核因子κB在精氨酸血管升压素诱导大鼠心肌成纤维细胞一氧化氮合成中的作用

本文探讨了精氨酸血管升压素(AVP)刺激下体外培养的大鼠心肌成纤维细胞(CFs)内一氧化氮(NO)含量、一氧化氮合酶(NOS)活性、诱导型一氧化氮合酶基因表达的变化及其与核因子κB(NF-κB)的关系。用胰酶消化法分离培养Sprague Dawley仔鼠的CFs,分别采用硝酸还原酶法、分光光度法、逆转录-聚合酶链式反应(RT-PCR)、免疫荧光-共聚焦显微镜和蛋白质印迹检测AVP干预下CFs的NO含量、NOS活性、iNOS mRNA表达和NF-κB的活化。结果显示,AVP浓度依赖性(0．001—0．1μmol／L)地增加CFs的NO含量,提高NOS活性,增加iNOS mRNA表达;AVP能够活化NF—κB,使其由细胞浆转位于细胞核;NF-κB特异性抑制剂吡咯啉烷二甲基硫脲(PDTC)能够抑制AVP诱导的CFs NO含量增加、NOS活性提高和iNOS mRNA表达增加。上述结果提示,AVP干预下CFs iNOS mRNA表达增加、NOS活性增高、NO合成增多可能通过NF-κB激活途径,NF-κB激活参与心肌纤维化的发生和发展。     

适宜浓度短链脂肪酸混合物对小胶质细胞炎症 抑制及机制研究

本研究的主要目的是探讨适宜浓度短链脂肪酸(short chain fatty acids,SCFAs)混合物对炎症环境下小胶质细胞的抑炎作用及其机制.采用脂多糖(LPS)刺激小鼠小胶质细胞系BV-2细胞建立神经炎症模型,并利用CCK8试剂盒检测不同浓度单一的乙酸钠、丙酸钠、丁酸钠处理后的细胞活力.设计选取这三种SCFAs对细胞活力无影响、且有抑炎效果的特定浓度进行组合(SCFAs mix),进一步检测SCFAs mix对LPS刺激下BV-2细胞炎症反应的影响及机制,包括:a.用一氧化氮(NO)试剂盒检测NO的释放;b.用ELISA检测炎症因子TNF-α、IL-6的释放;c.用qRT-PCR和Western blot检测炎症因子TNF-α、IL-6、炎症小体NLRP3、炎症通路相关蛋白TLR4、NF-κB等的表达变化.结果表明LPS刺激BV-2细胞4 h后,在体系中添加特定浓度的单一SCFA处理12 h后,不能缓解BV-2细胞的炎症反应,而将上述SCFAs配制成同等终浓度的SCFAs mix处理12 h却能显著降低细胞培养上清液中NO、TNF-α和IL-6 (均P0.001)的量,还能抑制BV-2细胞内iNOS、TNF-α、IL-6和NLRP3 mRNA的升高(均P0.001);通过对炎症信号通路关键分子的检测发现,SCFAs mix可以抑制LPS诱导的BV-2细胞内TLR4、MyD88、TRAF6和NF-κB蛋白的表达升高.综上可见:适宜浓度的混合SCFAs可通过调控TLR4/MyD88/TRAF6/NF-κB炎症通路抑制LPS诱导的小胶质细胞的炎症反应,而起到抗炎的保护作用.     

核因子κB、NO 与肺纤维化的研究进展

国内外对导致肺纤维化的肺部疾病中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)基因在NF-κB参与诱导活化下,催化合成的一氧化氮(nitric oxide,NO)在肺纤维化过程中发挥细胞保护性及细胞毒性双重作用的研究已取得一些进展。本文主要阐述iNOS基因在NF-κB诱导活化下合成的NO与肺纤维化的关系,从而为NO作用的双重性和网络性及NO与肺纤维化关系的研究提供一些线索。     

