降低了15.7%和28.1%(P<0.01)。结论 Con A免疫性肝损伤与肝细胞线粒体膜通透性增加、肝细胞线粒体结构完整性破坏、生成高能磷酸化合物能力下降和肝细胞能量储备减少有关。     

空心莲子草醇提物抗CCl4肝损伤的实验研究

目的观察空心莲子草醇提物对CCl4肝损伤的保护作用.方法给小鼠腹腔注射CCl4制成肝损伤模型,分为3组,每组10只.分别经口给予生理盐水、空心莲子草醇提物(0.02 g/g)和联苯双酯(0.2 mg/g).连续用药6天后,第7天将小鼠断头处死,取血和肝脏,分别测定空白对照组、空心莲子草组和联苯双酯组小白鼠的血清ALT和血清AST、肝指数及肝细胞镜检,并进行比较.结果空心莲子草组能明显对抗CCl4引起的血清ALT和血清AST增高,减轻CCl4对肝细胞的损伤.空白对照组与联苯双酯组和空心莲子草组比较均有显著性差异(P＜0.01).结论空心莲子草醇提物对CCl4致肝损伤有保护作用.     

姜黄素保护刀豆蛋白A诱导的小鼠肝损伤作用的研究

目的：研究姜黄素（curcumin,Cur）对刀豆蛋白A（concanavalin A,Con A）诱导的小鼠肝损伤的保护作用及可能机制。方法：40只ICR小鼠被随机均分为正常对照组、模型组、Cur100 mg/kg组和200 mg/kg组。受试物组每天灌胃姜黄素液,模型组和正常对照组每天灌胃同体积生理盐水,连续10 d。末次给药后4 h,除正常对照组外均采用尾静脉注射Con A 20 mg/kg,8 h后检测被测试动物肝匀浆液的丙二醛（MDA）和超氧化物歧化酶（SOD）指标及血清ALT、AST指标,观察肝组织病理学改变。结果：与模型组相比,受试物组ALT、AST活力均显著降低,肝脏MDA水平降低,SOD活力提高,肝组织病理改变减轻。结论：姜黄素具有减轻肝损伤的作用,该作用可能通过减轻肝脏的氧化应激损伤来实现。     

Study on the liver - protective effect of alcohol extracts from Polygonum chinense

目的 研究火炭母醇提物对大鼠急性肝损伤的保护作用.方法 采用CC14诱导大鼠急性肝损伤模型,观察火炭母醇提物对大鼠血清中丙氨酸氨基移换酶(ALT)、天门冬氨酸氨基移换酶(AST)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平的影响.结果 火炭母醇提物各剂量组均可抑制急性肝损伤大鼠血清中ALT、AST的活性,降低MDA的水平,升高SOD的活性.结论 火炭母醇提物对CCl4致大鼠急性肝损伤具有较好的保护作用.     

六味五灵片对刀豆蛋白A诱导的小鼠急性免疫性肝损伤的保护作用研究

目的研究六味五灵片(Liuweiwuling Tables,LWWL)对刀豆蛋白A(concanavalin A,Con A)诱导的小鼠急性免疫性肝损伤的保护作用及机制。方法将小鼠随机分成正常组对照组、模型组、双环醇组(Bicyclol,39 mg·kg~(-1))、六味五灵片低剂量组(8 g·kg~(-1))、六味五灵片高剂量组(16 g·kg~(-1)),各给药组每天给药1次,连续7 d,末次给药1 h后,除正常对照组外,其他各组尾静脉注射Con A(15 mg·kg~(-1))制备小鼠急性免疫性肝损伤模型。HE染色观察小鼠肝组织病理变化;比色法检测小鼠血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBIL);实时定量RT-q PCR测定法检测肝组织中白介素-12(IL-12)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白介素-4(IL-4)、白介素-10(IL-10)mRNA的表达;流式细胞术(FCM)观察脾脏Th1(IFN-γ)/Th2(IL-4)细胞的变化;免疫印迹(Western blot)法检测肝组织中Th1/Th2转录因子T-bet/GATA-3的表达。结果与正常对照组相比,模型组小鼠血清中ALT、AST、TBIL明显升高,小鼠肝组织病理损伤严重,肝细胞大量坏死、凋亡,表明造模成功。与模型组比较,六味五灵片低、高剂量组小鼠血清中ALT、AST、TBIL的水平明显降低;脾脏中Th1细胞减少,Th2细胞增多。肝组织中IL-12、IFN-γ、TNF-α的mRNA表达下降,IL-4、IL-10的mRNA表达上升,GATA-3蛋白表达上调,T-bet蛋白表达无明显变化。结论六味五灵片通过调节Th1/Th2的平衡,保护Con A诱导的急性免疫性肝损伤。     

