促肾上腺皮质释放因子对HT-29细胞中Toll样受体4/核因子-κB信号通路的影响

目的 观察促肾上腺皮质释放因子(corticotrophin-releasing factor CRF)对结肠上皮细胞系HT-29细胞中Toll样受体(TLR)4/核因子(NF)-κB的表达调控作用。方法 将HT-29细胞分为4组,正常对照组,脂多糖(LPS)组（IPS 20μg/ml刺激24 h）,CRF组(CRF 20 ng/ml刺激24h),CRF+LPS组（预先CRF孵育12h,更换细胞液后再与LPS孵育12h）。刺激结束后,RT-PCR法检测各组细胞中TLR4 mRNA的表达,提取细胞总蛋白,免疫印迹法检测各组细胞中TLR4和NF-κB p65蛋白表达水平。收集上清液,ELISA法检测各组细胞上清液中IL-8的表达。结果 LPS组TLR4 mRNA和蛋白表达水平为0.31±0.04和0.48±0.17,与正常对照组比较差异无统计学意义[0.28±0.02和0.45±0.12,t值分别＝0.216和0.712,P值均＞0.05],CRF组为1.05±0.06和1.08±0.21,与正常对照组相比明显增高（t值分别＝3.721和3.802,P值均＜0.05）,而CRF+ LPS组为1.68±0.05和1.81±0.18,更高于CRF组（t值分别＝4.816和3.918,P值均＜0.05）。各组HT-29细胞中NF-κB p65蛋白表达水平和细胞上清液中IL-8表达水平,与TLR4 mRNA和蛋白表达水平结果一致。结论 CRF不仅能直接刺激肠上皮细胞中TLR4/NF-κB通路活化,还可促进结肠上皮对LPS的反应增加,导致IL-8释放增多。     

促肾上腺皮质激素释放因子对肠上皮细胞紧密连接相关蛋白的调节

目的:探讨促肾上腺皮质激素释放因子(corticotrophin-releasing factor,CRF)对肠上皮细胞紧密连接相关蛋白的调节作用.方法:以CRF(20 ng/mL)刺激HT-29细胞24 h后再以脂多糖(lipopolysaccharides,LPS)(100 ng/mL)刺激24 h,收集细胞,ELISA法检测细胞提取物中胞质附着蛋白(zonula occludins protein,ZO)-1、ZO-2、Occludin、cldn1、cldn2、cldn3和cldn4的表达.再次将HT-29细胞以CRF 0、1、10、20 ng/mL的浓度刺激24 h和核因子B(nuclear factor B,NF-B)抑制剂马来酸二乙酯(diethylmaleate,DEM)1 mmol/L预孵1 h后加CRF 20 ng/mL刺激24 h,然后更换各组细胞液后继续再以LPS 100 ng/mL的浓度孵育24 h,收集各组HT-29细胞,提取蛋白,免疫印迹法检测cldn2表达水平.结果:ELISA检测结果显示以CRF刺激结肠上皮细胞24 h后再以LPS刺激后ZO-1、ZO-2、Occludin、cldn1和cldn3表达水平较单纯LPS刺激有轻度的下降,但是cldn2的表达则较前明显升高(P<0.01).免疫印迹法检测结果显示以LPS孵育前以CRF(1 ng/mL)刺激即可引起cldn2表达增高,随后加大CRF刺激浓度,分别以10和20 ng/mL刺激,则cldn2的表达随着刺激浓度加大而逐渐增高(P<0.05),呈现剂量依赖效应,而以CRF(20 ng/mL)刺激同时加入NF-B抑制剂DEM后再以LPS孵育HT-29细胞后,CRF则不能诱导cldn2表达增高(P>0.05).结论:CRF对肠上皮细胞中的cldn2表达刺激呈剂量依赖效应,并且CRF对肠上皮细胞的这种诱导作用可能与NF-B激活有关.     

