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.     

Delayed and differential induction of p38 MAPK isoforms in microglia and astrocytes in the brain after transient global ischemia

The p38 MAPK signaling pathway has been implicated in various pathological conditions of neuronal and non-neuronal cells. Here we report the differential induction of p38 MAPK isoforms, p38alpha and p38beta, in the adult gerbil brain following transient global ischemia. The p38alpha and p38beta kinase activities were gradually enhanced with the peak activity occurring around 2-4 days after ischemic insult. Immunohistochemical analysis revealed that p38alpha expression was increased as early as 4 h after ischemic insult and enhanced further reaching maximum induction around 4 days after ischemia. The induced p38alpha was concentrated in microglia in hippocampus as well as in frontal and parietal cortices of the brain, where significant neuronal damage was occurred. By contrast, immunostaining with anti-p38beta antibody indicated that p38beta was markedly induced in astrocytes in hippocampus around 4 days after ischemic insult, which lasted for the next several days. The differential induction of p38 MAPK isoforms following transient global ischemia, especially the induction of p38alpha and p38beta MAPKs in microglia and astrocytes, respectively, in different time points after ischemic insult suggest distinct roles of p38 MAPK isoforms in post-ischemic brain.     

Constitutive activity and differential localization of p38alpha and p38beta MAPKs in adult mouse brain

To understand the roles of p38 mitogen-activated protein kinase (p38 MAPK) isoforms in adult mouse brain, in vivo activities and detailed expression patterns of two p38 isoforms, p38alpha and p38beta, were examined by using biochemical and immunohistochemical analyses. The result indicated that the activity of both p38alpha and p38b MAPKs in normal adult mouse brain was remarkably high, and the nuclear pool of the p38 isoforms was primarily responsible for most of the constitutive p38 MAPK activity in brain. Both p38alpha and p38beta were highly expressed in brain areas including cerebral cortex, hippocampus, cerebellum, and few nuclei of the brainstem. At the subcellular level, p38alpha was distributed in dendrites and in cytoplasmic and nuclear regions of cell body of neurons, which is in contrast to p38beta, since p38beta was preferentially expressed in nucleus of neurons. These results suggest that the p38 pathway may play an important role, not only in inflammation and neuronal cell death as previously suggested, but also in normal physiology of adult mouse brain.     

p38 mitogen-activated protein kinase modulates expression of tumor necrosis factor-related apoptosis-inducing ligand induced by interferon-gamma in fetal brain astrocytes

This study describes the involvement of the p38 mitogen-activated protein kinase (MAPK) during interferon-gamma (IFN-gamma) signaling in fetal brain astrocytes. In some pathological conditions of brain, p38 MAPK transduces stress-related signals, increases expression of proinflammatory cytokines, and induces cellular damage or apoptosis. In astrocytes, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression level was increased by IFN-gamma. AG490, a JAK inhibitor, blocked TRAIL expression induced by IFN-gamma. SB203580, a specific p38alpha and p38beta2 MAPK inhibitor, decreased the TRAIL expression induced by IFN-gamma. The phosphorylation of the Ser727 site of STAT1, but not the Tyr701 site, was inhibited by SB203580. These results suggest that p38 MAPK modulates STAT1 phosphorylation in IFN-gamma signaling in fetal brain astrocytes.     

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。     

Delayed induction of p38 MAPKs in reactive astrocytes in the brain of mice after KA-induced seizure

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated in pathological changes in inflammatory and apoptotic processes in various cell types including neurons. Here we report the delayed induction of p38 MAPKs in the brain of mice following kainic acid (KA)-induced seizure. The immunoreactivities of p38alpha and p38beta MAPKs were markedly increased in the brain 4 days after KA administration, especially in the areas undergoing selective neuronal loss. In particular, p38beta was dramatically increased in reactive astrocytes of CA3 and CA1 regions of hippocampus with its enriched localization in the nucleus of astrocytes. The induction of p38beta was sustained for more than 10 days after KA-treatment. Pre-administration of the selective neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI), which suppressed the delayed neuronal death as well as astrogliosis in hippocampus of seizure-experienced animals, dramatically repressed the delayed induction of p38beta MAPK in astrocytes. The repression was reversed by the co-injection with L-arginine (L-arg), a substrate for NOS, which coincided with the aggravation of neuronal death. Together, these data suggested a role of p38 MAPK signal pathway in delayed neuronal death and/or in reactive gliosis in mice with KA-induced seizure.     

