丹参对大鼠全脑缺血再灌注后海马CA1区AP-1 DNA结合活性的影响

目的　探讨丹参对活化蛋白 1(AP 1)在缺血性脑损伤中的影响及其神经保护作用的机制。方法　采用颈动脉负压分流法制备大鼠全脑缺血再灌注模型 ,用EMSA法和组织化学法观察海马CA1区AP 1的DNA结合活性以及神经元形态改变。结果　 (1)缺血再灌 3h ,丹参能不同程度的增加AP 1的结合活性 ,其中R6 0组较NS组明显增强 (P <0 0 5 ) ,R3 0组也有增强 ,活性由假手术组的 2 8倍增加为 3 0倍 ,但较NS组无显著性差异。同时RSM能不同程度的减轻再灌 72h和 7d的神经元损伤 ,CA1区存活细胞数目较NS组明显增加 (P <0 0 5 )。结论　丹参能明显提高缺血后海马CA1区AP 1的DNA结合活力 ,丹参的神经保护作用可能与其调节AP 1的DNA结合活力有关。     

Modulation of AP-1 by Natural Chemopreventive Compounds in Human Colon HT-29 Cancer Cell Line

PURPOSE: Activator protein-1 (AP-1) has been implicated as playing important roles in apoptosis and cancer development. In this work, we studied several natural chemopreventive compounds for their potential chemopreventive properties in the modulation of AP-1 signaling pathway in HT-29 colon cancer cells. METHODS: The HT-29 cells were transfected with AP-1-luciferase reporter gene, and one of the stable clones (C-4) was used for subsequent experiments. The HT-29 C-4 cells were treated for 1 h with various natural chemopreventive agents and challenged with AP-1 stimulators such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or hydrogen peroxide (H2O2) for 6 h. The c-Jun N-terminal kinase (JNK) was examined to understand the effect of these compounds on the upstream signaling activator of AP-1. The protein expression level of endogenous cyclin D1, a gene that is under the control of AP-1, was also analyzed after treatments with the agents. In addition, cell death induced by these compounds was evaluated by MTS assay [3-(4.5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. RESULTS: TPA and H2O2 treatments strongly induced AP-1-luciferase activity as expected. Phenethyl isothiocyanate, sulforaphane, curcumin, and resveratrol increased AP-1-luciferase activity dose-dependently and then decreased at higher doses in the presence or absence of TPA. Allyl isothiocyanate and (-)-epigallocatechin-3-gallate (EGCG) increased AP-1-luciferase activity dose-dependently up to 50 and 100 microM. Other tea catechins and procyanidin dimers, however, had little or no effect on AP-1-luciferase activity. The JNK activity was induced by the isothiocyanates and EGCG. Most of the chemopreventive compounds induced cell death in a dose-dependent manner, with the exception of epicatechin (EC) and the procyanidins, which had little effect. The expression of endogenous cyclin D1 protein was well correlated with those of AP-1-luciferase assay. CONCLUSION: Taken together, these results suggest that natural chemopreventive compounds may have differential biological functions on the signal transduction pathways such as AP-1 in the intervention of colon cancer progression and carcinogenesis.     

Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway

Aim:

To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).

Methods:

Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.

Results:

Thrombin (0.3–3.0 U/mL) caused a concentration- and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH₂ (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH₂ (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH₂ (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3–30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3–3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1–10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.

Conclusion:

Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.     

AP-1在胰腺癌组织中的表达

目的 探讨AP-1在胰腺癌组织中的表达情况及意义.方法 选取35例胰腺癌患者术后肿瘤组织石蜡包埋标本(观察组)及35例健康人胰腺组织(对照组),采用双抗体夹心ABC-ELISA法进行AP-1检测,观察两组标本C-JUN和C-FOS表达情况并进行比较.结果 C-JUN和C-FOS表达阳性率观察组分别为80.00％、71.43％,对照组分别为22.86％、28.57％,两组差异均有统计学意义(x2=22.88、12.86,均P＜0.05).结论 AP-1在胰腺癌组织中高度表达,可指导临床采取合理的干预措施减缓肿瘤发展.     

