ERK/P38MAPK信号转导通路在人脐动脉血管平滑肌细胞表型转化中的作用

目的探讨血小板源生长因子-BB（PDGF-BB）及米诺环素是否可通过调节ERK／P38MAPK信号通路,从而影响人脐动脉血管平滑肌细胞（HASMC）的表型转化。方法建立HASMC体外培养模型,分为无血清的DMEM、PDGF-BB（20ng／ml）、PDGF-BB（20ng／ml）＋PD98059（30μmol／L）＋SB203580（20μmol／L）、PDGF-BB（20ng／ml）＋米诺环素（15μmol／L）、PDGF-BB（20ng／ml）＋米诺环素（30μmol／L）五组,Western Blot法检测ERK1／2、P-ERK1／2、P38、P—P38蛋白表达。结果体外培养的HASMC在PDGF-BB（20ng／ml）＋米诺环素（15μmol／L）培养液孵育24h后,P-P38蛋白表达较PDGF-BB组明显下降（P〈0．01）;在PDGF-BB（20ng／ml）＋米诺环素（30μmol／L）组,P—ERK1／2和P—P38蛋白表达均较PDGF-BB组明显下降（P〈0．01）,表明米诺环素显著抑制ERK/P38MAPK信号通路。结论（1）PDGF-BB诱导HASMC的去分化与ERK／P38MAPK信号通路有关,如抑制ERK／P38MAPK信号通路的活性,则HASMC保持分化表型;（2）米诺环素对PDGF-BB诱导HASMC去分化的抑制作用是通过抑制ERK／P38MAPK信号通路的活性,下调PDGF-BB诱导的ERK1／2和P38磷酸化水平而实现的,与其对HASMC的细胞毒性无关。     

Neuroprotective effects of silibinin: an in silico and in vitro study

Aim of the study: Astrogliosis is a key contributor for many neurological disorders involving apoptosis, neuroinflammation and subsequent neuronal death. Silibinin, a polyphenol isolated from milk thistle (Silybum marianum), has been shown to suppress the astrocyte activation in various neurodegenerative disorders and also exhibit a neuroprotective role in neuroinflammation-driven oxidative damage. The present study was designed with an aim to investigate the neuroprotective effects of Silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation.

Materials and methods: We have used in-silico molecular modelling techniques to study the interaction and binding affinity of silibinin with chemokine receptors associated with neuroinflammation. We have also tested silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation in C6 glia cell lines.

Results: In the present study, we found that treatment with silibinin significantly attenuates LPS-oxidative-nitrosative stress in C6 astrocytoma cells. We also observed the significant inhibition of induced astrocyte activity after treatment with silibinin. Moreover, molecular modelling studies have proposed a binding pose of silibinin with binding sites of p38 MAPK, CX3CR1 and P2X4 which is an important downstream cascade involved in glia cell activation and neuroinflammation.

Conclusions: Overall, the findings from the current study suggests that silibinin exhibits neuroprotective activity by attenuating oxidative damage and astrocytes activation.     

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.     

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.     

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。     

Cadmium-induced IL-6 and IL-8 expression and release from astrocytes are mediated by MAPK and NF-κB pathways

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24 h post-exposure to 1 and 10 μM, levels of intracellular cadmium in U-87 MG cells were 11.89 ± 3.59 and 53.08 ± 7.59 μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.     

局灶脑缺血再灌流后ICAM-1表达与白细胞浸润

目的　探讨大鼠局灶性脑缺血再灌流后细胞间粘附分子 - 1(ICAM- 1)表达规律与白细胞浸润。方法　采用大鼠 MCA线栓闭塞 /再通法建立局灶性缺血 /再灌流模型 ,动态观察 ICAM- 1、髓过氧化物酶 (MPO)变化情况。结果　 (1)鼠脑缺血 /再灌流后 MCA供血区皮质及梗死周边区的微血管 ICAM- 1表达于再灌流 6 h已增强 ,36～ 48h达高峰 ,以后逐渐减弱 ,但第 7天表达仍强于假手术组。(2 )坏死周边区神经元也表达 ICAM- 1。(3)再灌流 6 h出现白细胞浸润 ,浸润高峰在 2 4～ 36 h,6 d后恢复正常。结论　 (1)缺血再灌流使微血管表达 ICAM- 1上调 ,同时也伴随坏死周边区的神经元表达 ICAM- 1。 (2 )白细胞浸润与 ICAM- 1表达规律同步。     

Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3

Oxidative stress in the brain has been increasingly associated with the development of numerous human neurological diseases. Microglia, activated upon neuronal injury or inflammatory stimulation, are known to release superoxide anion (*O(2) (-)), hydrogen peroxide (H(2)O(2)), and nitric oxide (NO), thereby further contributing to oxidative neurotoxicity. The reaction of NO and *O(2) (-), forming the toxic peroxynitrite (ONOO(-)), has been proposed to play a pathogenic role in neuronal injury. However, the interactions between H(2)O(2) and NO during oxidative stress, which may promote or diminish cell death, is less clear. In this study, we explored oxidative neurotoxicity induced by H(2)O(2) plus NO in primary cultures of rat cerebral cortex neurons. As the mechanisms may involve reactions between H(2)O(2) and NO, we monitored the production of ONOO(-)and reactive oxygen species (ROS) throughout the experiments. Results indicated that the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and H(2)O(2) by themselves elicited neuronal death in a concentration- and time-dependent manner. Sublytic concentrations of H(2)O(2) plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. Transient ONOO(-)increase was accompanied by rapid H(2)O(2) decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation and the cleavage of caspase-3 were observed. Conversely, inhibition of p38 MAPK and caspase-3 significantly reduced apoptotic death induced by H(2)O(2) plus SNAP. These data suggest that H(2)O(2) and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase-3 is involved.     

p38MAPK对耐苯妥英钠和卡马西平癫痫模型大鼠电生理和行为的影响

目的 观察p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38MAPK)信号通路抑制剂SB202190对耐苯妥英钠和卡马西平大鼠电生理指标和行为学的影响.方法 建立慢性杏仁核点燃癫痫模型,筛选出耐药大鼠,通过侧脑室注射SB202190,对照组注射生理盐水.观察其对各组大鼠后放电阈值(after discharge threshold,ADT)和行为学的影响.结果 相对于对照组,耐药组大鼠在给予SB202190后,ADT明显高于对照组(P＜0.05);Racine行为分级明显下降(P＜0.05).结论 阻断p38MAPK信号通路可以协助AEDs改善耐药大鼠的电生理活动,降低点燃后发作分级,提示p38MAPK信号通路参与了难治性癫痫的耐药.     

Abnormal microglial activation in the Cstb−/− mouse,a model for progressive myoclonus epilepsy,EPM1

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1) is an autosomal‐recessively inherited neurodegenerative disorder characterized by severely incapacitating myoclonus, seizures, and ataxia, and caused by loss‐of‐function mutations in the cystatin B gene (CSTB). A central neuropathological finding in the Cstb^?/? mouse, an animal model for EPM1, is early microglial activation, which precedes astroglial activation, neuronal loss, and onset of myoclonus, thus implying a critical role for microglia in EPM1 pathogenesis. Here, we characterized phenotypic and functional properties of microglia from Cstb^?/? mice utilizing brain tissue, microglia directly isolated from the brain, and primary microglial cultures. Our results show significantly higher Cstb mRNA expression in microglia than in neurons and astrocytes. In Cstb^?/? mouse brain, expression of the inflammatory marker p‐p38 MAPK and the proportion of both pro‐inflammatory M1 and anti‐inflammatory M2 microglia is higher than in control mice. Moreover, M1/M2 polarization of microglia in presymptomatic Cstb^?/? mice is, compared to control mice, skewed towards M2 type at postnatal day 14 (P14), but towards M1 type at P30, a time point associated with onset of myoclonus. At this age, the high expression of both pro‐inflammatory inducible nitric oxide synthase (iNOS) and anti‐inflammatory arginase 1 (ARG1) in Cstb^?/? mouse cortex is accompanied by the presence of peripheral immune cells. Consistently, activated Cstb^?/? microglia show elevated chemokine release and chemotaxis. However, their MHCII surface expression is suppressed. Taken together, our results link CSTB deficiency to neuroinflammation with early activation and dysfunction of microglia and will open new avenues for therapeutic interventions for EPM1. GLIA 2015;63:400–411     

CX3CL1-mediated macrophage activation contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy

Painful peripheral neuropathy is a dose-limiting side effect of paclitaxel therapy, which hampers the optimal clinical management of chemotherapy in cancer patients. Currently the underlying mechanisms remain largely unknown. Here we showed that the clinically relevant dose of paclitaxel (3 × 8 mg/kg, cumulative dose 24 mg/kg) induced significant upregulation of the chemokine CX3CL1 in the A-fiber primary sensory neurons in vivo and in vitro and infiltration of macrophages into the dorsal root ganglion (DRG) in rats. Paclitaxel treatment also increased cleaved caspase-3 expression, induced the loss of primary afferent terminal fibers and decreased sciatic-evoked A-fiber responses in the spinal dorsal horn, indicating DRG neuronal apoptosis induced by paclitaxel. In addition, the paclitaxel-induced DRG neuronal apoptosis occurred exclusively in the presence of macrophage in vitro study. Intrathecal or systemic injection of CX3CL1 neutralizing antibody blocked paclitaxel-induced macrophage recruitment and neuronal apoptosis in the DRG, and also attenuated paclitaxel-induced allodynia. Furthermore, depletion of macrophage by systemic administration of clodronate inhibited paclitaxel-induced allodynia. Blocking CX3CL1 decreased activation of p38 MAPK in the macrophage, and inhibition of p38 MAPK activity blocked the neuronal apoptosis and development of mechanical allodynia induced by paclitaxel. These findings provide novel evidence that CX3CL1-recruited macrophage contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy.     

