乳源酪蛋白糖巨肽对NF-κB信号通路中关键蛋白的调控作用

以结肠癌细胞HT-29为细胞系,在确定乳源性酪蛋白糖巨肽(casein glycomacropeptide,CGMP)对脂多糖(lipopolysaccharides,LPS)诱导的HT-29细胞核因子-κB(nuclear factor-κB,NF-κB)亚单位p65蛋白影响的基础上,在最适作用时间条件下,利用Western blotting技术进一步检测乳源CGMP对NF-κB信号通路上关键蛋白IκBα、p-IκBα、E3RSIκB、UBC5表达水平的影响,以阐述乳源CGMP调控NF-κB信号通路中关键蛋白的作用机制。结果表明:乳源CGMP组的3种质量浓度(0.001、0.010、0.100?μg/m L)均可在一定程度上抑制LPS诱导的HT-29细胞NF-κB信号通路上IκBα蛋白的降解,0.100?μg/m L作用较为明显,与空白对照组比较有显著性差异(P0.01)。研究明确地证实了乳源CGMP可通过抑制p-IκBα、E3RSIκB、UBC5蛋白的表达来抑制IκBα蛋白的降解,进而抑制NF-κB信号通路的激活。结论:乳源CGMP可显著降低NF-κB信号通路关键蛋白IκBα的降解,其机制是抑制了IκBα的磷酸化和泛素化,使p-IκBα和泛素化关键蛋白E3RSIκB和UBC5的表达均有所下降,进而减少了IκBα的降解,增加了IκBα-p65-p50蛋白三聚体的数量,使p65蛋白核移位效应降低,进而减少下游基因的表达。因此,研究结果科学地阐释了乳源CGMP是通过调控NF-κB信号通路发挥抗炎的作用。     

乳源酪蛋白糖巨肽作用人结肠癌细胞HT-29调控炎症活力研究

为进一步阐释乳源酪蛋白糖巨肽(casein glycomacropeptide,CGMP)的抗炎及修复肠道炎症的功效,本研究在确定了乳源CGMP对脂多糖(lipopolysaccharides,LPS)诱导的结肠癌细胞HT-29NF-κB亚单位p65蛋白的影响和乳源CGMP调控核转录因子(nuclear factor,NF)-κB信号通路的基础上,通过构建表达乳源CGMP和NF-κB必需调节蛋白(NF-κB essential modulator,NEMO)的质粒,共转HT-29细胞,免疫共沉淀测定两个蛋白的相互作用。最后采用线粒体膜电位法测定乳源CGMP对HT-29细胞的促凋亡作用。结果表明,乳源CGMP与NEMO(NF-κB essential modulator)30 h共转HT-29细胞中,乳源CGMP与NEMO蛋白均表达,且表现出二者的相互作用。乳源CGMP可与NEMO相互作用影响NF-κB信号通路;同时乳源CGMP还促进HT-29细胞凋亡,呈时间依赖性,其中0.1μg/m L的CGMP作用效果最好。乳源CGMP与NEMO相互作用是间接作用于IκB激酶(IκB kinase,IKK)而作用于?IκB,进而影响NF-κB信号通路。研究揭示了乳源CGMP调控NF-κB信号通路的另一种途径,也充分说明乳源CGMP可通过作用多种抗炎途径发挥其调控作用。     

