TLR4/MAPKs信号通路在ox-LDL诱导的血管平滑肌细胞分泌MCP-1中的作用

目的:探讨Toll样受体4/丝裂原活化蛋白激酶(TLR4/MAPKs)信号通路在氧化性低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞分泌单核细胞趋化因子-1(MCP-1)中的作用。方法:在ox-LDL刺激下采用逆转录聚合酶链技术(RT-PCR)和酶联免疫吸附试验(ELISA)检测血管平滑肌细胞MCP-1的表达,用Western blotting检测细胞外信号调节激酶(ERK1/2)、p38促分裂原活化蛋白激酶(p38MAPK)磷酸化水平的变化。同时,分别应用TLR4中和抗体(TLR4单克隆抗体、TLR4阻断剂)、PD98059(ERK1/2特异性抑制剂)、SB23015(p38MAPK特异性抑制剂)、SP600125(JNK特异性抑制剂),观察其对ox-LDL诱导的MCP-1的表达和ERK1/2、p38MAPK磷酸化水平的影响。结果:ox-LDL刺激血管平滑肌细胞上调MCP-1mRNA和其蛋白的表达(P0.05);用TLR4中和抗体、PD98059、SB23015预孵育后MCP-1mRNA和其蛋白的表达较单独ox-LDL刺激情况下降低(P0.05),而用SP600125预孵育后降低不明显(P0.05);TLR4调节了ERK1/2和p38MAPKs的磷酸化水平。结论:ox-LDL是TLR4的内源性配体;ox-LDL通过或部分通过TLR4/ERK1/2和TLR4/p38MAPK信号通路介导血管平滑肌细胞MCP-1的表达。     

三七皂苷Rg1抑制脂多糖诱导小胶质细胞激活的机制研究

目的:探讨中药有效成分三七皂苷Rg1(Ginsenoside Rg1,Rg1)对抑制脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞株BV-2细胞激活的机制。方法:用LPS刺激BV-2细胞构建激活模型,采用四甲基偶氮唑蓝比色法(MTT)检测Rg1对BV-2细胞的活力影响,蛋白质免疫印迹(Western Blot)方法检测不同浓度Rg1(10、20和40μmol/L)对磷酸化的核因子-κB抑制蛋白-α(inhibitorκB-α,IκB-α)和反应结合蛋白(cAMP-responseelement binding protein,CREB)以及促分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)家族的细胞外信号调节激酶(extracellular signal-regulated kinase 1/2,ERK1/2)、c-Jun氨基端激酶(c-Jun N-terminal kinase,JNK)和p38促分裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)等细胞信号通路蛋白的表达及其变化规律。结果:不同浓度的Rg1明显抑制了LPS诱导的磷酸化IκB-α和CREB蛋白表达以及MAPKs通路(ERK1/2,JNK,p38 MAPK)磷酸化蛋白表达,并且对p38 MAPK表达的影响呈剂量依赖性。结论:Rg1可能通过抑制MAPKs的磷酸化来调控LPS诱导的小胶质细胞株BV-2细胞激活,发挥其神经抗炎的作用。     

梅毒螺旋体膜蛋白Tp0971激活MAPKs和NF-κB诱导巨噬细胞分泌TNF-α和IL-1β

目的:探讨梅毒螺旋体(Tp)膜蛋白Tp0971诱导巨噬细胞分泌TNF-α和IL-1β的分子机制。方法:课题组前期表达的Tp0971重组蛋白为研究对象,将不同浓度的重组Tp0971刺激巨噬细胞,分别采用ELISA和实时定量PCR检测TNF-α和IL-1β的分泌及其mRNA的表达。采用Western blot检测丝裂原活化蛋白激酶(MAPKs) p38,ERK1/2以及JNK1/2的磷酸化,并用相应的抑制剂处理观察TNF-α和IL-1β的变化。同时采用Western blot观察核转录因子κB(NF-κB)的核转位情况,并采用抑制剂处理观察其在介导TNF-α和IL-1β分泌中的作用。结果:ELISA结果显示,重组Tp0971能在0.5～10μg/ml剂量范围内诱导巨噬细胞分泌TNF-α和IL-1β。并能促进其mRNA表达,采用转录抑制剂放线菌素D(Act D)或翻译抑制剂放线菌酮(CHX)处理后,TNF-α和IL-1β的转录和分泌水平显著降低。Tp0971也可诱导p38,ERK1/2以及JNK1/2磷酸化,采用其相应的抑制剂处理后,TNF-α和IL-1β分泌明显减少。Western blot结果也显示Tp0971能诱导NF-κB p65亚基核转位,采用NF-κB抑制剂PDTC处理后,TNF-α和IL-1β分泌水平降低。结论:Tp0971激活MAPKs和NF-κB诱导巨噬细胞分泌TNF-α和IL-1β。     

