N-甲基-N''''-硝基-N-亚硝基胍诱发的vero细胞JNK/SAPK通路的激活

目的和方法 :为了研究DNA损伤剂N -甲基 -N -硝基 -N -亚硝基胍 (MNNG)诱发vero细胞非定标性突变发生的信号转导机制 ,我们观察了MNNG诱发vero细胞c -JunNH2 -terminalkinase/stressactivatedproteinkinase(JNK/SAPK)通路激活的情况 :分别用Western印迹法和固相激酶活性测定法检测JNK1磷酸化和JNKs激酶活性。结果 :发现用 0 2 μmol/LMNNG和 1mg/L放线菌素酮 (CHM )分别处理vero细胞 2 5h和 1h后 ,都引起细胞抽提液中磷酸化JNK1的比例明显增高。同时通过测定JNKs的底物c -Jun的磷酸化程度 ,发现这两种处理也都可引起JNKs激酶活性显著增高 (分别增高 6 7和 3 0倍 )。结论 :证明 0 2 μmol/LMNNG和 1mg/LCHM分别处理vero细胞 2 5h和 1h都可引起vero细胞内JNK/SAPK通路被激活。     

MNNG对哺乳类细胞JNK/SAPK及p38MAPK作用及其信号源研究

目的:研究低浓度烷化剂N-甲基-N' -硝基-N-亚硝基胍(MNNG)对JNK/SAPK及p38 MAPK通路的作用及其信号源。方法: 分别测定完整Vero细胞和脱核Vero细胞的JNK/SAPK及p38 MAPK酶活性,并比较其结果。 结果:低浓度MNNG在完整Vero细胞和脱核Vero细胞中均抑制JNK/SAPK酶活性;在p38 MAPK通路中,完整Vero细胞表现酶活性升高,而脱核Vero细胞该激活作用消失。 结论: 低浓度MNNG抑制JNK/SAPK的作用不依赖于核内信号,而对p38 MAPK的激活作用依赖与于核内信号。     

Anti-IgM诱导人B淋巴瘤Daudi细胞凋亡的信号传导通路

目的 研究B细胞抗原受体（BCR）介导的细胞凋亡,探索其可能的信号传导通路。阐明B细胞凋亡的分子机制。方法 ^3H掺入法检测细胞生长,应用FACS方法分析细胞周期和细胞凋亡,Western blot检测anti-IgM刺激引起Daudi细胞信号分子的变化。结果 anti-IgM可导致人B淋巴瘤Daudi细胞发生凋亡。细胞凋亡的数量与anti-IgM浓度呈正相关,Western blot结果显示,anti-IgM刺激引起Daudi细胞内氨酸蛋白分子磷酸化水平的变化速度与浓度相关;但随时间的延长,变化状况趋于稳定;在48-72h,许多被激活的,呈现磷酸化的栈氨酸蛋白分子又回复到低磷酸化或去磷酸化的静止状态,另外,虽然JNK1和ERK2蛋白水平未发生明显改变,但JNK/SAPK的重要下游分子之一,c-Jun的蛋白表达水平及其63和73位点的丝氨酸磷酸化水平立即长高并维持在高水平状态。结论 anti-IgM刺激激活JNK/SAPK,JNK/SAPK通路可能参与了anti-IgM引起的Daudi细胞的凋亡过程。     

应激活化的蛋白激酶在神经酰胺介导细胞凋亡中的作用

第二信使神经酰胺激活SAPK/JNK,活化的SAPK/JNK导致AP-1转录因子磷酸化,AP-1刺激自身表达,增强自身活性,介导细胞凋亡。神经酰胺及其代谢产物鞘氨醇和鞘氨醇-1磷酸酯分别激活JNK-p38路径和ERK路径,调节细胞增殖、分化或凋亡。     

