p38MAPK信号通路与肝缺血再灌注损伤的关系研究

p38MAPK通路参与诸如细胞氧化应激、细胞凋亡和炎症等多种生理和病理过程并在其中起着重要作用。肝缺血再灌注损伤(HIRI)是肝脏外科手术面临的一大难题,由于HIRI发病机制复杂,临床上尚未发现更好的应对HIRI的防治策略。本文就p38MAPK信号通路的主要功能及其在HIRI中的相关作用作一综述,以便为防治HIRI提供一个新的思路。     

N-acetylcysteine reduces chemokine release via inhibition of p38 MAPK in human airway smooth muscle cells.

Reactive oxygen species are involved in the activation of several mitogen-activated protein kinases (MAPKs), key-players in the production of several cytokines. Therefore the current study investigated whether N-acetylcysteine (NAC), an antioxidative agent, inhibits the interleukin (IL)-1beta-induced expression and production of eotaxin and monocyte chemotactic protein (MCP)-1 in human airway smooth muscle cells (HASMC). NAC (10 mM) decreased the expression of eotaxin and MCP-1, by 46 +/- 11% (n=7) and 87 +/- 4% (n=6), respectively; the eotaxin release was inhibited by 75 +/- 5% (n=7), whereas the MCP-1 release was decreased by 69 +/- 41% (n=10). NAC (1 mM) also decreased the IL-1beta-induced activation of p38 MAPK. Compared with unstimulated cells, a four-fold increase in 8-isoprostane production in IL-1beta-stimulated HASMC was observed, which could be inhibited by NAC in a concentration-dependent way, with a maximum inhibition of 39 +/- 12%, with 1 mM NAC. The present study demonstrated that N-acetylcysteine inhibits the interleukin-1beta-induced eotaxin and monocyte chemotactic protein 1 expression and production due to a decreased activation of p38 mitogen-activated protein kinase. This study has also shown that N-acetylcysteine decreases the interleukin-1beta-induced production of reactive oxygen species, as suggested by a reduction in the 8-isoprostane production.     

Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability

The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-alpha on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-alpha lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10(-8) to 10(-6) mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-alpha-induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-alpha-induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-alpha-induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1-induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.     

Chemokine CCL5/RANTES inhibition reduces myocardial reperfusion injury in atherosclerotic mice

Although beneficial for cardiomyocyte salvage and to limit myocardial damage and cardiac dysfunction, restoration of blood flow after prolonged ischemia exacerbates myocardial injuries. Several deleterious processes that contribute to cardiomyocyte death have been proposed, including massive release of reactive oxygen species, calcium overload and hypercontracture development or leukocyte infiltration within the damaged myocardium. Chemokines are known to enhance leukocyte diapedesis at inflammatory sites. The aim of the present study was to investigate the effect of chemokine CCL5/RANTES antagonism in an in vivo mouse model of ischemia and reperfusion. ApoE^−/− mice were submitted to 30 min ischemia, by ligature of the left coronary artery, followed by 24 h reperfusion. Intraperitoneal injection of 10 μg of CCL5/RANTES antagonist [⁴⁴AANA⁴⁷]-RANTES, 5 min prior to reperfusion, reduced infarct size as well as Troponin I serum levels compared to PBS-treated mice. This beneficial effect of [⁴⁴AANA⁴⁷]-RANTES treatment was associated with reduced leukocyte infiltration into the reperfused myocardium, as well as decreased chemokines Ccl2/Mcp-1 and Ccl3/Mip-1α expression, oxidative stress, and apoptosis. However, mice deficient for the CCL5/RANTES receptor Ccr5 did not exhibit myocardium salvage in our model of ischemia-reperfusion. Furthermore, [⁴⁴AANA⁴⁷]-RANTES did not mediate cardioprotection in these ApoE^−/− Ccr5^−/− deficient mice, probably due to enhanced expression of compensatory chemokines. This study provides the first evidence that inhibition of CCL5/RANTES exerts cardioprotective effects during early myocardial reperfusion, through its anti-inflammatory properties. Our findings indicate that blocking chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce reperfusion injuries in patients during acute coronary syndromes.     

p38MAPK抑制剂与急性肺损伤的保护

急性肺损伤可由多种诱因引起,是急性呼吸衰竭最主要部分,死亡率高.p38MAPK信号转导途径参与急性肺损伤的发生、发展.p38MAPK抑制剂是一类合成的小分子有机化合物,通过特异性阻断p38减少急性肺损伤时炎症因子表达,改善血流动力学,减轻组织损伤,有望成为急性肺损伤治疗的新途径.     

p38MAPK inhibition enhances basal and norepinephrine-stimulated p42/44MAPK phosphorylation in rat pinealocytes

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4beta phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.     