铁观音茶提取物对脂多糖诱导RAW264.7细胞炎症反应的抑制作用及机制

研究铁观音茶提取物对脂多糖(LPS)诱导的RAW264.7细胞炎症反应的抑制作用及机制。用脂多糖作用于RAW264.7细胞,建立炎症模型,并用吲哚美辛和不同浓度铁观音提取物处理,检测NO和IL-6的分泌情况,qPCR检测一氧化氮合酶(iNOS)、环氧合酶2(COX-2)、肿瘤坏死因子α(TNF-α)、单核细胞趋化蛋白1(MCP-1)、白细胞介素6(IL-6)mRNA相对表达,Western Blot检测炎症相关蛋白激酶(IKKβ),核转录因子κB抑制因子(IκB)、核转录因子κB p65(NF κB p65)及其磷酸化产物的相对表达。结果显示,铁观音茶提取物能显著抑制炎症介质NO分泌和IL-6蛋白表达量(p<0.05),抑制炎症相关基因iNOS、COX-2、TNF-α和MCP-1等表达,并极显著抑制NF-κB信号通路相关蛋白IKKβ、IkB和p65的磷酸化(p<0.01)。以上结果表明,铁观音茶提取物可明显抑制LPS诱导的RAW264.7细胞炎症反应,其机制可能与抑制NF-κB信号通路激活有关。     

羊栖菜多酚通过核转录因子-κB/丝裂原活化蛋白激酶通路缓解脂多糖诱导的RAW264.7细胞炎症反应

目的：研究羊栖菜多酚对脂多糖（lipopolysaccharide,LPS）诱导小鼠单核巨噬细胞白血病细胞RAW264.7细胞炎症反应的影响。方法：噻唑蓝法测定细胞活力;Griess法测定一氧化氮（NO）水平;实时荧光定量聚合酶链式反应测定白细胞介素（interleukin,IL）-6、IL-1β、肿瘤坏死因子（tumor necrosis factor,TNF）-α、环氧合酶2（cyclooxygenase-2,COX-2）和诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的基因相对表达量;流式细胞术测定细胞吞噬能力;蛋白免疫印迹法测定信号通路丝裂原活化蛋白激酶（mitogen-activated protein kinases,MAPKs）和核转录因子（nuclear factor,NF）-κB信号通路关键蛋白表达水平。结果：羊栖菜多酚对RAW264.7细胞的安全质量浓度范围为0～160 μg/mL。与LPS组相比,羊栖菜多酚剂量依赖性降低巨噬细胞吞噬能力并抑制NO的生成。同时,羊栖菜多酚下调促炎细胞因子（IL-1β、IL-6、TNF-α）和炎症诱导酶（iNOS、COX-2）的mRNA水平,且作用效果与给药剂量及LPS刺激时间相关。这些炎性介质的表达与羊栖菜多酚抑制p38 MAPK和NF-κB p65的激活有关。结论：羊栖菜多酚可通过减弱p38 MAPK和NF-κB p65信号通路的激活水平,抑制下游炎症介质的转录表达,从而缓解LPS诱导的巨噬细胞炎症反应。     

大蒜素对脂多糖诱导腹腔巨噬细胞炎症反应的抑制作用及机制

目的:探讨大蒜素对脂多糖(LPS)诱导小鼠腹腔巨噬细胞炎症反应的抑制作用及机制。方法:建立LPS诱导小鼠腹腔巨噬细胞炎症反应细胞模型,并用地塞米松和不同浓度大蒜素处理,MTT法检测细胞活力,中性红吞噬实验检测吞噬能力,Griess法检测一氧化氮(NO)及ELISA法检测COX-2酶活性和IL-6的分泌,qPCR检测环氧合酶2(COX-2)、一氧化氮合酶(iNOS)和IL-6的mRNA表达水平,Western Blot检测COX-2、iNOS和IL-6的蛋白表达以及核转录因子NF-κB p65及其磷酸化产物的相对表达。结果:大蒜素浓度在40~160 μg/mL范围内对腹腔巨噬细胞均无细胞毒性;与LPS组比较,大蒜素处理组能促进腹腔巨噬细胞的吞噬能力,能显著(P<0.05)抑制炎症因子COX-2酶活性、NO和IL-6的分泌,能显著(P<0.05)抑制基因COX-2、iNOS和IL-6 mRNA和蛋白的相对表达,并极显著(P<0.01)抑制NF-κB p65信号通路的磷酸化。结论:大蒜素能显著抑制LPS诱导的小鼠腹腔巨噬细胞的炎症反应,其机制可能与抑制NF-κB信号通路激活有关。     

