利拉鲁肽通过调控HSP70-TLR4轴抑制db/db小鼠肾脏的炎症性损伤

目的探讨胰高血糖素样肽(GLP-1)类似物利拉鲁肽对糖尿病肾病(diabetic nephropathy,DN)炎症性损伤及高糖刺激下肾小管上皮细胞的作用及其可能机制。方法将实验小鼠随机分为对照组(db/m组)、糖尿病肾病模型组(db/db组)和利拉鲁肽治疗组(db/db+Li组),分别给予生理盐水或利拉鲁肽(1 mg·kg~(-1)·d~(-1))腹腔注射,持续给药8周。实验结束时,检测各组小鼠体质量、血糖、24 h尿白蛋白以及血肌酐水平。体外培养TCMK-1细胞,无血清DMEM培养基同步培养24 h,分为对照组(control)、高糖组(high glucose,HG)、HG+Li组、HG+VER、HG+HSP70组、HG+HSP70+NC si RNA(negative control si RNA,NC si RNA)组及HG+HSP70+TLR4 si RNA组,TLR4的小干扰RNA(si RNA)转染TCMK-1细胞后采用Western blot法检测TLR4蛋白表达。Real-time PCR检测肾组织及细胞中TNF-α、IL-6、IL-1β和CCL2 m RNA表达,Western blot分析TLR4及HSP70蛋白表达。结果实验结束时,各组小鼠均存活。与db/m组相比,db/db组小鼠体质量、空腹血糖、24 h尿白蛋白及血肌酐水平均显著升高(P0.05),利拉鲁肽治疗能显著逆转db/db小鼠上述指标的升高(P0.05)。db/db小鼠及高糖刺激下TCMK-1细胞炎症相关因子TNF-α、IL-6、IL-1β及CCL2 m RNA相对表达显著增加(P0.05),TLR4及HSP70蛋白表达显著升高(P0.05),利拉鲁肽治疗能显著降低db/db小鼠及高糖刺激下TCMK-1细胞炎症因子TNF-α、IL-1β及CCL2表达,TLR4 si RNA干扰TLR4蛋白表达能显著降低高糖刺激下TCMK-1细胞炎症相关因子TNF-α、IL-1β及CCL2 m RNA相对表达量(P0.05)。结论利拉鲁肽通过调控HSP70-TLR4轴活性抑制DN肾小管炎症性损伤,继而抑制DN病程进展。     

巨噬细胞过氧化物酶体增殖物激活受体α激活抑制巨噬细胞炎症反应诱导的心脏成纤维细胞活化及迁移

目的:研究巨噬细胞过氧化物酶体增殖物激活受体α(PPARα)激活对巨噬细胞炎症反应诱导的心脏成纤维细胞活化及迁移的影响。方法:将小鼠骨髓来源巨噬细胞随机分为4组:对照组、PPARα激动剂WY14643(10μmol/L)组、血管紧张素Ⅱ(Ang Ⅱ; 1μmol/L)组和Ang Ⅱ+WY14643组。培养24 h后收集上述各组巨噬细胞的培养上清作为条件培养基(CM),并利用RT-qPCR检测巨噬细胞PPARα及促炎因子白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子(TNF-α)的mRNA表达,Western blot检测IL-6和IL-1β的蛋白表达。用上述4组CM培养心脏成纤维细胞24 h,RT-qPCR检测心脏成纤维细胞纤维化标志物I型胶原α2链(Col1a2)、Ⅲ型胶原α1链(Col3a1)和肌动蛋白α2(Acta2)的mRNA表达,Western blot检测心脏成纤维细胞中collagen I、collagen ⅡI和α-平滑肌肌动蛋白(α-SMA;由Acta2基因编码)的蛋白水平。心脏成纤维细胞加入上述4组CM后用划痕实验观察迁移情况。结果:Ang Ⅱ显著增加巨噬细胞炎症因子IL-6、IL-1β和TNF-α表达的同时明显降低PPARα的表达,而WY14643显著降低Ang Ⅱ诱导的巨噬细胞炎症因子的表达。AngⅡ也可显著增加巨噬细胞中IL-6和pro-IL-1β的蛋白表达,而WY14643明显降低Ang Ⅱ诱导的巨噬细胞IL-6和pro-IL-1β的蛋白表达。Ang Ⅱ处理后的CM显著促进心脏成纤维细胞迁移及Col1a2、Col3a1和Acta2的mRNA表达,而WY14643处理后的CM则抑制心脏成纤维细胞的迁移以及Col1a2、Col3a1和Acta2的mRNA表达。Ang Ⅱ处理后的CM也促进心脏成纤维细胞collagen I、collagen ⅡI及α-SMA的蛋白表达,而WY14643处理后的CM则抑制心脏成纤维细胞collagen I、collagen ⅡI及α-SMA的蛋白表达。结论:WY14643激活的PPARα通过减轻Ang Ⅱ诱导的巨噬细胞炎症反应而抑制心脏成纤维细胞的活化及迁移。     

