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.     

转染热休克蛋白70对p38MAPK信号通路的影响

目的：探讨热休克蛋白（HSP70）在人胶质瘤细胞BT-325p38MAPK信号通路中的作用。方法：用脂质体介导法将hsp70基因导入人胶质瘤细胞BT-325中，倒置显微镜观察转染细胞的形态学及粘附性变化，紫外线照射30min后，采用免疫组化和Western-blot方法测定转染前后HSP70的表达水平及照射前后p38MAPK表达情况。结果：免疫组化和Western-blot证实hsp70基因成功转染入BT-325中，转染细胞受到紫外线照射后p38MAPK表达减弱。结论：体外转染hsp70基因可抑制紫外线照射后BT-325细胞p38MAPK的表达。     

Upregulation of astrocytes protein phosphatase-2A stimulates astrocytes migration via inhibiting p38 MAPK in tg2576 mice

One of the earliest neuropathological changes in Alzheimer disease (AD) is the accumulation of astrocytes at sites of β-amyloid (Aβ) deposits, but the cause of this cellular response is unclear. As the activity of protein phosphatase 2A (PP2A) is significantly decreased in the AD brains, we studied the role of PP2A in astrocytes migration. We observed unexpectedly that PP2A activity associated with glial fibrillary acidic protein, an astrocyte marker, was significantly upregulated in tg2576 mice, demonstrated by an increased enzyme activity, a decreased demethylation at leucine-309 (DM-PP2Ac), and a decreased phosphorylation at tyrosine-307 of PP2A (pY307-PP2Ac). Further studies by using in vitro wound-healing model and transwell assay demonstrated that upregulation of PP2A pharmacologically and genetically could stimulate astrocytes migration. Activation of PP2A promotes actin organization and inhibits p38 mitogen-activated protein kinases (p38 MAPK), while simultaneous activation of p38 MAPK partially abolishes the PP2A-induced astrocytes migration. Our data suggest that activation of astrocytes PP2A in tg2567 mice may stimulate the migration of astrocytes to the amyloid plaques by p38 MAPK inhibition, implying that PP2A deficits observed in AD may cause Aβ accumulation via hindering the astrocytes migration.     

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.     

p38MAPK在体外培养星形胶质细胞缺氧/复氧损伤中的表达

目的 建立SD大鼠星形胶质细胞缺氧复氧损伤模型,探讨p38MAPK活性变化与星形胶质细胞损伤的关系.方法 体外培养新生SD大鼠星形胶质细胞,实验设正常对照组（N）、SB203580组（SB组,10 μmol/L）、缺氧/复氧组（H/R组）和缺氧/复氧组＋SB203580阻断p38MAPK组（H/R＋SB组）.应用MTT法、WB法、ELISA法检测缺氧4 h、8 h、复氧6 h、12 h、24 h、48 h时细胞存活率,p38MAPK、p-p38（磷酸化p38MAPK）及TNF-α的变化.结果 培养星形胶质细胞GFAP阳性表达率大于97%.缺氧/复氧使星形胶质细胞活力降低,SB203580阻断p38MAPK细胞活力高于H/R组,各组星形胶质细胞总p38MAPK水平无显著变化,缺氧复氧干预后p-p38表达上调,TNF-α水平显著增高.用SB203580阻断p38MAPK通路后,SB＋H/R组较H/R组p-p38、TNF-α水平降低.SB组总p38MAPK、p-p38、TNF-α水平与N组比较无显著变化.结论 p38MAPK信号通路参与了星形胶质细胞缺氧复氧损伤过程.     

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.     

p38 mitogen-activated protein kinase inhibitor reduces neurocan production in cultured spinal cord astrocytes

Chondroitin sulfate proteoglycans are formed in scar tissue after a spinal cord injury and inhibit axon regrowth. The production of neurocan, one of these chondroitin sulfate proteoglycans, in cultured spinal cord astrocytes increased after the addition of epidermal growth factor (EGF) in a dose-dependent manner (2-200 ng/ml). In astrocytes stimulated by 20 ng/ml of EGF, neurocan production was inhibited after the addition of the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580: 3-10 μM) in a dose-dependent manner. These results suggest that the activation of p38 MAPK is one of the mechanisms of neurocan production in EGF-stimulated astrocytes. The p38 MAPK inhibitor may reduce neurocan production and accelerate axonal regrowth after a spinal cord injury.     

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.     

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.     

Inhibition of delayed induction of p38 mitogen-activated protein kinase attenuates kainic acid-induced neuronal loss in the hippocampus

The activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in the pathological changes accompanying inflammatory and apoptotic processes of various cell types including neurons. In a kainic acid (KA)-induced mouse seizure model, p38 MAPK is induced in reactive astrocytes in the CA3 region of the hippocampus where severe neuronal loss occurs. Here we report the delayed and protracted activation of p38 MAPK in the CA3 region of the hippocampus of mice treated with KA. In this model, the inhibition of p38 MAPK isoforms by SB203580, a specific inhibitor, attenuated neuronal loss in the CA3 and CA1 regions of the hippocampus, which was accompanied by the suppression of the p38 MAPK activation as well as astrogliosis. Thus, the delayed and sustained induction of p38 MAPK plays a crucial role in the neuronal damage of KA-induced brain seizures.     