Activation of G protein-coupled receptor 30 protects neurons by regulating autophagy in astrocytes

Ischemic stroke leads to neuronal damage induced by excitotoxicity, inflammation, and oxidative stress. Astrocytes play diverse roles in stroke and ischemia-induced inflammation, and autophagy is critical for maintaining astrocytic functions. Our previous studies showed that the activation of G protein-coupled receptor 30 (GPR30), an estrogen membrane receptor, protected neurons from excitotoxicity. However, the role of astrocytic GPR30 in maintaining autophagy and neuroprotection remained unclear. In this study, we found that the neuroprotection induced by G1 (GPR30 agonist) in wild-type mice after a middle cerebral artery occlusion was completely blocked in GPR30 conventional knockout (KO) mice but partially attenuated in astrocytic or neuronal GPR30 KO mice. In cultured primary astrocytes, glutamate exposure induced astrocyte proliferation and decreased astrocyte autophagy by activating mammalian target of rapamycin (mTOR) and c-Jun N-terminal kinase (JNK) and inhibiting p38 mitogen-activated protein kinase (MAPK) pathway. G1 treatment restored autophagy to its basal level by regulating the p38 pathway but not the mTOR and JNK signaling pathways. Our findings revealed a key role of GPR30 in neuroprotection via the regulation of astrocyte autophagy and support astrocytic GPR30 as a potential drug target against ischemic brain damage.     

Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance

Compelling evidence has suggested that spinal glial cells were activated by chronic morphine treatment and involved in the development of morphine tolerance. However, the mechanisms of glial activation were still largely unknown in morphine tolerance. In present study, we investigated the role of p38 mitogen-activated protein kinase (p38 MAPK) in the spinal cord in the development of chronic morphine antinociceptive tolerance. We found that intrathecal administration of morphine (15 microg) daily for 7 consecutive days significantly induced an increase in number of phospho-p38 (p-p38) immunoreactive cells in the spinal cord compared with chronic saline or acute morphine treated rats. Double immunofluorescence staining revealed that p-p38 immunoreactivity was exclusively restricted in the activated spinal microglia, not in astrocytes or neurons. Repeated intrathecal administration of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (10 microg or 2 microg), a specific p38 inhibitor, 30 min before each morphine injection for 7 consecutive days significantly attenuated tolerance to morphine analgesia assessed by tail flick test. However, a single intrathecal administration of SB203580 (10 microg) did not antagonize the established tolerance to morphine analgesia. Taken together, these findings suggested that p38 MAPK activation in the spinal microglia was involved in the development of morphine antinociceptive tolerance. Inhibition of p38 MAPK by SB203580 in the spinal cord attenuated but not reversed the tolerance to morphine analgesia. The present study provides the first evidence that p38 activation in spinal microglia played an important role in the development of tolerance to morphine analgesia.     

Forced retraction of spinal root injury enhances activation of p38 MAPK cascade in infiltrating macrophages

Root‐rupture injury is a type of preganglionic brachial plexus injury resulting from traction force, where a small section of the spinal root is usually left behind. We have established experimental models of both root‐rupture injury with traction force and rhizotomy without traction force in rats and we examined the activation of microglia/macrophages in both conditions. LGP107 and LGP96, which are rat homologs of lysosome‐associated membrane proteins, were most useful as immunohistochemical markers of mononuclear phagocytes. The metabolic activation of macrophages was analyzed by immunohistochemistry with a series of antibodies against tumor necrosis factor‐alpha (TNF‐alpha), cathepsin B, p38 mitogen‐activated protein kinase (MAPK), and mitogen‐activated kinase kinase 3 (MKK3). Both root‐rupture injury and rhizotomy rapidly induced the aggregation of numerous macrophages from the injured dorsal root to the dorsal funiculus and TNF‐alpha was highly expressed by the macrophages in the injured dorsal root at 48 h. Activation of p38 MAPK was preferentially observed in the macrophages at the ruptured dorsal root; however, only slight activation of p38 MAPK was observed at the rhizotomized dorsal root. These findings suggest that traction injury of the spinal root might induce activation of the p38 MAPK cascade and production of TNF‐alpha in the infiltrating macrophages, both of which might participate in aggravation of the root injury.     

Recombinant human TNF-binding protein-1 (rhTBP-1) treatment delays both symptoms progression and motor neuron loss in the wobbler mouse

TNF-α overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-α in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-α and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-α was further confirmed by the protective effect of treatment with rhTNF-α binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-α and TNFR1 levels. rhTBP-1 might possibly bind TNF-α and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.