β-石竹烯通过作用于HMGB1/TLR4/NF-κB通路减轻小鼠局灶性脑缺血再灌注损伤

目的:研究β-石竹烯(β-caryophyllene,BCP)对脑缺血再灌注损伤(Cerebral ischemia-reperfusion,CIR)小鼠的保护作用及可能的机制。方法:将C57BL/6小鼠随机分组为假手术组(Sham)、模型组(CIR)、β-石竹烯组(62、124、248mg/kg)。采用线栓法建立小鼠CIR损伤模型,缺血1 h,再灌注24 h后,进行神经行为学评分和脑梗死体积测定;TUNEL法观察CIR后缺血区神经细胞的死亡情况;Western blot法检测脑组织中TLR4和NF-κB(p65)蛋白表达情况;免疫组织化学法检测缺血侧脑NF-κB(p65)的表达;ELISA法检测CIR小鼠血清中HMGB1和缺血侧脑组织中IL-1β、TNF-α的含量变化。结果:与模型组相比,BCP(248 mg/kg)可明显改善小鼠神经功能,减少脑梗死体积,降低缺血区神经元的死亡率(P0.01);降低血清中HMGB1浓度、TLR4的蛋白表达量(P0.01),抑制炎症通路NF-κB的激活并减少TNF-α、IL-1β的释放(P0.01)。结论:BCP能够减轻CIR诱导的小鼠脑组织损伤,其保护作用可能是通过抑制HMGB1/TLR4/NF-κB介导的炎症反应。     

硫化氢抑制大鼠急性心肌缺血引起的细胞炎症反应

 目的：探讨硫化氢（H2S）能否抑制大鼠急性心肌缺血引起的细胞炎症反应。方法：结扎大鼠左冠状动脉前降支4 h,引起心肌缺血损伤。健康雄性SD大鼠随机分为假手术组（sham）、缺血组（ischemia）以及硫氢化钠(NaHS) 5 μmol/L、10 μmol/L和20 μmol/L组。NaHS组分别于心肌急性缺血2 h时更换为5 μmol/L、10 μmol/L和20 μmol/L NaHS灌流液。缺血后4 h记录心功能变化。实时荧光定量PCR检测离体心肌组织中TNF-α、IL-1β、IL-6、IL-10和ICAM-1 mRNA表达;Western blotting检测离体心肌组织中NF-κB的表达。结果：Ischemia组离体心肌功能下降（与sham组相比P<0.01）,NaHS组离体心肌功能比ischemia组明显改善（P<0.05或P<0.01）。Ischemia组离体心肌组织中TNF-α、IL-1β、IL-6和ICAM-1 mRNA表达显著增强,IL-10 mRNA表达显著降低（与sham组相比P<0.01）;NaHS组离体心肌组织中TNF-α、IL-1β、IL-6和ICAM-1 mRNA表达比ischemia组明显降低,NaHS 10 μmol/L,20 μmol/L组离体心肌组织中IL-10 mRNA表达比ischemia组明显升高（P<0.05或P<0.01）。与sham组相比,ischemia组NF-κB表达显著增加（P<0.01）,而NaHS 10 μmol/L和20 μmol/L组NF-κB的表达明显低于ischemia组（P<0.05或P<0.01）。结论：H2S可通过阻断NF-κB相关信号通路的转导,抑制炎症反应,明显改善大鼠急性心肌缺血损伤。     

