紫草素通过内质网应激蛋白抑制人骨肉瘤U-2 OS细胞增殖的研究

目的研究紫草素对人骨肉瘤U-2 OS细胞存活的抑制和对内质网应激蛋白的调控机制。方法采用CCK-8法检测0.156 25～4μmol/L紫草素对人骨肉瘤U-2 OS细胞抑制率;取对数生长期U-2 OS细胞,分别给予0～4μmol/L紫草素诱导U-2 OS细胞凋亡,24 h后用流式细胞术检测细胞凋亡率;通过Western blotting检测紫草素对内质网应激蛋白ATF6、IRE1α、ATF4、GRP78的表达调控作用;将混有质粒或小干扰RNA的Lipofectamine 2000试剂处理U-2 OS细胞6 h换液,加入紫草素处理,检测ATF4或GRP78对紫草素处理的细胞活力的影响;将ATF4或空白质粒(3μg)、GRP78报告质粒(3μg)和海肾内参质粒(4μg)均匀混合,用Lipofectamine 2000试剂对人骨肉瘤U-2 OS细胞进行转染,6 h换液,加入0.8μmol/L紫草素继续孵育24h,实验结果用各孔海肾荧光做参比进行校正,检测紫草素对ATF4介导的GRP78转录的调控作用。结果紫草素显著抑制人骨肉瘤细胞的活力并能浓度相关性促进细胞凋亡;Westernblotting结果显示,紫草素能特异性增加ATF4和GRP78蛋白的表达;过表达ATF4能显著抑制U-2 OS细胞活力(P0.01);敲低ATF4能部分逆转紫草素对细胞活力的抑制(P0.01);过表达GRP78并不影响紫草素对细胞存活的抑制效果,但敲低GRP78可以显著增加紫草素对U-2 OS细胞活力的抑制(P0.01);报告基因结果显示紫草素能增加ATF4对GRP78基因的转录调控。结论紫草素能显著抑制人骨肉瘤U-2 OS细胞活力,诱导内质网应激蛋白的表达,敲低GRP78能进一步增加紫草素的细胞毒性。     

内质网应激信号通路在棕榈酸诱导的血管内皮细胞凋亡中的作用

目的探讨内质网应激(endoplasmic reticulum stress,ERS)信号通路在棕榈酸诱导的人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)凋亡中的作用。方法不同浓度(0.1、0.2、0.4、0.8 mmol·L~(-1))棕榈酸刺激HUVECs(0、12、24、48 h),CCK-8法检测血管内皮细胞增殖能力;免疫印迹法检测血管内皮细胞GRP78、CHOP、PERK、IRE1、ATF6等ERS信号通路蛋白的表达水平;免疫荧光法检测细胞内凋亡水平。结果 0.4 mmol·L~(-1)棕榈酸刺激血管内皮细胞24 h,细胞增殖率明显降低;GRP78、CHOP、PERK、IRE1、ATF6等ERS信号通路蛋白的表达明显升高(P<0.05),细胞凋亡水平明显增高(P<0.05);ERS抑制剂4-苯基丁酸(4-PBA,10 mmol·L~(-1))预处理组血管内皮细胞凋亡水平明显下调(P<0.05)。结论 ERS信号通路在棕榈酸所致血管内皮细胞凋亡的防治中有重要意义。     

内质网应激与酒精性肝病研究进展

内质网应激(ERS)由各种因素刺激所引起的,钙离子稳态失衡,ER管腔内错误折叠蛋白或未折叠蛋白堆积的病理状态,ERS通过激活UPR重建稳态。在非应激状态下,PERK、IRE1、ATF6三种跨膜感受蛋白与GPR78形成复合体,处于无活性状态。应激状态下时,累积下来的非折叠蛋白将竞争GRP78的结合,使得三者和GRP78解离并激活,引起GRP78表达增加。当处于ERS状态下的时间过长或者应激反应程度过强时,UPR通过激活ERS介导的细胞凋亡程序促进细胞死亡。其中PERK反向自身磷酸化,p-eIF-2a通过活化一种转录因子,诱导GRP78、GRP94等内质网分子伴侣蛋白表达增加,Caspase-12活化,触发了一个包括Caspase-12,9,3特异级联反应,从而导致凋亡。酒精性肝病从炎症向纤维化进展的一个重要特征就是含硫氨基酸的代谢紊乱。肝脏是其代谢的主要场所。同型半胱氨酸(Hcy)作为应激原,可以引起强度过高的ERS,最终导致肝细胞凋亡。甲硫氨酸代谢障碍所致的高半胱氨酸血症(HHcy)引起的ERS可能是酒精性肝损伤(Alcoholic liver disease,ALD)重要的发病机制之一。     