化瘀祛痰方药及其拆方对内皮细胞TLR4/NF-κB炎症信号通路干预的研究

目的探讨化瘀祛痰方药及其拆方含药血清对脂多糖(LPS)诱导人脐静脉内皮细胞EA.hy926 TLR4/NF-κB信号通路活化的干预作用。方法 40只SD大鼠随机分为5组,即空白对照组、全方组、补气组、化瘀组、祛痰组,各组大鼠分别以生理盐水和相应中药煎剂连续灌胃9 d,末次灌胃给药2 h后,腹主动脉采血,离心后分离血清。体外培养EA.hy926细胞,随机分为7组,即①正常对照组、②LPS刺激组、③全方组、④补气组、⑤化瘀组、⑥祛痰组、⑦空白血清对照组。其中②组加入终浓度为10μg/ml的LPS,③～⑦组用各组含药血清(浓度为10%)预处理24 h后加入终浓度为10μg/ml的LPS,各组细胞培养24 h后进行各项指标测定。采用实时定量反转录-聚合酶链反应(Real-time PCR)定量分析TLR4、NF-κB和TNF-αmRNA表达;ELISA法检测TNF-α含量;Western印迹检测TLR4、NF-κB蛋白表达。结果与正常对照组相比,LPS刺激后TLR4、NF-κB和TNF-α表达显著增加(P<0.01),全方组TLR4、NF-κB和TNF-α的表达与刺激组相比显著减少(P<0.01);拆方各组中,化瘀组TLR4、NF-κB和TNF-α水平均显著减少(P<0.01),而补气组和祛痰组TLR4、NF-κB和TNF-α水平降低不明显。结论化瘀祛痰方药及化瘀拆方可显著抑制内皮细胞TLR4/NF-κB炎症信号通路的活化,这可能是其抗AS的作用机制之一。     

NF-κB decoy寡核苷酸对结肠癌SW480细胞TLR4表达的影响

目的 探讨核转录子κB(NF-κB)decoy寡核苷酸(ODNs)对结肠癌SW480细胞Toll样受体4(TLR4)表达的影响. 方法 体外培养SW480细胞,LPS(10μL)刺激3 h后,用脂质体Lipofectin2000介导NF-κB decoy ODNs转染6 h,RT-PCR法检测细胞的TLR4 mRNA.设对照组、LPS组、SODN组、Lipofectin2000组. 结果 转染NF-κB decoy ODN的SW480细胞的TLR4 mRNA表达明显高于对照组(P<0.01),但明显低于其他组(P均<0.01). 结论 NF-κB decoy ODNs明显抑制SW480细胞TLR4的表达.     

血管紧张素1-7对雨蛙素诱导的胰腺腺泡细胞Toll样受体4/核因子-κB通路的影响

目的探讨血管紧张素1-7[Ang(1-7)]对雨蛙素诱导的胰腺腺泡细胞AR42J中Toll样受体(TLR)4/核因子(NF)-κB通路的影响。方法 AR42J细胞随机分为4组:对照组、模型组(10 nmol/L雨蛙素刺激)、Ang(1-7)组(10-7mol/L、10-6mol/L、10-5mol/L Ang(1-7)+10 nmol/L雨蛙素)及Ang(1-7)受体抑制剂A779组(10-7mol/L、10-6mol/L、10-5mol/L A779+10 nmol/L雨蛙素)。对照组为正常生长的AR42J细胞,模型组用10 nmol/L的雨蛙素刺激AR42J分别于15 min、30 min、2 h、6 h、12 h及24 h收集细胞,Ang(1-7)组为不同浓度Ang(1-7)作用30 min后再用雨蛙素刺激12 h收集细胞,A779组为不同浓度A779作用30 min后再用雨蛙素刺激12 h收集细胞。免疫荧光技术检测TLR4及NF-κB在AR42J中的表达部位,Western Blot技术检测各组细胞中TLR4及NF-κB的蛋白表达水平。计量资料组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 AR42J细胞中可见TLR4及NF-κB均于细胞胞浆中表达。与对照组相比,模型组TLR4随雨蛙素造模时间呈先增高后减少的动态变化:在造模后30 min、2 h及6 h显著升高,12 h显示降低,差异均有统计学意义(P值均0.05);NF-κB随雨蛙素造模时间呈现逐渐升高的动态变化:在造模后30 min、2 h、6 h、12 h及24 h显著升高,差异均有统计学意义(P值均0.05)。Ang(1-7)浓度为10-5mol/L时TLR4及NF-κB蛋白表达下降,与模型组比较差异均有统计学意义(0.570±0.046 vs 0.893±0.057,0.520±0.071 vs 0.750±0.057,P值均0.05)。A779浓度为10-5mol/L时TLR4及NF-κB蛋白表达升高,与模型组比较差异均有统计学意义(0.680±0.045 vs 0.563±0.088,0.617±0.071 vs 0.453±0.054,P值均0.05)。结论雨蛙素刺激的AR42J细胞中,Ang(1-7)能够下调TLR4,抑制NF-κB激活发挥作用。     