雷公藤多苷联合地塞米松对EAE中TLRs/NF-κB信号通路的作用

 目的：探讨雷公藤多苷联合地塞米松对实验性变态反应性脑脊髓炎（EAE）的治疗效果及其作用途径。方法：所有实验动物被分为空白对照组、EAE组、治疗组1(地塞米松)和治疗组2（雷公藤多苷联合地塞米松）。比较各组平均临床评分;分别采用实时定量RT-PCR和免疫组化法检测不同组别脑组织中TLR4和TLR9 mRNA和蛋白含量,免疫组化法检测NF-κB p65蛋白的表达;采用ELISA法检测外周血中TNF-α、IL-1β和IL-6含量的变化。结果：各治疗组平均临床评分较EAE组均降低（P<0.05）;治疗组2较治疗组1平均临床评分降低（P<0.05）。在发病高峰期,EAE组TLR4和TLR9 mRNA及蛋白表达较空白对照组增高,而各治疗组较EAE组降低（均P<0.05）;EAE组NF-κB p65阳性表达率较各治疗组升高（P<0.05）;各治疗组炎症细胞因子TNF-α、IL-1β和IL-6含量较EAE组降低（P<0.05）。治疗组2与治疗组1比较,上述各指标差异显著（均P<0.05）,且雷公藤多苷与地塞米松具有交互作用（F=75.749,P<0.01）。
结论：TLRs/NF-κB信号通路可能参与了EAE的发病过程;雷公藤多苷联合地塞米松具有明显改善EAE临床症状的效果,其可能通过抑制TLRs/NF-κB信号通路发挥抗炎及免疫抑制双重疗效。     

鬼针草总黄酮对大鼠佐剂性关节炎的治疗作用及机制

目的 探讨鬼针草总黄酮(TFB)对大鼠佐剂性关节炎(AA)关节肿胀指数、炎症因子水平、toll样受体4（TLR4）信号转导通路及病理评分的影响。方法 选取50只雄性SD大鼠，随机分为空白组、模型组、TFB(40 mg/kg、80 mg/kg、160 mg/kg)组，每组各10只。比较各组大鼠关节肿胀指数；采用放射免疫法检测血清白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）的水平；Western blotting检测巨噬细胞中TLR4、核因子κB（NF-κB）的表达水平；HE染色进行大鼠关节病理评分。结果 模型组关节肿胀指数、炎症因子水平、TLR4及NF-κB表达均明显高于空白组，不同剂量的TFB组（40 mg/kg、80 mg/kg、160 mg/kg）关节肿胀指数、炎症因子水平、TLR4、NF-κB表达及病理评分均显著低于模型组(P<0.05)，且TFB剂量越大，所有指标表达水平越低(P<0.05)。结论 TFB对AA大鼠具有治疗作用，其作用机制可能与降低关节肿胀指数及炎症因子水平、抑制TLR4和NF-κB表达、阻碍血管翳形成有关。     

血管生成素4对脂多糖诱导的人脐静脉内皮细胞损伤的影响

 目的：观察血管生成素4（Ang-4）对脂多糖(LPS）诱导的人脐静脉内皮细胞（HUVECs）损伤的影响,并探讨其可能的作用机制。方法：EnVision法免疫组化鉴定HUVECs。LPS诱导细胞损伤,不同剂量的Ang-4干预。显微镜下观察细胞形态,MTT法检测细胞活力,ELISA法检测von Willebrand因子（vWF）和肿瘤坏死因子（TNF-α）含量,real-time PCR检测Toll样受体4（TLR4）、核因子κB（NF-κB） p65和TNF-α mRNA含量变化。结果：免疫组化示胞浆内人Ⅷ因子相关抗原呈阳性;与正常组比,LPS组细胞增殖活力降低（P<0.01）,vWF及TNF-α分泌增多(P<0.01),且TLR4、NF-κB p65和TNF-α mRNA表达升高(P<0.01);Ang-4（100 μg/L）组可恢复细胞活力,降低vWF及TNF-α含量(P<0.01),抑制LPS所致TLR4、NF-κB p65和TNF-α mRNA的表达(P<0.01)。结论：Ang-4可拮抗LPS诱导的HUVECs损伤,这可能与抑制TLR4-NF-κB p65-TNF-α信号通路的活化有关。     

