Activation of MAPKs in the anti-β(2)GPI/β(2)GPI-induced tissue factor expression through TLR4/IRAKs pathway in THP-1 cells

Our previous study has demonstrated that the Toll-like receptor 4 (TLR4) signaling pathways contribute to the induction of tissue factor (TF) expression by anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI) in human acute monocytic leukemia cell line THP-1. In this study, we focused on the identification of the downstream targets of the TLR4 pathways. When THP-1 cells were treated with anti-β₂GPI/β₂GPI complex, enhanced TF expression was observed, along with induced phosphorylation of p38, ERK1/2 and JNK1/2 MAPKs. When the activity of MAPKs was blocked by their corresponding inhibitors (SB203580: p38; U0126: ERK; SP600125: JNK), the expression of TF was reduced significantly. Furthermore, the anti-β₂GPI/β₂GPI-induced phosphorylation of p38, ERK1/2 and JNK1/2 was inhibited significantly by TAK-242, a blocker of the signaling transduction mediated by the intracellular domain of TLR4; sc-204013, a specific inhibitor of IRAKs, was also able to partially inhibit the phosphorylation of the MAPKs. Our results demonstrated that MAPKs (p38, ERK1/2 and JNK1/2) were the crucial downstream targets of the anti-β₂GPI/β₂GPI-triggered TLR4 signaling pathways in THP-1 cells. This essential role of MAPKs may also promote better understanding of the pathogenesis of antiphospholipid syndrome (APS).     

Activation of mitogen-activated protein kinases after permanent cerebral artery occlusion in mouse brain.

The purpose of this study was to examine the activation, topographic distribution, and cellular location of three mitogen-activated protein kinases (MAPKs) after permanent middle cerebral artery occlusion (MCAO) in mice. Phosphorylated MAPKs expression in the ischemic region was quantified using Western blot analysis and localized immunohistochemically using the diaminobenzide staining and double-labeled immunostaining. Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38 mitogen-activated protein (p38), and c-Jun NH2-terminal kinase or stress-activated protein kinase (SAPK/JNK) were initially activated at 30 minutes, 10 minutes, and 5 minutes, respectively, after focal cerebral ischemia. Peak expression represented a 2.7-fold, 3.7-fold, and 4.8-fold increase in each of these MAPKs, respectively. The immunohistochemical expressions of ERK1, ERK2, p38, and SAPK/JNK protein paralleled the Western blot analysis results. Double-labeled immunofluorescent staining demonstrated that the neurons and astrocytes expressed ERK1, ERK2, p38, and SAPK/JNK during the early time points after MCAO. The current results demonstrate that brain damage after ischemia rapidly triggers time-dependent ERK1, ERK2, p38, and SAPK/ JNK phosphorylation, and reveals that neurons and astrocytes are involved in the activation of the MAPK pathway. This very early expression of MAPKs suggests that MAPKs may be closely involved in signal transduction during cerebral ischemia.     

The muscle mitogen-activated protein kinase is altered in sporadic inclusion body myositis

OBJECTIVE: To examine the origin of hyperphosphorylated proteins within the vacuolated myofibers in sporadic inclusion body myositis (s-IBM) and search for dysregulated intracellular protein phosphorylation. BACKGROUND: s-IBM is morphologically characterized by primary endomysial inflammation and vacuolated myofibers containing tubulofilaments that originate from cytoskeletal proteins. Mitogen-activated protein kinases (MAPKs) play a role in regulating phosphorylation and maintaining the stability of the cytoskeletal architecture. METHODS: Muscle biopsies from seven patients with s-IBM and 15 controls were examined for the expression of the active components of the various MAPKs, including p44/42MAPK, p38MAPK, p46JNK1, p54JNK2, and p54JNK3, using immunocytochemistry and Western blot analysis. The expression of selected phosphorylated components was also examined in the same specimens. RESULTS: In s-IBM, but not the disease controls, the vacuolated muscle fibers express active p42MAPK but not JNK or p38MAPK. Western blots of cell lysates confirmed the hyperexpression of p42MAPK and demonstrated a novel 35 kD phosphoprotein. Antibodies against phosphoepitopes of the 35 kD protein preferentially immunostained antigens within the vacuolated muscle fibers of s-IBM but not disease controls. CONCLUSION: In s-IBM, there is increased p42MAPK activation and abnormal intracellular protein phosphorylation with selective accumulation of a 35 kD phosphoprotein within the vacuolated fibers. Although the hyperexpression of 35kD protein may represent cytoskeletal by-products due to heightened p42MAPK activation, its abundant expression only in s-IBM implies that hyperphosphorylated myofibrillar proteins may be involved in the primary disease process.     