泽泻水醇提取物改善千里光碱致小鼠急性肝损伤的作用与机制研究

近年来,含吡咯里西啶生物碱(pyrrolizidine alkaloid,PA)的中草药所致药源性肝损伤引起了国内外的广泛关注,但缺乏有效的临床治疗药物。因此,本文研究中药泽泻对代表性PA千里光碱(senecionine,SEN)所致急性肝损伤的改善作用,并初步探讨其潜在的药理作用机制。实验方案经上海中医药大学实验动物福利与伦理委员会审查,符合实验动物福利与伦理相关规范。小鼠单次灌胃SEN(50 mg·kg^-1)造成急性肝损伤模型,并设泽泻水提物(18 g·kg^-1)保护组、泽泻醇提物(18 g·kg^-1)保护组和空白对照组。结果表明,泽泻对SEN致小鼠急性肝损伤有明显的保护作用:泽泻提取物可显著降低急性肝损伤小鼠血清谷丙转氨酶、谷草转氨酶活性及总胆汁酸水平,炎性细胞浸润、肝窦淤血、肝组织坏死等病理变化均有所缓解,且醇提物药效优于水提物。进一步测定血清中主要胆汁酸的含量和肝脏胆汁酸代谢相关因子的mRNA和蛋白表达,发现泽泻可调控胆汁外排转运体和肝脏代谢酶的表达,维护胆汁酸代谢平衡,修复受损的肝细胞。本研究为临床上应用泽泻防治PA致肝损伤提供了理论依据。     

五指毛桃水提物对拘束应激性肝损伤的保护作用

目的:研究五指毛桃水提物对拘束负荷诱发小鼠应激性肝损伤的保护作用.方法:将雄性昆明种小鼠随机分为正常对照组,拘束应激模型组,破壁灵芝孢子粉(GL)组(500 mg·kg^-1)和五指毛桃水提物(RFH)低、中、高剂量组(125,250及500 mg·kg^-1),共6组.连续灌胃给药4 d后,除正常对照组外,其余各组小鼠拘束负荷18 h诱发小鼠应激性肝损伤,分别用赖氏法测定小鼠血浆丙氨酸氨基转移酶(ALT)活性、硫代巴比妥酸法测定肝组织丙二醛(MDA)含量、Griess化学法测定一氧化氮(NO)含量、荧光酶标仪测定肝组织抗氧化能力指数(ORAC)、HPLC法测定谷胱甘肽(GSH)含量、比色法测定谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽硫转移酶(GST)活性.结果:与拘束应激模型组相比,五指毛桃水提物可以明显降低拘束负荷小鼠血浆ALT活性、肝组织MDA和NO含量,有效提高肝组织的ORAC指数、GSH含量、GSH-Px和GST活性.结论:五指毛桃水提物对拘束负荷诱发小鼠应激性肝损伤有一定的保护作用,其作用可能与缓解拘束负荷小鼠的氧化应激状态相关.     

马栗种子提取物通过抑制活性氧及JNK途径保护刀豆蛋白A诱导的小鼠急性肝损伤

目的观察马栗种子提取物(AH)对刀豆蛋白A(Con A)诱导的小鼠急性肝损伤的保护作用,并探讨其相关机制是否与AH抑制氧化应激及其介导的c-Jun氨基末端激酶(JNK)途径有关。方法小鼠尾静脉一次性注射Con A(20mg·kg~(-1))制备急性肝损伤模型,AH保护组分别给予12.5、25、50 mg·kg~(-1)AH连续灌胃20 d预保护;全自动生化分析仪检测血清ALT、AST、TP、Alb、A/G;ELISA法检测小鼠血清IFN-γ、TNF-α水平;肝组织HE染色并进行病理学损伤分级评估;试剂盒检测肝组织MDA、SOD、GSH水平;分光光度法检测caspase-3活性;Western blot检测caspase-3、Bax、Bcl-2、p-JNK、p-Akt的蛋白表达情况。结果与Con A模型组相比,不同浓度的AH处理组小鼠血清ALT、AST、IFN-γ、TNF-α水平明显降低,TP、Alb、A/G比明显增高;肝组织的MDA水平明显降低,SOD、GSH水平明显升高。肝组织病理学与血清学指标改变相一致,AH处理组相比Con A模型组病理损伤明显减轻,病理损伤分级降低;与模型组相比,AH保护组细胞色素C、caspase-3、Bax/Bcl-2、p-JNK均明显降低,而pAkt蛋白表达升高。结论 AH可明显改善Con A诱导的小鼠急性肝损伤,其机制与AH抗氧化应激,进而抑制JNK/线粒体凋亡途径有关。     