红霉素对吸烟大鼠肺组织激活蛋白-1铜锌超氧化物歧化酶表达的影响

目的通过研究红霉素对吸烟大鼠肺组织激活蛋白-1(AP-1)、铜锌超氧化物歧化酶(CuZn-SOD)表达的影响,探讨其在慢性阻塞性肺疾病(COPD)抗氧化治疗中的作用及其机制。方法Wistar大鼠30只,随机分为对照组、吸烟组和红霉素组。分别采用免疫组织化学法、免疫细胞化学法和原位杂交半定量技术检测支气管上皮细胞及肺泡巨噬细胞中AP-1(c-Jun和c-Fos亚单位)和CuZn-SOD蛋白及其mRNA的表达水平。结果吸烟组大鼠支气管上皮细胞和肺泡巨噬细胞c-Jun、c-Fos、CuZn-SOD蛋白及其mRNA表达均高于对照组(P均<0.05);红霉素组c-Jun、c-Fos表达水平明显低于吸烟组(P均<0.01),而CuZn-SOD表达水平明显高于吸烟组(P均<0.01)。结论红霉素可能通过影响AP-1、CuZn-SOD的表达而在COPD治疗中发挥一定抗氧化作用。     

8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages

8-Prenylkaempferol is a prenylflavonoid isolated from the roots of Sophora flavescens, a Chinese herb with anti-inflammatory properties. However whether 8-prenylkaempferol itself displayed an anti-inflammatory activity remained unclear. In this study, we evaluated the effect of 8-prenylkaempferol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. 8-Prenylkaempferol inhibited significantly LPS-induced NO production through suppressing inducible NO synthase (iNOS) expression at both protein and mRNA levels but failed to affect sodium nitroprusside-triggered NO production, iNOS enzyme activity, and cell viability. Further investigation of the mechanisms revealed that 8-prenylkaempferol inhibited LPS-induced c-Jun phosphorylation (a major component of activator protein-1, AP-1), but did not attenuate IkB-alpha degradation nor NF-kappaB nuclear translocation. Cellular signaling analysis using mitogen-activating protein kinase (MAPK) inhibitors including 2'-amino-3'-methoxyflavone (PD98059, MEK1/2 inhibitor), 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580, p38 kinase inhibitor) and anthra[1-9-cd]pyrazol-6(2H)-one (SP600125, c-Jun N-terminal kinase inhibitor) demonstrated that extracellular signal-regulated kinase1/2 (ERK1/2), p38 and JNK all participated in LPS-stimulated iNOS expression and NO production, but 8-prenylkaempferol interfered selectively with JNK phosphorylation. On the other hand, LPS-induced c-Jun phosphorylation was attenuated in the presence of SP600125. We suggested that interfering with JNK-mediated c-Jun phosphorylation and thus blocking AP-1 activation might contribute to the suppression effects of 8-prenylkaempferol on iNOS. These findings provided the first molecular basis that 8-prenylkaempferol is an effective agent for attenuating pro-inflammatory NO induction.     

Suppression of phorbol ester-induced NF-kappaB activation by capsaicin in cultured human promyelocytic leukemia cells

Capsaicin, a major pungent constituent of red pepper (Capsicum annuum L.) possesses a vast variety of pharmacologic and physiologic activities. Despite its irritant properties, the compound exerts anti-inflammatory and anti-nociceptive effects. Previous studies from this laboratory revealed that capsaicin, when topically applied onto dorsal skin of female ICR mice, strongly attenuated activation of NF-kappaB and AP-1 induced by the typical tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), which may account for its anti-tumor promoting activity in mouse skin. In the present work, we have found that capsaicin suppresses TPA-stimulated activation of NF-kappaB through inhibition of IkappaB alpha degradation and blockade of subsequent nuclear translocation of p65 in human promyelocytic leukemia HL-60 cells. Methylation of the phenolic hydroxyl group of capsaicin abolished its inhibitory effect on NF-kappaB DNA binding. Likewise, TPA-induced activation of AP-1 was mitigated by capsaicin treatment.     