Comparative study of vanadate- and phorbol ester-induced cyclo-oxygenase-2 expression in human endothelial cells

Our previous study showed that vanadate, an inhibitor of protein tyrosine phosphatases, induced the expression of cyclo-oxygenase (COX)-2 in a protein-tyrosine-kinase (PTK)-dependent manner in human umbilical vein endothelial cells (HUVEC). Here, we further compared the actions of vanadate and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), on induction of COX-2 with special reference to mitogen-activated protein kinases (MAPKs) in HUVEC. Vanadate induced activation of three families of MAPKs, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun amino-terminal kinase (JNK) 1, while activation of ERK 1/2 alone was induced by PMA. The former activation by vanadate and the latter one by PMA were inhibited by tyrphostin-47, an inhibitor of PTKs, and by Ro31-8220, a PKC inhibitor, respectively. Either tyrphostin-47, PD98059, a specific inhibitor of the upstream kinase toward ERK1/2, or SB203580, a specific inhibitor of p38, completely suppressed vanadate-induction of COX-2 mRNA and protein. On the other hand, PMA-induction of COX-2 mRNA and protein was abolished by Ro31-8220 or PD98059 but not by SB203580. These data indicate that PMA-induced and PKC-dependent expression of COX-2 requires mainly activation of ERK 1/2 among MAPKs, while activation of both ERK1/2 and p38 or possibly of all three families of MAPKs is necessary for vanadate-induced and PTK-dependent expression of COX-2.     

Evidence that p38 mitogen-activated protein kinase contributes to neonatal hypoxic-ischemic brain injury

We tested the response of stress-activated mitogen-activated protein kinases (MAPKs) - p38 MAPK and c-JUN NH2-terminal kinase (JNK) - following hypoxia-ischemia (H-I) induced by unilateral carotid artery ligation and hypoxia (8% O2 and 92% N2) for 2.5 h in postnatal-day-7 rats. Phosphorylation of p38 MAPK increased in the hippocampus and cortex immediately following H-I and returned to a basal level 6 h later. In contrast to p38 MAPK, phosphorylation of JNK decreased in the hippocampus and cortex immediately following H-I. Intracerebroventricular administration of two different p38 MAPK inhibitors prior to H-I significantly protected the neonatal brain from H-I injury. Interestingly, p38 MAPK inhibitors did not attenuate caspase-3 activation 24 h after H-I. Thus, these data suggest that p38 MAPKs contribute to the rapid, early component of brain injury following neonatal H-I.     

Magnolol protects against trimethyltin-induced neuronal damage and glial activation in vitro and in vivo

Trimethyltin (TMT), an organotin with potent neurotoxic effects by selectively damaging to hippocampus, is used as a tool for creating an experimental model of neurodegeneration. In the present study, we investigated the protective effects of magnolol, a natural biphenolic compound, on TMT-induced neurodegeneration and glial activation in vitro and in vivo. In HT22 murine neuroblastoma cells, TMT induced necrotic/apoptotic cell death and oxidative stress, including intracellular reactive oxygen species (ROS), protein carbonylation, induction of heme oxygenase-1 (HO-1), and activation of all mitogen-activated protein kinases (MAPKs) family proteins. However, magnolol treatment significantly suppressed neuronal cell death by inhibiting TMT-mediated ROS generation and activation of JNK and p38 MAPKs. In BV-2 microglial cells, magnolol efficiently attenuated TMT-induced microglial activation via suppression of ROS generation and activation of JNK, p38 MAPKs, and nuclear factor-κB (NF-κB) signaling. In an in vivo mouse study, TMT induced massive neuronal damage and enhanced oxidative stress at day 2. We also observed a concomitant increase in glial cells and inducible nitric oxide synthase (iNOS) expression on the same day. These features of TMT toxicity were reversed by treatment of magnolol. We observed that p-JNK and p-p38 MAPK levels were increased in the mouse hippocampus at day 1 after TMT treatment and that magnolol blocked TMT-induced JNK and p38 MAPK activation. Magnolol administration prevented TMT-induced hippocampal neurodegeneration and glial activation, possibly through the regulation of TMT-mediated ROS generation and MAPK activation.     

