p38MAPK通路相关因素与妊娠免疫耐受

妊娠是母体免疫系统对胎儿父系抗原发生免疫耐受的过程。母胎免疫耐受平衡破坏,将导致子痫前期、流产、胎儿宫内生长迟缓等病理性妊娠。文中从吲哚胺2,3-过氧化物酶、血红素加氧酶-1调控p38丝裂原活化激酶(p38MAPK)调节树突细胞功能的角度阐述免疫系统在妊娠中的作用。     

大鼠创伤失血性休克复合内毒素打击模型中p38丝裂原活化蛋白激酶磷酸化水平的改变

目的　探讨创伤失血性休克复合内毒素打击模型中大鼠腹腔巨噬细胞 (M)内p38丝裂原活化蛋白激酶(p38MAPK)磷酸化水平的改变。方法　制作大鼠创伤失血性休克复合内毒素打击动物模型 ,检测伤后 90min、4h血清中TNF-α、IL - 1β含量的改变及大鼠腹腔M内p38MAPK磷酸化水平的变化 ,并观察致伤组大鼠肺及小肠组织的病理改变。 结果　大鼠在创伤失血性休克复合内毒素打击后血清TNF -α、IL - 1β的含量较对照组明显升高 (P <0 .0 1) ,腹腔M内p38MAPK磷酸化水平较对照组明显升高 (P <0 .0 1) ,致伤组大鼠肺及小肠组织炎症病理改变显著。结论　p38MAPK的激活状态可能在大鼠创伤失血性休克复合内毒素打击模型的炎症反应中占有重要地位。     

Contribution of allosteric disulfide bonds to regulation of hemostasis

Summary.  Protein disulfide bonds are covalent links between pairs of Cys residues in the polypeptide chain. Acquisition of disulfide bonds is an important way that proteins have evolved and are continuing to evolve. These bonds serve either a structural or functional role. There are two types of functional disulfide: the catalytic bonds that reside in the active sites of oxidoreductases and the allosteric bonds. Allosteric disulfides are defined as bonds that have evolved to control the manner in which proteins function by breaking or forming in a precise way. The known allosteric bonds have a particular configuration known as the −RHStaple. Several hemostasis proteins contain −RHStaple disulfides and there is increasing evidence that some of these bonds may be involved in the functioning of the protein in which they reside. The best studied of these to date is the −RHStaple disulfide in tissue factor and its role in de-encryption of the cofactor.     

应激后大鼠下丘脑磷酸化p38MAPK的变化及电针足三里穴的调节作用

目的 观察应激对大鼠下丘脑磷酸化p38MAPK的影响及电针足三里穴的调节作用。方法将28只Wistar大鼠随机分为对照组、束缚应激组和束缚应激 电针组，后2组再根据束缚解除后及电针足三里穴位治疗后不同时间点分别分为3个亚组。电针治疗采用频率为5～12Hz的疏密波，治疗总时间30min。于相应时间点处死各组大鼠，采用Western印迹杂交法观察其下丘脑内磷酸化p38MAPK的变化。结果束缚应激组大鼠下丘脑内p38MAPK磷酸化水平明显高于对照组，且在束缚应激解除后3h仍保持在较高水平；应激大鼠经电针足三里穴处理后，下丘脑内磷酸化p38MAPK与束缚应激组相比明显回落，这种调节作用可持续到电针治疗结束后3h。结论束缚应激能引起下丘脑应激信号转导系统中p38MAPK的磷酸化，这可能是应激活动过程中的重要事件之一。电针足三里穴对下丘脑内磷酸化p38MAPK的调节作用可能与下丘脑-垂体-肾上腺轴的活动有关。     

Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance.