CPSIT_p7通过激活JNK/ERK信号通路诱导宿主细胞炎症

目的初步探讨鹦鹉热嗜衣原体蛋白CPSIT_p7对宿主细胞炎症反应的调节作用及其分子机制。方法佛波酯(PMA)处理THP-1细胞过夜,诱导其分化为贴壁的巨噬细胞,之后用CPSIT_p7蛋白刺激贴壁细胞,或先用30μmol/L ERK抑制剂PD98059、JNK抑制剂SP600125和p38抑制剂SB202190分别预处理贴壁细胞,再用CPSIT_p7蛋白处理贴壁细胞;Western blot检测ERK、JNK和p38磷酸化水平,ELISA检测各种炎症因子的表达水平。结果 0~10μg/ml CPSIT_p7蛋白刺激PMA诱导的THP-1细胞24 h后,随着CPSIT_p7质量浓度升高,IL-6、IL-1β、IL-8及TNF-α的含量呈剂量依赖性增加;10μg/ml的CPSIT_p7处理细胞0、6、12、24和36 h,在24 h时IL-6、IL-8及IL-1β表达水平达到高峰,而TNF-α在12 h就达到高峰;CPSIT_p7蛋白处理细胞后其ERK和JNK磷酸化水平显著升高,p38磷酸化水平改变不明显;JNK和ERK抑制剂能明显降低CPSIT_p7蛋白诱导的IL-6、IL-1β、IL-8及TNF-α表达。结论 CPSIT_p7通过JNK/MAPKs和ERK/MAPKs信号传导途径诱导THP-1产生IL-1β、IL-6、IL-8及TNF-α炎症因子,与p38/MAPKs信号传导通路无关。     

TGF-β1激活的p38MAPK在TGF-β1上调人卵巢癌细胞PAI-1表达中的作用

目的:纤溶酶原激活物抑制剂1(PAI-1)在凝血、创伤修复、炎症和肿瘤转移中起重要作用。已有报道转化生长因子β1(TGF-β1)能通过Smad通路诱导PAI-1表达,但TGF-β1能否通过激活非Smad通路诱导PAI-1表达尚不清楚,因此本研究探讨了在卵巢癌细胞中TGF-β1激活的非Smad通路p38丝裂原活化蛋白激酶(p38MAPK)和细胞外信号调节激酶(ERK)与TGF-β1上调PAI-1表达的关系。方法:用10μg/L TGF-β1处理卵巢癌SKOV3细胞和HO-8910细胞后,采用real-time PCR和Western blotting的方法检测PAI-1的表达,用磷酸化p38MAPK的抗体和磷酸化ERK的抗体检测p38 MAPK和ERK的激活情况,用p38 MAPK和ERK的特异性抑制剂SB203580和PD98059分别抑制其活性后,检测PAI-1的表达。结果:TGF-β1在卵巢癌细胞中可明显上调PAI-1mRNA和蛋白的表达,并可快速激活p38 MAPK和ERK。用p38 MAPK的抑制剂可以明显抑制TGF-β1上调PAI-1表达,但是抑制ERK活性对TGF-β1上调PAI-1表达没有明显影响。结论:TGF-β1激活的p38 MAPK通路参与了TGF-β1上调PAI-1的表达。     