化瘀祛痰方药及其拆方对内皮细胞TLR4/NF-kB炎症信号通路干预的研究

目的 探讨化瘀祛痰方药及其拆方含药血清对脂多糖(LPS)诱导人脐静脉内皮细胞EA.hy926 TLR4/NF-kB信号通路活化的干预作用.方法 40只SD大鼠随机分为5组,即空白对照组、全方组、补气组、化瘀组、祛痰组,各组大鼠分别以生理盐水和相应中药煎剂连续灌胃9d,末次灌胃给药2h后,腹主动脉采血,离心后分离血清.体外培养EA.hy926细胞,随机分为7组,即①正常对照组、②LPS刺激组、③全方组、④补气组、⑤化瘀组、⑥祛痰组、⑦空白血清对照组.其中②组加入终浓度为10μg/ml的LPS,③～⑦组用各组含药血清(浓度为10％)预处理24 h后加入终浓度为10 μg/ml的LPS,各组细胞培养24 h后进行各项指标测定.采用实时定量反转录-聚合酶链反应(Real-time PCR)定量分析TLR4、NF-kB和TNF-α mRNA表达;ELISA法检测TNF-α含量;Western印迹检测TLR4、NF-kB蛋白表达.结果 与正常对照组相比,LPS刺激后TLR4、NF-kB和TNF-α表达显著增加(P＜0.01),全方组TLR4、NF-kB和TNF-α的表达与刺激组相比显著减少(P＜0.01);拆方各组中,化瘀组TLR4、NF-kB和TNF-α水平均显著减少(P＜0.01),而补气组和祛痰组TLR4、NF-kB和TNF-α水平降低不明显.结论 化瘀祛痰方药及化瘀拆方可显著抑制内皮细胞TLR4/NF-kB炎症信号通路的活化,这可能是其抗AS的作用机制之一.     