羚羊角与钩藤联合用药抑制热性惊厥大鼠脑损伤作用机制研究

目的:探讨羚羊角与钩藤联合用药对热性惊厥所致发育期大鼠脑损伤的保护作用及其作用机制。方法:采用热水浴建立热性惊厥大鼠模型,实验分为5 组,空白对照组、模型组、羚羊角组、钩藤组、羚羊角与钩藤联合用药组(简称联用组),每组10 只,共50 只。通过HE 染色法观察组织病理情况;ELISA 法检测各组大鼠脑组织TNF-α、IL-1β、Glu 及Asp 含量;免疫组化法检测大鼠海马区TNF-α、IL-1β表达情况。结果:联用组、羚羊角组、钩藤组脑组织Asp、Glu、TNF-α及IL-1β及海马区TNF-α、IL-1β含量显著低于模型组(P<0.01,P<0.05)。联用组脑组织Asp、TNF-α及IL-1β含量较钩藤组显著降低(P<0.05)。联用组海马区TNF-α较羚羊角组、钩藤组显著降低(P<0.05),IL-1β较钩藤组表达显著降低(P<0.05)。结论:羚羊角与钩藤联合用药抑制热性惊厥所致大鼠脑损伤,效果优于羚羊角及钩藤单用,其作用机制可能与其抑制促炎症因子TNF-α、IL-1β及兴奋性氨基酸Asp、Glu 表达有关。     

妊娠期脂多糖接触激活体内TLR4信号通路引起胚胎脑内炎症应激

目的: 观察孕鼠腹腔脂多糖 (LPS) 注射后母体外周血单个核细胞 (PBMNC) 上Toll样受体4 (TLR4) 信号通路激活情况和胚胎脑组织中炎症性细胞因子的水平。方法: 10.5 d孕鼠腹腔注射LPS 0、3、6、12、24、48 和72 h后,通过蛋白免印迹测定孕鼠PBMNC 上TLR4、分化抗原14 (CD14)、髓样分化蛋白2 (MD-2)、p65和p50蛋白水平,Luminex 100免疫荧光法测定孕鼠血清、羊水、脑组织及胚胎脑组织细胞因子TNF-α、IL-1β和IL-10蛋白水平,实时RT-PCR测定孕鼠PBMNC和胚胎脑组织TNF-α、IL-1β和IL-10 mRNA水平。结果: LPS腹腔注射诱导激活孕鼠PBMNC上TLR4信号通路,TLR4、CD14和MD-2蛋白水平短暂增高,核因子κB (NF-κB) 激活片段p65 和p50蛋白水平增高,TNF-α、IL-1β和IL-10 mRNA水平增高(P<0.01);孕鼠外周血和羊水TNF-α、IL-1β和IL-10水平短暂增高(P<0.01),脑内IL-1β蛋白水平短暂升高(P<0.01);胚胎脑内TNF-α和IL-1β蛋白和mRNA水平显著增高(P<0.01)。结论: 妊娠期母体LPS接触可激活母体外周免疫细胞TLR4信号通路,引发胚胎脑内炎症应激状态,可能增加出生后相关疾病的发生风险。     

RNAi沉默NF-κB p65对小鼠巨噬细胞细胞因子表达的影响

目的: 探讨RNA干扰(RNAi)技术在抑制NF-κB p65表达、调控LPS活化后巨噬细胞细胞因子表达中的应用。方法: 利用阳离子脂质体将NF-κB p65小干扰RNA(siRNA)瞬时转染入Ana-1细胞,RT-PCR及Western blotting法检测其沉默效率,ELISA法检测LPS(1 mg/L)刺激下0 h、4 h、12 h和24 h Ana-1细胞培养上清中TNF-α、IL-1β、IL-6和IL-10浓度。结果: NF-κB p65 siRNA转染24 h后,NF-κB p65在基因水平及蛋白水平表达均被明显抑制(P<0.05)。RNA干扰组Ana-1细胞培养上清中细胞因子TNF-α、IL-1β和IL-6表达在相应时点内均较对照组明显降低(P<0.05),IL-10表达显著升高,在12 h和24 h差异显著(P<0.05)。结论: 体外实验初步证实RNAi技术能有效沉默小鼠巨噬细胞NF-κB p65 基因的表达,下调其下游调控的促炎症细胞因子TNF-α、IL-1β、IL-6及上调抑炎症细胞因子IL-10的表达,从而抑制过度的炎症反应。     

