聚合度7-15的壳寡糖抑制脂多糖刺激的单核细胞产生TNF-α和IL-8的作用研究

本文通过建立脂多糖刺激的单核细胞炎症损伤模型,观察聚合度7-15的壳寡糖对炎性单核细胞白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)表达的影响,及对p38丝裂原激活的蛋白激酶(p38 mitogen-activated protein kinases,p38MAPK)信号通路磷酸化的影响。采用p38信号通路抑制剂(SB203580)验证抑制p38信号通路对脂多糖诱导的单核细胞表达IL-8和TNF-α的作用,从而探索壳寡糖抑制单核细胞炎性损伤的分子机制。结果表明壳寡糖可抑制脂多糖诱导的单核细胞表达IL-8和TNF-α,并且抑制p38信号蛋白的磷酸化水平。因此,初步认为壳寡糖可能通过抑制炎性U937细胞中p38MAPK信号通路抑制IL-8和TNF-α的表达。     

齐墩果酸抑制肿瘤坏死因子-α诱导的成纤维细胞样滑膜细胞炎症因子表达及其机制研究

探讨齐墩果酸(Oleanolic acid,OA)对肿瘤坏死因子-α(TNF-α)诱导成纤维细胞样滑膜细胞的炎症因子表达的影响及其机制。首先复苏培养人成纤维细胞样滑膜细胞(FLS),通过RT-PCR检测细胞IL-6及IL-1βmRNA表达,采用Western blot方法检测p38MAPK及NF-κB蛋白表达变化,通过ELISA法检测细胞上清液中IL-6及IL-1β浓度。与对照组比较,TNF-α明显诱导FLS细胞IL-6及IL-1βmRNA的表达及上清液中IL-6及IL-1β的分泌(P0.05),同时磷酸化p38蛋白和核NF-κB明显增加(P0.05),且p38MAPK阻断剂SB203580能抑制TNF-α诱导的核NF-κB增加。OA呈浓度依赖性抑制TNF-α诱导的FLS细胞p38蛋白磷酸化和核NF-κB增加(P0.05)。且OA、p38MAPK通路抑制剂SB203580或NF-κB阻断剂BAY 11-7082均能抑制TNF-α诱导的IL-6及IL-1β分泌增加(P0.05)。综上所述,OA能抑制TNF-α诱导的FLS细胞炎症因子IL-6及IL-1β的产生,其机制可能与抑制p38MAPK/NF-κB信号通路有关。     

A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响

为了观察A-Raf的突变体A-Raf-S214C对MAPK(mitogen-activated protein kinase,丝裂原活化蛋白激酶)信号通路的影响,并探讨其诱发肺癌和朗格汉斯增生症的可能分子机制。通过定点突变构建pc DNA5-FRT-TO-A-Raf-S214C突变质粒,并利用该质粒构建稳定表达A-Raf-S214C突变体的HEK293细胞系,之后通过检测下游ERK磷酸化程度,确定该突变体对MAPK信号通路的影响。结果显示,通过实验成功构建了稳定表达A-Raf-S214C突变体的细胞系,在不同浓度的多西环素诱导下,其表达量有所不同。与野生型相比,A-Raf-S214C突变体可明显增强MAPK信号通路中下游信号分子ERK的磷酸化。结果表明A-Raf-S214C突变体是A-Raf激酶的激活突变体,可以导致MAPK信号通路下游信号分子ERK磷酸化明显增强。     

木犀草素通过抑制p65 NF-κB、促进p85 PI3K调节微血管内皮细胞VCAM-1表达

通过抑制微血管内皮细胞血管细胞黏附分子(VCAM)-1的表达,木犀草素可阻遏中性粒细胞与微血管内皮细胞的黏附,起到抗炎作用。木犀草素调节VCAM-1表达与三条信号通路有关：丝裂原活化蛋白激酶(MAPK)、核因子kappa B (NF-κB)/IκB和磷脂酰肌醇3激酶(PI3K)/Akt通路。其中,MAPK和NF-κB/IκB通路参与VCAM-1正向调节,PI3K/Akt通路参与VCAM-1负向调节。本文研究了木犀草素对微血管内皮细胞该三条通路中的关键蛋白p38 MAPK、p65 NF-κB、p85 PI3K磷酸化。结果表明：木犀草素在反应的30s和1min促进p38 MAPK磷酸化,在30 s、1 min和5 min促进p85 PI3K磷酸化,而在30 s、1 min、5 min和30 min抑制p65 NF-κB磷酸化。阻抑p38 MAPK通路导致VCAM-1表达下调,而p38 MAPK抑制剂SB203580可通过抑制p38 MAPK磷酸化也下调VCAM-1,提示木犀草素对微血管内皮细胞VCAM-1的调节作用独立于p38 MAPK磷酸化。由此可知,木犀草素通过抑制p65 NF-κB磷酸化或促进p85 PI3K磷酸化调节微血管内皮细胞VCAM-1表达。本文为木犀草素抗炎作用的分子机制研究提供了新的线索。     