杨桃根DMDD对高糖诱导的肾小管上皮细胞HK-2内质网应激IRE1α通路及炎症反应的抑制作用北大核心CSCD

目的研究杨桃根活性成分2-十二烷基-6-甲氧基-2,5-二烯-1,4-环己二酮(DMDD)对高糖诱导肾小管上皮细胞HK-2内质网应激及炎症反应的抑制作用。方法体外培养HK-2细胞,并分为正常组、高糖组、内质网应激抑制剂4-PBA组(5 mmoL·L^(-1))和DMDD高、中、低浓度组(8、4、2μmol·L^(-1))。各组细胞培养结束后,采用CCK-8法检测细胞存活率;ELISA检测细胞上清液中TNF-α、IL-6的水平;AO/EB染色法检测细胞凋亡;Western blot法检测细胞中凋亡相关蛋白Bcl-2、Bax的表达水平、内质网应激标志蛋白GRP78、CHOP的表达水平以及IRE1α通路相关蛋白(IRE1α、JNK)的磷酸化水平。结果与高糖组比较,DMDD各浓度组细胞存活率明显升高;TNF-α、IL-6水平,Bax/Bcl-2、GRP78、CHOP蛋白表达水平和IRE1α、JNK蛋白磷酸化水平均明显降低,细胞中橙红色荧光明显减少。结论DMDD可能部分通过抑制内质网应激及IRE1α通路相关蛋白的表达,减少炎症反应,达到HK-2细胞的保护作用。     

昆布多糖对肾纤维化大鼠肾组织内质网应激的保护作用

目的研究昆布多糖对肾纤维化大鼠肾组织内质网应激(ERS)分子伴侣GRP78、GRP94蛋白表达的影响。方法采用单侧输尿管梗阻(UUO)诱导大鼠肾间质纤维化的动物模型,将大鼠随机分为假手术组、模型组、依那普利组、昆布多糖高、中、低剂量组。术后第7天处死大鼠,收集血清测定肌酐(Scr)、尿素氮(BUN)水平。采用Western免疫印迹法检测大鼠肾组织GRP78、GRP94的蛋白表达;采用HE、Masson染色检测肾小管损伤及肾间质纤维化程度。结果各治疗组与模型组比较大鼠肾小管间质损伤指数、肾间质纤维化程度、血清Scr、BUN水平及肾组织GRP78、GRP94蛋白表达有差异(P<0.05;P<0.01),昆布多糖与依那普利组比较,大鼠肾组织GRP78、GRP94表达升高(P<0.05),肾小管间质损伤及肾间质纤维化程度明显降低(P<0.05),肾功能Scr、BUN明显降低(P<0.05)。结论UUO早期昆布多糖可能通过上调ERS分子伴侣GRP78、GRP94蛋白表达,协助变性蛋白进行重新折叠、装配及跨膜转运,抑制未折叠蛋白反应,阻断ERS应激信号传导通路,从而减轻肾间质纤维化的发生和发展。     