姜黄素对脂多糖刺激的肾小管近端上皮细胞分泌的相关炎症因子的影响

目的:本研究旨在观察姜黄素对脂多糖(LPS)刺激下的人肾小管近端上皮细胞分泌的单核细胞趋化因子(MCP-1)及白细胞介素8(IL-8)的表达水平变化,并初步探讨其作用机制. 方法:将肾小管上皮细胞(HK-2细胞)培养于KSF培养液中,设正常对照组给予正常培养液,LPS刺激组(1 ng/ml,100 ng/ml和10μg/ml),姜黄素干预组(LPS 100 ng/ml+姜黄素5 μM或50μM),分别培养4～24h后收集细胞,应用Real-time PCR检测各组细胞MCP.-及IL-8的表达.ELISA检测细胞培养上清液中MCP-1及IL-8的表达. 结果:在不同浓度的LPS刺激HK-2细胞24h后即可明显升高MCP-1及IL-8的mRNA表达水平,随着LPS浓度(1 ng/ml,100 ng/ml和10 μg/ml)增加,MCP-1 mRNA表达分别上升为对照组的1.74、2.15和14.7倍(P<0.01);IL-8 mRNA表达上升为对照组的2.74、5.4和16.45倍(P<0.01).而以100 ng/ml的LPS刺激HK-2细胞4～24h,观察姜黄素的干预作用,可见不同浓度的姜黄素(5μM和50 μM)均可显著抑制LPS所诱导的HK-2细胞MCP-1及IL-8 mRNA的表达,且以50μM姜黄素干预组为明显.同时ELISA检测细胞上清液中MCP-1蛋白水平,可见5μM姜黄素组在24h时较相应对照组下降9.5%(P<0.05),而50 μM姜黄素组在8h时即较相应对照组下降19.5%(P<0.01).而在4h和12h时,5μM的姜黄素干预组较相应对照组IL-8的表达下降的11..2%(P<0.05)和7.58%(P<0.01),在50 μM姜黄素干预组则为32.90%和18.79%(P<0.01). 结论:姜黄素可抑制LPS所诱导的肾小管上皮细胞MCP-1及IL-8的表达,提示姜黄素在肾脏炎症过程中具有抑制肾小管上皮细胞炎症因子分泌及潜在抗纤维化作用.     

脂多糖介导的TLR4信号通路在膀胱癌免疫逃逸中的作用及其机制

目的探讨脂多糖介导的Toll样受体(TLR)4信号通路活化在膀胱癌(BC)T24细胞系免疫逃逸中的作用及其机制。方法取对数生长期T24细胞,使用1μg/ml的脂多糖分别刺激该细胞0、6、12、24 h,采用流式细胞仪检测细胞表面TLR4的表达量,采用RT-PCR检测细胞中程序性死亡配体-1(PD-1,又称B7-H1)mRNA的表达,采用酶联免疫吸附法(ELISA)检测细胞中B7-H1蛋白的表达,分析TLR4表达与B7-H1 mRNA及其蛋白的相关性。结果 TLR4阳性率随刺激时间的增长而上调,且在12 h时达到最高值,24 h时表达略有降低,但仍较高。6、12、24 h时TLR4阳性率与0 h时比较差异有统计学意义(P<0.05)。RT-PCR及ELISA结果显示B7-H1 mRNA及蛋白的表达随刺激时间的增长而上调,且在12 h时达到最高值,24 h时表达略有降低,但仍较高。与0 h时比较,6 h和24 h T24细胞B7-H1 mRNA及蛋白表达量增高(P<0.05),12 h显著增高(P<0.01)。TLR4与B7-H1 mRNA呈正相关(r=0.785,P=0.002),TLR4与B7-H1蛋白呈正相关(r=0.825,P=0.012)。结论脂多糖能活化TLR4信号通路,并上调B7-H1 mRNA及其蛋白的表达,可能参与BC细胞免疫逃逸,为BC的靶向治疗提供新思路。     