Baicalein ameliorates cognitive deficits in epilepsy-like tremor rat

Baicalein has been shown to possess various pharmacological actions. The current work was designed to assess the neuroprotection of baicalein against cognitive deficits in epilepsy-like tremor rat (TRM). Epileptic characteristics and memory functions were assessed by electroencephalograms recording and Morris water maze test, respectively. The changes of oxidative indicators including malondialdehyde (MDA), catalase (CAT), Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-SOD, glutathione (GSH), glutathione peroxidase (GSH-PX) and 8-isoprostane were measured using corresponding commercial kits. Real-time RT-PCR and immunoassay were employed to detect activities of various inflammatory mediators such as NF-κB p65, TNF-α, IL-1β, IL-6 and IL-10. Western blot analysis was performed to determine heat shock protein (HSP) 70 and mitogen-activated protein kinases (MAPKs) (including ERK, JNK and p38) proteins. Our results illustrated that baicalein significantly ameliorated epileptiform activity and cognitive deficits in TRM. Besides, reduced oxidative stress and inflammatory responses were also found in TRM treated with baicalein. Furthermore, there were evident alterations of HSP70 and MAPK cascades at protein levels after 14-day pretreatment with baicalein. It was concluded that the neuroprotective effect of baicalein against cognitive dysfunction might be associated with suppressing oxidative stress, inhibiting inflammation and mediating HSP70 as well as MAPK cascades in absence-like TRM.     

Beta-amyloid-induced inflammation and cholinergic hypofunction in the rat brain in vivo: involvement of the p38MAPK pathway

Injection into the nucleus basalis of the rat of preaggregated Abeta(1-42) produced a congophylic deposit and microglial and astrocyte activation and infiltration and caused a strong inflammatory reaction characterized by IL-1beta production, increased inducible cyclooxygenase (COX-2), and inducible nitric oxide synthase (iNOS) expression. Many phospho-p38MAPK-positive cells were observed around the deposit at 7 days after Abeta injection. Phospho-p38MAPK colocalized with activated microglial cells, but not astrocytes. The inflammatory reaction was accompanied by cholinergic hypofunction. We investigated the protective effect of the selective COX-2 inhibitor rofecoxib in attenuating the inflammatory response and neurodegeneration evoked by Abeta(1-42). Rofecoxib (3 mg/kg/day, 7 days) reduced microglia and astrocyte activation, iNOS induction, and p38MAPK activation to control levels. Cholinergic hypofunction was also significantly attenuated by treatment with rofecoxib. We show here for the first time in vivo the pivotal role played by the p38MAPK microglial signal transduction pathway in the inflammatory response to the Abeta(1-42) deposit.     

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。     

Long-term potentiation of neuronal excitation by neuron-glia interactions in the rat spinal dorsal horn

By imaging neuronal excitation in rat spinal cord slices with a voltage-sensitive dye, we examined the role of glial cells in the P2X receptor agonist alphabeta-methylene ATP (alphabetameATP)-triggered long-term potentiation (LTP) in the dorsal horn. Bath application of alphabetameATP potentiated neuronal excitation in the superficial dorsal horn. The potentiation was inhibited in the presence of the P2X receptor antagonists TNP-ATP, PPADS and A-317491, and was not induced in slices taken from rats neonatally treated with capsaicin. These results suggest that alphabetameATP acts on P2X receptors, possibly P2X(3) and/or P2X(2/3), in capsaicin-sensitive primary afferent terminals. Furthermore, the potentiation was inhibited by treatment with the glial metabolism inhibitor monofluoroacetic acid. Results obtained with the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6, and antibodies to TNF-alpha and IL-6, as well as by double immunolabelling of activated p38 MAPK with markers of astrocytes and microglia, demonstrated that alphabetameATP activated p38 MAPK in astrocytes, and that the presence of proinflammatory cytokines and p38 MAPK activation were necessary for the induction of alphabetameATP-triggered LTP. These findings indicate that glial cells contribute to the alphabetameATP-induced LTP, which might be part of a cellular mechanism for the induction of persistent pain.     

Transforming growth factor-activated kinase 1 induced in spinal astrocytes contributes to mechanical hypersensitivity after nerve injury

Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain.     

Role of p38MAPK in S1P receptor‐mediated differentiation of human oligodendrocyte progenitors

FTY720 is a sphingosine 1‐phosphate receptor (S1PR) modulator used as a daily therapy to reduce disease activity in the relapsing form of multiple sclerosis (MS). FTY720 readily accesses the CNS. Previous studies have shown that phosphorylated FTY720 (FTY720‐p) enhances oligodendrocyte progenitor cell (OPC) survival, differentiation, and remyelination following experimentally induced demyelination in rodents. To elucidate the underlying mechanism, human fetal OPCs alone or in co‐culture with rat dorsal root ganglia neurons (DRGN) were treated daily with FTY720‐p, a condition that desensitizes cellular responses to S1P, the natural ligand of S1PR. In co‐cultures, FTY720‐p and S1P given daily or every three days increased the number of O1/MBP double positive cells and axonal ensheathment. In cultures composed of PDGFRα‐antibody selected cells alone, daily application of FTY720‐p also increased the number of O4/GC double positive cells. At an early time point (day 2), FTY720‐p activated ERK1/2, CREB and p38MAPK in O4‐positive cells, as well as in β‐III Tubulin positive neurons and GFAP positive astrocytes. In later cultures (day 6), FTY720‐p activated p38MAPK in O4 positive cells, p38MAPK and ERK1/2 in neurons, and p38MAPK, ERK1/2 and CREB in astrocytes. A MEK inhibitor (U0126) prevented the differentiation of OPCs into O4‐positive cells, while a p38MAPK inhibitor (PD169316) blocked progression into O4‐positive and into GC‐positive stages of differentiation. Our results demonstrate that FTY720‐p, under conditions that model daily clinical use, can act directly on OPCs to impact differentiation, and also indirectly via neurons and astrocytes by activating ERK1/2 and p38MAPK. GLIA 2014;62:1361–1375     

Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.