槲皮素通过抑制NF-κB减弱类风湿关节炎大鼠软骨细胞基质降解和细胞凋亡

目的探讨槲皮素(quercetin,Quer)对NF-κB活性的影响及对类风湿关节炎大鼠软骨细胞基质降解和细胞凋亡的作用。方法建立Ⅱ型胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠模型,设立CIA模型组、Quer治疗组并以正常大鼠作为对照组,比较各组大鼠AI指数和足趾容积;ELISA检测关节腔液中IL-1β、MMP-13含量;Western blot检测关节软骨组织中p-p65、MMP-13蛋白的表达。体外培养大鼠软骨组织细胞,分为control组、IL-1β处理组,检测软骨组织培养液中MMP-13、Hyp和蛋白多糖含量。将体外培养的软骨细胞分为3组:control组、IL-1β组、IL-1β+Quer组,检测软骨细胞内p-p65、MMP-13、COL2A1蛋白表达,流式检测细胞凋亡。结果与control组相比,CIA组大鼠AI指数和足趾容积明显增加,关节腔液中IL-1β、MMP-13含量以及软骨组织中p-p65、MMP-13蛋白表达明显增加;给予Quer治疗可显著降低大鼠的AI指数和足趾容积,使软骨组织内p-p65、MMP-13蛋白表达量明显减少,关节腔液中IL-1β、MMP-13含量明显降低。IL-1β处理可明显升高软骨组织培养基中MMP-13、Hyp含量,使蛋白多糖含量明显减少。IL-1β处理明显上调软骨细胞内p-p65、MMP-13蛋白表达,使COL2A1蛋白表达明显减少,软骨细胞凋亡明显增加;给予Quer处理则明显抑制软骨细胞内p-p65、MMP-13蛋白表达,使COL2A1表达量增加,细胞凋亡明显减少。结论 Quer可通过抑制NF-κB激活,减弱炎症环境下软骨细胞内MMP-13产生、基质降解和细胞凋亡,起到保护软骨的作用。     

IL-12单克隆抗体通过调节肠黏膜免疫缓解克罗恩病模型小鼠肠道炎症

目的观察白细胞介素12单克隆抗体(IL-12 mAb)对IL-10敲除(IL-10 KO)的克罗恩病(CD)小鼠模型肠道炎症的治疗作用并分析可能的机制。方法选取15周龄雄性IL-10 KO小鼠16只,随机分为对照组及IL-12 mAb处理组。IL-12 mAb处理组小鼠给予腹腔注射IL-12 mAb(25 mg/kg,每周1次),对照组给予0. 2 m L生理盐水腹腔注射。处理4周后,采用炎症性肠病疾病活动度指数(DAI)及HE染色结合Spencer结肠炎组织学评分评估两组小鼠肠道炎症程度及组织学改变;采用活体肠黏膜异硫氰酸荧光素标记的葡聚糖(FITC-dextran)通透性实验评估两组小鼠肠黏膜屏障功能,并采用Western blot法检测肠黏膜密封蛋白1(claudin-1)的蛋白水平;采用流式细胞术检测肠黏膜CD3、CD4、γ干扰素(IFN-γ)和IL-4,评估Th1细胞/Th2细胞平衡。采用ELISA检测两组小鼠肠黏膜IL-13及肿瘤坏死因子α(TNF-α)水平,Western blot法检测肠黏膜磷酸化的信号转导子与转录激活子6(p-STAT6)的蛋白水平。结果 IL-12 mAb治疗后第3周及第4周,IL-12 mAb处理组小鼠DAI及肠道炎症评分均显著低于对照组。同时,IL-12 mAb处理组小鼠肠黏膜通透性显著低于对照组,且肠黏膜claudin-1表达显著高于对照组。同时,IL-12 mAb治疗抑制肠黏膜Th1细胞比例,上调Th2细胞比例。信号通路分析中发现,IL-12 mAb治疗提高肠黏膜p-STAT6及IL-13水平,而抑制TNF-α水平。结论 IL-12 mAb可有效缓解CD小鼠模型的肠道炎症并保护肠黏膜屏障,可能与抑制肠黏膜Th1细胞免疫反应及上调STAT6信号有关。     