白藜芦醇通过减缓内质网应激及增加eNOS的表达改善高热量高胆固醇饮食引起的内皮损伤

目的探讨白藜芦醇改善高热量高胆固醇饮食引起的血管内皮损伤的机制是否与内质网应激(endoplasmic reticulum stress,ERS)及内皮型一氧化氮合酶(e NOS)表达的改变有关。方法将24只♂C57BL/6小鼠随机分为标准饮食组(SCD)、高热量高胆固醇饮食组(HCD)及高热量高胆固醇饮食+白藜芦醇组(HCD+RES,白藜芦醇,400 mg·kg-1·d-1),共12周。观察胸主动脉内皮的病理学变化,检测eNOS的表达和ERS上下游相关基因的表达。将血管内皮细胞(MAEC)分为正常对照组(NC组)、模型组(棕榈酸组,PA)、中浓度RES药物对照组(RES)、低中高剂量RES治疗组(RES dose+PA),观察其对棕榈酸(PA)诱导的小鼠MAEC增殖的影响,检测葡萄糖调节蛋白78(GRP78)、转录因子GADD153(CHOP)蛋白和eNOS蛋白的表达。结果与SCD组相比,HCD组小鼠胸主动脉管壁明显增厚,弹力纤维排列紊乱,ERS上下游基因表达明显升高(P<0.05),eN OS蛋白表达减少;与HCD组相比,HCD+RES组血管弹力纤维排列明显改善,ERS相关基因的表达水平明显降低(P<0.05),eNOS蛋白表达增加。与NC组相比,PA组细胞增殖明显减少(P<0.05),GRP78和CHOP表达明显增加(P<0.05),eNOS表达降低,中、高剂量RES干预后MAECs增殖明显增强(P<0.05),GRP78和CHOP表达明显减少(P<0.05),eNOS蛋白表达增加。结论白藜芦醇有明显的改善高脂饮食引起的内皮损伤和PA诱导的抑制内皮细胞增殖作用,可能与其减缓ERS、增加eNOS的表达有关。     

人参皂苷Rg1对单侧输尿管梗阻大鼠肾脏内质网应激的影响

目的观察人参皂苷Rg1(G-Rg1)对单侧输尿管梗阻(UUO)模型大鼠肾组织内质网应激反应(ERS)的影响。方法 22只SD大鼠随机分为假手术组、模型组、治疗组。治疗组在UUO的基础上给予G-Rg120 mg·kg-1·d-1腹腔注射,模型组及对照组均给予等剂量的生理盐水腹腔注射。14 d后处死大鼠,收集血标本检测肾功能,肾组织行肾脏病理染色及蛋白印迹法检测各组大鼠内质网应激相关蛋白GRP78、凋亡蛋白CHOP等的表达情况。结果模型组大鼠肾功能恶化明显,肾间质纤维化、炎细胞浸润等明显加重;蛋白印迹法结果表明GRP78、CHOP的表达明显增多;治疗组大鼠肾功能明显好转,肾脏病理损害减轻,同时ERS相关蛋白GRP78、CHOP的表达下降。结论 G-Rg1可以通过抑制内质网应激反应减轻UUO模型的肾间质纤维化,发挥肾脏保护作用。     

黄芪甲苷保护大鼠缺血/再灌注心脏的内质网应激机制研究

目的研究黄芪甲苷保护缺血/再灌注心脏的内质网应激(ERS)机制。方法建立离体心脏缺血/再灌注模型,♂Wistar大鼠结扎冠状动脉进行心肌缺血30 min/再灌注120 min,随机分成假手术组、缺血/再灌注组、ERS抑制剂牛磺熊脱氧胆酸(TUDCA)组、黄芪甲苷组;Western blot检测缺血期及再灌注期ERS分子伴侣葡萄糖调节蛋白78(GRP78)的表达;激光扫描共聚焦显微镜免疫荧光化学染色法检测GRP78的表达;TTC法测定心肌梗死面积;HE染色观察心肌组织形态学改变。结果与假手术组相比,缺血期GRP78表达没有明显增加,再灌注期GRP78表达明显增加,黄芪甲苷能够模拟TUDCA明显抑制再灌注期GRP78的表达、减少心肌梗死面积、改善心肌组织的形态,差异具有统计学意义(P<0.05)。结论 ERS发生于再灌注期而不是缺血期,黄芪甲苷通过抑制ERS保护大鼠缺血/再灌注心脏。     