红景天苷对脂多糖诱导小鼠巨噬细胞炎性介质分泌的影响

本文旨在探讨红景天苷(salidroside,Sal)对脂多糖(lipopolysaccharide,LPS)诱导小鼠巨噬细胞系J774.1炎性活化的影响及其可能机制。J774.1细胞分为PBS对照组、LPS(0.5μg/m L)刺激组和不同剂量(5、25、125μg/m L)Sal预处理+LPS组。CCK-8比色法检测细胞活性,ELISA测定培养上清中TNF-α、MCP-1和MIP-2含量,硝酸还原酶法测定上清中NO含量,RT-PCR检测细胞i NOS m RNA表达,Western blot检测胞浆i NOS蛋白和胞浆与胞核NF-κB/p65蛋白表达,Trans AMTM NF-κB/p65活性检测试剂盒测定NF-κB/p65 DNA结合活性。结果显示,0.5μg/m L LPS以及不同剂量(5、25、125μg/m L)Sal处理细胞12 h对J774.1细胞活力无影响;与LPS刺激组比较,LPS刺激前Sal预处理J774.1细胞,培养上清中TNF-α、MCP-1、MIP-2和NO含量呈剂量依赖性降低(P0.05),细胞i NOS m RNA和蛋白表达水平下调(P0.05),胞核NF-κB/p65蛋白表达降低(P0.05)而胞浆NF-κB/p65蛋白相应增加(P0.05),且NF-κB/p65 DNA结合活性呈剂量依赖性降低(P0.05)。以上结果提示,Sal预处理能够降低LPS诱导的巨噬细胞炎性活化,其机制可能通过干扰LPS/TLR4/NF-κB信号通路,从而降低炎性介质及细胞因子的过度表达和分泌。     

miR-33s在NF-κB抑制ABCA1表达及胆固醇流出中的作用

为探讨mi R-33s在核因子κB(NF-κB)抑制三磷酸腺苷结合盒转运体A1(ABCA1)表达及胆固醇流出中的作用,THP-1巨噬细胞源性泡沫细胞经不同浓度脂多糖(LPS)处理,活化NF-κB,或以PDTC(NF-κB抑制剂)预处理细胞后再加入LPS,实时荧光定量PCR检测细胞mi R-33s及其宿主基因胆固醇调节元件结合蛋白(SREBPs)的表达,蛋白质印迹法检测SREBPs的蛋白质表达,染色体免疫共沉淀检测NF-κB p65与SREBPs启动子区结合情况;LPS处理基础上,转染mi R-33s抑制物或mi R-33s模拟物,RT-PCR检测ABCA1 m RNA表达水平,蛋白质印迹法检测ABCA1蛋白水平,液体闪烁计数仪检测细胞内的胆固醇流出.结果显示,NF-κB活化促进mi R-33s及SREBPs的表达,使用PDTC抑制NF-κB,细胞内mi R-33s和SREBPs的表达下降;NF-κB p65可与SREBPs启动子区直接结合;转染mi R-33s抑制剂后,NF-κB活化对ABCA1的抑制作用减弱,胆固醇流出增强;相反,转染mi R-33s抑制物,NF-κB活化对ABCA1的抑制作用增强,胆固醇流出减弱.结果提示,NF-κB活化可促进mi R-33s表达,抑制ABCA1及胆固醇流出.     

延龄草苷对过氧化氢诱导的PC12细胞氧化损伤和炎症因子表达的影响

探讨延龄草苷对过氧化氢(H_2O_2)诱导的PC12细胞氧化损伤和炎症因子表达的影响。采用MTT法和LDH活性测定观察延龄草苷对PC12细胞模型的影响,采用相关试剂盒检测活性氧(ROS)和丙二醛(MDA)含量,超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,Western blot检测Sirt1、NF-κB和TNF-α的蛋白表达。延龄草苷(5~20μM)能够显著提高H_2O_2损伤的PC12细胞的活力,提高细胞抗氧化能力,并上调Sirt1的表达,下调NF-κB和TNF-α的表达,其保护作用可能与提高抗氧化能力,降低炎症因子损伤,调控Sirt1/NF-κB信号通路有关。     

抗氧化剂芦丁对星形胶质细胞iNOS基因表达的影响

研究抗氧化剂芦丁对星形胶质细胞可诱导型一氧化氮合酶基因表达的抑制作用,并探讨其作用的机制,以脂多糖和γ-干扰素诱导星形胶质细胞表达可诱导型一氧化氮合酶,应用蛋白质印迹分析以及ESR自旋捕集技术研究了芦丁对星形胶质细胞产生内源性一氧化氮的作用,研究结果表明芦丁能够有效地抑制星形胶质细胞可诱导型一氧化氮合酶基因的表达、减少内源性一氧化氮的生成,而这一抑制作用是通过NF-κB信号通路实现的,芦丁对星形胶质细胞的活化具有明显的调控作用。     

白术提取物苍术酮对脂多糖诱导的BV2细胞神经炎性影响及相关机制研究

研究旨在白术提取物苍术酮(AT)对脂多糖(LPS)诱导的BV2细胞抗炎作用,并探讨其相关机制。提取苍术酮并进行结构鉴定;以LPS诱导BV2细胞建立炎症模型;四甲基偶氮唑蓝(MTT)法检测细胞存活率;Griess反应检测细胞上清液中NO水平;ELISA法检测细胞上清中PGE2、IL-6、TNF-α水平;Western Blot法检测COX-2、iNOS、MAPK、NF-κB蛋白表达。结果表明LPS诱导BV2细胞后NO、PGE_2、IL-6、TNF-α水平和COX-2及iNOS蛋白表达显著增高,苍术酮预处理后均显著降低。进一步研究表明苍术酮可显著降低LPS诱导BV2细胞ERK、JNK、NF-κB蛋白表达。说明苍术酮能够有效预防LPS诱导BV2细胞神经炎性反应,机制与抑制炎症通路相关。     