p38丝裂原活化蛋白激酶在实验性蛛网膜下腔出血后早期脑损伤中的作用

目的探讨p38丝裂原活化蛋白激酶(p38MAPK)在蛛网膜下腔出血(SAH)后早期脑损伤(EBI)中的作用。方法成年雄性SD大鼠随机分配至对照组、SAH组及p38MAPK干预组,每组18只。采用血管内穿刺法制作SAH模型,干预组于术前30 min经侧脑室注射p38MAPK特异性抑制剂SB203580,造模后24 h处死。观察各组大鼠脑含水量和神经功能评分,RT-PCR及免疫组化检测脑组织p38MAPK表达。结果与对照组相比,SAH组大鼠脑含水量(t=-196.35,P0.01)及p38 MAPK的mRNA水平(t=-24.75,P0.01)均明显升高,神经功能评分明显减低(t=201.08,P0.01)。与SAH组相比,干预组脑含水量(t=75.67,P0.01)及p38 MAPK的mRNA水平(t=9.43,P0.01)均明显下降,神经功能评分明显升高(t=-81.68,P0.01)。免疫组化示SAH组及干预组均有p38MAPK表达,但干预组较SAH组表达水平明显下降(t=-3.37,P0.01)。结论 p38 MAPK在EBI形成机制中起重要作用,有望成为防治EBI的药物作用新靶点。     

Inhibitors of p38 mitogen-activated protein kinase enhance proliferation of mouse neural stem cells

The p38 mitogen-activated protein kinase (MAPK) is induced in response to environmental stress. Although p38 MAPK has been implicated in diverse cellular processes, including cell proliferation, differentiation, and survival of differentiated cells in the central nervous system (CNS), the expression profile and roles of p38 MAPK in the developing brain remain largely unknown. In the present study, we demonstrate that p38 MAPK is expressed predominantly in nestin-positive cells in the cerebral cortex in embryonic day 10 (E10) brain and that expression of the protein decreases gradually during development. To investigate the roles of p38 MAPK in the embryonic brain, two selective p38 MAPK inhibitors, SB202190 and SB203580, were added to the primary neuronal cultures from E10-E14 brains. After 7 days of exposure to these inhibitors, but not SB202474, a negative analog of SB203580, numerous large neurospheres were present. MAPK inhibitors also selectively increased the growth rate of neural stem cells (NSCs) purified from secondary neurospheres and the number of bromodeoxyuridine-positive NSCs. Thus, p38 MAPK inhibitors are potent stimulators of NSC proliferation, and p38 MAPK may be an intrinsic negative regulator of NSC proliferation during early brain development.     

实验性大鼠尾壳核脑出血后p38MAPK、ICAM-1的动态表达

目的探讨脑出血后血肿周围组织p38丝裂原活化蛋白激酶(p38M APK)和细胞间粘附分子-1(I-CAM-1)的动态变化。方法健康雄性W istar大鼠42只,将动物随机分成假手术组和脑出血组,采用免疫组织化学方法观察术后不同时间点p38M APK和ICAM-1的动态变化。结果脑出血组各时间点血肿周围组织均有不同程度磷酸化p38M APK阳性细胞表达,脑出血后3h周围组织即有表达,于6h出现广泛性表达,24h时达最高峰,持续至5d仍有表达。ICAM-1的表达在48h达高峰,随后渐下降。结论脑出血后脑组织损伤诱导p38M APK和ICAM-1的表达,二者可能参与了脑出血后脑组织损伤的病理机制。     

