p38丝裂原活化蛋白激酶途径对大鼠脑缺血再灌注后脑组织基质金属蛋白酶-9表达及脑水肿形成的影响

目的探讨p38丝裂原活化蛋白激酶(MAPK)途径对大鼠脑缺血再灌注后脑组织基质金属蛋白酶-9(MMP-9)表达及脑水肿形成的影响。方法 54只SPF级雄性SD大鼠,随机分为假手术组(Sham组)、缺血再灌注组(I/R组)和p38抑制剂组(SB组)。采用改良线栓法制备大鼠大脑中动脉缺血再灌注模型。再灌注24 h后对大鼠进行神经功能缺损评分,Evans Blue法测定血-脑屏障通透性;干湿比重法测定脑组织含水量,采用Western blot检测缺血周边区脑组织磷酸化p38(p-p38)和MMP-9的表达。结果与Sham组相比,I/R组大鼠神经功能缺损加重(P0.05);与I/R组相比,SB组大鼠神经功能缺损明显减轻(P0.05)。与Sham组比较,I/R组血-脑屏障通透性及脑含水量明显增加(均P0.05);与I/R组相比,SB组血-脑屏障通透性及脑含水量降低(均P0.05)。与Sham组相比,I/R组大鼠缺血周边区脑组织p-p38、MMP-9的表达明显上调(均P0.05);与I/R组相比,SB组大鼠缺血周边区脑组织p-p38、MMP-9的表达明显下调(均P0.05)。结论 p38 MAPK参与了大鼠脑缺血再灌注后脑水肿的形成,机制可能为大鼠脑缺血再灌注后激活p38MAPK使缺血周边区脑组织MMP-9的表达上调,破坏血-脑屏障通透性,导致脑水肿发生。     

Activation of mitogen-activated protein kinases in experimental cerebral ischemia

OBJECTIVES: Mitogen-activated protein kinases (MAPK) regulate cell survival and differentiation. The aim of the present study is to investigate the activation pattern of different MAPKs [extracellular signal-regulated kinase (ERK), c-jun-N-terminal kinase (JNK) and p38] after cerebral ischemia. MATERIAL AND METHODS: Rats were subjected to cerebral ischemia using a model for transient (2 h) and permanent middle cerebral artery occlusion (MCAO). The rats were allowed 6 h to 1 week of survival before immunohistochemical evaluation with phospho-specific antibodies, recognizing activated MAPKs. RESULTS: ERK was activated in ipsilateral blood vessels, neurons and glia, but also in contralateral vessels. JNK activation was absent in neurons but appeared in arterial blood vessels and glia at the lesion side. Active p38 was observed in macrophages in maturing infarcts. CONCLUSIONS: ERK and JNK may participate in the angiogenic response to cerebral ischemia. ERK, but not JNK, was activated in neurons, possibly indicating a pathophysiologic role. Active p38 might be involved in the inflammatory reaction.     

基于p38丝裂原活化蛋白激酶通路的胰高血糖素样肽-1对大鼠脑缺血再灌注损伤的影响及机制

目的基于p38丝裂原活化蛋白激酶(p38MAPK)通路探讨胰高血糖素样肽-1(GLP-1)对大鼠脑缺血再灌注(I/R)损伤的影响及机制。方法将雄性SD大鼠分为假手术组、模型组、GLP-1组和p38MAPK抑制剂组,每组12只。模型组、GLP-1组和p38MAPK抑制剂组通过大脑中动脉栓塞及再灌注建立脑I/R损伤模型,GLP-1组给予利拉鲁肽(70μg/kg)、p38MAPK抑制剂组给予p38MAPK抑制剂SB202190(10μmol/L、5μl)干预。比较四组大鼠的脑梗死体积、水迷宫行为参数及梗死脑组织细胞凋亡率、氧化应激指标、炎症细胞因子、p38MAPK通路分子的差异。结果与模型组比较,GLP-1组和p38MAPK抑制剂组大鼠的脑梗死体积明显降低,逃避潜伏期明显缩短、穿越平台次数明显增多,梗死脑组织中的细胞凋亡率及丙二醛(MDA)、超氧化物歧化酶(SOD)、TNF-α、IL-1β、IL-6、p-p38水平显著减少,SOD、谷胱甘肽过氧化物酶(GPx)水平明显增加(均P<0.05),p-ERK1/2、p-JNK的表达水平无明显变化。与假手术组比较,模型组大鼠的逃避潜伏期明显延长、穿越平台次数明显减少,梗死脑组织的细胞凋亡率及MDA、ROS、TNF-α、IL-1β、IL-6、p-p38水平明显增高,SOD、GPx水平明显减少(均P<0.05),p-ERK1/2、p-JNK的表达水平无明显变化。结论GLP-1能够通过抑制p38介导的氧化应激及炎症反应减轻大鼠脑I/R损伤。     