小檗碱对内毒素刺激致Caco-2细胞间高通透性的保护作用

目的探讨小檗碱(BBR)对脂多糖(LPS)刺激致Caco-2细胞间高通透性的保护作用。方法体外培养肠上皮细胞Caco-2,分为四组:(1)对照组(培养基);(2)LPS组(培养基+LPS 2μg/ml);(3)BBR组(培养基+BBR 10μM);(4)BBR/LPS组(培养基+LPS 2μg/ml+BBR 10μM)。在倒置显微镜下直接观察细胞生长形态,采用荧光黄透过率检测细胞间通透性,采用Western-blot法检测细胞间紧密连接主要结构蛋白occludin、细胞膜Toll样受体-4(TLR4)表达,采用免疫荧光法检测细胞TLR4下游分子MyD88表达。结果在倒置显微镜下可见对照组和BBR组细胞连接完整,LPS组细胞间出现锯齿样断裂,但在BBR/LPS组细胞间连接毛糙,但无明显片状中断;对照组和BBR组荧光黄透过浓度分别为(683.3±98.9)μg/ml和(849.0±89.7)μg/ml)以及细胞occludin、TLR4和MyD88蛋白表达差异均无统计学意义(P0.05);与对照组比,LPS组透过荧光黄浓度(1886±176.0μg/ml)显著增加,TLR4蛋白表达增加,occludin表达减少,胞浆MyD88荧光信号增强;与LPS组比,BBR/LPS组透过荧光黄浓度(1071.0±65.9μg/ml)显著下降,TLR4蛋白表达减弱,occludin表达增加,胞浆MyD88荧光信号减弱。结论 BBR通过减弱TLR4/MyD88活性,增加occludin表达,进而减轻LPS刺激致Caco-2细胞间高通透性。     

银杏酮酯对心肌细胞Toll样受体4/核转录因子κB及血管紧张素原、血管紧张素Ⅱ1型受体的抑制作用

目的观察银杏酮酯50(GBE50)对脂多糖激活的心肌细胞 Toll 样受体4(TLR4)/核转录因子κB(NF-κB)信号及血管紧张素原(ATG)、血管紧张素Ⅱ 1型受体(AT_1R)表达的影响,探讨 GBE50防治左室重构的作用机制。方法体外培养新生大鼠心肌细胞(NRVM),分4组:1)对照组:DMEM 培养基中不加任何干预因素;2)脂多糖组:DMEM 培养基中加入脂多糖1 mg/L;3)脂多糖+GBE50组:预先加入 GBE50 80 mg/L,1 h 后加入脂多糖1 mg/L;4)脂多糖+咖啡酸苯乙酯组:预先加入咖啡酸苯乙酯20 mg/L30 min 后加入脂多糖1mg/L。干预24 h 后免疫细胞化学方法观察 NF-κB p65核易位情况;RT-PCR 检测 TLR4、ATG 及 AT_1R、心肌肌球蛋白重链β-MHC mRNA 的表达情况,考马斯亮蓝法测定心肌细胞蛋白含量。结果 LPS 刺激下 NRVM TLR4 mR-NA 明显升高(P<0.01),NF-κB p65核易位增多(P<0.01),ATG 和 AT_1R、心肌肌球蛋白重链β-MHC mRNA表达以及细胞蛋白含量明显增高(P<0.01)...     

Role of corticotropin-releasing factor receptors type 1 and 2 in modulating the rat adrenocorticotropin response to stressors

We investigated the contribution of corticotropin-releasing factor (CRF) receptors types 1 and 2 (CRF(1) and CRF(2)) in mediating the ACTH response to shock, alcohol injection, or endotoxemia in the rat. Peptidic (Astressin B and Astressin(2)-B) and nonpeptidic (NBI 30775) CRF antagonists were injected iv before the stressors at doses previously shown to be effective in blocking the corresponding receptors. Because NBI 30775, which specifically blocks CRF(1), penetrates the brain following systemic injection, we also compared its effect with that of Astressin B, which primarily, though not exclusively, targets CRF(1) but does not cross the blood-brain barrier. Shocks, alcohol (4.5 g/kg, intragastrically) or lipopolysaccharide (LPS, 1 micro g/kg, iv) all significantly released ACTH. Astressin B or NBI 30775 markedly decreased the effect of shocks or alcohol and also interfered, though less significantly so, with the influence of LPS. In contrast, specific blockade of CRF(2) with Astressin(2)-B, although not significantly altering the overall ACTH response to shocks, alcohol, or LPS, slightly enhanced ACTH levels during the early phase of some of these responses. Interestingly, combined administration of NBI 30775 and Astressin(2)-B decreased ACTH levels more than NBI 30775 alone, although this difference did not reach statistical significance. Finally, blockade of CRF(1) and/or CRF(2) augmented LPS- induced TNF-alpha and IL-6 release. Collectively, there results confirm the critical role played by CRF(1) in mediating the ACTH response to shocks, alcohol and LPS, whereas the influence of CRF(2) remains subtle. Finally, we showed that peripheral endogenous CRF restrains the ability of LPS to release cytokines.     