p38MAPK activation and DUSP10 expression in meningiomas

The mitogen activated protein kinase (MAPK) p38MAPK has been implicated in regulation of cell proliferation and apoptosis. However, expression, activation and regulation has not been studied in meningiomas, to our knowledge. p38MAPK is regulated, in part, by dual specificity phosphatases (DUSP) that inactivate signaling by dephosphorylation. DUSP10 is also a likely participant in regulating meningioma proliferation. Five fetal and an adult human leptomeninges and 37 meningioma cultures (MC) were evaluated for DUSP10 as well as phosphorylation of its substrates p38MAPK and p44/42MAPK by western blot and DUSP10 expression by polymerase chain reaction. Platelet derived growth factor-BB (PDGF-BB), transforming growth factor B1 (TGFB1) and cerebrospinal fluid effects on DUSP10 and signaling were also studied in vitro. DUSP10 and phospho-p38MAPK and phospho-p44/42MAPK were detected in all six leptomeninges. DUSP10 was detected in 13 of 17 World Health Organization grade I, 11 of 14 grade II and four of six grade III meningiomas. Phospho-p38MAPK was detected in nine of 17 grade I, two of six grade II, and four of six grade III meningiomas. In the majority of meningiomas DUSP10 expression correlated inversely with phosphorylation of p38MAPK. PDGF-BB increased DUSP10 in MC2 and MC4 and weakly in MC3. TGFB1 increased phosphorylation of p38MAPK and caspase 3 activation. Thus p38MAPK and DUSP10 likely participate in the pathogenesis of meningiomas.     

Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia-reperfusion.

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1beta and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1beta, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.     

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。     

Activation of p38 MAPK participates in brain ischemic tolerance induced by limb ischemic preconditioning by up-regulating HSP 70

This study investigates whether activation of p38 MAPK by the up-regulation of HSP 70 participates in the induction of brain ischemic tolerance by limb ischemic preconditioning (LIP). Western blot and immunohistochemical assays indicated that p38 MAPK activation occurred earlier than HSP 70 induction in the CA1 region of the hippocampus after LIP. P-p38 MAPK expression was up-regulated at 6 h and reached its peak 12 h after LIP, while HSP 70 expression was not significantly increased until 1 day and peaked 2 days after LIP. Neuropathological evaluation by thionin staining showed that quercetin (4 ml/kg, 50 mg/kg, intraperitoneal injection), an inhibitor of HSP 70, blocked the protective effect of LIP against delayed neuronal death that is normally induced by lethal brain ischemic insult, indicating that HSP 70 participates in the induction of brain ischemic tolerance by LIP. Furthermore, SB 203580, an inhibitor of HSP 70, inhibited HSP 70 activation in the CA1 region of the hippocampus induced by LIP either with or without the presence of subsequent brain ischemic insult. Based on the above results, it can be concluded that activation of p38 MAPK participates in the brain ischemic tolerance induced by LIP at least partly by the up-regulation of HSP 70 expression.     

MAPK信号通路在创伤性脑损伤中的作用

目的通过建立小鼠创伤性脑损伤(TBI)模型,研究丝裂原活化蛋白激酶(MAPKs)通路中的细胞外调节蛋白激酶1/2(ERK1/2)通路、JNK通路和p38通路的激活及在TBI中的作用及机制。方法建立小鼠TBI模型,通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,确定TBI后MAPK通路的激活情况;分别加入ERK1/2通路抑制剂(PD98059,500μmol/L)、JNK通路抑制剂(SP600125,500μmol/L)和p38通路抑制剂(SB203580,500μmol/L),通过脑干湿重检测、神经功能学评分和TUNEL染色评估不同抑制剂对TBI的作用,并通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,明确ERK1/2通路、JNK通路和p38通路之间的相互调节作用。结果 TBI可分别引起ERK1/2通路、JNK通路和p38通路的激活;抑制ERK通路和JNK通路可减轻TBI引起的脑水肿、神经功能损伤和细胞凋亡,而抑制p38通路则加重TBI引起的脑水肿、神经功能损伤和细胞凋亡;抑制JNK通路可减少ERK1/2的相对磷酸化水平,而抑制p38通路可增加ERK1/2的相对磷酸化水平。结论 TBI后,ERK1/2通路和JNK通路的激活发挥促进损伤形成的作用,而p38通路的激活则起到神经保护的作用;ERK1/2通路的激活受到JNK通路的促进和p38通路的抑制,表明MAPK通路之间存在相互调节。     

Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

The pathological basis of neonatal hypoxia–ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI‐induced neuronal cell death. The p38 mitogen‐activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase‐3 and the appearance of apoptotic neuronal cells. Pre‐treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I‐1‐driven adeno‐associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD‐mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.     