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endotoxin, possibly involving changes in guanine nucleotide regulatory (G) protein-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibit cross tolerance and altered hemodynamic responses to thromboxane (Tx)A2 mimetics, suggesting potential changes in vascular responsiveness. We tested the hypothesis that endotoxin tolerance results in vascular hyporesponsiveness to a TxA2 mimetic via alterations in the TxA2 receptor, G protein function, and/or second messenger production. Rats were rendered endotoxin tolerant by increasing sublethal consecutive doses of Salmonella enteritidis endotoxin (100 to 5000 micrograms/kg, i.p.) for 4 days. The animals were sacrificed 2 days after the final dose of endotoxin for removal of aortas. Contractile responses of aortic rings to U46619, a TxA2 agonist, were assessed in control and tolerant rats. The EC50 values for U46619 were 14.8 +/- 6.6 nM and 32.3 +/- 3.1 nM (n = 5-7), (P < 0.05) for control and tolerant rats, respectively. Crude membranes were prepared from aortas of control and tolerant rats, and binding of I-BOP TxA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha (1E, 3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7- oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), a TxA2 agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA2 receptor was saturable and revealed a single class of TxA2 receptors for both groups. There was no significant difference in control (n = 7) compared with tolerant (n = 5) Kd values (2.1 +/- 0.2 vs. 2.4 +/- 0.9 nM, respectively), or Bmax (31 +/- 6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase activity was determined. GTPase activity in tolerant membranes was significantly reduced (P < 0.05) compared with control membranes (309 +/- 23 (n = 5) vs. 440 +/- 32 (n = 7) pmol/mg/protein/min, respectively). However, U46619-stimulated phosphoinositide production was similar in vascular tissue from control and tolerant rats. These observations suggest that the decreased contractile response to TxA2 mimetics in endotoxin tolerance does not result from a change in receptor number, affinity of TxA2 receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascular G protein function.     

p38 MAPK inhibitors ameliorate target organ damage in hypertension: Part 1. p38 MAPK-dependent endothelial dysfunction and hypertension

Numerous mediators, believed to play a role in endothelial dysfunction (e.g., neurohormones, cytokines, hypoxia, and stretch), have been shown to activate p38 mitogen-activated protein kinase (MAPK) in a variety of cell types. The purpose of the present study was to examine the regulation of p38 MAPK in endothelium and its role in endothelial dysfunction and salt sensitivity. In cultured human umbilical vein endothelial cells (HUVECs), tumor necrosis factor-alpha and lipopolysaccharide increased phosphorylation of p38 MAPK (P-p38 MAPK) and increased ICAM-1 expression. Preincubation with highly selective p38 MAPK inhibitors, 1-(1,3-dihydroxyprop-2-yl)-4-(4-fluorophenyl)-5-[2-phenoxypyrimidin-4-yl] imidazole (SB-239063AN) or SB-239063, dose dependently reduced intercellular adhesion molecule-1 expression in HUVECs. In spontaneously hypertensive-stroke prone rats (SHR-SP), P-p38 MAPK was localized by immunohistochemistry to the aortic endothelium and adventitia but was undetectable in aortae from normotensive rats. Introduction of a salt/fat diet (SFD) to the SHR-SP strain induced endothelial dysfunction (ex vivo vascular reactivity analysis), albuminuria, and an increase in blood pressure within 4 weeks. Chronic dietary dosing (approx. 100 mg/kg/day) with SB-239063AN inhibited the SFD diet-induced hypertension. In addition, delayed treatment also significantly improved survival and restored nitric oxide-mediated endothelium-dependent relaxation in SFD-SHR-SPs with established endothelial dysfunction. These results suggest an important role for p38 MAPK in endothelial inflammation and dysfunction as well as providing the first evidence for p38 MAPK-dependent hypertension.     