Bone mesenchymal stem cell-conditioned medium induces the upregulation of Smad6, which inhibits the BMP-4/Smad1/5/8 signaling pathway

Objectives: In this study, we aimed to investigate the effect of bone mesenchymal stem cells (BMSCs) conditioned medium on BMP4-induced activation of the Smad signaling pathway during the process of neural stem cells differentiation.

Methods: We designed four experimental groups: (A) control group, (B) BMP-4 treatment group, (C) BMSC-CM group, and (D) BMSC-CM (BMSC-conditioned medium) combined with BMP-4 group. The expression of microtube-associated protein-2 (MAP-2) detected by immunocytochemical staining and western blot. The expression of glial fibrillary acidic protein (GFAP) and BMP receptors (BMPRII and BMPRIa) was determined by western blot, while SMAD1/5/8 and SMAD6 mRNA expression was detected by quantitative real-time polymerase chain reaction (Q-RT-PCR).

Results: We found that the expression of microtube-associated protein-2 (MAP-2) increased in group B compared to group A. In group C, the expression of GFAP was increased and the expression of MAP-2 was decreased compared to group A. This phenomenon was weaker in group D. We also demonstrated that BMSC-CM could inhibit the activation of BMP signaling by downregulating the expression of BMPRII and BMPRIa proteins. Moreover, BMSC-CM could upregulate the expression of Smad6 mRNA and inhibit the activation of Smad1/5/8 mRNA.

Conclusion: These observations suggest that BMSC-CM could neutralize the effect of BMP-4 on the differentiation of NSCs by upregulating the expression of Smad6 and decrease the expression of GFAP by inhibiting the BMP-4-SMAD1/5/8 signaling pathway, conversely increasing the generation of neurons.     

c-Jun N-Terminal Kinase (JNK) and p38 Play Different Roles in Age-Related Purkinje Cell Death in Murine Organotypic Culture

Several studies have shown that Purkinje cells die by apoptosis in organotypic slice cultures from postnatal 3-day-old (P3) mice. This cell death is age-dependent and has been proposed as indirect evidence for the programmed Purkinje cell death occurring in in vivo cerebellum. Here, we studied whether c-jun N-terminal kinase (JNK) and p38 kinase pathways contribute to the Purkinje cell death observed in cerebellar slice cultures obtained from P3 mice. Slice culture treatment with D-JNKI1 or SB203580, respectively inhibitors of JNK and p38 MAP kinases, results in a better survival of Purkinje cells. Interestingly, the combined treatment with the two inhibitors potentiated single treatment effects. These results suggest that p38 and JNK pathways might be differently implicated in this Purkinje cell death. Time course experiments found p38 activation immediately post-slicing, whereas JNK activation was detected only 2 h after the culture. We hypothesize that p38 activation might be due to the “sliced condition,” and JNK activation might be more specific to P3 age-dependent cell death. The study of JNK and p38 activation in cerebellar lysates from P0 slice culture confirmed JNK activation being specific for the P3 explants, whereas p38 is activated both from P0 and P3 cerebellar slice culture lysates. These results suggest that p38 is activated by the slicing, whereas JNK activation is related to developmental Purkinje cell death.     

Activated glia induce neuron death via MAP kinase signaling pathways involving JNK and p38

Chronic glial activation in neurodegenerative diseases contributes to neuronal dysfunction and neuron loss through production of neuroinflammatory molecules. However, the molecular mechanisms, particularly the signal transduction pathways involved in glia-dependent neuron death, are poorly understood. As a first step to address this question, we used a neuron-glia co-culture system that allows diffusion of soluble molecules between glia and neurons to test the potential importance of mitogen-activated protein kinase (MAPK) signaling pathways in the glia-induced neuron death. Activation of glia in co-culture by lipopolysaccharide (LPS) induced apoptotic-like neuron death. The MAPKs tested (p38, JNK, ERK1/2) were activated in both glia and neurons following LPS treatment, suggesting their involvement in both glial activation and neuronal response to diffusible, glia-derived neurotoxic molecules. Inhibitors of p38 and JNK partially blocked neuron death in the LPS-treated co-culture, whereas an ERK1/2 pathway inhibitor did not protect neurons. These results show that p38 and JNK MAPKs, but not ERK1/2 MAPK, are important signal transduction pathways contributing to glia-induced neuron death.     