乳源酪蛋白糖巨肽对结肠癌HT-29细胞中COX-2、iNOS、GST-π表达的影响

目的:研究乳源酪蛋白糖巨肽(casein glycomacropeptide,CGMP)对人结肠癌HT-29细胞增殖及细胞中环氧合酶-2(cyclooxyenase-2,COX-2)、诱导性一氧化氮合酶(induc ible nitric oxide synthase,iNOS)及谷胱甘肽-S-转移酶π(glutathione-S-transferaseπ,GST-π)表达的影响,为探讨CGMP作为功能性食品提供可靠的依据。方法:采用噻唑蓝(methyl thiazolyl tetrazolium,MTT)实验测定不同剂量CGMP作用12、24、48 h对结肠癌HT-29细胞增殖的抑制情况;在不同剂量CGMP作用HT-29细胞24 h后,通过逆转录聚合酶链反应扩增细胞中提取的COX-2 mRNA、iNOS mRNA、GST-πmRNA,用凝胶成像自动分析系统检测各扩增带COX-2 mRNA、iNOS mRNA、GST-πmRNA水平。结果:1)CGMP组对细胞的增殖均有抑制作用,其中10-4 mg/mL抑制效果最强,且呈现时间依赖性。2)低剂量的CGMP(10-5、10-4、10-3 mg/mL)可显著降低3种基因的表达。结论:CGMP可在一定程度上抑制HT-29细胞的增殖,并呈时间依赖性,同时CGMP能在mRNA水平上抑制COX-2、iNOS、GST-π的表达,进而降低3种基因蛋白的表达,从而进一步改善结直肠癌。     

竹节参总提物对LPS诱导BV2小胶质细胞炎症反应的保护作用

本文研究了竹节参总提物对脂多糖（lipopolysaccharide, LPS）刺激所致的BV2小胶质细胞炎症反应的保护作用及其机制。MTT筛选不同浓度竹节参总提取对小胶质细胞增殖影响;一氧化氮试剂盒检测NO表达水平;PCR检测IL-1β、TNF-α和iNOS的mRNA表达;免疫荧光检测NF-κB表达及定位。竹节参总提物在3.125~50 mg/L对正常状态及LPS 1 μg/mL诱导的炎症状态下小胶质细胞生长均无明显影响;与正常组比较,模型组NO释放量是其11.82倍,TNF-α、IL-1β和iNOS的mRNA表达量分别是正常组的1.35、1.78和2.50倍,并且NF-κB核移位明显;与模型组比较,竹节参总提物在3.125~50 mg/L均能不同程度抑制NO释放量,并减少IL-1β、TNF-α和iNOS的mRNA表达水平;25、50 mg/L竹节参总提物有效抑制了小胶质细胞中NF-κB的核移位。竹节参总提物可减轻LPS诱导的BV2小胶质细胞炎症反应,其主要机制为抑制NF-κB的核移位,进而降低了炎症因子的分泌。     