MAPKs在anti-β2GPI/β2GPI刺激THP-1细胞表达TF过程中的作用探讨

目的:探讨丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPKs)在anti-β2 GPI/β2 GPI复合物诱导单核细胞株THP-1表达组织因子(TF)中的活化及其作用。方法:利用荧光定量PCR(Real-time PCR)、TF活性试剂盒等分别检测anti-β2 GPI/β2 GPI复合物诱导THP-1细胞表达TF mRNA及TF活性,Western blot检测细胞表达p38、磷酸化-p38(p-p38)、ERK1/2、磷酸化-ERK1/2(p-ERK1/2)、JNK、磷酸化-JNK(p-JNK)的情况。进一步采用p38、ERK1/2、JNK抑制剂(SB203580、U0126、SP600125)观察是否能阻断anti-β2 GPI/β2 GPI复合物诱导THP-1细胞表达TF。结果:Anti-β2 GPI/β2 GPI复合物(100μg/ml)能够显著增强THP-1细胞表达TF,并使p-p38、p-ERK1/2、p-JNK水平显著升高(P<0.05 vs control);其引发的MAPKs磷酸化具有时间效应性,均在刺激30分钟时达到高峰;对应的特异抑制剂SB203580(10μmol/L)、U0126(5μmol/L)、SP600125(90 nmol/L)单独或合并处理THP-1细胞后,anti-β2 GPI/β2 GPI复合物诱导细胞TF mRNA表达及TF活性的效应明显被阻断(P<0.01 vs control)。结论:Anti-β2 GPI/β2 GPI复合物诱导THP-1细胞表达TF过程中,MAPKs被激活进而发挥重要作用。     

盐酸戊乙奎醚抑制脂多糖致急性肺损伤大鼠肺组织p38丝裂原活化蛋白激酶的活化

目的:观察盐酸戊乙奎醚(PHC)对脂多糖(LPS)致急性肺损伤(ALI)大鼠肺组织p38丝裂原活化蛋白激酶(p38MAPK)、c-jun氨基末端激酶(JNK)活化的影响。方法:SD大鼠随机分为对照组、LPS模型组（5 mg/kg LPS,iv）和LPS+PHC高、中、低(3.0、1.0和0.3 mg/kg)3个剂量组,每组6只,进行PHC对肺组织p38MAPK、JNK表达的量效性分析;另取大鼠在注入NS后即刻0（对照组）和注射LPS后2 h、4 h、6 h和12 h共5个时点,每时点6只,进行肺组织p38MAPK、JNK表达的时效性分析。蛋白免疫印迹法检测肺组织p38MAPK、JNK的表达。结果:LPS模型组大鼠肺组织磷酸化p38MAPK、JNK的表达显著高于对照组（P＜0.05);PHC高剂量组显著抑制LPS诱导的大鼠肺组织磷酸化p38MAPK表达（P＜0.05);PHC在造模后6 h时最能有效抑制磷酸化p38MAPK上调。与LPS模型组相比,PHC高、中、低剂量组磷酸化JNK的表达均无显著差异(均P＞0.05);造模后不同时点,PHC对磷酸化JNK的表达均无抑制作用。结论:PHC抑制LPS诱导的ALI大鼠肺组织p38MAPK活化,但不能抑制JNK活化,PHC对LPS诱导大鼠ALI的拮抗作用可能与其抑制p38MAPK的活化有关。     

肺炎克雷伯菌夹膜多糖激活AP-1 诱导人支气管上皮细胞表达 β-防御素-3

目的:观察肺炎克雷伯菌夹膜多糖(CPS)对人支气管上皮细胞表达β-防御素-3(hBD-3)的影响,并探讨其作用机制。方法:用肺炎克雷伯菌CPS刺激人支气管上皮细胞BEAS-2B。使用实时定量PCR和ELISA分别检测hBD-3 mRNA和蛋白表达的影响。测定CPS处理前后丝裂原活化蛋白激酶(MAPKs)以及c-Jun的磷酸化。采用MAPKs和NF-κB抑制剂或RNA干扰TLR4表达,观察其在介导hBD-3表达中的作用。采用染色质共沉淀技术检测核转录因子AP-1与hBD-3启动子的结合情况。结果:CPS处理细胞后,hBD-3 mRNA的表达呈剂量依赖性升高,ELISA获得类似结果。CPS能激活MAPKs,采用p38、ERK及NF-κB抑制剂SB203580、PD98059和PDTC处理细胞后,不影响hBD-3的表达,而JNK抑制剂SP600125处理后,hBD-3分泌明显降低。CPS可诱导AP-1亚基c-Jun磷酸化及能增强AP-1与hBD-3启动子的结合。结论:肺炎克雷伯菌夹膜多糖激活AP-1诱导人支气管上皮细胞表达β-防御素-3。     