Inhibition of p38 mitogen-activated protein kinase: dose-dependent suppression of leukocyte and endothelial response after endotoxin challenge in humans

OBJECTIVE: We studied the activity of a single oral dose of RWJ-67657, a synthetic p38 mitogen-activated protein kinase inhibitor, in preventing dual leukocyte/endothelial activation after endotoxin infusion in healthy volunteers. DESIGN: Prospective placebo-controlled study. SETTING: Intensive care unit at a university medical center. SUBJECTS: Twenty-one healthy male volunteers. INTERVENTIONS: Endotoxin (4 ng/kg) as a 1-min infusion. According to randomization, the volunteers received placebo (n = 6) or 1400 mg (n = 4), 700 mg (n = 6), or 350 mg (n = 5) of RWJ-67657. MEASUREMENTS AND MAIN RESULTS: Neutrophil activation was investigated by analyzing the extent of membrane expression of adhesion markers by calibrated flow cytometry. Circulating intercellular adhesion molecule-1 and E-selectin were measured by enzyme-linked immunosorbent assays. The endotoxin-induced shedding of L-selectin was diminished in a dose-dependent manner (p <.0001). High-dose RWJ-67657 prevented up-regulation of the integrins CD11b (p <.01) and CD 66b (p <.01) on neutrophils. The endotoxin-induced increase in circulating intercellular adhesion molecule-1 and circulation E-selectin was almost completely prevented by high-dose RWJ-67657. CONCLUSION: A single oral dose of RWJ-67657 prevented neutrophil and endothelial activation after endotoxin infusion.     

Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences

Endotoxin tolerance was first described in a study that exposed animals to a sublethal dose of bacterial endotoxin. The animals subsequently survived a lethal injection of endotoxin. This refractory state is associated with the innate immune system and, in particular, with monocytes and macrophages, which act as the main participants. Several mechanisms are involved in the control of endotoxin tolerance; however, a full understanding of this phenomenon remains elusive. A number of recent reports indicate that clinical examples of endotoxin tolerance include not only sepsis but also diseases such as cystic fibrosis and acute coronary syndrome. In these pathologies, the risk of new infections correlates with a refractory state. This review integrates the molecular basis and clinical implications of endotoxin tolerance in various pathologies.     

Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride

We have previously shown that in renal cortex, COX-2 expression is localized to macula densa and surrounding cortical thick ascending limb of Henle (cTALH). Dietary salt restriction increases local expression of COX-2, which mediates renin production and secretion. Given that decreased luminal chloride [Cl(-)] at the level of the macula densa increases renin production and secretion, we investigated the role of extracellular ion concentration on COX-2 expression. Quiescent rabbit cTALH cells were incubated in a physiological salt solution containing high or low levels of NaCl. Immunoreactive COX-2 expression increased significantly in the low NaCl solution. COX-2 expression also increased after administration of the Na(+)/K(+)/2Cl(-) cotransport inhibitor, bumetanide. Selective substitution of chloride led to increased COX-2 expression, whereas selective substitution of sodium had no effect. The p38 MAP kinase inhibitor PD169316 decreased low NaCl-induced COX-2 expression. Low-salt or low-chloride medium induced cultured cTALH to accumulate >/= 3-fold higher levels of pp38, the activated (phosphorylated) form of p38; low-salt medium also increased pJNK and pERK levels. Feeding rats a low-salt diet for 14 days induced a significant increase in renal cortical pp38 expression, predominantly in the macula densa and cTALH. These results suggest that reduced extracellular chloride leads to increased COX-2 expression, which may be mediated by activation of a p38-dependent signaling pathway.     

Tolerance to tumor necrosis factor in rats and the relationship to endotoxin tolerance and toxicity

Treatment of rats with recombinant human TNF initially causes a marked decrease in food intake, a loss of body weight, and a negative nitrogen balance. These alterations normalize with continued twice daily intraperitoneal injections of the same dose. Rats tolerized to TNF in this manner are refractory to a lethal dose of TNF. Also, TNF-pretreated and -tolerized rats have prolonged survival and reversed histopathologic changes after injection of a lethal dose of endotoxin compared with control animals. The TNF-tolerant state is dependent on the dose of TNF used and the length of TNF pretreatment. TNF-induced tolerance is relatively short lived, being present 2-4 d after TNF pretreatment and dissipating by 2 wk. Rats made tolerant to endotoxin are also tolerant to a lethal dose of TNF. A bidirectional crossreacting tolerance exists between TNF and endotoxin. The mechanism of TNF tolerance is unclear, but it does not appear to be due to a humoral immune response or a perturbation of the uptake and clearance of injected TNF.     