脂多糖对大鼠肺泡巨噬细胞Toll样受体4和髓样分化蛋白-2基因表达及炎性因子分泌的影响

目的 探讨脂多糖刺激大鼠肺泡巨噬细胞株NR8383细胞Toll样受体4(TLR4)、髓样分化蛋白-2(MD-2)mRNA表达和炎性因子分泌的影响,以及MD-2小干扰RNA(MD-2 siRNA)对脂多糖刺激下NR8383细胞炎性因子分泌的作用.方法 体外培养NR8383细胞,以不同浓度脂多糖(0.01～10 mg/L)刺激2 h,以1 mg/L脂多糖刺激2～24 h.运用脂质体Lipofectamine 2000将MD-2siRNA转染至细胞.半定量RT-PCR方法检测细胞TLR4和MD-2 mRNA的表达,酶联免疫吸附法检测细胞培养上清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β的含量.统计学处理采用单因素方差分析、独立样本t检验和Pearson相关分析.结果 对照组NR8383细胞TLR4和MD-2mRNA的相对表达量分别为0.52±0.05和0.44±0.09,0.01 mg/L浓度脂多糖刺激前后表达量无明显改变,0.1 mg/L浓度时表达增加,随脂多糖浓度的升高表达量进一步增加,10 mg/L刺激后相对表达量分别为0.72±0.06和0.65±0.10(F=17.26、6.04,P<0.01);TNF-αIL-6和IL-1β含量具有类似改变趋势,对照组分别为(25.8±3.4)ng/L、(62.4±4.7)ng/L和(31.6±1.7)ng/L;在10 mg/L脂多糖刺激下分别增加至(58.9±5.3)ng/L、(96.5±3.9)ng/L和(55.4±5.4)ng/L(F=29.55、54.47、31.45,P<0.01).在1 mr/L脂多糖刺激下,TLR4和MD-2 mRNA的表达量在2 h后明显增加,6 h达高峰,8 h开始回落,至24 h时仍高于基础值(F=5.28、4.11,P<0.01);TNF-α、IL-6和IL-1β含量具有类似变化(F=10.64、11.23、17.58,P<0.01),其中TNF-α和IL-1β含量在6 h达高峰,持续至8 h,IL-6含量在8 h达高峰,持续至12 h.TLR4和MD-2 mRNA表达呈正相关(r=0.513,P<0.01).MD-2 siRNA对NR8383细胞MD-2基因的干扰效率为67%,在脂多糖刺激下干扰组细胞培养上清液中TNF-α、IL-1β和IL-6水平未见明显升高.结论 高浓度脂多糖可较长时间上调大鼠肺泡巨噬细胞株NR8383细胞TLR4和MD-2基因的表达,并促进TNF-α、IL-6和IL-1β的分泌.MD-2 siRNA可抑制脂多糖刺激下NR8383细胞分泌TNF-α、IL-1 β和IL-6.     

Attenuation of Colonic Inflammation by PPARγ in Intestinal Epithelial Cells: Effect on Toll-like Receptor Pathway