Region-specific effects of acute and repeated restraint stress on the phosphorylation of mitogen-activated protein kinases

The mitogen-activated protein kinases (MAPKs) are a family of signal transduction mediators that regulate a host of cellular activities, including cell growth and proliferation, and differentiation and survival, via sequential phosphorylation and activation of a cassette of three protein kinases. MAPKs are also recruited when the brain undergoes synaptic plasticity and remodeling (e.g., during induction of long-term potentiation, learning and memory consolidation). The activities of some of these kinases are altered in response to various acute stimuli such as ischemic insult, visceral pain and electroconvulsive shock. In the present study we used immunoblotting techniques to examine the effects of acute and repeated restraint stress on the phosphorylation state of three MAPKs, the extracellular signal-regulated kinase Erk1/2, c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK, in different brain regions. A single exposure to 30 min of restraint stress-elevated phospho-Erk1/2 (P-Erk1/2) levels in all three brain regions examined (hippocampus, medial prefrontal cortex and cingulate cortex), but did not alter the phosphorylation pattern of the other two MAPKs in any region. In marked contrast, exposure to restraint for 11 days (30 min/day) reduced the levels of all three MAPKs, but only in the prefrontal cortex. The results are compared to the reported effects of acute and chronic stress on other biochemical and functional measures.     

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.     

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction

Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects.

Materials and Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs.

Conclusions

CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.     

Thrombin-activated microglia contribute to death of dopaminergic neurons in rat mesencephalic cultures: dual roles of mitogen-activated protein kinase signaling pathways

This study evaluated the role of thrombin-activated microglia in the neurodegeneration of mesencephalic cultures. Immunocytochemical and biochemical evidence indicated that in co-cultures consisting of rat cortical microglia and mesencephalic neurons, thrombin led to nonselective loss of mesencephalic neurons. Accompanying neurodegeneration, microglial activation was obvious, evidenced by expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and by increasing production of TNF-alpha and nitric oxide (NO). In mesencephalic neurons treated with conditioned media (CM) taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. The neurotoxicity of the CM was diminished when it was derived from microglia co-treated with thrombin and either an extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor (PD98059) or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB203580). Moreover, jun N-terminal kinase (JNK) and p38-MAPK were activated in mesencephalic neurons treated with CM of thrombin-activated microglia. Inhibition of JNK and p38-MAPK rescued the dopaminergic neurons. Collectively, these results indicate that thrombin-activated microglia induce neurodegeneration in cultured mesencephalic neurons and that the MAPKs actively participate in both microglial activation and neurodegeneration. The present data carefully suggest that microglial activation triggered by thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.     

Contribution of mitogen-activated protein kinases to NMDA-induced neurotoxicity in the rat retina

We examined the contributions of the mitogen-activated protein kinases (MAPKs) family [extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK)] to N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Detection of apoptotic cell death in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining began 6 h after intravitreal NMDA (100 nmol) injection and continued to increase thereafter. Western blot analysis showed that phosphorylated MAPKs (p-MAPKs) were expressed in the retina following a temporal manner: maximal expression of phosphorylated ERK (p-ERK) at 1 h, maximal expression of phosphorylated p38 (p-p38) at 6 h, and beginning of phosphorylated JNK (p-JNK) significant increase at 6 h after injection. An immunohistochemical/TUNEL co-localization study showed that p-JNK- and p-p38-positive cells in the RGCL were frequently TUNEL-positive, whereas few p-ERK-positive cells were TUNEL-positive. Moreover, co-injection of inhibitors for JNK (0.2 nmol SP600125) and/or p38 (2.0 nmol SB203580) with NMDA was effective in ameliorating NMDA-induced apoptotic cell loss in the RGCL 12 h after injection, as shown by TUNEL-positive cell counts. These inhibitors also protected the inner retina as shown by morphometric studies such as cell counts in the RGCL and measurement of the IPL thickness 7 days after injection. On the other hand, an ERK inhibitor (2.0 nmol U0126) did not suppress NMDA-induced cell death in the RGCL nor thinning of the IPL. These findings suggest that JNK and p38 are proapoptotic in NMDA-induced cell death in the RGCL, but not ERK.     

Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland

In rodent pineal glands, sympathetic innervation, which leads to norepinephrine release, is a key process in the circadian regulation of physiology and certain gene expressions. It has been shown that gene expression of the rate-limiting enzyme in the melatonin synthesis arylalkylamine N-acetyltransferase (Aa-Nat), circadian clock gene Period1, and mitogen-activated protein kinase (MAPK) phosphtase-1 (MKP-1), is controlled mainly by a norepinephrine-beta-adrenergic receptor-cAMP signaling cascade in the rat pineal gland. To further dissect the signaling cascades that regulate those gene expressions, we examined whether MAPKs are involved in cAMP-induced gene expression. Western blot and immunohistochemical analyses showed that one of the three MAPKs, c-Jun N-terminal kinase (JNK), was expressed in the pineal, and was phosphorylated by cAMP analogue stimulation with a peak 20 min after start of the stimulation, in vitro. A specific JNK inhibitor SP600125 (Anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone), but not its negative control (N1-Methyl-1,9-pyrazoloanthrone), significantly reduced cAMP-stimulated Aa-Nat, Period1, and MKP-1 mRNA levels. Although another MAPK, p38(MAPK), has also been shown to be activated by cAMP stimulation, a p38(MAPK) inhibitor, SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, HCl), showed no effect on cAMP-induced Aa-Nat and Period1 mRNA levels; whereas SB203580, but not its negative analogue SB202474 (4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole, DiHCl), significantly reduced cAMP-induced MKP-1 mRNA levels. Taken together, our data suggest that cAMP-induced Aa-Nat and Period1 are likely to be mediated by activation of JNK, whereas MKP-1 may be mediated by both p38(MAPK) and JNK activations.     

Immune activation, viral gene product expression and neurotoxicity in the HIV-1 transgenic rat

The HIV-1 transgenic (TG) rat has been shown to be a useful model of nervous system disease that occurs in human HIV-1 infection. Studies were, therefore, performed to examine characteristics of the immune response in the periphery and brain of the animals and expression of factors in the nervous system that might be associated with neurotoxicity. Activated splenocytes from wild-type (WT) and TG rats were stimulated with either CD3/CD28 or with lipopolysaccharide (LPS) and examined for proliferative responses and for proinflammatory cytokine (IFN-γ, TNF-α and IL-1β) secretion. Brain tissue lysates from the rats were also examined for proinflammatory cytokine levels and tissue sections were stained by immunofluorescence for class II MHC+, ED1+ or Iba1+ (for macrophages and microglial cells), and for GFAP+ (for astrocytes) cells and for co-labeling of these cells for TNF-α. Co-labeling was also performed to identify cells expressing HIV-1 gp160, tat, nef and vif. Finally, on Western blots brain tissue lysates were examined for phosphorylation of Erk1/2, p38, JNK-SAPK and Erk5. TG rat splenocyte proliferative responses were higher than for WT with CD3/CD28-stimulation but lower than WT with LPS stimulation. CD3/CD28-stimulated TG rat splenocytes also secreted higher levels of IFN-γ, TNF-α and IL-1β whereas LPS-stimulated TG rat splenocytes secreted higher levels of only TNF-α than cultures from WT rats. Levels of all three cytokines were higher in brain lysates from TG rats than for WT rats. On immunofluorescence staining of corresponding sections of brain, TG rats contained increased numbers of class II MHC+ and ED1+ cells, and there was also increased co-labeling or these cells as well as astrocytes for TNF-α. Iba1+ cells showed positive staining for all of the HIV proteins whereas astrocytes showed significant positive staining for only nef and vif. Phosphorylation of Erk1/2, p38 and JNK/SAPK was detected for both TG and WT rat tissues with higher levels of phosphorylation forms of these proteins detected in the TG rat brain. Phosphorylation of Erk5, a marker that is associated with specifically neuronal repair, was detected only in TG rat brain. These studies suggest that activated nervous system mononuclear phagocytes and astrocytes expressing HIV-1 gene products in specific patterns are associated with neurodegeneration in the HIV-1 TG rat.     

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。