Prevention of JNK phosphorylation as a mechanism for rosiglitazone in neuroprotection after transient cerebral ischemia: activation of dual specificity phosphatase

Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist, prevents cell death after cerebral ischemia in animal models, but the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone protects neurons against ischemia. Mice treated with rosiglitazone were subjected to 60 minutes of focal ischemia followed by reperfusion. Rosiglitazone reduced infarct volume after ischemia and reperfusion. We show that this neuroprotective effect was reversed with a PPARγ antagonist. Western blot analysis showed a significant increase in expression of phosphorylated stress-activated protein kinases (c-Jun N-terminal kinase (JNK) and p38) in ischemic brain tissue. Rosiglitazone blocked this increase. Furthermore, we observed that rosiglitazone increased expression of the dual-specificity phosphatase 8 (DUSP8) protein and messenger RNA in ischemic brain tissue. Dual-specificity phosphatase 8 is a mitogen-activated protein kinase phosphatase that can dephosphorylate JNK and p38. Another key finding of the present study was that knockdown of DUSP8 in primary cultured cortical neurons that were subjected to oxygen–glucose deprivation diminished rosiglitazone''s effect on downregulation of JNK phosphorylation. Thus, rosiglitazone''s neuroprotective effect after ischemia is mediated by blocking JNK phosphorylation induced by ischemia via DUSP8 upregulation.     

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

Electroacupuncture(EA) has anti-oxidative and anti-inflammatory actions,but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion(I/R) injury involves modulation of the extracellular regulated kinase 1/2(ERK1/2) signaling pathway is unclear.Middle cerebral artery occlusion(MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours.A 30-minute period of EA stimulation was applied to both Baihui(DU20) and Dazhui(DU14) acupoints in each rat(10 mm EA penetration depth,continuous wave with a frequency of 3 Hz,and a current intensity of 1–3 m A) when reperfusion was initiated.EA significantly reduced infarct volume,alleviated neuronal injury,and improved neurological function in rats with MCAO.Furthermore,high m RNA expression of Bax and low m RNA expression of Bcl-2 induced by MCAO was prevented by EA.EA substantially restored total glutathione reductase(GR),glutathione(GSH) and glutathione peroxidase(GSH-Px) levels.Additionally,Nrf2 and glutamylcysteine synthetase(GCS) expression levels were markedly increased by EA.Interestingly,the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059(a specific MEK inhibitor).Collectively,our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA.Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.     

颅脑损伤后p38丝裂原活化蛋白激酶在挫伤皮层中的表达及其意义

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）在颅脑损伤后挫伤皮层中的表达。方法挫伤皮层标本来自24例颅脑损伤患者，取样时间为伤后5h-5d，将患者按伤后取标本时间平均分为4组，即〈24h组、24～48h组、48-72h组和〉72h组。所有病例常规行开颅血肿清除术，应用免疫组化技术测定挫伤皮层中磷酸化p38MAPK的表达。结果在颅脑损伤后挫伤皮层中．p38MAPK的表达明显上调（P〈0．05），表达高峰为伤后24h内（P〈0．01），主要在血管内皮细胞中表达，在神经细胞及神经胶质细胞中极少表达。结论p38MAPK在人颅脑损伤后挫伤皮层中的表达上调，提示其可能在颅脑损伤后的病理生理过程中起重要作用。     

Administration of the p38 MAPK inhibitor SB203580 affords brain protection with a wide therapeutic window against focal ischemic insult

We have reported previously the delayed and differential induction of p38alpha and p38beta mitogen-activated protein kinases (MAPKs) in microglia and astrocytes, respectively, in brain after transient global ischemia. We report here the sustained induction and activation of p38alpha MAPK in activating microglia in rat brain after transient middle cerebral artery occlusion (MCAO). The intraventricular administration of SB203580, a p38 MAPK inhibitor, 30 min before MCAO reduced the infarct volume to 50% of the control, which was accompanied by the significant improvement of neurological deficits. More interestingly, the infarct volume was reduced to 72% and 77% when SB203580 was administered 6 hr and 12 hr after MCAO, respectively. The induction of various factors involved in inflammatory processes, such as inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and cyclooxygenase-2 (COX-2), was suppressed by the administration of SB203580 at 6 hr after MCAO. These results suggest that sustained activation of p38 MAPK pathway and p38 MAPK-associated inflammatory processes play a crucial role in postischemic brain.     