银杏叶提取物对肝纤维化大鼠肝组织GRP78及CHOP表达的影响

目的：观察银杏叶提取物（GbE）对肝纤维大鼠内质网应激通路相关分子GRP78、CHOP表达的影响,探讨GbE抗肝纤维的作用机制。方法：采用40％的CCl4皮下注射（3mL.Kg-1.d-1,2次/周,首剂加倍） 6周诱导大鼠肝纤维化模型,同时分别以200、100、50mg.Kg-1.d-1的GbE灌胃干预6周,空腹取肝组织,RealTime-PCR及免疫组化法分别检测肝组织GRP78及CHOP基因的mRNA及蛋白表达。结果：肝组织GRP78和CHOP的mRNA及蛋白的表达较正常组明显增加（P〈0.05 P〈0.01）;应用高、中、低剂量GbE干预后,大鼠肝组织GRP78和CHOP基因的mRNA及蛋白表达显著下降（P〈0.05 P〈0.01）。结论：肝纤维化大鼠肝脏发生了ERS反应,GbE下调肝纤维化大鼠肝组织内质网应激通路相关分子GRP78和CHOP的mRNA及蛋白的表达可能是其抗肝纤维化作用的机制之一。     

Apocynin改善TNF-α诱导肝细胞胰岛素抵抗的机制研究

目的：探讨NADPH氧化酶（NOX）的P47PHOX亚基膜转位的抑制剂Apocynin对TNF-α诱导的胰岛素抵抗以及氧化应激水平的影响。方法：实验分为4组：对照组、TNF-α（4ng/mL）处理组、TNF-α＋Apocynin（100μmol/L）处理组和Apocynin（100μmol/L）处理组。蒽酮法检测细胞内糖原合成;用DCFH-DA探针标记,流式细胞术检测细胞内活性氧的水平;Westernblotting检测JNK、p-JNK、IRS1、p-IRS1的表达。结果：与对照组比较,TNF-α刺激HepG2后活性氧水平显著增加,细胞内糖原合成障碍。Apocy-nin能显著降低TNF-α诱导的细胞内活性氧的水平,并促进细胞内糖原合成。TNF-α激活JNK,同时抑制胰岛素信号通路,Apocynin能抑制TNF-α对JNK的激活,并且促进胰岛素信号通路敏感性。单独用Apocynin处理对细胞内活性氧和糖原合成以及下游信号通路均无显著影响。结论：Apocynin能降低TNF-α诱导的细胞内活性氧的水平,改善TNF-α诱导肝细胞胰岛素抵抗状态。     

Role of glutathione in reduction of arsenate and of gamma-glutamyltranspeptidase in disposition of arsenite in rats

Arsenate (AsV), the environmentally prevalent form of arsenic, is converted sequentially in the body to arsenite (AsIII), monomethylarsonic acid (MMAsV), monomethylarsonous acid (MMAsIII), and dimethylarsinic acid (DMAsV) and some trimethylated metabolites. Although the biliary excretion of arsenic in rats is known to be glutathione (GSH)-dependent, involving transport of arsenic-GSH conjugates, the role of GSH in the reduction of AsV to the more toxic AsIII in vivo has not been defined. Therefore, we studied how the fate of AsV is influenced by buthionine sulfoximine (BSO), which depletes GSH in tissues. Control and BSO-treated rats were given AsV (50 micromol/kg, i.v.) and arsenic metabolites in bile, urine, blood and tissues were analysed by HPLC-HG-AFS. BSO increased retention of AsV in blood and tissues and decreased appearance of AsIII in blood, bile (by 96%) and urine (by 63%). The biliary excretion of MMAsIII was also nearly abolished, the appearance of MMAsIII and MMAsV in the blood was delayed and the renal concentrations of these monomethylated arsenicals were decreased by BSO. Interestingly, appearance of DMAsV in blood and urine remained unchanged and the concentrations of this metabolite in the kidneys and muscle were even increased in response to BSO. To test the role of gamma-glutamyltranspeptidase (GGT) in arsenic disposition, the effect of the of the GGT inhibitor acivicin was investigated in rats injected with AsIII (50 micromol/kg, i.v.). Acivicin lowered the hepatic and renal GGT activities and increased the biliary as well as urinary excretion of GSH, but failed to alter the disposition (i.e. blood and tissue concentrations, biliary and urinary excretion) of AsIII and its metabolites. In conclusion, shortage of GSH decreases not only the hepatobiliary transport of arsenic, but also reduction of AsV and the formation of monomethylated arsenic, while not hindering the production of dimethylated arsenic. While GSH plays an important role in the disposition and toxicity of arsenic, GGT, which hydrolyses GSH and GSH conjugates, apparently does not influence the fate of the GSH-reactive trivalent arsenicals in rats.     