NF-κB介导哮喘过敏原刺激后支气管上皮细胞的凋亡

目的研究核转录因子κB(NF-κB)在哮喘过敏原刺激后支气管上皮细胞中的表达及对细胞凋亡的影响。方法以300U/L尘螨抗原提取物作为过敏原刺激支气管上皮细胞系16HBE细胞,应用RT-PCR和Western blot检测尘螨抗原提取物刺激对16HBE细胞NF-κB(p65)表达水平的影响;用NF-κB(p65)sh RNA沉默16HBE细胞内NF-κB表达后,应用流式细胞术和Western blot检测NF-κB(p65)在过敏原刺激诱导16HBE细胞凋亡中的作用。结果过敏原刺激后使16HBE细胞内NF-κB(p65)m RNA和蛋白水平明显上调,明显诱导16HBE细胞凋亡和活化型caspase-3水平上调,沉默NF-κB可使过敏原刺激诱导的16HBE细胞凋亡率降低和活化型caspase-3水平上调减少。结论 NF-κB可介导哮喘过敏原刺激后的支气管上皮细胞凋亡。     

阿魏酸对脂多糖诱导的小鼠小胶质细胞炎性反应的抑制作用

阿魏酸是川芎、当归等中药的有效成分之一,具有较强的抗氧化活性和抗炎作用。小胶质细胞是脑内常驻的免疫效应细胞,极易被激活而导致脑内发生慢性神经性炎症反应,与阿尔茨海默病等神经退行性疾病的发生发展密切相关。该研究采用脂多糖(LPS)刺激小胶质细胞(BV-2)活化,研究阿魏酸对炎症反应的抑制作用。结果表明,2.5～22.5μg/mL的阿魏酸浓度依赖性的抑制一氧化氮(NO)、前列腺素E2(PGE2)、白介素-1β(IL-1β)等炎症因子的产生,以及一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)蛋白的表达,其作用机制可能与其抑制Toll样受体4(TLR4)表达有关。     

Phenelzine (Monoamine Oxidase Inhibitor) Increases Production of Nitric Oxide and Proinflammatory Cytokines via the NF-κB Pathway in Lipopolysaccharide-Activated Microglia Cells

Phenelzine is a potent monoamine oxidase inhibitor that is used in patients with depression. It is also well known that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, which suggests that NO is involved in the pathogenesis of depression. The purpose of this study was to determine if phenelzine affects the production of NO and tumor necrosis factor-alpha (TNF-α) in activated microglia cells. BV-2 microglia cells and primary microglia cells were cultured in DMEM and DMEM/F12 and then cells were treated with LPS or LPS plus phenelzine for 24?h. The culture medium was collected for determination of NO, TNF-α, and IL-6 and cells were harvested by lysis buffer for Western blot analysis. Phenelzine increased the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS), as well as the release of TNF-α and IL-6 in BV-2 microglia cells. It is also confirmed that phenelzine increased the levels of NO, TNF-α and IL-6 in LPS-activated primary microglia cells. Phenelzine increased nuclear translocation of NF-κB by phosphorylation of IκB-α in LPS-activated microglia cells. These findings suggest that high doses of phenelzine could aggravate inflammatory responses in microglia cells that are mediated by NO and TNF-α.     

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)?[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.     

Inhibitory effect of sulphated polysaccharide porphyran on nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophages

Porphyran, extracted from an edible red alga (Porphyra yezoensis), is a sulphated polysaccharide with a wide variety of biological activities including anti-tumour, antioxidant and immuno-modulating activities. In this study, we examined the effect of porphyran on nitric oxide (NO) production in mouse macrophage cell line RAW264.7 cells. Although no significant activity of porphyran to induce NO or tumour necrosis factor-α (TNF-α) production in RAW264.7 cells was observed at the concentration range tested (10-500 μg/ml), it was found for the first time that porphyran inhibited NO production and expression of inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). In the presence of 500 μg/ml porphyran, NO production and expression of iNOS in LPS-treated RAW264.7 cells were completely suppressed. On the other hand, porphyran showed only a marginal effect on the secretion of TNF-α from LPS-stimulated RAW264.7 cells. Electrophoretic mobility shift assay (EMSA) using infrared dye labelled oligonucleotide with nuclear factor-κB (NF-κB) consensus sequence suggested that porphyran inhibited the LPS-induced NF-κB activation. The LPS-inducible nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also inhibited by the pre-treatment with porphyran. Our results obtained in in vitro analysis suggest that porphyran suppresses NO production in LPS-stimulated macrophages by the blocking of NF-κB activation.     

Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50?~?200?μg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.     

Nitric oxide augments oridonin-induced efferocytosis by human histocytic lymphoma U937 cells via autophagy and the NF-κB-COX-2-IL-1β pathway

Abstract We previously demonstrated that oridonin-induced autophagy enhanced efferocytosis (phagocytosis of apoptotic cells) by macrophage-like U937 cells through activation of the inflammatory pathways. In this study, exposure of U937 cells to 2.5 μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase 1400 W (dihydrochloride) or L-NAME (hydrochloride). NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation and iNOS expression. Moreover, oridonin-induced stimulation of efferocytosis was significantly suppressed by 1400 W or L-NAME. In addition, 1400 W or L-NAME impaired oridonin-induced autophagy. Inhibition of autophagy with 3-methyladenine (3MA) or Beclin-1 siRNA attenuated the uptake of apoptotic cells with a slight increase in the production of NO. The pro-inflammatory cytokine interleukin-1β (IL-1β) has been reported to be involved in oridonin-induced efferocytosis in U937 cells and interact with NO to contribute to inflammatory responses. 1400 W or L-NAME blocked the secretion of IL-1β and the activation of NF-κB and COX-2. Provision of SNP or LPS in place of oridonin resulted in the similar enhancement of efferocytosis, autophagy, the release of IL-1β and the expression of signal protein. NO augmented the oridonin-induced efferocytosis by mediating autophagy and activating the NF-κB-COX-2-IL-1β pathway. Inhibition of NF-κB or COX-2 in turn decreased the production of NO and the expression of iNOS. There exists a positive feedback loop between NO generation and NF-κB-COX-2-IL-1β pathway.     

Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-κB pathway in microglia cells

Microglial activation plays an important role in neuroinflammation, which contributes to neuronal damage, and inhibition of microglial activation may have therapeutic benefits that could alleviate the progression of neurodegeneration. Recent studies have indicated that the antimalarial agent artemisinin has the ability to inhibit NF-κB activation. In this study, the inhibitory effects of artemisinin on the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-stimulated primary microglia. Our results show that artemisinin significantly inhibited LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO). Artemisinin significantly decreased both the mRNA and the protein levels of these pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) and increased the protein levels of IκB-α, which forms a cytoplasmic inactive complex with the p65-p50 heterodimeric complex. Artemisinin treatment significantly inhibited basal and LPS-induced migration of BV-2 microglia. Electrophoretic mobility shift assays revealed increased NF-κB binding activity in LPS-stimulated primary microglia, and this increase could be prevented by artemisinin. The inhibitory effects of artemisinin on LPS-stimulated microglia were blocked after IκB-α was silenced with IκB-α siRNA. Our results suggest that artemisinin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The data provide direct evidence of the potential application of artemisinin for the treatment of neuroinflammatory diseases.     

黑色素及谷胱甘肽通过抑制NF-κB途径的激活可有效抑制H 5N1禽流感病毒感染

我国是H5N1高致病性禽流感频发区.为筛选天然药物用于H5N1禽流感的防治并研究其抗病毒感染的作用机制,本研究选取黑色素及谷胱甘肽（GSH）为候选药物,通过Hoechst 33258荧光染色,观察了禽流感病毒(AIV)感染后的细胞形态变化;细胞凋亡流式细胞术定量分析表明,H5N1 AIV感染可诱导宿主狗肾上皮细胞(MDCK)凋亡,而黑色素及GSH对其具有显著抑制作用(P<0.01);当20 μg/ml黑色素及10 mmol/LGSH共同作用时,抑制率高达89% (P <0.01);采用MTT法检测,黑色素及GSH可增强AIV感染后的宿主细胞存活率;病毒血凝素(Ｈa)基因的RT-PCR检测结果表明,黑色素及GSH可显著降低AIV在宿主细胞内的增殖(P<0.01);一氧化氮（NO）毛细管电泳（CE）激光诱导荧光检测显示,黑色素及GSH均可显著降低病毒感染诱导宿主细胞内的NO释放量(P<0.05);RT-PCR技术结合ELISA方法,分别分析了病毒感染后宿主细胞的诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）、环氧化酶(cyclooxygenase-2, COX-2)及核因子κB(nuclear factor-kappa B,NF-κB）在mRNA和蛋白水平表达的差异.结果表明,黑色素及GSH可抑制病毒感染诱导的iNOS与COX-2的基因及蛋白的表达 (P<0.05),并进而抑制NF-κB途径的激活(P<0.01),从而可有效抑制H5N1 AIV感染.