大鼠短暂局灶性大脑中动脉缺血后calpain的表达

目的：研究calpain在缺血性脑损伤中的作用，进一步探讨缺血性脑血管病的分子机制，为治疗研发提供理论依据。方法：用Belayev改良的Langa线栓法制备大鼠局灶性大脑中动脉(MCA)缺血／再灌注模型，TTC染色观察梗死灶的形成，分别用原位杂交及免疫组化技术检测鼠脑中calpain mRNA与活性蛋白的表达。结果：缺血2h再灌注24h,TTC染色见明显的梗死灶形成，正常脑组织、假手术组及：MCAO缺血对侧脑中有少量的calpain mRNA表达，但活性蛋白几无表达；缺血脑组织calpain mRNA表达及蛋白质活化均显著增加，呈双峰式，MCA缺血2h增加，再灌注4h减少，至24h更明显增高，而48h又有所下降。结论：Calpain参与了缺血性脑损伤过程，尤其在迟发性神经元死亡中起重要作用。     

Antioxidant CR-6 protects against reperfusion injury after a transient episode of focal brain ischemia in rats

Oxidative and nitrosative stress are targets for intervention after ischemia/reperfusion. The aim of this study was to explore the effect of CR-6, a vitamin-E analogue that is antioxidant and scavenger of nitrogen-reactive species. Sprague–Dawley rats had the middle cerebral artery (MCA) occluded either for 90 mins or permanently. Cortical perfusion was continuously monitored by laser–Doppler flowmetry. CR-6 (100 mg/kg) was administered orally either at 2 and 8 h after MCA occlusion, or at 2 h only. Infarct volume, neurological deficit, and signs of reperfusion injury were evaluated. CR-6 was detected in plasma and brain by HPLC. CR-6 reduced glutathione consumption in the ischemic brain and superoxide generation in the isolated MCA. CR-6 decreased infarct volume and attenuated the neurological deficit at 1 and 7 days after ischemia/reperfusion, but not after permanent ischemia. Immediately after reperfusion, cortical blood flow values returned to their baseline (±20%) in several animals, whereas others showed hyper-perfusion (>20% of baseline). Reactive hyperemia was associated with adverse events such as increased cortical BBB leakage, edema, protein nitrotyrosination, COX-2 expression, and neutrophil accumulation; and with a poorer outcome, and CR-6 attenuated these effects. In conclusion, oral CR-6 administration after transient ischemia protects the brain from reperfusion injury.     

Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury. To further iden-tify the involved mechanisms, we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa's method. At 30 minutes before model establishment, p38 MAPK blocker SB20358 was injected into the left lateral ventricles. At 1.5 hours after model establishment, electroacupuncture was administered at acupoints of Chize (LU5), Hegu (LI4), Zusanli (ST36), and Sanyinjiao (SP6) for 20 minutes in the affected side. Results showed that the combination of EA and SB20358 injec-tion significantly decreased neurologic impairment scores, but no significant differences were determined among different interventional groups. Hematoxylin-eosin staining also showed reduced brain tissue injuries. Compared with the SB20358 group, the cells were regularly arranged, the structures were complete, and the number of viable neurons was higher in the SB20358 + electroacupuncture group. Termi-nal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay showed a decreased apoptotic index in each group, with a significant decrease in the SB20358 + electroacupuncture group. Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 + electroacupuncture group compared with the ischemia/reperfusion group. There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group. These find-ings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway. A time period of 3 days could promote the repair of ischemic cerebral nerves.     

Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia

We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours+22- to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed ∼15,000 leukocytes (CD45^+high cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising ∼40% lymphoid cells and ∼60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to ∼5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8⁺ T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4⁺ T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were ∼50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.     

蛛网膜下腔出血后大鼠基底动脉p38MAPK信号传导途径研究

目的探讨蛛网膜下腔出血(SAH)后大鼠基底动脉中p38丝裂原活化蛋白激酶(p38MAPK)信号传导通路的活化情况以及与脑血管痉挛(CVS)的关系。方法通过枕大池二次注血方法制作大鼠SAH模型,以免疫组化方法和逆转录酶-多聚酶链反应分析,分别从蛋白、基因水平分析SAH后基底动脉中p38MAPK信号传导通路的活化情况。结果 SAH后大鼠基底动脉逐渐出现痉挛。基底动脉磷酸化p38MAPK表达逐渐增加,3 d时达高峰并持续至第5 d,14 d时恢复正常。p38MAPK基因表达在注血后1 d明显增加,逐渐增加,于5 d时达高峰,14 d仍维持较高水平。结论SAH后大鼠基底动脉中p38MAPK信号传导通路激活,可能诱导CVS的发生。     

Neurotoxicity of pneumolysin,a major pneumococcal virulence factor,involves calcium influx and depends on activation of p38 mitogen-activated protein kinase