木犀草素及其黄酮苷的抗炎、抗氧化作用

目的:以木犀草素及其3种黄酮苷(木犀草苷、荭草素、异荭草素)为研究对象,基于HTP-1细胞探究其抗炎与抗氧化活性。方法:采用荧光定量聚合酶链式反应法和Western Blot法分析4种黄酮类化合物预处理后细胞因子表达情况,核转录因子κB(nuclear factor κB,NF-κB)信号通路调控蛋白κB抑制因子α亚基(α inhibitor of κB,IκBα)、IκB激酶β亚基(IκB kinaseβ,IKKβ)、P65的表达情况,以及抗氧化相关核因子E2相关性因子2(nuclear factor erythroid 2 related factor 2,Nrf2)及血红素氧合酶1(heme oxygenase 1,HO-1)的表达情况。结果:4种黄酮类化合物对肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、促炎因子白细胞介素(interleukin,IL)-6均具有极显著抑制作用(P0.01),荭草素可以进一步抑制促炎因子IL-1β的表达(P0.01);木犀草素及3种黄酮苷均可以显著抑制炎性细胞中IKKβ、IκBα的磷酸化,3种黄酮苷还可减少细胞核中P65的表达量,进而较好地抑制NF-κB信号通路发挥抗炎活性;荭草素可极显著促进炎性细胞中Nrf2的表达(P0.01),促进Nrf2发生核移位(P0.01),使HO-1的表达量处于较低水平(P0.01),保持较低的氧化应激水平。结论:木犀草素及3种黄酮苷具有较好的抗炎作用,其中荭草素可通过抑制NF-κB信号通路调控蛋白IKKβ、IκBα、P65的表达发挥较强的抗炎活性,显著降低细胞中TNF-α、IL-6、IL-1β的表达,同时通过促进核转录因子Nrf2的表达与核移位,激活Nrf2发挥较好的抗氧化作用,使细胞中HO-1酶表达量处于较低水平。     

枸杞多糖对LPS诱导BV2小胶质细胞的抗炎活性及NF-κB信号通路的调控作用

为探究枸杞多糖（Lycium barbarum polysaccharide,LBP）对脂多糖（lipopolysaccharide,LPS）诱导的BV2小胶质细胞的保护作用。本研究利用MTT法检测细胞活性,ELISA检测炎症因子的分泌,Western Blot检测蛋白表达情况。结果显示,单独LPS处理BV2小胶质细胞后,细胞的活性无显著变化而细胞上清液中的炎症因子PGE2、IL-1β、IL-6及TNF-α释放显著增多;将枸杞多糖梯度与LPS诱导的BV2小胶质细胞共孵育24 h后,有效地提升了LPS诱导的BV2小胶质细胞的活性,同时显著抑制了LPS激活的BV2小胶质细胞炎症因子的释放（P<0.05）;流式结果表明,LPS组小胶质细胞可产生大量ROS,小胶质细胞经不同浓度LBP处理12 h后,ROS含量显著降低（P<0.05）;Western Blot检测结果显示,与对照组相比,LPS组和LBP组中NF-κB信号通路相关蛋白表达及细胞核内p65蛋白表达水平均显著上调（P<0.05）,且与LBP浓度成反比,而细胞质内p65蛋白表达显著降低（P<0.05）,且随着LBP浓度增加而降低。本研究表明,枸杞多糖对LPS激活的BV2小胶质细胞有显著的保护作用,对于LPS激活的BV2小胶质细胞引起的炎症反应具有抑制作用,同时能抑制小胶质细胞中ROS的含量以发挥抗氧化应激作用,同时有效地抑制LPS刺激后细胞内p-TAK1、iκB、p-iκB及p-p65的表达。     

DPA体外抗炎活性及机制研究

以脂多糖(LPS)诱导的RAW264.7巨噬细胞为炎症的体外模型,以炎症介质【NO和前列腺素E2(PGE2)】和炎症因子(TNF-α,IL-1β,IL-6和IL-10)为指标,研究二十二碳五烯酸(DPA)的体外抗炎活性,并从NF-κB代谢通路的角度探究DPA体外抗炎活性的作用机制。结果表明,DPA可以显著抑制iNOS和COX-2的表达,抑制炎症介质NO和PGE2的分泌,并且,DPA通过调控促炎因子(TNF-α,IL-1β,IL-6)和抑炎因子(IL-10)的平衡发挥抗炎作用。Western blot结果表明,DPA显著抑制NF-κB代谢通路中p50和p65的磷酸化,限制p50/p65核内转移,抑制炎症级联反应,发挥抗炎作用。     