DADS诱导人胃癌细胞分化作用中ERK/AP-1通路的改变

目的　探讨ERK/AP 1通路在二烯丙基二硫 (DADS)诱导人胃癌细胞分化中的作用 ,阐明DADS诱导人胃癌细胞分化的分子机制。方法　采用免疫细胞化学、图像分析及WesternBlot等方法 ,观察DADS作用于体外培养的人胃癌MGC80 3细胞前后AP 1成员c fos与c jun表达的改变以及ERKMAPK激酶的变化。结果　免疫细胞化学及图像分析结果显示 ,对照组c fos、c jun表达呈强阳性 ,而处理组呈阴性或弱阳性 ,阳性率明显降低 ,处理组光密度值较对照组明显低 (P <0 0 5 )。WesternBlot结果显示 :从 2 0、2 5、30到 35mg·L-1DADS处理癌细胞 ,DADS呈浓度依赖性抑制人胃癌细胞中ERK的活化 (P <0 0 5 ) ;并且DADS +MEK抑制剂PD980 5 9能完全阻断ERK活化 (P <0 0 5 ) ;时间效应上 ,在DADS刺激后 15～ 30min抑制作用最强 ,2h左右恢复至接近正常 (P <0 0 5 )。结论　ERK/AP 1通路抑制是DADS诱导人胃癌细胞分化的重要分子机制     

Possible Involvements of Nuclear Factor-κB and Activator Protein-1 in the Tumor Necrosis Factor-α–Induced Upregulation of Matrix Metalloproteinase-12 in Human Alveolar Epithelial A549 Cell Line

Matrix metalloproteinase-12 (MMP-12) has been suggested to play an important role in airway inflammatory diseases. Tumor necrosis factor-α (TNF-α ) is known to cause an upregulation of MMP-12 via an activation of activator protein-1 (AP-1) in monocytes. In the present study, we investigated the effect of TNF-α on the expressions of MMP-12 in airway epithelial cells, one of the sources of MMP-12 in the airway, and its underlying mechanism. MMP-12 mRNA and protein expressions induced by TNF-α in the absence or presence of BMS-345541 (a selective IκB kinase inhibitor) or SP600125 [a selective c-Jun N-terminal kinase (JNK) inhibitor] were measured by quantitative real-time PCR and Western blotting, respectively. Furthermore, siRNAs for p65 and JNK2 were used to confirm the involvements of nuclear factor-κB (NF-κB) and AP-1 in the MMP-12 mRNA expression induced by TNF-α in A549 cells. Both MMP-12 mRNA and protein were upregulated by the treatment with TNF-α in time- and concentration-dependent manners. Both BMS-345541 and SP600125 inhibited the upregulation of MMP-12 induced by TNF-α. Furthermore, both the depletion of p65 and JNK2 by siRNAs significantly attenuated the upregulation of MMP-12 induced by TNF-α. These findings suggest that both NF-κB and JNK / AP-1 pathways are important for the MMP-12 upregulation induced by TNF-α in A549 cells.     

TOLL样受体 9/激活蛋白-1信号通路在过敏性鼻炎中的作用

目的 探讨TOLL样受体9/激活蛋白-1(TLR9/AP-1)信号通路在过敏性鼻炎中的作用机制.方法 48只SD大鼠采用随机数字表法分为对照组、实验组、干预组,每组16只.实验组、干预组制备过敏性鼻炎大鼠的动物模型.干预组造模成功后给予治疗药物干预.将各组16只大鼠采用随机数字表法分为两组,每组8只,分别于末次干预后6、12 h处死后采集血液标本和鼻黏膜标本.检测各组大鼠干预后免疫球蛋白E(IgE)、白细胞介素-4(IL-4)、肿瘤坏死因子-α(TNF-α)、症状评分、鼻黏膜组织中C-Fos、C-Jun表达.结果 实验组大鼠IgE[(18.65±1.09)IU/mL比(11.39±1.56)IU/mL]、IL-4[(75.33±7.16)ng/L比(56.55±6.08)ng/L]、TNF-α[(57.25±5.15)ng/L比(32.33±2.26)ng/L]、症状评分、C-Fos[(0.850±0.012)比(0.425±0.050)]、C-Jun[(0.950±0.052)比(0.425±0.030)]均明显高于对照组,差异有统计学意义(P<0.01).干预组大鼠IgE[(15.95±1.02)IU/mL]、IL-4[(65.12±5.47)ng/L]、TNF-α[(48.53±4.18)ng/L]、症状评分、C-Fos[(0.575±0.047)]、C-Jun[(0.615±0.047)]均明显低于实验组,差异有统计学意义(P<0.01).结论 药物治疗过敏性鼻炎的机制与抑制TLR9/AP-1信号通路及其下游因子的释放和分泌有关.     