Neuronal injury in bacterial meningitis is caused by the interplay of host inflammatory responses and direct bacterial toxicity. We investigated the mechanisms by which pneumolysin, a cytosolic pneumococcal protein, induces damage to neurons. The toxicity after exposure of human SH-SY5Y neuroblastoma cells and hippocampal organotypic cultures to pneumolysin was time- and dose-dependent. Pneumolysin led to a strong calcium influx apparently mediated by pores on the cell membrane formed by the toxin itself and not by voltage-gated calcium channels. Buffering of intracellular calcium with BAPTA-AM [1, 2-bis (o-aminophenoxy) ethane N, N, N', N'-tetraacetic acid tetra(acetomethoxyl) ester] improved survival of neuronal cells following challenge with pneumolysin. Western blotting revealed increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) as early as 30 min after challenge with pneumolysin. SB 203580, a potent and selective inhibitor of p38 MAPK, rescued human neuronal cells from pneumolysin-induced death. Inhibition of the mitochondrial permeability transition pore using bongkrekate and caspase inhibition also improved survival following challenge with the toxin. Modulation of cell death pathways activated by pneumolysin may influence the outcome of pneumococcal meningitis.     

Dynamic expression of p38beta MAPK in neurons and astrocytes after transient focal ischemia

Here we report the dynamically regulated expression of p38beta MAPK isoform in specific subsets of cells in postischemic brain. The activity of p38beta MAPK in the postischemic brain revealed biphasic induction at 30 min and 4 days after 1 h MCAO. During the early surge period, p38beta MAPK was preferentially localized in the nucleus and dendrites of neurons in the future infarction area, while during the delayed surge p38beta MAPK was heavily induced in reactive astrocytes in penumbra. The temporally and spatially regulated pattern of p38beta MAPK expression in the postischemic brain suggests distinct roles of p38beta MAPK in neuronal death and in the astrocyte activation.     

Neuroprotection by dimethyloxalylglycine following permanent and transient focal cerebral ischemia in rats

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1α levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.     

Inhibiting p38 mitogen-activated protein kinase attenuates cerebral ischemic injury in Swedish mutant amyloid precursor protein transgenic mice

Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB239063 into Swedish mutant amyloid precursor protein (APP/SWE) transgenic and non-transgenic mice. The number of surviving neurons in the penumbra was quantified using Nissl staining, and the activity of p38 MAPKs was measured by western blotting. The number of surviving neurons in the penumbra was significantly reduced in APP/SWE transgenic mice compared with non-transgenic controls 7 days after cerebral ischemia, but the activity of p38 MAPKs was significantly elevated compared with the non-ischemic hemisphere in the APP/SWE transgenic mice. SB239063 prevented these changes. The APP/SWE mutation exacerbated ischemic brain injury, and this could be alleviated by inhibiting p38 MAPK activity.     

GluR6-containing KA receptor mediates the activation of p38 MAP kinase in rat hippocampal CA1 region during brain ischemia injury

Our previous study showed that kainate (KA) receptor subunit GluR6 played an important role in ischemia-induced MLK3 and JNK activation and neuronal degeneration through the GluR6-PSD95-MLK3 signaling module. However, whether the KA receptors subunit GluR6 is involved in the activation of p38 MAP kinase during the transient brain ischemia/reperfusion (I/R) in the rat hippocampal CA1 subfield is still unknown. In this present study, we first evaluated the time-course of phospho-p38 MAP kinase at various time-points after 15 min of ischemia and then observed the effects of antagonist of KA receptor subunit GluR6, GluR6 antisence oligodeoxynucleotides on the phosphorylation of p38 MAP kinase induced by I/R. Results showed that inhibiting KA receptor GluR6 or suppressing the expression of KA receptor GluR6 could down-regulate the elevation of phospho-p38 MAP kinase induced by I/R. These drugs also reduced the phosphorylation of MLK3, MKK3/MKK6, MKK4, and MAPKAPK2. Additionally, our results indicated administration of three drugs, including p38 MAP kinase inhibitor before brain ischemia significantly decreased the number of TUNEL-positive cells detected at 3 days of reperfusion and increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion after 15 min of ischemia. Taken together, we suggest that GluR6-contained KA receptors can mediate p38 MAP kinase activation through a kinase cascade, including MLK3, MKK3/MKK6, and MKK4 and then induce increased phosphorylation of MAPKAPK-2 during ischemia injury and ultimately result in neuronal cell death in the rat hippocampal CA1 region.     

白果内酯抑制持续性脑栓塞大鼠水通道蛋白1，4及胶质纤维酸性蛋白的表达

实验结果发现白果内酯预处理可以降低永久性大脑中动脉闭塞大鼠脑组织含水量和梗死面积;下调水通道蛋白1,4 mRNA在水肿脑组织中的表达,继而抑制其合成,特别是在缺血的早期阶段（8 h）;抑制胶质纤维酸性蛋白的表达,减轻反应性胶质增生。说明白果内酯可通过抑制水通道蛋白的表达减轻脑水肿。