人参、甘草、五味子复合醋提物对老年小鼠急性炎性肝损伤保护作用的研究

目的:本研究旨在通过脂多糖（Lipopolysaccharide,LPS）诱导肝损伤小鼠考察复合醋提物（MVE）对老年小鼠炎性肝损伤的保护效果。方法:将小鼠随机分为正常对照组、MVE组、LPS组、LPS+MVE组。通过苏木精-伊红染色评估肝组织病理情况和免疫组织化学染色判断髓过氧化物酶的表达变化。同时,检测肝组织中白介素-1β、白介素-17、CXC趋化因子配体1、CXC趋化因子配体17等生化因子水平。免疫印迹法测定肝脏中Toll样受体4（Toll-like receptor 4,TLR4）和p-NF-κB p65蛋白表达量变化。结果:复合醋提物能减少中性粒细胞及炎症细胞浸润,极显著降低LPS模型小鼠肝脏谷丙转氨酶（ALT）、谷草转氨酶（AST）、活性氧（ROS）水平（P<0.01）,并降低促炎因子和中性粒细胞趋化因子水平（P<0.05）,降低炎症通路TLR4和p-NF-κB p65的蛋白表达水平（P<0.001）。结论:MVE能改善LPS诱导急性炎症小鼠的肝损伤,此外,MVE还能通过平衡促炎和抗炎因子水平变化、抑制TLR4-NF-κB信号通路,最终达到发挥护肝作用效果。     

榆干离褶伞溶栓酶对脂多糖诱导的血管内皮细胞损伤的保护作用

目的：探讨榆干离褶伞溶栓酶对脂多糖（lipopolysaccharide,LPS）诱导的血管内皮细胞炎性损伤的保护作用。方法：以LPS诱导人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）炎性损伤。将HUVEC分为空白对照组、模型组和榆干离褶伞溶栓酶（Lyophyllum ulmarium fibrinolytic enzyme,LUFE）低、中、高剂量组。采用噻唑蓝法测定HUVEC存活率,通过酶联免疫吸附测试法检测细胞上清液乳酸脱氢酶（lactate dehydrogenase,LDH）、肿瘤坏死因子α（tumor necrosis factor α,TNF-α）、白介素6（interleukin 6,IL-6）、E-选择素和单核细胞趋化因子1（monocyte chemoattractant protein 1,MCP-1）水平。流式细胞术检测细胞间黏附分子1（intercellular cell adhesion molecule 1,ICAM-1）表达水平,采用Hoechst染色法观察HUVEC与人急性单核细胞白血病细胞系（human acute monocytic leukemia cell line-1,THP-1）的黏附作用。用蛋白印迹实验检测HUVEC的Toll样受体4（toll-like receptor 4,TLR4）、丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）以及核因子-κB（nuclear factor κB,NF-κB）通路中主要蛋白（髓样分化因子88（myeloid differentiation factor 88,MyD88）、转化生长因子β激活激酶1（transforming growth factor β activated kinase 1,TAK1）、磷酸化TAK1（phosphorylated TAK1,p-TAK1））的表达和活化情况。结果：LUFE能够抑制LPS所诱导的HUVEC培养上清液LDH、TNF-α、IL-6、E-选择素和MCP-1水平的升高,降低细胞ICAM-1表达水平并减弱HUVEC与THP-1的黏附作用。与模型组比较,LUFE各剂量组TLR4、MyD88、p-TAK1/TAK1、磷酸化c-Jun氨基末端激酶（phosphorylated c-Jun N-terminal kinase,p-JNK）/JNK、p-p38/p38、p-NF-κB/NF-κB水平显著降低（P＜0.05）。结论：LUFE对血管内皮细胞的炎性损伤具有保护作用,其作用机制可能是通过抑制TLR4/MyD88/TAK1/NF-κB信号通路及MAPK通路,进而降低炎症因子水平,从而保护血管内皮细胞。     

Isoflavone content and apoptotic effect in HT-29 cancer cells of a soy germ extract