微透析取样技术在中药体内分析中的应用研究进展

本文综述了微透析取样技术在中药体内分析中的应用,介绍微透析取样技术的原理、组成、探针类型、特点,重点阐述了微透析取样技术在测定脑、血液、皮肤等组织器官中中药有效成分浓度的应用实例。表明微透析取样技术在中药药效研究中具有广阔的前景。     

应用PASS系统分析我院2010年住院医嘱

目的:了解我院2010年住院患者的合理用药情况,探讨如何利用合理用药监测系统( PASS)提高合理用药水平.方法:利用PASS对我院2010年15 966例住院患者的1 184 997条用药医嘱进行监测,以黑色警示医嘱为依据,收集不合理用药信息,并对监测结果进行统计、分析.结果:不合理用药医嘱50 261条,发生率为4.24％.绝对禁止黑色医嘱5441条,主要为药物相互作用(66.54％)、注射液体外配伍(17.86％)、用法用量(15.46％)、儿童警告(1.14％).结论:应用PASS系统能有效监测医嘱中的不合理用药情况,有利于提高临床合理用药水平,但PASS系统尚存在局限性,有待进一步完善.     

Changes in levels of dependency and predictors of mortality in elderly people in institutional care in Dunedin

The 1983 study of dependency of subjects in institutional care in Dunedin was repeated two years later. A significant increase in levels of dependency in residential homes, particularly in the Religious and Welfare sector was found. In 1983 there were 29 high dependency residents and 73 medium dependency residents in residential homes. In 1985 these numbers had increased to 55 and 86 respectively. There was no change in the number of low dependency residents. In 1983, 6 high dependency residents had been admitted to residential home care in the year prior to the study. In 1985 the number of high dependency residents recently admitted had increased to 23. There had also been a significant increase in the dependency of patients in Religious and Welfare continuing care hospitals. Of the 933 subjects in institutional care in 1983 who were able to be followed, 354 (37.9%) died in the following 2 years. Mortality rate was higher for those in hospital care (48.1%) than for those in residential home care (29.6%). Mortality rates were higher in more dependent subjects and this was evident for each measure of dependency.     

应用PASS系统对我院2008年住院医嘱的分析

目的监测分析2008年我院住院患者用药情况。方法将PASS系统嵌入医生工作站、临床药学工作站等子系统,构建合理用药计算机网络系统,对住院医嘱进行及时监测,将监测结果向医生反馈,并对其进行统计、分析。结果2008年共监测医嘱3 620 241条,不合理医嘱908条,占0.02%。不合理医嘱中,配伍禁忌(381条)占41.96%,用法用量(381条)占41.96%,药物相互作用(108条)占11.89%,儿童用药(38条)占4.19%。经与医生沟通后,更改不合理医嘱856条,占94.27%。结论PASS系统可有效监测医嘱中的不合理用药,通过与医生交流,大大减少药物不良事件的发生,值得临床推广应用,也为临床药师开展工作带来了极大的便利。但PASS系统尚存在局限性,有待进一步完善。     

Role of desacetylation in the detoxification of cephalothin in renal cells in culture