靶向IL-6慢病毒介导RNA干扰实验性基因治疗类风湿关节炎

目的:探讨靶向小鼠IL-6基因RNA干扰慢病毒载体颗粒(LV-IL-6-RNAi)对小鼠巨噬细胞株J774A.1细胞IL-6表达的影响,及其对胶原诱导性关节炎(CIA)的治疗作用。方法:设计合成3对特异性针对小鼠IL-6基因的小干扰RNA(siRNA)序列,筛选最高效siRNA序,构建高效LV-IL-6-RNAi。LV-IL-6-RNAi感染小鼠巨噬细胞株J774A.1细胞,并设慢病毒阴性对照(LV-NC-RNAi)、培养基空白对照,RT-qPCR检测J774A.1细胞的IL-6、肿瘤坏死因子-α(TNF-α)和IL-1β mRNA相对表达水平,以检测LV-IL-6-RNAi体外干扰效率。取DBA/小鼠构建CIA模型并随机分为4组:LV-IL-6-RNAi组、LV-NC-RNAi组、甲氨蝶呤(MTX)阳性对照组、PBS空白对照组,分别关节腔注射LV-IL-6-RNAi、LV-NC-RNAi、腹腔注射MTX、PBS溶液,记录CIA小鼠关节炎评分,ELISA检测小鼠IL-6、IL-1β和TNF-α血清水平,组织病理检测炎症关节的炎症细胞浸润数。结果:成功构建了高效LV-IL-6-RNAi。...     

TLR2信号在沙眼衣原体生殖道感染中介导了早期炎症细胞因子的产生

目的探讨TLR2和TLR4在小鼠沙眼衣原体生殖道感染免疫应答中的作用。方法用沙眼衣原体小鼠肺炎株(Mo Pn,1×104IFUs)经生殖道感染野生型小鼠(WT,11只)、TLR2基因缺陷小鼠(TLR2 KO,14只)和TLR4基因缺陷小鼠(TLR4 KO,11只),复制生殖道沙眼衣原体感染模型。于感染后不同时间点取生殖道分泌物,部分用于免疫荧光法检测衣原体包涵体数量,部分用于炎症细胞因子IL-1α、IL-6和MIP-2水平检测。在感染后第70天,处死小鼠,无菌分离腹腔巨噬细胞,于体外衣原体Mo Pn感染,培养24 h后,测定上清液中IL-1α、IL-6和MIP-2含量。细胞因子含量测定均采用ELISA法。结果 TLR2 KO或TLR4 KO小鼠与WT小鼠在每一个检测时间点下生殖道带菌量无差异,且带菌持续时间相同,截止到感染后第38天,3种基因型所有小鼠均清除了下生殖道感染的衣原体。TLR2基因缺失小鼠巨噬细胞产生的炎症细胞因子IL-1α、IL-6和MIP-2水平均明显低于野生型WT小鼠(P0.01),而TLR4基因缺失小鼠巨噬细胞产生的3种炎症细胞因子水平均与野生型WT小鼠无差异(P0.05);TLR2基因缺失小鼠棉拭子标本同样具有较低水平的炎症细胞因子(P0.05)。结论在沙眼衣原体生殖道感染中,TLR2介导了早期炎症细胞因子的产生。沙眼衣原体诱导的早期免疫应答部分依赖于TLR2,而不依赖TLR4。     

甲氨蝶呤对类风湿关节炎模型大鼠的疗效及对ACCP和RF的影响

目的 探讨甲氨蝶呤(MTX)治疗大鼠类风湿关节炎(RA)的疗效及对抗环瓜氨酸肽抗体(ACCP)、类风湿因子(RF)的影响.方法 选择60只SD大鼠建立类风湿关节炎模型,并随机分为对照组、模型组和治疗组.治疗组大鼠每周给予甲氨蝶呤灌胃3.8 mg/kg,连续6周.大鼠关节炎评分和足趾容积的测定RA疾病程度;苏木精-伊红(HE)染色和甲苯胺蓝染色检测类风湿关节炎的病理改变程度;ELISA检测组织中ACCP、RF、TNF-α,IL-1β和IL-17水平的表达;Western blot检测RANKL和OPG蛋白表达.结果 随着实验时间的延长,治疗组大鼠体质量于实验第4周和第6周明显高于模型组(P<0.05);治疗组大鼠关节炎评分和足趾容积明显低于模型组(P<0.05).与模型组比较,治疗组RANKL蛋白含量显著降低,OPG蛋白含量明显上升(P<0.05);治疗组ACCP和RF含量显著降低,TNF-α、IL-1β和IL-17含量明显下降(P<0.05).结论 甲氨蝶呤治疗大鼠类风湿关节炎具有较好的疗效,且能抑制细胞因子的表达,降低ACCP、RF水平.     