衰老标记蛋白30介导四氢姜黄素抗心肌缺血再灌注损伤的机制研究

目的 探讨衰老标记蛋白30（SMP30）在四氢姜黄素（THC）抗心肌缺血再灌注（MI/R）损伤中的作用。 方法 构建H9c2细胞缺氧/复氧模型和小鼠MI/R模型,利用腺病毒和短发夹RNA（shRNA）,在细胞和动物水平下调心肌的SMP30的表达,通过CCK8细胞活力试剂盒、ANNEXIN V- FITC/PI凋亡试剂盒、乳酸脱氢酶（LDH）活性试剂盒、肌酸激酶同功酶（CK-MB）试剂盒、心脏超声等方法,评价细胞活性和心脏功能。 结果 敲低SMP30水平后,THC对H9c2细胞的保护作用被部分逆转,THC抗H/R诱发的H9c2细胞死亡能力被削弱;SMP30通过Bax/Blc2途径介导THC对H9c2细胞的保护;THC能够显著改善小鼠MI/R后心脏功能,敲低小鼠心脏SMP30水平后,THC的心脏保护作用被部分逆转。 结论 SMP30参与介导THC抗I/R诱发的心肌细胞凋亡。     

Activation of p38 MAPK induced by a multi-cycle ischaemic preconditioning protocol is associated with attenuated p38 MAPK activity during sustained ischaemia and reperfusion

The role of p38 mitogen-activated protein kinase (MAPK) in ischaemic preconditioning remains controversial. Since most previous studies focussed on events only during sustained ischaemia, the aim of this study was to establish the activation pattern of p38 MAPK during a multicycle preconditioning protocol, sustained ischaemia as well as reperfusion and to correlate these events with functional recovery of the isolated perfused rat heart. Isolated perfused rat hearts were preconditioned by 3x5 min global ischaemia followed by 25 min global ischaemia and 30 min reperfusion. Non-preconditioned hearts were subjected to 25 min global ischaemia and 30 min reperfusion. Hearts were freeze-clamped and p38 MAPK activation in tissue lysates was assessed by standard Western blotting techniques, using a dual phospho-p38 MAPK antibody as well as a non-radioactive IP-kinase assay. The results showed that transient dual phosphorylation and activation of p38 MAPK occurs during a 3x5 min preconditioning protocol: the activation was maximal during the first episode, becoming progressively lower during the second and third episodes. p38 MAPK activation was significantly less during both sustained ischaemia and reperfusion in preconditioned hearts, when compared with non-preconditioned hearts. Attenuation of p38 MAPK activity during sustained ischaemia and reperfusion was associated with improved functional recovery. The effect of inhibition of p38 MAPK activation on cardioprotection was further evaluated in adult, isolated cardiomyocytes. Administration of SB 203580 (1-10 microM) before and during the preconditioning protocol, had no effect on cell morphology and viability after 2 h hypoxia, compared to untreated preconditioned cardiomyocytes. When administered to non-preconditioned cells before the onset of 2 h hypoxia, it caused a significant improvement in both morphology and viability. In summary, the results suggest that attenuation of the kinase activity during sustained ischaemia and reperfusion may be an essential element of the preconditioning process.     

Asymmetric dimethylarginine induces apoptosis via p38 MAPK/caspase-3-dependent signaling pathway in endothelial cells

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributor for endothelial dysfunction and its effects on endothelium are not yet completely defined. The aim of this study was to investigate ADMA-induced apoptosis and its mechanisms in human umbilical vein endothelial cells (HUVECs). Apoptosis was evaluated by in situ terminal uridine nick end labeling (TUNEL) assay and DNA fragmentation analysis. Caspase-3 activity was measured using a colorimetric protease assay kit. Activations of mitogen-activated protein kinases (MAPKs) were characterized by Western blot and immunofluorescence. Intracellular oxidant production was measured using H(2)DCF-DA, an oxidant-sensitive fluorescent indicator. ADMA (3-30 microM) induced apoptosis of HUVECs in a dose- and time-dependent manner. Caspase-3 was activated during apoptosis and its specific inhibitor DEVD-CHO significantly attenuated ADMA-induced apoptosis. Phosphorylation of p38 MAPK was induced by ADMA, and p38 MAPK specific inhibitor SB203580 concentration-dependently prevented ADMA-induced caspase-3 activation and cell apoptosis. ADMA increased intracellular oxidant production, which was significantly suppressed by intracellular antioxidant PDTC, l-arginine or antisense endothelial NOS mRNA. They also markedly prevented ADMA-induced phosphorylation of p38 MAPK and cell apoptosis. In conclusion, our present results demonstrate that ADMA induces apoptosis of endothelial cell via elevation of intracellular oxidant production, which involves p38 MAPK/caspase-3-dependent signaling pathway.     

p38MAPK表达在冠心病患者冬眠心肌细胞凋亡的意义

目的:探讨冠心病患者冬眠心肌细胞内活化的丝裂素活化蛋白激酶(p38MAPK)对心肌细胞凋亡的影响.方法:行冠状动脉搭桥术(CABG)的冠心病患者10例,术前1周内用多巴酚丁胺超声负荷试验结合多普勒组织成像确定冬眠心肌及正常心肌的存在部位,CABG术中根据检测结果进行取材(分别取正常心肌和冬眠心肌),并经电镜证实.取材心肌用Tunel法检测心肌细胞凋亡情况,免疫印迹法(Western-blot)检测磷酸化的p38的表达情况.结果:冬眠心肌细胞内磷酸化p38、心肌细胞凋亡数较正常心肌高;p38与心肌细胞凋亡数相关(P＜0.05,r=0.816).结论:心肌慢性缺血时,心肌细胞内p38MAPK信号活化,活化的p38MAPK介导冬眠心肌细胞凋亡.