The toxicity of three cephalosporin antibiotics to rabbit kidney cells in culture was compared to their known nephrotoxic potential in vivo (cephaloridine greater than cefazolin greater than cephalothin). While cephalothin is considered to be a relatively nonnephrotoxic cephalosporin when administered to many species including humans and rabbits, in several in vitro systems involving rabbit renal tissue, cephalothin was comparatively more toxic than anticipated based on in vivo data. Cephalothin is extensively desacetylated in rabbits to a less microbiologically active metabolite, desacetylcephalothin. When a microsomal S9 fraction from rabbit kidney was added to the in vitro assay in cultured rabbit renal cells, cephalothin was desacetylated and its toxicity to kidney cells was reduced. The addition of S9 in vitro provided a toxicity ranking of the cephalosporins that correlated with their known in vivo nephrotoxic potentials (cephaloridine greater than cefazolin greater than cephalothin). The in vitro detoxification of cephalothin by S9 was blocked by the coadministration of the esterase inhibitor, aminocarb. Desacetylcephalothin was relatively nontoxic to rabbit renal tissue in vitro. These results suggest that the desacetylation of cephalothin in vivo represents a previously unrecognized mechanism of detoxification of this cephalosporin antibiotic. Furthermore, this mechanism of detoxification may be applicable to other acetylated cephalosporins.     

2009年某院住院患者抗真菌药用药调查

目的:分析讨论某院抗真菌药使用的合理性,为临床安全有效地使用抗真菌药提供参考。方法:回顾性统计分析某院2009年住院患者抗真菌药用药信息。结果:2009年某院住院患者抗真菌药DDDs排名前3名分别为:氟康唑、制霉菌素和伊曲康唑;使用金额排名前3名分别为:氟康唑、米卡芬净及卡泊芬净;更换一种抗真菌药进行治疗的患者数为176人,在全部患者中占13.4%。结论:应进一步强化用药指征的意识,提高标本送检率,同时改善某些抗真菌用药不合理更换的现象,以避免耐药性发生,从而更好更长远地体现抗真菌药的治疗价值。     

Kinetics of in vitro metabolism of methoxyphenamine in rats

1. Methoxyphenamine (MP) was metabolized in vitro by rat liver preparations to O-desmethylmethoxyphenamine (O-desmethyl-MP), N-desmethylmethoxyphenamine (N-desmethyl-MP) and 5-hydroxymethoxyphenamine (5-hydroxy-MP). These metabolic pathways were inhibited by SKF 525-A and carbon monoxide, which indicates that these reactions were mediated at least partly by an NADPH-dependent cytochrome P-450 system. 2. Strain differences in the metabolism of this drug in vitro were observed in female Lewis and Dark Agouti (DA) rats, which are proposed models for human debrisoquine phenotypes. Methoxyphenamine O-demethylase and 5-hydroxylase activity in DA rats were lower than those in Lewis rats. 3. The metabolic transformation of methoxyphenamine in vitro to O-desmethyl-MP was inhibited competitively by debrisoquine and sparteine. This indicates that the cytochrome P-450 isoenzyme mediating the metabolism of MP to O-desmethyl-MP is similar to that mediating metabolism of debrisoquine and sparteine. However, no inhibition was observed with methenytoin.     

Comparison of in vitro cell models in predicting in vivo brain entry of drugs

Although several in vitro models have been reported to predict the ability of drug candidates to cross the blood-brain barrier, their real in vivo relevance has rarely been evaluated. The present study demonstrates the in vivo relevance of simple unidirectional permeability coefficient (P(app)) determined in three in vitro cell models (BBMEC, Caco-2 and MDCKII-MDR1) for nine model drugs (alprenolol, atenolol, metoprolol, pindolol, entacapone, tolcapone, baclofen, midazolam and ondansetron) by using dual probe microdialysis in the rat brain and blood as an in vivo measure. There was a clear correlation between the P(app) and the unbound brain/blood ratios determined by in vivo microdialysis (BBMEC r=0.99, Caco-2 r=0.91 and MDCKII-MDR1 r=0.85). Despite of the substantial differences in the absolute in vitro P(app) values and regardless of the method used (side-by-side vs. filter insert system), the capability of the in vitro models to rank order drugs was similar. By this approach, thus, the additional value offered by the true endothelial cell model (BBMEC) remains obscure. The present results also highlight the need of both in vitro as well as in vivo methods in characterization of blood-brain barrier passage of new drug candidates.