c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.     

作用于AP-1信号转导通路的抗肿瘤天然产物

激活蛋白-1(activator protein-1,AP-1)是由DNA结合蛋白组成的一类重要的转录调控因子,参与细胞存活、增殖和分化等重要的生物学过程。AP-1的过度激活使下游与肿瘤浸润和转移相关的蛋白表达增加,在恶性肿瘤浸润和转移中发挥关键作用。因此,靶向AP-1并抑制其活性是近年来发展的一种极具吸引力的恶性肿瘤防治策略。本文综述了当前对AP-1结构和功能的认识,并探讨了最近几年报道的几种天然活性化合物通过调节AP-1相关信号通路、发挥抗肿瘤效果的作用机制。     

凝血酶通过AP-1信号途径刺激人肾小球系膜细胞表达MMP-9 mRNA(英文)

目的:利用体外培养人肾小球系膜细胞观察凝血酶对系膜细胞表达MMP-9 mRNA的诱导作用及其可能的细胞内信号传导机制。方法:体外分离并培养人肾小球系膜细胞,以不同浓度凝血酶(500,1500和4500u/L)刺激后采用Northern杂交及凝胶阻滞试验(EMSA)分别观察MMP-9 mRNA表达水平及转录因子AP-1活性变化;同时观察了凝血酶特异性抑活物(水蛭素)、AP-1药理阻断剂(curcumin)和c-fos反义寡核苷酸对凝血酶上述作用的影响。结果:Northern分析表明凝血酶可以剂量依赖性地提高人系膜细胞表达MMP-9 mRNA,与对照组相比,分别提高1.1,3.3和4.8倍;EMSA实验结果发现凝血酶在促进MMP-9 mRNA表达的同时,系膜细胞AP-1的结合能力也相应地提高,与对照组相比分别增加2.5,4.9和6.1倍,与MMP-9 mRNA呈平行性变化;凝血酶抑活物水蛭素可明显地抑制凝血酶上调MMP-9mRNA及AP-1的DNA结合活性;同时AP-1药理阻断剂curcumin和c-fos反义寡核苷酸在有效抑制AP-1结合活性的同时,也明显地抑制MMP-9 mRNA表达水平。结论:凝血酶在体外培养的人肾小球系膜细胞具有上调MMP-9 mRNA表达的作用,转录因子AP-1介导了凝血酶的这一作用。     

Cyclic AMP inhibits stretch-induced overexpression of fibronectin in glomerular mesangial cells

Glomerular hypertension is proposed to play an important role in the progression of various glomerular diseases. Glomerular mesangial cells are considered to be exposed to the stretch stress due to glomerular hypertension and are found to produce the excess amount of extracellular matrix (ECM) proteins including fibronectin when exposed to the mechanical stretch. Herein, we provide the evidence that cAMP-generating agents inhibit the stretch-induced overexpression of fibronectin through the inhibition of the stretch-induced activation of mitogen-activated protein kinases (MAPKs) in protein kinase-A-dependent manner. We also found that the mechanical stretch enhanced the binding of nuclear extracts to activator protein-1 (AP-1)-like sequences in the promoter region of rat fibronectin gene and this enhancement was also prevented by the cAMP-generating agent. These results indicate that the agents, which activate cAMP/protein kinase-A axis, may work protectively against the injury from glomerular hypertension in mesangial cells.