白细胞介素23在呼吸道合胞病毒感染致Th1、Th2及Th17细胞分化中的作用及机制

本研究旨在探讨白细胞介素23(interleukin 23,IL-23)在呼吸道合胞病毒(respiratory syncytial virus,RSV)感染支气管上皮细胞BEAS-2B后对Th1、Th2和Th17细胞分化的影响及作用机制。将RSV感染BEAS-2B后的上清液与淋巴细胞共孵育,并分别阻断IL-23受体(IL-23 receptor,IL-23R)、IL-23p19亚基及p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)信号通路。应用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测上清液中细胞因子γ干扰素(interferon γ,IFN-γ)、IL-4、IL-17的浓度。同时,应用实时聚合酶链反应(polymerase chain reaction,PCR)检测相关转录因子(t-bet、gata3、rorγt)和信号转导子(stat4、stat6、stat3)的表达。结果显示,RSV感染后IFN-γ、IL-4和IL-17蛋白表达上调,转录因子及信号转导子的表达也有所增加。阻断IL-23和p38 MAPK信号通路后,Th1、Th2和Th7细胞分泌的细胞因子及转录因子表达均明显下降。结果提示,阻断IL-23后可在基因转导层面抑制RSV感染上皮细胞后诱导的Th1、Th2和Th17细胞分化,此过程可能与p38 MAPK信号通路有关。     

黄芪对白介素1β诱导的大鼠系膜细胞增殖和细胞外基质产生的影响

目的:观察黄芪(Astragalus Membranaceus,AM)注射剂对5/6肾切除大鼠模型的疗效,及体外对系膜细胞(Mesangial cells,MCs)增殖和细胞外基质(Intracellular Matrix,ECM)产生的影响。方法:建立5/6肾切除大鼠模型后,将大鼠分为四组:对照组、未治疗组、低剂量/高剂量AM治疗组。观察比较16周后各组大鼠肾功能和尿蛋白排泄量情况。体外以白介素1β(IL-1β)作为刺激物,诱导MCs增殖,给予不同剂量AM干预。二苯基四氮唑溴盐(MTT)法检测MCs增殖情况,流式细胞仪检测细胞周期,酶联免疫吸附(ELISA)法检测细胞上清中纤连蛋白(fibronectin)含量,RT-PCR检测细胞p38 mRNA表达,Western免疫印迹法检测胶原Ⅳ、总p38MAPK、磷酸化p38MAPK、磷酸化MKK3/MKK6、磷酸化MKK4等蛋白水平。结果:在体外AM可显著抑制IL-1β诱导的MCs增殖和阻止细胞进入合成周期,显著降低fibronectin、胶原Ⅳ产生,磷酸化p38蛋白和磷酸化MKK3/MKK6的表达显著受抑;在体内AM治疗能显著改善5/6肾切除大鼠的肾功能情况,降低蛋白尿排泄量,延缓肾功能恶化。结论:AM治疗可通过抑制磷酸化MKK3/6和磷酸化p38 MAPK蛋白的表达,发挥抑制IL-1β诱导的MCs增殖作用,能抑制MCs的ECM积聚,有效的治疗进展性肾脏病,为慢性肾纤维化提供了有前景的治疗策略。     

p38MAPK介导的胶质细胞iNOS的转录激活机制

丝裂原激活蛋白激酶(MAPK)酶级联反应系统参与胶质细胞中iNOS的合成.通过瞬时转染p38MAPK途径中上游激酶,MAPK激酶3(MKK3)和MAPK激酶6 (MKK6 )表达质粒,进一步了解p38MAPK级联传导信号系统调节iNOS基因在胶质细胞中的转录激活机制.MKK3或MKK6表达质粒与接有荧光素酶(luciferase ,Luc)的大鼠iNOS启动基因质粒(iNOS Luc)联合转染C6星形胶质细胞株引起iNOS Luc的激活,并且使细胞因子诱导的iNOSmRNA的表达增强.这两种效应都能够被p38MAPK抑制剂SB2 0 35 80所抑制.MKK3 6也可以诱导核因子κB(NFκB Luc)依赖的转录活性.这些分子水平的研究结果为p38MAPK信号级联传导途径在调节大鼠胶质细胞中iNOS基因转录激活中的重要作用,包括转录因子NFκB的作用提供了证据.通过阻断iNOS表达或NO的生成,抑制细胞炎症发生,为防治神经细胞炎症反应性疾病提供实验依据.     