Notch通路在大鼠肾脏缺血再灌注损伤TLR4介导的炎症反应中的作用

 目的: 探讨Notch通路对大鼠肾脏缺血再灌注损伤(IRI)中Toll样受体4(TLR4)介导的炎症反应的作用。方法: 雄性SD大鼠75只随机分为假手术组(sham组)、缺血再灌注组(IRI组)和γ-分泌酶抑制剂DAPT干预组(DAPT组)。分别于再灌注6 h、12 h、24 h、48 h、72 h时点观察各组肾脏病理改变,检测血尿素氮(BUN)和血清肌酐(Scr)水平,ELISA检测血清肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平,免疫组化和Western blot分别检测大鼠肾脏Notch1、TLR4和NF-κB p65蛋白表达水平。结果: 在IRI组,呈现不同程度的以肾小管上皮细胞和间质损伤为主的肾脏病理改变,BUN、Scr及血清炎性因子TNF-α、IL-6含量在各时点均显著高于sham组(P<0.05),而在DAPT干预组,在各时点肾脏病理损伤明显减轻,BUN、Scr和血清TNF-α、IL-6水平均显著低于IRI组(P<0.05)。Notch1、TLR4和NF-κB p65主要表达于肾小管上皮细胞胞质中,在sham组仅有微量表达,在IRI组则有高表达,在各时点表达与sham组相比均显著增强(P<0.05),而在DAPT组,各因子的表达水平在各时点均较IRI组显著降低(P<0.05)。结论: 大鼠肾脏IRI出现显著的肾功能及肾脏病理改变,血清炎性因子TNF-α和IL-6水平升高,Notch1、TLR4及NF-κB p65在肾组织中表达增强;而DAPT可通过抑制Notch1活化和TLR4/NF-κB通路,抑制TLR4所介导的炎症反应,从而发挥肾脏保护作用。     

瑞舒伐他汀预处理对缺血再灌注大鼠大脑中动脉血管平滑肌细胞炎症因子的影响及其机制

目的 探讨瑞舒伐他汀预处理对大鼠局灶性脑缺血再灌注后大脑中动脉血管平滑肌细胞（VSMCs）中炎性细胞因子白细胞介素（IL）-1β、IL-6、肿瘤坏死因子ɑ（TNF-α）表达的影响以及可能的机制。 方法 36只健康成年SD大鼠,雌雄不限,随机分为假手术组,局灶性脑缺血再灌注组,瑞舒伐他汀预处理组,每组12只。大脑中动脉缺血2 h再灌注24 h后, Real-time PCR及Western blotting法检测大鼠大脑中动脉VSMCs中IL-1β、IL-6和TNF-α以及核因子κB（NF-κB）mRNA和蛋白的表达。 结果 再灌注24 h后,模型组VSMCs IL-1β、IL-6 以及TNF-αmRNA和蛋白的表达明显增加,而给予瑞舒伐他汀预处理后,可明显抑制大脑中动脉VSMCs中IL-1β、IL-6和TNF-α mRNA和蛋白的高表达,同时也可发现,VSMCs中NF-κB mRNA和蛋白的表达也明显减少。 结论 瑞舒伐他汀预处理可有效抑制大脑中动脉VSMCs中IL-1β、IL-6和TNF-α mRNA和蛋白的表达,从而减轻缺血再灌注后炎症反应对脑组织的进一步损伤作用。瑞舒伐他汀预处理对IL-1β、IL-6及TNF-α的作用可能与其减少VSMCs中NF-κB的表达有关。     

Évaluation des risques microbiologiques hydriques associés à Stenotrophomonas maltophilia et Pseudomonas aeruginosa au CHU d’Amiens

Background

Pseudomonas aeruginosa (Psa) and Stenotrophomonas maltophilia (Smalto) are major opportunistic waterborne pathogens causing hospital-acquired infections. This study aimed to assess the biocontamination level of cold water used in Amiens’ university hospital wards, from March to June 2008.