Contribution of peripheral blood pooling to central hemodynamic disturbances during endotoxin insult in intact dogs

The aim of the present study was to determine possible effects of Escherichia coli endotoxin on peripheral vascular compliance and relate them to concomitant central hemodynamic disturbances. Endotoxin was infused at 0.25 micrograms/kg.min during 2 h in six anesthetized dogs, while six additional animals served as controls. Vascular compliance of the systemic circulation was calculated in intact animals from the changes in CVP after known changes in systemic blood volume. In control dogs, vascular compliance averaged 2.3 ml/mm Hg.kg body weight. During slow endotoxin infusion, cardiovascular effects were measurable only after a certain period of time had elapsed from the start of endotoxin insult and consisted of hypotension associated with systemic vasodilation. Systemic BP decreased gradually from 124 to 68 mm Hg while vascular compliance was finally increased by 100%, when compared to control values. This latter rise was responsible for a reduction in the cardiac preloads. Pulmonary wedge pressure and CVP were decreased from 7.1 to 3.4 and from 4.5 to 2.6 mm Hg, respectively. However, parallel to the decrease in left ventricular preload, endotoxin induced a progressive decrease in left ventricular afterload. Because of the balance in ventricular loading, cardiac output remained almost unchanged. After volume loading (dextran 30 ml/kg), cardiac output was remarkably increased from 3.28 to 6.24 L/min.m2 while peripheral vasodilation was not affected by this maneuver. It is concluded that low dose endotoxin infusion induces in dogs a hemodynamic pattern similar to human sepsis. The left ventricular loading changes are related to an enhanced systemic vascular compliance from 2.3 to 4.5 ml/mm Hg.kg. High flow shock state is encountered provided peripheral blood pooling is compensated by adequate volume replacement.     

抑制NHE1和p38 MAPK通路增加白血病细胞对伊马替尼的敏感性

本研究旨在探讨抑制NHE1活性和细胞内酸化能否逆转白血病细胞对伊马替尼的耐药,以及慢性髓系白血病（CML）患者细胞的BCR／ABL下游分子网络。对CML患者原代白血病细胞或K562／DOX,K562／G01耐药细胞株进行酸化处理,检测细胞内P-糖蛋白（Pgp）基因及蛋白表达,药物在细胞内蓄积。考察细胞酸化后原代白血病细胞ERK1／2、p38MAPK磷酸化水平的变化。结果表明：细胞酸化后进展期患者细胞内药物蓄积增加,对伊马替尼的敏感性增强。随着细胞内pH的降低,进展期CML患者的细胞内p38MAPK磷酸化水平呈下降趋势,ERK1／2磷酸化水平3min时升高、30min后下降。特异性p38MAPK抑制剂SB203580可与NHEl抑制剂卡立泊来德协同下调Pgp蛋白的表达。结论：抑制NHE1活性能够显著降低耐药细胞中Pgp表达,增加CML疾病进展期患者细胞中罗丹明123及阿霉素的累积,增加细胞对伊马替尼的敏感性,p38MAPK信号传导通路参与其中。     

Microcirculatory alterations of hepatic and mesenteric microcirculation in endotoxin tolerance

LPSs getting access to the circulation of mammalian organisms cause typical systemic inflammatory reactions with symptoms characteristic for acute sepsis. One possibility to attenuate LPS effects is to expose a host to a challenge with low LPS doses, which results in the establishment of "endotoxin tolerance" (ET). Because the microcirculation is of particular importance in LPS action, it seemed of interest to analyze leukocyte-endothelial interactions in the mesentery and liver once endotoxin tolerance has been established and are challenged with LPS. The mesenteric and hepatic microcirculation was investigated by intravital microscopy. After induction of ET LPS, shock was induced by i.v. injection of LPS, and microcirculation of the mesentery and liver was examined. Endotoxin tolerance resulted in reduced ex vivo TNF-alpha synthesis of whole blood. In vivo LPS caused no increase of body temperature. In sinusoids, LPS challenge increased adherence of leukocytes in naive rats, which was almost completely prevented by ET induction. In contrast, in postsinusoidal venules, leukocyte adherence was more intense after ET induction and subsequent to LPS application. Similarly, in postcapillary mesenteric venules, increased adherence of leukocytes after LPS challenge in the ET group was observed. After LPS injection, the endothelial barrier was more disturbed in the nontolerant group when compared with the ET group. Soluble L-selectin and intercellular adhesion molecule were elevated in both ET and untreated rats. Endotoxin tolerance influences leukocyte-endothelial interaction differentially depending on organ and vessel area.     