内皮高表达脂多糖相关因子1抑制巨噬细胞炎症反应

目的 本实验旨在观察内皮高表达脂多糖相关因子1(endothelial overexpressed lipopolysaccharide-associated factor1,EOLA1)对巨噬细胞炎症反应的影响及可能机制。方法 培养小鼠巨噬细胞,脂多糖（lipopolysaccharide, LPS）刺激激活炎症信号通路,检测肿瘤坏死因子（TNF-α）、白介素6(IL-6)和EOLA1转录水平变化,采用化学阻断剂在不同位点阻断炎症反应信号通路,再检测TNF-α、IL-6和EOLA1转录变化。巨噬细胞过表达mEOLA1,RT-qPCR检测细胞M1、M2极化类型标志基因mRNA水平,以流式细胞术检测巨噬细胞表型改变,Western blot检测M1型巨噬细胞标记物iNOS表达。结果 LPS刺激后巨噬细胞释放炎症因子TNF-α、IL-6增加,而EOLA1表达下调;不同位点阻断炎症反应信号通路后炎症介质的表达明显受抑制,而EOLA1的表达上升;在巨噬细胞中高表达EOLA1,再行LPS刺激,检测到TNF-α、IL-6增加并不明显,流式细胞检测显示高表达EOLA1的巨噬细胞在LPS刺激后仍保持非...     

苦参碱抑制LPS诱导巨噬细胞IL-1β、TNF-α分泌及机制研究

目的:探讨苦参碱抑制脂多糖(lipopolysaccharide,LPS)诱导巨噬细胞分泌炎症因子包括肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)对Toll样受体4(TLR4)、c-Jun表达的影响。方法:购买并培养小鼠RAW264.7巨噬细胞株,分成四组,即空白对照组、苦参碱组、LPS组、苦参碱干预组,通过浓度为100μg/L的LPS DMEM孵育1 h,之后将LPS弃去,然后加入不含血清的DMEM或125.25 mg/L苦参碱DMEM继续培养。分别收集上述四组细胞处理完毕后5、30、60、120min的细胞及培养液。通过PCR法检测小鼠RAW264.7巨噬细胞TLR4及c-Jun mRNA的表达,通过免疫细胞化学法检测小鼠RAW264.7巨噬细胞c-Jun蛋白的表达;通过ELISA法测定各组培养液中TNF-α、IL-1β的分泌。结果:苦参碱诱导组与空白对照组相比,TLR4 mRNA、c-Jun mRNA及c-Jun蛋白的表达量和TNF-α及IL-1β分泌量各项指标差异无统计学意义(P0.05);LPS组各个时间点TLR4 mRNA、c-Jun mRNA及c-Jun蛋白的表达量和TNF-α及IL-1β分泌量明显高于空白对照组,且高水平能够维持2 h以上(P0.05);苦参碱干预组能够有效的抑制LPS诱导的巨噬细胞TLR4 mRNA、c-Jun mRNA及c-Jun蛋白的表达量,降低炎症因子TNF-α、IL-1β的分泌量。结论:苦参碱可能通过抑制LPS诱导的巨噬细胞丝裂原活化的蛋白激酶信号通路上TLR4、c-Jun的过表达从而有效的降低终末炎症因子TNF-α及IL-1β的释放,进而有效的抑制炎症反应,减轻内毒素炎症反应的程度。     