脂联素调节糖脂代谢相关信号通路的研究进展

脂联素是一种主要由脂肪组织分泌的脂肪细胞因子,具有调节糖脂代谢、增强胰岛素敏感性、抗炎和抗动脉粥样硬化等多种作用。在脂联素介导的信号通路中,脂联素首先与脂联素受体(AdipoR)位于膜外的羧基端结合,再通过AdipoR膜内的氨基端与信号接头蛋白结合,进而激活下游的多条信号通路,其中腺苷酸活化蛋白激酶(AMPK)是脂联素信号通路中的关键分子,活化的AMPK可以使其下游的乙酰辅酶A羧化酶(ACC)、p38丝裂原活化蛋白激酶(p38 MAPK)、磷脂酰肌醇3激酶(PI3K)等多种胞质信号分子磷酸化,介导细胞能量代谢。本文重点综述了脂联素通过AMPK调节糖脂代谢的信号通路的研究进展。     

MAPK/ERK 和 MAPK/P38 信号通路的活化参与沙眼衣原体诱导前炎因子的产生

沙眼衣原体感染可导致沙眼、性传播性疾病、不孕症等疾病,主要病理表现是炎症反应引起的组织损伤和瘢痕.因此,沙眼衣原体诱导产生的炎症因子是导致疾病的关键,沙眼衣原体可直接感染内皮细胞产生各种前炎因子,但其机制目前还不清楚.通过ELISA和免疫印迹等方法,检测到沙眼衣原体感染HeLa229细胞可产生IL-8,IL-1α,IL-1β,IL-6等前炎因子,并且沙眼衣原体感染可以主要激活宿主细胞MAPK/ERK和MAPK/P38信号通路.抑制MAPK/ERK和MAPK/P38信号通路显示,两条通路在沙眼衣原体感染过程中参与调节不同的炎症因子产生.MAPK/P38信号通路的活化参与调控IL-1α,IL-6的产生,而IL-8则同时受MAPK/ERK和MAPK/P38两条通路的调控.     

Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1.

Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-regulated by another small G protein Rap. Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1. Transient transfection of cells with constitutively active forms of the known IL-1 signaling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK. Dominant negative forms of these were found to inhibit activation of p38 MAPK by IL-1. Dominant negative RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies downstream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway. Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominant negative mutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominant negative MKK3 or MKK6. In the same manner, the inhibitory effect of Rap on the activation of p38 by IL-1 occurred at a point downstream of MyD88, IRAK, and TRAF6, since the activation of p38 MAPK by these components was inhibited by overexpressing active Rap1AV12, while neither MKK3 nor MKK6 were affected. Active RasVHa associated with IRAK, IRAK2, and TRAF6, but not MyD88. In addition we found a role for TAK-1 in the activation of p38 MAPK by IL-1, with TAK-1 also associating with active Ras. Our study suggests that upon activation Ras becomes associated with IRAK, Traf-6, and TAK-1, possibly aiding the assembly of this multiprotein signaling complex required for p38 MAPK activation by IL-1.     

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.     

p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mphi). We used synthetic inhibitors and dominant-negative Mphi mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3(-/-) Mphi, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38alpha MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38alpha MAPK association with adaptor protein, TGF-beta-activated protein kinase 1-binding protein-1. We observed TGF-beta-activated protein kinase 1-binding protein-1-p38alpha MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mphi. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38alpha MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.     

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.     

Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8

Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. Stress-induced overproduction of inflammatory cytokines, such as interleukin-8 (IL-8), is one of the early events of inflammation. The objective of this study was to elucidate mechanistic links between oxidative stress and overproduction of IL-8 in retinal pigment epithelial (RPE) cells. We found that exposure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased expression and secretion of IL-8. All of these oxidative stressors also inactivated the proteasome in RPE cells. In contrast, tert-butylhydroperoxide (TBH), a lipophilic oxidant that did not stimulate IL-8 production, also did not inactivate the proteasome. Moreover, prolonged treatment of RPE cells with proteasome-specific inhibitors recapitulated the stimulation of IL-8 production. These data suggest that oxidative inactivation of the proteasome is a potential mechanistic link between oxidative stress and up-regulation of the proinflammatory IL-8. The downstream signaling pathways that govern the production of IL-8 include NF-kappaB and p38 MAPK. Proteasome inhibition both attenuated the activation and delayed the turnoff of NF-kappaB, resulting in biphasic effects on the production of IL-8. Prolonged proteasome inhibition (>2 h) resulted in activation of p38 MAPK via activation of MKK3/6 and increased the production of IL-8. Chemically inhibiting the p38 MAPK blocked the proteasome inhibition-induced up-regulation of IL-8. Together, these data indicate that oxidative inactivation of the proteasome and the related activation of the p38 MAPK pathway provide a potential link between oxidative stress and overproduction of proinflammatory cytokines, such as IL-8.     

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.     

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.