羟基喜树碱对环孢素A药物动力学参数的影响

目的 :研究羟基喜树碱 (HCPT)对家兔环孢素 A(Cs A)药物动力学的影响。方法 :采用荧光偏振免疫分析法 (FPIA)对单用 Cs A及 Cs A与 HCPT合用后 ,家兔体内 Cs A全血药物浓度进行测定 ,并对两组药动学参数进行了统计学处理。结果 :HCPT可使 Cs A的 a,V(c) ,K12及 CL(s)显著性降低 ,AUC略有增加 ,T1/2 (a)显著性增加 (P<0 .0 5 ) ,合并用药组的 Cs A血药浓度在给药后的 0 .2 5 ,0 .5和 1.0 h高于对照组 ,且在 1.0 h时差异有显著性 (P<0 .0 5 ) ,其余药动学参数无显著变化。结论 :Cs A与 HCPT合用可行     

Novel 1,5-Benzodiazepines as CCK-B Ligands. Effect of Aryl-Carbamic Substituents at the C-3 Position Together with Halogen Substitution on the Benzo-Fused Ring

The synthesis and biological evaluation as potential CCK-B receptor ligands of a number of 1-isopentyl-3-aryloxycarbamoyl-5-aryl-1,5-benzodiazepines substituted with halogen atoms on the benzo-fused ring is here briefly discussed.     

前列腺素E对实验性大鼠急性肾缺血血栓素A的影响

目的:观察前列腺素E(PGE)对实验性大鼠急性肾缺血时血栓素A(TXA)的影响。方法:Wistar雄性大鼠40只随机分为两组,模型对照组20只,实验组20只。用戊巴比妥钠40 mg/kg麻醉,5~8 min后,开腹钝性分离出左肾动脉,夹闭40 min后放开,治疗组在夹闭40 min后血管内给予PGE12 ml/kg,两组分别于夹闭前及夹闭后40 min及开放后1 h取血,测定血栓素B2(TXB2)、6-酮-前列腺素F1a(6-keto-PGF1a)。结果:治疗组注入PGE11 h后血浆中的TXB2、6-keto-PGF1a的浓度显著下降,而对照组无明显下降,治疗组与对照组比较有显著性差异。结论:PGE能显著降低大鼠急性肾缺血TXB2水平。     

The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing cys269 with glycine

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60 MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC₅₀ values for 21 MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21 MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC₅₀ values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC₅₀ values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC₅₀ values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC₅₀ values were determined under the same experimental conditions, ensuring the quality of the data. The IC₅₀ values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.     

具有UVA、UVB双重防护效果的防晒唇膏的研究

目的 针对大多数唇膏对全波长紫外线照射无防护作用的现状,研制一种兼具UVA和UVB防护功能的防晒唇膏。方法 以黏稠度和耐热性为评价指标,采用正交实验,优化处方中蜂蜡、蓖麻油、液体石蜡的最佳配比。评价最优处方的涂展性、稳定性及防紫外线效果。结果 蜂蜡∶蓖麻油∶液体石蜡为8∶5∶4（w/w/w）时为最优处方,黏稠度较大,易于涂展、稳定性好,对UVA、UVB波段紫外线防护效果好。结论 该防晒唇膏制备简便、成本低、质量稳定,有望成为一种新型的全效防护防晒唇膏。     

Cyclosporin A analogues: recent advances

Cyclosporin A (Sandimmune^®, Neoral^®; Novartis) is a cyclic hydrophobic undecapeptide used to prevent and treat organ transplantation rejection. Cyclosporin A has potential therapeutic applications in the treatment of diseases such as asthma, psoriasis, atopic dermatitis and rheumatoid arthritis, but its wide use is limited by its poor bioavaliability and toxicity, the most significant of which is nephrotoxicity. There has been a tremendous research effort in producing cyclosporin analogues with better pharmacological profiles. This paper will give a general overview of the recent advances (1999 – 2002) made in producing novel semisynthetic cyclosporin analogues with improved bioavailability and therapeutic index.     