抑制钠氢交换蛋白下调IL-8表达和促进p38磷酸化

我们前期的研究证明,抑制钠氢交换蛋白1(NHE1)可以减少VEGF表达从而抑制K562细胞诱导的血管生成。本研究探讨抑制NHE1后其他可能的血管生成因子变化及相关机制。用NHE1特异性抑制剂卡立泊来德10μmol/L处理K562细胞;蛋白芯片筛选NHE1抑制后血管生成因子的表达变化,实时定量PCR验证卡立泊来德处理后白介素-8(IL-8)的表达水平;构建K562稳定干扰NHE1细胞株,实时定量PCR验证干扰NHE1后IL-8的表达变化,Western blot检测卡立泊来德处理后细胞内p38磷酸化水平;p38抑制剂SB203580处理K562细胞,实时定量检测IL-8表达变化。蛋白芯片筛选结果显示,卡立泊来德处理后K562细胞中IL-8表达显著下调;实时定量结果进一步验证了这种抑制效应;卡立泊来德处理后p38磷酸化水平显著上调;卡立泊来德处理后加入SB203580抑制p38,IL-8表达可以部分恢复。结论:抑制NHE1可能通过促进p38磷酸化,下调促血管生成因子IL-8的表达。     

长链非编码RNA TTN-AS1通过miR-3928调节p38MAPK参与宫颈癌进展的机制研究

目的 探究长链非编码RNA(LncRNA)TTN-AS1通过miR-3928调节p38MAPK参与宫颈癌进展的机制.方法 分析肿瘤基因组图谱数据库中宫颈癌患者TTN-AS1水平与MAPK14水平和患者生存之间的关系.双荧光素酶报告验证靶向关系.通过转染miR-3928模拟物和TTN-AS1构建miR-3928和/或TT...     

Heat shock factor 1 inhibits nuclear factor-kappaB nuclear binding activity during endotoxin tolerance and heat shock

Rationale

Sepsis, endotoxin tolerance, and heat shock (HS) all display down-regulation of innate immunity. We hypothesize that HS factor 1 (HSF-1) induces competitive inhibition of nuclear factor–κB (NF-κB)–induced signal transduction in both endotoxin tolerance and HS.

Objectives

We compared endotoxin tolerance and HS in RAW 264.7 cells. We transfected cells with an HS protein 70 (HSP70) plasmid to test whether HSP70 is the mediator of HS-induced NF-κB inhibition. We studied the effects of endotoxin stimulation and HS, both separately and together, on “wild-type” cells, cells transfected with the HSP70 plasmid, and cells transfected with vehicle.

Findings

Heat shock protein 70 plasmid–transfected cells had increased HSP70 expression and demonstrated decreased nitric oxide (NO) release and inducible NO synthase messenger RNA expression in response to endotoxin compared with wild-type and empty plasmid–transfected cells. Heat shock completely abolished subsequent NO and inducible NO synthase messenger RNA expression in wild-type cells. Heat shock factor 1 reached maximum expression 60 to 90 minutes after HS. Heat shock protein 70–transfected cells still displayed endotoxin-induced NF-κB nuclear binding, whereas endotoxin tolerance, HS, and exposure to HSF-1, but not exposure to an unrelated promoter, inhibited NF-κB nuclear binding.

Conclusions

Endotoxin tolerance and HS appear to share a common immune suppressive effect, possibly through HSF-1–mediated competitive inhibition of NF-κB nuclear binding.