Methods

We cultivated 122 pairs of cold water first jet and taps cotton-swabs on Cetrimide agar for Psa, on Stenotrophomonas maltophilia selective medium with coloured indicator (SM2i) for Smalto, on Mueller Hinton agar used as isolation medium reference for both, 48 h at 30 °C. Data analysed with Épi-Info 6.04dFr were compared with chi² test, significant at p < .05.

Results

Psa and Smalto were isolated in 26.2 and 14.8% of water samples and in 21.3 and 10.7% of swab samples respectively. They were associated in 11.5% of water samples and 5% of swab samples. Psa was alone in 13.1% of water samples and 7.4% of swab samples whereas Smalto was found in 6.6% of water and 2.5% of swabs. Psa and Smalto were isolated from 14.8% of water samples and 8.2% of swab samples of the same tap. Finally, respectively 35.2 and 17.2% of the cold water taps were biocontaminated by Psa and Smalto. In fact, microbiologic water taps contamination risk was two-fold higher for Psa than for Smalto, p < .001, without variation between wards.

Conclusion

Sm2i and Cetrimide are suited and efficient medium respectively for Smalto and Psa isolation. Cold-water samples are sufficient for waterborne pathogens biocontamination risk appraisal. Our results urged healthcare workers on efficient water fittings microbiologic risk control to prevent healthcare associated waterborne infections, notably due to Psa and Smalto.     

γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

Deoxygenation of ferret erythrocytes stimulates Na⁺–K⁺–2Cl⁻ cotransport by 111% ( s.d. , 46) compared to controls in air. Half-maximal activation occurs at a P _O2 of 24 mmHg ( s.d. , 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25–35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 ( s.d. , 0.07) to 1.40 m m ( s.d. , 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.     

Thymic differentiation of TCRαβ+ CD8αα+ IELs

Summary:  Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)αβ⁺ CD8αα⁺ IELs has been particularly controversial. Here we provide a synthesis, based on recent work, that attempts to unify the divergent views. The intestine has a primordial function in lymphopoiesis, and precursors with the potential to differentiate into T cells are found both in the epithelium and underlying lamina propria. Moreover, the thymus has been reported to export cells to the intestine that are not fully differentiated. TCRαβ⁺ CD8αα⁺ IELs can differentiate in the intestine from each of these sources, but in normal euthymic mice, the thymus appears to be the major source for TCRαβ⁺ CD8αα⁺ IELs. This unique IEL subset is a self-reactive population that requires exposure to self-agonists for selection in the thymus, similar to other regulatory T-cell populations. IELs transition through a double-positive (DP) intermediate in the thymus, but they originate from a subset of the DP cells that can be identified by its expression of CD8αα homodimers. The agonist-selected cells in the thymus are TCRβ⁺ but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8αα and downregulation of CD5 occur after thymus export, perhaps in the intestine under the influence of interleukin-15. As a result of agonist exposure, a new gene expression program is activated. Therefore, the increased understanding of the developmental origin of TCRαβ⁺ CD8αα⁺ IELs may help us to understand how they participate in immune regulation and protection in the intestine.     

ÜBer die auswertung viskosimetrischer messungen für polymer/polymer/lösungsmittel-systeme

A critical discussion of the recommended methods for compatibility determinations in polymer mixtures by viscosimetry measurements shows, that in the case of the systems PVC/PVAc/cyclohexanone the best results are obtained by comparison of the parameters of interaction. In this case it is possible to estimate the optimal rapport of mixture. Extending the thermodynamic methods to polymer/polymer/solvent systems, it can be shown, that the systems studied here are characterized by far-reaching interactions, indicating an interaction of the functional groups of the polymer chains. This confirms our previous conclusion, that the polymers show two kinds of compatibility, that is, true compatibility of the macromoleculare chains and pseudo-compatibility due to the interactions of the functional groups.