氧化应激在乙醛引起的心肌细胞凋亡中的作用

目的： 探讨活性氧（ROS）的氧化损伤机制在经乙醛诱导心肌细胞凋亡过程中的作用，阐明氧化应激水平增高导致细胞凋亡可能是乙醇性心肌病（AHMD）的主要原因之一。 方法: 体外培养大鼠心肌细胞，分别用乙醇（100 μmol/L）和乙醛（100 μmol/L）干预24 h，比较细胞凋亡程度；检测48 h内细胞胞内ROS水平变化、胞外分泌SOD活性变化；并用Western blotting技术检测ROS介导MAPK信号途径相关蛋白磷酸化水平变化；并与氧化剂H2O2处理组和预先加入抗氧化剂乙酰半胱氨酸（NAC）的乙醛处理组比较。 结果: 乙醇和乙醛分别干预心肌细胞24 h后，凋亡途径激活因子caspase 3均被激活(P<0.05)，乙醛较乙醇具有更强的诱导凋亡作用（P<0.05）。乙醛诱导心肌细胞ROS水平增高呈时间依赖性，相应抗氧自由基SOD酶活性也随之增高，分别于18-24 h达到峰值。同时，ROS通过JNK和ERK磷酸化水平增高而激活MAPK途径，诱导心肌细胞凋亡，这种效应可以被NAC逆转，等浓度乙醛弱于H2O2的诱导作用。结论: 乙醛通过增高胞内ROS水平损伤心肌细胞，激活ROS介导JNK途径诱导细胞凋亡。其作用弱于H2O2等强氧化剂，提示降低细胞ROS 水平，阻断凋亡信号通路可有效抑制乙醛诱导的心肌凋亡，对于AHMD临床预防和治疗具有指导意义。     

氧化应激性肠上皮细胞损伤与MAPK信号通路的关系研究

目的观察氧化应激引起肠上皮细胞（IEC-6）损伤后丝裂原活化蛋白激酶（MAPK）信号通路活化的情况,从而探讨氧化应激损伤的机制。方法采用MTT法检测不同浓度（100、200、300、400、500、800、1000、2000μmol/L）H2O2对IEC-6生存率的影响;用WesternBlot法检测H2O2作用不同时间（0.25、0.5、1、2、3、4h）下ERK、JNK以及p38磷酸化的激活情况。结果与正常组相比,随着H2O2刺激浓度的增加,IEC-6的生存率逐渐降低,呈浓度依赖性。当H2O2刺激浓度为200μmol/L时细胞的生存率约为50%。ERK1/2、JNK1/2、p38在H2O2刺激0.25h开始磷酸化,在刺激0.5h达到最高,随后磷酸化水平恢复到基础值。结论氧化应激早期可通过激活ERK、JNK以及p38的磷酸化引起IEC-6的损伤。     

异氟醚和七氟醚对新生大鼠皮质凋亡以及JNK和p38表达的不同影响

目的: 研究同等剂量的异氟醚和七氟醚对新生大鼠皮质神经元凋亡的影响以及对JNK和p38蛋白表达的不同影响。方法: 55只出生后7 d(P7)的新生大鼠(共5窝,每窝取11只),随机均分为异氟醚组(Ⅰ组)、七氟醚组(S组)和对照组(C组),各组分别吸入1.1%异氟醚、1.8%七氟醚和空气4 h。在麻醉结束后2 h每窝每组各取1只幼鼠灌注取脑,免疫组织化学法检测皮质压部后区caspase-3表达(n=5);另外,每窝C组在麻醉处理0 h,Ⅰ组和S组分别在麻醉2 h、4 h取新鲜脑皮质,Western blotting检测磷酸化SAPK/JNK、磷酸化p38,以及SAPK/JNK、p38表达的变化(n=5)。结果: Ⅰ组和S组caspase-3表达分别较对照组增加441%(P<0.01)和151%(P<0.01),Ⅰ组比S组增加115%(P<0.05);Ⅰ组磷酸化SAPK/JNK表达在麻醉2 h和4 h较对照组分别增加219%(P<0.05)和181%(P<0.05),S组在2 h和4 h均与对照组无显著差异;Ⅰ组磷酸化p38表达在麻醉2 h和4 h较对照组分别增加38.9%(P<0.05)和36.9%(P<0.05),S组在2 h和4 h较对照组分别增加32.6%(P<0.05)和128.0%(P<0.01)。结论: 0.5最低肺泡有效浓度(MAC)异氟醚比七氟醚诱导更多新生大鼠大脑皮质神经元凋亡,异氟醚诱导凋亡可能与激活SAPK/JNK磷酸化有关,而七氟醚诱导凋亡可能与激活p38磷酸化有关。     

13-甲基十四烷酸诱导膀胱癌细胞株T24 凋亡的机制研究

目的： 研究13-甲基十四烷酸诱导膀胱癌T24细胞凋亡的作用,探讨其可能的机制。方法：将不同浓度的13-甲基十四烷酸作用于膀胱癌T24细胞,用MTT法检测细胞增殖, 流式细胞仪（FCM）检测细胞周期,TUNEL法检测细胞凋亡指数,蛋白免疫印迹法分析参与凋亡的蛋白。结果：药物处理2 h后p38、JNK磷酸化增强,AKT磷酸化减弱,8 h后线粒体中细胞色素C释放,FADD及磷酸化FADD没有明显变化,caspase酶底物 PARP、lamin B、RB在12 h出现明显的裂解片段,且药物诱导T24细胞凋亡的过程有时间浓度依赖效应。结论：在13-MTD诱导T24细胞凋亡的过程中p38MAPK和JNK/SAPK信号通路被激活,PI3K/AKT信号通路被抑制,进而激活线粒体凋亡途径,使线粒体内的细胞色素C释放到胞浆,激活caspase酶,裂解相应的凋亡底物lamin B、Rb、PARP,促进T24细胞凋亡。     