Colon cancer is one of the leading causes of death and a major public health problem in western countries. We examined the isoflavone content of 70% ethanol extract of soy germ (SG) and its apoptotic effect in HT-29 colon cancer cells. Our results showed that the major isoflavones of the SG extract were daidzein and genistein, and it effectively induced apoptosis in HT-29 cancer cells. In addition, nuclear factor-kappa B (NF-κB) expression was reduced in cells treated with the SG extract, which reduced cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) expression. These combined effects ultimately resulted in apoptosis via caspase-3 activation. In addition, daidzein and genistein, the major isoflavones of SG extract, also exerted the apoptotic effect against HT-29 cancer cells. These results suggest that the inhibition of NF-κB expression is the most important factor in the induction of apoptosis in HT-29 cells treated with the SG extract.     

In vitro screening of probiotics and synbiotics according to anti-inflammatory and anti-proliferative effects

There is emerging evidence of the efficiency of probiotic, prebiotic and synbiotic treatments in inflammatory bowel diseases (IBDs) and one of their long-term complications, colorectal cancer (CRC). In this study, various strains of probiotic lactic acid bacteria, prebiotic glucooligosaccharides (GOS) or a synbiotic combination of the two were screened for anti-inflammatory and anti-proliferative effects in different in vitro models in the context of such diseases. To mimic IBD response to Gram negative bacteria, HT-29 cells were sensitised to inflammatory response to lipopolysaccharide (LPS) by IFNγ which increased expression of TLR4, the LPS biosensor, and were then treated by probiotics, prebiotics and synbiotics. Secreted IL-8 and activated NF-κB were monitored as inflammation biomarkers. A selection of active strains were then subjected to a second inflammatory cell culture model consisting of inflammatory activated transgenic Caco-2 cells transfected by a reporter gene under the control of NF-κB inducible promoter. Quantification of reporter gene expression allowed us to demonstrate some probiotic inhibitory properties or to confirm such characteristics in two different models. Proliferation of cancerous HT-29 cells was monitored by XTT assay. Only three probiotic strains induced a proliferation decrease, but with a lack of reproducibility. Binary or ternary probiotic associations, complemented or not by prebiotic GOS, significantly decreased proliferation, especially with a synbiotic association of Bifidobacterium breve, Lactococcus lactis and oligoalternan, a GOS. This combination was selected for the following experiments. We showed the involvement of both bacterial and carbohydrate compounds of this synbiotic in the observed effect by dose range tests. We demonstrated that this decrease in proliferation may be due to an induction of a differentiated phenotype, as shown by the up-regulation of intestinal alkaline phosphatase, a biomarker of differentiation, monitored by real-time RT-PCR in HT-29 cells treated by the selected synbiotics. Thus, this study demonstrates the ability of probiotics to exert anti-inflammatory effects and shows some anti-proliferative characteristics for a specific synbiotics. These products should be further evaluated in animal models to confirm the in vitro results.     

Anti-inflammatory effect of lactobacilli bacteria on HepG2 cells is through cross-regulation of TLR4 and NOD2 signalling

Pathogen peptidoglycans are detected via host’s innate immune system. This course of action is executed by nucleotide-binding oligomerization domain (NOD) proteins, which have been identified inducing inflammation through nuclear factor-kappa B (NF-κB). Excessive stimulation of liver cells by endotoxin leds to severe inflammatory symptoms. In this research, seven strains of lactobacilli bacteria were prior processed into crude cell wall extracts, followed by testing if crude cell wall extracts could lessen pro-inflammatory processes launched by lipopolysaccharides. The results showed lactobacilli bacteria activated NOD2 expression in mild degrees, and did not lead to serious inflammation. Prior exposure of HepG2 cells to lactobacilli bacteria rendered them desensitized to subsequent LPS challenge. The mechanism by which lactobacilli bacteria attenuated inflammation might be because of the fact that lactobacilli bacteria induced interleukin-10, suppressor of cytokine signalling 1/3 and peroxisome proliferator-activated receptors alpha via NOD2-NF-κB pathway, and then cross-regulated Toll-like receptor 4 (TLR4) downstream signal transduction.