The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor highly expressed in the colon and playing an anti-inflammatory role through inhibition of the NF-κB pathway. Toll-like receptor 4 (TLR4) has been known to mediate LPS-induced cellular signaling through activation of NF-κB pathway in intestinal epithelial cells. The aims of this study were to evaluate attenuation of inflammation by PPARγ in intestinal epithelial cells and to study the possible relation between PPARγ and TLR4. HT-29 human epithelial cells were stimulated with LPS (20 μg/ml) and PPARγ ligand, 15d-PGJ₂ (10 μM), or with LPS (20 μg/ml) alone for 24 hr. COX-2, IL-8, TLR4, and PPARγ mRNA expression was assessed by RT-PCR. IL-8 protein levels and TLR4 protein expression were analyzed by ELISA and Western blot, respectively. To evaluate the action mechanisms of PPARγ ligand, Western blot analysis for IκBα degradation was performed. Costimulation with LPS and PPARγ ligand in comparison to LPS stimulation alone (1) decreased COX-2, IL-8 mRNA expression and IL-8 protein secretion, (2) decreased TLR4 mRNA and protein expression, and (3) decreased PPARγ mRNA expression. PPARγ ligand delayed LPS-induced IκBα degradation. These findings suggest that PPAR-γ ligands suppress inflammation in intestinal epithelial cells. PPARγ and TLR, these two antagonistic signaling pathways in intestinal epithelial cells may be partially cross-linked.     

辛伐他汀对缺氧/复氧诱导的心肌细胞凋亡的拮抗作用及其作用机制

目的: 探讨辛伐他汀对缺氧/复氧诱导的心肌细胞损伤的拮抗作用及潜在机制。方法: 分离培养 Sprague-Dawley(SD)大鼠(乳鼠)心肌细胞,随机分为对照组、缺氧2h/复氧4h(H/R4h)组、不同浓度(0.1、1.0及10 μmol/L)的辛伐他汀干预组及Toll样受体4(TLR4)中和性抗体MTS510组(浓度为10 μg/L)。H/R4h组给予缺氧2 h后，随即复氧4 h。细胞处理后，进行PI-AnnexinV染色用流式细胞仪检测心肌细胞的凋亡率，用ELISA法检测心肌乳酸脱氢酶(LDH)的活性；用免疫印迹法测TLR4蛋白的含量。结果:与H/R4h组相比,辛伐他汀干预组可显著降低心肌细胞的凋亡率(16.0% vs. 28.6%,P<0.01)及LDH 的活性(P<0.01)，并呈剂量依赖性。中浓度的辛伐他汀组开始出现拮抗作用，峰值出现在高浓度辛伐他汀组, 加入MTS510阻断剂可降低心肌细胞的凋亡率及LDH的活性(P<0.01)。结论:辛伐他汀对H/R造成的心肌细胞损伤具有拮抗作用,并呈剂量依赖性,其作用机制可能与TLR4信号通路有关。     

Dissociation of the adrenocorticotropin secretory responses to corticotropin-releasing factor (CRF) and vasopressin or oxytocin by using a specific cytotoxic analog of CRF

Control of ACTH secretion in the pituitary in the absence of target cells for CRF, the most potent ACTH secretagogue, was studied in dissociated bovine anterior pituitary cells treated with a potent selective cytotoxin. The cytotoxin is a conjugate of the CRF analog [Nle21,38, Arg36]rat (r) CRF and the plant toxin gelonin. Dissociated bovine anterior pituitary cells were pretreated with vehicle, 2 nM ovine CRF, 2 nM cytotoxic conjugate, or unconjugated [Nle21,38,Arg36]rCRF and gelonin in amounts equivalent to that of 2 nM cytotoxic conjugate for 12 h, then extensively washed and cultured for 3 days before acute secretion experiments. Unstimulated ACTH secretion was similar in all groups. ACTH secretion in response to CRF was attenuated by pretreatment with the cytotoxic conjugate; CRF (2.5 nM)-stimulated secretion was 7.0, 6.3, and 2.8 times the unstimulated rate in cells pretreated with vehicle, 2 nM CRF, or 2 nM cytotoxic conjugate, respectively. Likewise, the ACTH secretory response to a cAMP analog was attenuated by pretreatment with the conjugate; 8-bromo-cAMP (10 mM)-stimulated secretion was 6.8, 7.1, and 3.3 times the unstimulated rate in cells pretreated with vehicle, CRF, or conjugate, respectively. In contrast, the ACTH responses to vasopressin (VP) or oxytocin (OR) remained intact. VP stimulated the ACTH secretion rate by 4.2, 4.0, and 3.5 times, respectively, in the three groups. OT stimulated the ACTH secretion rate by 2.7, 2.6, and 2.3 times in the three groups. Pretreatment with the conjugate attenuated the response to CRF and VP in combination by the same amount as it attenuated the response to CRF alone. The ACTH secretory responses in cells pretreated with unconjugated [Nle21,38,Arg36]rCRF and gelonin were not different from responses in cells pretreated with vehicle. These results suggest that there is a separate mechanism or cell type for OT- and VP-stimulated ACTH secretion distinct from that responsible for the action of CRF on pituitary cells.     