多抗甲素对脐血树突状细胞体外扩增、成熟的影响

目的观察多抗甲素(polyactin A,PA)对脐血树突状细胞(dendritic cell,DC)体外扩增、成熟的影响。方法分别用加有PA、GM-CSF+TNF-α+IL-4(GTI)及GTI+PA(GTIP)的RPMI-1640培养液体外诱导培养脐血单核细胞,培养d7,Elivision免疫组化方法检测各组细胞CD1a、CD83、HLA-DR、CD34抗原表达情况,透射电镜观察细胞形态。结果PA组、GTI组及GTIP组均有一定数量的典型DC,电镜观察该细胞表面有大量粗细不等的树枝状突起;PA组CD1a+、CD83+细胞比例分别达19·63%±3·61%、14·52%±5·79%,高于对照组(即单纯培养液组);GTIP组CD1a+细胞比例升高最明显,高于GTI组。结论PA不仅能促进脐血DC体外扩增、成熟,还能协同GM-CSF、TNF-α、IL-4促进DC生成,PA是一种实用的DC活化剂。     

高效液相色谱法AtlantisC18反相色谱柱检测生物样品中儿茶酚胺的研究

采用了AtlantisC18反相色谱柱（适用于分离极性分子的反相色谱柱），成功地分离了去甲肾上腺素（NA），肾上腺素（A），多巴胺（DA）等儿茶酚胺（CAs）类物质，研究了CAs在AtlantisC18和NovlepakC18柱的保留特性，比较了流动相中离子对试剂辛烷基磺酸钠（B8）浓度对CAs容量因子k’的作用，确认采用AtlantisC18柱分离CAs，色谱条件优化空间更大，更适于复杂生物样品分析。色谱条件：AtlantisC18柱，电化检测，流动相：NaH2PO4 50mmol／L、柠檬酸钠50mmol／L缓冲液-乙腈（体积比（75：25），检测限（S／N=2）在0．93—2．43pg，绝对回收率均在98．02％以上，样品日内RSD1．5％以下。方法精密、准确。     

Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins

A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 μg/kg, respectively; both below the European legal limit of 160 μg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 μg/kg and 3.9% at 124 μg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.     

Comparison of protein phosphatase inhibition activities and mouse toxicities of microcystins.

Eight naturally purified microcystins (MCs), including MC-LR, -FR, -WR, -RR, [d-Asp(3)]MC-FR, -WR, -RR, and [Dha(7)]MC-RR were utilized to determine the effects of amino acid substitutions and modifications on MC-induced protein phosphatase inhibition activity and mouse toxicity. Catalytic subunits of protein phosphatase 1 (PP-1) and 2A (PP-2A) were purified and subjected to the inhibition assays, and intraperitoneal injection was used to administer MCs into mice for the toxicity assay. It is found that the replacement of the non-polar amino acid l-leucine at the second position of these heptacyclic peptide toxins by a polar l-arginine reduces their mouse toxicities and inhibitory activities against PP-1 and PP-2A to different extends. Demethylation of methyldehydroalanine (Mdha) at the seventh amino acid of MC-RR exhibits the least mouse toxicity and phosphatase inhibition. The loss of a methyl group on the common methylaspartic acid (MeAsp) at the third position of MC-FR, -WR, and -RR does not alter their toxicity levels, but dominantly reduces their activities in PP-1 inhibition compared to other substitutions or modifications. This suggests that the methyl group on MeAsp is also important for MCs inhibition. However, such a tendency is not observed for PP-2A. By comparing the LD(50) values of the mouse toxicity assay and IC(50) values of the PP-1 and PP-2A inhibition assay of eight MCs using linear regression, it is evident that the MC-induced toxicity is much more related to the inhibition of PP-2A than PP-1, which suggests that PP-2A inhibition may play a major role in the MC-induced mouse toxicity.