酪氨酸激酶在TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用

目的 研究在类风湿关节炎成纤维样滑膜细胞（RA FLS）信号转导中,酪氨酸激酶在TNF－α刺激下丝裂原活化蛋白激酶（mitogen-activated protein kinases,MAPKs)活化中的作用。方法 原代培养类风湿关节炎成纤维样滑膜细胞。应用Western blot检测TNF－α短时间内引起RA FLS蛋白质酪氨酸磷酸化状态改变,及其对MAPKs家族成员活化的浓度效应和时相特点;并应用genistein,酪氨酸激酶（PTK）抑制剂观察对MAPKs活化的抑制情况。结果 TNF－α可以瞬时引起RA FLS蛋白质酪氨酸磷酸化程度增加;并在短时间内激活MAPKs通路（ERK2、JNK2、P38）。不同浓度梯度TNF－α作用显示：10IU／ml时对ERK2、JNK2即可达到峰值活化,100IU／ml时P38达到最大活化。时间上,ERK2、JNK2、P38的活化分别在TNF－α作用后5min、15min、15min最明显;genistein对TNF－α诱导的ERK2活化抑制作用显著,而对于JNK2、P38的抑制则较弱。结论 TNF－α在RA FLS信号转导中,可以瞬时导致蛋白质酪氨酸磷酸化程度增加,并同时激活MAPKs 3条通路,但是对MAPKs 3个亚家族成员的活化具有异质性;PTK在TNF－α导致ERK活化中发挥作用,对JNK、P38活化无明显影响。     

多发性骨髓瘤中MAPK信号通路对BLyS表达的调控研究

目的 研究多发性骨髓瘤(MM)细胞中丝裂原活化蛋白激酶(MAPK)信号通路的表达及活化情况,探讨MAPK信号通路对MM细胞B淋巴细胞刺激因子(BLyS)表达变化的影响及对MM细胞增殖与存活的影响,并初步探讨MAPK信号通路在IFN-γ(MM重要的促生长因子)上调MM细胞BLyS表达过程中的作用.方法 应用Western blot方法检测MM细胞中蛋白ERK、p-ERK、JNK、p-JNK、p38及p-p38的表达情况;应用RT-PCR及Western blot检测MAPK信号通路对BLyS表达的影响;应用WST-1法检测靶向JNK的MAPK信号通路抑制剂SP600125对MM细胞增殖与存活的影响.结果 MM细胞株中,除了ERK、JNK及p38的表达外,还有活化蛋白p-JNK的表达;靶向JNK的MAPK信号通路抑制剂SP600125可下调MM细胞BLyS的表达,其激动剂茴香霉素(anisomycin)可上调BLyS的表达;IFN-γ可上调MM细胞BLyS的表达,SP600125可部分抵消IFN-γ对BLyS的上调作用;SP600125可抑制MM细胞的增殖与存活.结论 MM细胞中有JNK/SAPK信号通路的活化;JNK/SAPK信号通路的活化程度与BLyS的表达高低呈正相关;JNK/SAPK信号通路在IFN-γ上调MM细胞BLyS表达过程中发挥重要作用.     

LPS调节U937细胞上B7-H1表达的初步研究

目的了解脂多糖（LPS）刺激后U937细胞上B7-H1的表达变化情况。方法培养U937细胞,用荧光半定量实时PCR和流式细胞仪技术,分别观测未刺激和用LPS刺激的U937细胞B7-H1mRNA与蛋白的表达情况。结果未经刺激的U937细胞组成性表达B7-H1,LPS刺激可显著增强B7-H1基因mRNA的转录和蛋白的表达。结论LPS在转录与翻译两个环节均可上调U937细胞B7-H1的表达水平。     

脂多糖诱导小鼠肠组织ICAM-1表达的变化及p38MAPK在其中的作用

目的:研究脂多糖(LPS)诱导小鼠肠组织细胞间粘附分子-1(ICAM-1)表达的变化及p38丝裂原活化蛋白激酶(p38MAPK)在其中的调控作用。方法:用不同剂量的LPS或LPS加p38MAPK特异性抑制剂SB203580对小鼠进行不同时间的处理后, 分别采用Westernblot和RT-PCR检测ICAM-1蛋白和mRNA表达情况。结果:小鼠肠组织中ICAM-1蛋白和mRNA的表达在LPS剌激后显著高于对照组, LPS剌激后12-36h, ICAM-1表达增加最为显著。LPS剂量在20.0mg/kg时, 对ICAM-1的表达具有最大的剌激作用。SB203580预处理小鼠30min, 可显著抑制LPS诱导的ICAM-1蛋白和mRNA的表达。结论:LPS可诱导小鼠肠组织中ICAM-1蛋白和mRNA的表达增加, 并具有时间和剂量依赖性, p38MAPK信号转导通路可能在内毒素休克小鼠肠组织ICAM-1表达中起重要调节作用, 提示抑制p38MAPK通路可能对内毒素休克时肠损伤的防治有重要的意义。     

Porins from Salmonella enterica serovar Typhimurium activate the transcription factors activating protein 1 and NF-kappaB through the Raf-1-mitogen-activated protein kinase cascade.