肺炎克雷伯菌黏附素FimH蛋白通过TLR4/NF-κB途径诱导巨噬细胞分泌炎症因子

目的初步探讨肺炎克雷伯菌黏附素FimH蛋白诱导巨噬细胞分泌炎症因子及其作用机制,为阐明肺炎克雷伯菌的致病机制提供实验证据。方法使用肺炎克雷伯菌FimH蛋白刺激巨噬细胞,然后用ELISA法检测IL-1β、TNF-α和IL-6的含量,用Western blot检测iκB-α的表达量。分别用TAK-242、PDTC、SB203580和SP600125预处理巨噬细胞,抑制TLR4、NF-κB、p38和c-Jun活性,再使用ELISA法检测FimH蛋白刺激后IL-1β、TNF-α和IL-6的表达。结果使用FimH蛋白刺激人源性巨噬细胞,IL-1β、TNF-α和IL-6的分泌量明显高于对照。FimH蛋白刺激会够降低细胞中iκB-α的含量。当使用TAK-242抑制TLR4的活性后,FimH蛋白诱导的IL-1β、TNF-α和IL-6表达量下降。使用PDTC和SB203580分别抑制NF-κB和p38的活性,也发现FimH蛋白诱导炎症因子能力下降。但是抑制c-Jun的活性不影响FimH蛋白诱导IL-1β、TNF-α和IL-6的表达。结论肺炎克雷伯菌FimH蛋白能够通过TLR4/NF-κB途径诱导巨噬细胞分泌IL-1β、TNF-α和IL-6。     

Development in in vitro toxicology

There are three principal pressures driving the development of in vitro toxicology: (1) the need for more efficient testing systems to cope with the large number of xenobiotics currently being developed; (2) public pressure to reduce animal experimentation; and (3) a need for a better understanding of the mechanisms of toxicity. Within this, in vitro toxicology is focused on local, systemic, and target-organ toxicity. It is becoming increasingly apparent that a step or decision-tree approach using input of a variety of experimental data (physicochemical properties, biokinetics, cytotoxicity) provides the most efficient system for predicting toxicity. Examples of the use of in vitro toxicity systems for prediction of systemic toxicity and target-organ (liver) toxicity are presented.Originally presented at ECCP 93.     

Seasonal variations in acute toxoplasmosis in pregnant women in Slovenia

Between December 1999 and December 2004, 40 081 pregnant women were examined for toxoplasmosis with Toxo-IgG, Toxo-IgM enzyme immunoassay. Women with positive results were then retested with the Toxo-IgG avidity assay for recent toxoplasmosis. Recent acute toxoplasmosis in pregnant women was found to be significantly more frequent (p < 0.01) during winter than summer. The incidence of acute toxoplasmosis during winter-spring was also significantly more frequent (p < 0.025) than summer-autumn. This phenomenon should be taken into account when formulating preventive measures for toxoplasmosis, especially for pregnant women.     

Age-related changes in polyamines in memory-associated brain structures in rats

Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines and have important roles in maintaining normal cellular function, regulating neurotransmitter receptors and modulating learning and memory. Recent evidence suggests a role of putrescine in hippocampal neurogenesis, that is significantly impaired during aging. The present study measured the polyamine levels in memory-related brain structures in 24- (aged), 12- (middle-aged) and 4- (young) month-old rats using liquid chromatography/mass spectrometry and high performance liquid chromatography. In the hippocampus, the putrescine levels were significantly decreased in the CA1 and dentate gyrus, and increased in the CA2/3 with age. Significant age-related increases in the spermidine levels were found in the CA1 and CA2/3. There was no difference between groups in spermine in any sub-regions examined. In the parahippocampal region, increased putrescine level with age was observed in the entorhinal cortex, and age did not alter the spermidine levels. The spermine level was significantly decreased in the perirhinal cortex and increased in the postrhinal cortex with age. In the prefrontal cortex, there was age-related decrease in putrescine, and the spermidine and spermine levels were significantly increased with age. This study, for the first time, demonstrates age-related region-specific changes in polyamines in memory-associated structures, suggesting that polyamine system dysfunction may potentially contribute to aged-related impairments in hippocampal neurogenesis and learning and memory.     

休克过程中肾上腺髓质素变化的研究进展

Adrenomedullin (AM) is a new peptidergic regulator of vascular function. AM serves as a hormone, which has many biological properties, plays an important role in the many pathophysiological processes, especially shock. This review will highlight the structure, biological properties of AM and the relationship between AM and shock.     

Secular change in menarche in women in Madrid

The age at menarche was estimated by recollection in 1617 women between the ages of 18 and 60 in Madrid and a nearby suburb, Pinto. The population of Pinto is working-class and the Madrid group, taken from residential neighbourhoods , belongs to the upper middle class. In both groups we found a diminution in average age at menarche, from 14.04 to 13.02 years in Madrid and from 14.55 to 13.16 years from about 1935 to about 1965 in Pinto. These changes have been more intense in the group which is less well-off economically, where living conditions have varied much more drastically.     

Pitfalls in TRAP assay in routine detection of malignancy in effusions

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.