JNK/SAPK信号传递途径与细胞应激反应

c-JunNH_2-末端激酶(JNK)又称为应激活化蛋白激酶(SAPK),是有丝分裂原活化蛋白激酶(MAPK)家族成员之一。大量研究证实,JNK/SAPK信号传递途径在细胞应激反应中起重要作用。JNK/SAPK信号传递途径的激活促进细胞凋亡发生,其机制与诱导FasL表达、调控凋亡相关基因差异表达和改变细胞内Ca~(2 )环境与激活caspases家族有关;在某些情况下,JNK/SAPK信号传递途径的激活并不导致细胞凋亡,相反还可能与细胞增殖有关。JNK/SAPK信号传递途径在细胞应激反应中的作用表现出复杂性和多样性。     

细胞凋亡相关的信号通路

目前研究较多的细胞凋亡信号通路有wnt及JNK两条。Wnt信号通路有经典wnt通路,wnt/PCP通路和wnt/钙离子通路三个分支,引起细胞凋亡的机制主要有β-连环蛋白/tcf的转录调节途径,APC激活途径两种。JNK信号通路通过MKKKs-MKKs-MAPK转导。激活的JNK信号通路通过磷酸化转录因子、细胞骨架相关蛋白、酶等多种底物来调节细胞的生理过程,最终导致细胞凋亡。     

Inhibition of caspase 3 abrogates lipopolysaccharide-induced nitric oxide production by preventing activation of NF-kappaB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-kappaB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-kappaB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-kappaB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.     

Insulin-like growth factor-1 activates Akt and Jun N-terminal kinases (JNKs) in promoting the survival of T lymphocytes

Insulin-like growth factor 1 receptor (IGF-1R) expression is augmented on T cells upon ligation of CD28, and this promotes IGF-1-mediated protection from Fas-induced cell death for up to 6 days. To determine the mechanism of action of IGF-1R in T-cell expansion, we investigated the signalling pathways activated by IGF-1 in T cells and in Jurkat cells. We found that IGF-1 transiently induces Akt, jun N-terminal kinases (JNK), and c-Jun phosphorylation in activated T cells, with JNK and c-Jun phosphorylation occurring faster than Akt phosphorylation. To mimic IGF-1R expression levels in CD28-stimulated Jurkat cells these cells were stably transfected to over-express the IGF-1R. Jurkat/IGF-1R cells exhibited enhanced constitutive Akt phosphorylation compared with mock-transfected controls, but IGF-1 induced transient phosphorylation of MKK4, JNKs, and c-Jun. Inhibition of PI-3 kinase activity and Akt phosphorylation with LY294002 totally suppressed IGF-1-mediated protection from Fas killing in activated T cells, but only partially suppressed IGF-1-mediated protection in Jurkat/IGF-1R cells. However, either dicumarol in T cells or a dominant negative JNK1 (APF) in Jurkat/IGF-1R cells greatly suppressed IGF-1-mediated protection from Fas killing. Together, these data demonstrate that IGF-1-mediated activation of JNKs and PI-3 kinase contributes to normal T-cell survival, whereas the JNK pathway may be more important in Jurkat leukaemia cells.     

The involvement of a stress-activated pathway in equine influenza virus-mediated apoptosis