The effects of prenatal stress on expression of p38 MAPK in offspring hippocampus

The hippocampus, which has the highest density of GC receptors in the brain, is involved in the regulation of the HPA and the behavioral responses to stress. Overexposure to corticosteroid hormones is harmful to hippocampal neuron integrity. Our purpose is to investigate the effects of prenatal stress (PNS) on expression of p38 mitogen-activated protein kinase (p38 MAPK) in offspring hippocampal neurons using Western blotting and Immunohistochemistry. The prenatal restraint stress induces significant increase in the expression of p-p38 MAPK and total p38 MAPK in female offspring hippocampus. The level of p-p38 MAPK in PNS female offspring rats was significantly increased (126.41+/-3.937, n=6) compared with that in the control female offspring rats (101.35+/-3.468, n=6, P<0.01). Immunoblot analysis revealed there was significant difference in the level of total p38 MAPK between the female control and prenatal restraint stress offspring rats (101.70+/-3.162 vs. 128.111+/-2.724, respectively, P<0.01). Immunodensity of p38 MAPK was significantly increased above female control in PNS female offspring hippocampal CA3 and CA4 fields (P<0.001 vs. control group, CON). The data suggest that exposure of animals to a period of stressful experience during a critical phase could impose lasting effects on the offspring hippocampal neurons cellular signalling of offspring hippocampus.     

NOS、p38MAPK、Caspase-3介导大鼠脑缺血神经细胞凋亡可能通路的实验研究

目的探讨脑缺血后细胞凋亡发生的可能机制以及神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、p38丝裂原活化蛋白激酶(mitogen activated proteinkinasep38,p38MAPK)和半光氨酸蛋白酶-3(caspase-3)在脑缺血后神经细胞凋亡中的共同作用机制。方法采用线栓法闭塞大鼠大脑中动脉(middle cerebral artery occlusion,MACO)建立脑缺血SD大鼠模型,应用透射电镜观察脑缺血对脑组织超微结构的影响,流式细胞仪方法(FCM)分别定量检测细胞凋亡率,半定量RT-PCR检测nNOS、iNOS,p38MAPK和Caspase-3mRNA表达水平。结果透视电镜下脑缺血6h出现核固缩,缺血12h出现细胞核分裂,缺血24h出现凋亡小体;FCM检测细胞凋亡百分率随着缺血时间延长而增加,缺血72h达到高峰,约70.37%;RT-PCR产物的琼脂糖凝胶电泳显示nNOS、iNOS、p38MAPK和Caspase-3mRNA的特异性片段大小分别为501、342、250和342bp,但mRNA表达量不一致,nNOS mRNA主要在缺血早期表达,iNOS、p38MAPK和Caspase-3mRNA在缺血中晚期表达,并在缺血3~5d,后三种基因的表达量达到高峰。结论脑缺血区域发生典型的神经细胞凋亡现象,nNOS来源的NOS在缺血早期发挥神经毒性作用,iNOS来源的NOS在缺血晚期发挥神经毒性作用;NOS,p38MAPK和Caspase-3三种基因的相互关系可能构成介导缺血神经细胞凋亡的通路之一。     

精氨酸加压素对星形胶质细胞水孔蛋白-4表达的调节及脑水肿形成影响的研究

目的研究精氨酸加压素(AVP)对星形胶质细胞水孔蛋白-4(AQP4)表达的调节,以及p38 MAPK信号通路在AQP4表达过程的作用,明确AVP及AQP4在脑水肿发生过程中的作用。方法大鼠大脑皮质分离星形胶质细胞,星形胶质细胞经分别用AVP、V1a受体(V1aR)拮抗剂和SB 203580进行处理,采用免疫组织化学技术及RT-PCR对AQP4 mRNA进行检测,Western blot检测p38 MAPK信号通路在AVP诱导AQP4表达中的活化程度。结果500nmol/L的AVP处理6h后,AQP4 mRNA表达开始升高(P<0.01),到12h达高峰(P<0.01),24h后仍维持在较高的水平(P<0.05)。加入p38 MAPK抑制剂SB 203580干预后,AQP4 mRNA表达水平与对照组比较差异不显著(P>0.05);AVP处理15min后p38 MAPK磷酸化水平开始增加,30min达高峰,持续到60min开始下降。V1aR拮抗剂处理后p38 MAPK磷酸化水平整个时间段均未出现明显变化。结论AVP通过激活V1aR引起p38MAPK信号通路活化从而诱导AQP4 mRNA高表达,从基因水平对AQP4进行调节,可能在脑水肿发生中,尤其是在星形胶质细胞水肿形成中起重要作用。V1aR拮抗剂及p38 MAPK抑制剂能抑制AQP4 mRNA的表达,避免星形胶质细胞肿胀。