In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-kappaB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2'-amino-3'-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-kappaB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-kappaB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-kappaB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-kappaB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS.     

N-Methyl-D-aspartate receptors and p38 mitogen-activated protein kinase are required for cAMP-dependent cyclase response element binding protein and Elk-1 phosphorylation in the striatum

In vivo cyclic adenosine monophosphate (cAMP)-induced N-methyl-D-aspartate receptor and mitogen-activated protein kinase activation was investigated in the dorsal striatum by semiquantitative immunocytochemistry. Intracerebroventricular infusion of 8-bromo-adenosine 3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-cAMPS), increased phosphorylated cAMP-responsive element binding protein, phosphorylated Elk-1 and Fos immunoreactivity in a dose-dependent manner. Intracerebroventricular infusion of the N-methyl-D-aspartate antagonist, MK801, decreased, but tetrodotoxin or the mitogen-activated extracellular-regulated kinase inhibitor, PD98059, did not affect Sp-8-Br-cAMPS-induced phosphorylated c-AMP-responsive element binding protein, phosphorylated Elk-1, phosphorylated extracellular-signal-regulated kinase and Fos immunoreactivity. The p38 mitogen-activated protein kinase inhibitor, SB203580, decreased the Sp-8-Br-cAMPS-induced increase in all markers, except phosphorylated extracellular-signal-regulated kinase, in a dose-dependent manner.We suggest that N-methyl-D-aspartate receptors couple c-AMP to phosphorylation events and immediate early gene induction in the nucleus of striatal medium spiny neurons. These events are mediated by crosstalk between protein kinase A and mitogen-activated protein kinase cascades in vivo.     

The protein kinase C inhibitor,H7, inhibits tumor cell invasion and metastasis in mouse melanoma via suppression of ERK1/2

Protein kinase C (PKC) has been shown to be a signal transducer during tumorigenesis, tumor cell invasion, and metastasis. Recent studies have reported that the PKC inhibitor, 7-hydroxystaurosporine, inhibits tumor cell invasion. However, the molecular mechanisms of this inhibition of invasion and metastasis are not well understood. In the present study, we attempt to clarify the mechanism by which H7, a PKC inhibitor, inhibits tumor cell invasion and metastasis in the melanoma cell line B16BL6. It was found that H7 inhibits B16BL6 cell invasion and metastasis. We also observed that H7 inhibits the mRNA expression and protein activities of matrix metalloproteinase (MMP)-1, -2, -9 and MT1-MMP. Furthermore, H7 suppresses phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). However, other signal transduction factors, such as p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase 1/2 (JNK1/2), were unaffected. Moreover, U0126, a MEK1/2 inhibitor, also inhibited B16BL6 cell invasion and metastasis, as well as the mRNA expression and protein activities of MMP-1, -2, -9 and MT1-MMP. This indicates that H7 inhibits signal transduction through the PKC/MEK/ERK pathway, thereby inhibiting B16BL6 cell invasion and metastasis. These results suggest that PKC inhibitors have potential clinical applications in the treatment of tumor cell metastasis.     

Monocyte chemoattractant protein-1 production by intestinal epithelial cells in vitro: a role for p38 in epithelial chemokine expression.

The intestinal epithelial cell (IEC) represents the first cellular barrier to infection. Consistent with this sentinel role, IEC are known to produce a variety of chemokines in response to bacterial infection or proinflammatory cytokines. These chemokines act as potent leukocyte activators and chemoattractants in vivo. In this report, we begin to characterize the regulation of expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the rat small intestinal IEC-18 line. Following stimulation with either interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS), IEC-18 cells produced MCP-1, with IL-1 proving a more effective stimulus than LPS at both the mRNA and protein levels. Expression of MCP-1 due to either stimulus was inhibited by tyrosine kinase inhibitors, prompting us to investigate potential phosphotyrosine-dependent targets responsible for MCP-1 expression. We detected activation of p38, a member of the mitogen-activated protein kinase family, following either IL-1 or LPS treatment. Specific inhibition of this kinase using the compound SB203580 caused a destabilization of MCP-1 mRNA. These data point to a role for p38 in the regulation of MCP-1 mRNA expression by the IEC.