We have shown elsewhere that equine-2 influenza virus (EIV; subtype H3N8) induced pronounced cell death in infected cells through apoptosis as demonstrated by DNA fragmentation assay and a combined TUNEL and immunostaining scheme. In this study, we investigated the mechanism of EIV-mediated cytotoxicity on a permissive mammalian epithelial cell line, Madin-Darby canine kidney (MDCK) cells. EIV infection increased the cellular levels of oxidative stress and c-Jun/AP-1 protein (which is known to be affected by oxidative stress), as well as its DNA binding activity. Increased production of TGF-beta1, an inducer of c-Jun N-terminal kinase or stress-activated protein kinase (JNK/SAPK) activation, was also detected in EIV-infected MDCK cells. It has been reported that TGF-beta may initiate a signaling cascade leading to JNK/SAPK activation. Addition of c-Jun antisense oligodeoxynucleotide, antioxidant N-acetyl-cysteine (NAC), JNK/SAPK inhibitor carvedilol, or TGF-beta-neutralizing antibody effectively blocked c-Jun/AP-1 upregulation and TGF-beta1 production mediated by EIV infection. These treatments also attenuated EIV-induced cytopathogenic effects (CPE) and apoptosis. Our results suggest that a stress-activated pathway is involved in apoptosis mediated by EIV infection. It is likely that EIV infection turns on the JNK/SAPK cascade, which modulates the activity of apoptosis-promoting regulatory factor c-Jun/AP-1 and epithelial growth inhibitory cytokine TGF-beta.     

丝裂原激活蛋白激酶在内毒素脂多糖诱导大鼠施万细胞一氧化氮合酶表达中的作用

目的 主要研究丝裂原激活蛋白激酶(MAPK)信号途径在内毒素脂多糖(LPS)诱导大鼠施万细胞(Scs)诱导型一氧化氮合酶(iNOS)基因表达和一氧化氮(NO)产生中的作用.方法 先用3种MAPK的特异性抑制剂PD98059(ERK1/2)、SB202190(P38 MAPK)和SP600125(JNK)以不同浓度预处理细胞1h,再用LPS作用施万细胞4 h后,用RT-PCR检测细胞中iNOS mRNA、IL-6 mRNA和TNF-α mRNA的表达;Western blotting观察iNOS蛋白水平的表达变化;通过测定细胞培养液中亚硝酸盐含量来观察NO的水平.结果 LPS可显著激活施万细胞中MAPK信号通路诱导iNOS表达.MAPK的抑制剂预处理细胞后,可显著抑制细胞iNOS mRNA和NO的合成及IL-6 mRNA和TNF-α mRNA的表达.结论 MAPK信号通路参与了LPS介导的大鼠施万细胞iNOS基因表达和NO产生,通过阻断细胞内信号转导通路来减少iNOS及其他细胞因子的产生,为抑制周围神经损伤后的炎症以及免疫反应发生提供了一条新思路.     

Requirement of the JIP1 scaffold protein for stress-induced JNK activation

The c-Jun N-terminal kinase (JNK) signal transduction pathway is activated in response to the exposure of cells to environmental stress. Components of the JNK signaling pathway interact with the JIP1 scaffold protein. JIP1 is located in the neurites of primary hippocampal neurons. However, in response to stress, JIP1 accumulates in the soma together with activated JNK and phosphorylated c-Jun. Disruption of the Jip1 gene in mice by homologous recombination prevented JNK activation caused by exposure to excitotoxic stress and anoxic stress in vivo and in vitro. These data show that the JIP1 scaffold protein is a critical component of a MAP-kinase signal transduction pathway.     

藤黄酸增强人胃癌SGC7901/DDP细胞对顺铂的敏感性

目的 探讨藤黄酸（GA）对人胃癌SGC7901/DDP细胞顺铂敏感性的影响及其分子机制。方法 采用顺铂（DDP）浓度梯度递增法构建人胃癌顺铂耐药株SGC7901/DDP细胞,采用细胞计数盒-8（CCK-8）法检测藤黄酸和顺铂对SGC7901/DDP细胞的毒性作用,采用Chou-Talalay中效分析法定量评价藤黄酸和顺铂的联合作用效果,采用流式细胞术检测细胞凋亡,采用Western blotting方法检测Bcl-2、Bax、Survivin、多药耐药相关蛋白2（MRP2）、磷酸化氨基末端蛋白激酶（p-JNK）（Thr183/Tyr185）和JNK的蛋白水平。结果 藤黄酸与顺铂各自单独作用48 h的IC₅₀分别为2.94 μmmol/L和39.76 μmmol/L;当抑制率超过20%时,两者联合应用呈协同效应;藤黄酸可协同增强顺铂诱导的细胞凋亡（P<0.05）,下调Survivin和MRP2蛋白水平（P<0.05）,上调Bax蛋白水平（P<0.05）,抑制JNK磷酸化（P<0.05）;JNK特异性抑制剂SP600125可下调MRP2蛋白水平（P<0.05）。结论 藤黄酸可增强人胃癌SGC7901/DDP细胞对顺铂的敏感性,这可能与藤黄酸通过抑制JNK信号通路下调MRP2蛋白表达,以及上调Bax蛋白表达和下调Survivin蛋白表达有关。