Probiotics may reduce inflammation by enhancing peroxisome proliferator activated receptor gamma activation in HT-29 cells]

BACKGROUND/AIMS: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor highly expressed in the colon which plays an anti-inflammatory role through the inhibition of nuclear factor-kappaB (NF-kappaB) pathway. Probiotics have been shown to exert beneficial effects on inflammatory bowel diseases. However, the exact mechanism by which probiotics exert protection against intestinal inflammation is not well understood. The aims of this study were to evaluate the attenuation of inflammatory response by probiotics in intestinal epithelial cells and to study the association between probiotics and PPARgamma. METHODS: HT-29 human epithelial cells were stimulated with LPS (20 microg/mL) and probiotics, Lactobacillus casei (L. casei) (10(5)-10(7) cfu/mL), or with LPS (20 microg/mL) alone for 24 hours. Interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), toll-like receptor-4 (TLR-4) and PPARgamma mRNA expressions were assessed by RT-PCR. IL-8 protein secretion was measured by ELISA. HT-29 cells were transfected with tk promoter-luciferase plasmid containing a peroxisome proliferator response element (PPRE). After stimulation with L. casei or PPARgamma agonist (15d-PGJ2 or ciglitazone), luciferase activities were measured. RESULTS: LPS induced IL-8, COX-2, TLR-4 mRNA expression, and IL-8 protein secretion in HT-29 cells. Treatment with LPS and L. casei in comparison with LPS stimulation alone lowered IL-8, COX-2, TLR-4 mRNA expression, and IL-8 protein secretion. L. casei increased PPARgamma mRNA expression in dose-dependent manner. L. casei activated PPRE in HT-29 cells transfected with PPRE3-tk-luciferase construct. CONCLUSIONS: Probiotics, L. casei, suppresses the expression of inflammatory mediators in intestinal epithelial cells. The anti-inflammatory action of L. casei might be partially related to PPARgamma activation.     

Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft

AIM: To investigate the expression of toll-like receptor 4 (TLR4) and MD-2 gene and protein in Kupffer cells (KCs) and their role in ischemia-reperfusion (IR) injury of rat liver graft. METHODS: KCs were isolated at 0 (control group), 2, 12, 24 h (IR group) following IR in rat liver graft. mRNA expression of TLR4 and MD-2 was detected by RT-PCR analysis, protein expression of TLR4/MD-2 was detected by flow cytometric (FCM) analysis, and tumor necrosis factor-alpha (TNF-alpha) level in supernatant was measured by ELISA. Then isolated KCs were incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-alpha level was measured again. RESULTS: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-alpha in IR group increased significantly at 2 h following IR, and reached the maximum at 12 h, and slightly decreased at 24 h, but were still significantly higher than those in the control group (P<0.01). The expression of these factors was markedly decreased after anti-TLR4 antibody treatment as compared with the IR group (P<0.01). CONCLUSION: Lipopolysaccharide (LPS) following IR can up-regulate TLR4/MD-2 gene and protein expression in KCs, and synthesize cytokine TNF-alpha. Anti TLR4 antibody can inhibit the production of TNF-alpha induced by LPS. TLR4 and its partner molecule MD-2 may play an important role in Kupffer cell activation and IR injury.