外周P物质受体介导伤害性刺激所致脊髓背角c-fos基因的表达

用免疫组化染色方法,观察了Ｐ物质受体在外周对伤害性刺激信息的介导作用。于福尔马林注入双侧后肢足底前１０ｍｉｎ,将不同浓度的ＳＰ受体特异性拮抗剂Ｌ６６８,１６９注入一侧足底,另一侧注入生理盐水。结果：１０＾－４ｍｏｌ／Ｌ的Ｌ６６８,１６９明显抑制了该侧脊髓背角浅层ｃ－ｆｏｓ基因的表达而对深层影响不大;     

Lateral spinal nucleus of the rat: NADPH-diaphorase activity and Fos expression after noxious peripheral stimulation

Results of electrophysiological studies suggest a significant role of the lateral spinal nucleus (LSpN) in the transmission of nociceptive signals. In our study, the presence of Fos immunoreactivity and NADPH-diaphorase positivity was observed in the rat LSpN following noxious peripheral subcutaneous stimulation. Formalin-induced unilateral hindpaw stimulation in the rat caused bilateral NADPH-d reactivity and ipsilateral Fos expression in this nucleus. In the LSpN of the L3–L5 segments of stimulated rats, on average, 4.1 ± 1.2 NADPH-d-positive, NADPH-d(+), 5.1+1.8 Fos-immunoreactive, Fos(+), and 3.0 ± 1.1 double-labeled neurons per 25-μm-thick section were found unilaterally. A close anatomical relationship between NADPH-d(+) processes and Fos(+) cell nuclei in the LSpN was also observed following noxious peripheral stimulation. These neuroanatomical findings support the hypothesis that the LSpN is involved in pain processing and suggest an important role of nitric oxide-mediated signal transduction in this nucleus. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 38–42, January–February, 2008.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fospositive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

Increased expression of spinal cord Fos protein induced by bladder stimulation after spinal cord injury

These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after spinal cord injury (SCI). In urethan-anesthetized Wistar rats after SCI for 6 wk, intravesical saline distension significantly (P 相似文献   

The ERK mitogen-activated protein kinase pathway contributes to Ebola virus glycoprotein-induced cytotoxicity

Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates. Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of expression of a mutant form of GP lacking a mucin-like domain (GPDeltamuc). GP-induced cell death is nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways, including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2, was reduced in cells expressing GP compared to those expressing GPDeltamuc as determined by flow cytometry, in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown potentiated the decrease in alphaV integrin expression associated with toxicity. Conversely, activation of the pathway through the expression of a constitutively active form of ERK2 significantly protected against this effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a role in pathogenicity induced by this gene product.     

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.     

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors

Background

Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies.

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Conversely, intrathecal injection of the TLR3 agonist polyinosine–polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine–polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.     

Inflammation enhances reflex and spinal neuron responses to noxious visceral stimulation in rats

To improve understanding of sensory processes related to visceral inflammation, the effect of turpentine-induced inflammation on reflex (cardiovascular/visceromotor) and extracellularly recorded lumbosacral dorsal horn neuron responses to colorectal distension (CRD) was investigated. A 25% solution of turpentine, applied to the colorectal mucosa, produced inflammation, decreased compliance of the colonic wall, and enhanced reflex responses in unanesthetized rats within 2-6 h. At 24 h posttreatment, pressor responses to CRD (80 mmHg, 20 s) were 20% greater, and intraluminal pressures needed to evoke visceromotor reflexes were 30% lower than controls. Parallel electrophysiological experiments in spinal cord-transected, decerebrate rats demonstrated that two neuronal subgroups excited by CRD were differentially affected by turpentine administered 24 h before testing. During CRD, abrupt neurons were 70% less active and sustained neurons were 25% more active than similar neurons in controls. In summary, reflex and neuronal subgroup (sustained neurons) responses to CRD were both potentiated by chemical inflammation. This suggests that the neurophysiological basis for inflammation-induced increases in reflex responses to CRD is increased activity of this neuronal subgroup.     

Angiotensin II contributes to renal fibrosis independently of Notch pathway activation

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.     

SUMOylation of the kainate receptor subunit GluK2 contributes to the activation of the MLK3-JNK3 pathway following kainate stimulation

Protein SUMOylation has been implicated in the pathogenesis of ischemic stroke. However, the underlying mechanisms remain unclear. Here, we found that global brain ischemia evokes a sustained elevation of GluK2 SUMOylation in the rat hippocampal CA1 region. Over-expression of wild-type GluK2, but not SUMOylation-deficient mutant, significantly increased the activity of MLK3 and JNK3 after kainate stimulation. SUMOylation deficiency attenuated the kainate-stimulated interaction between MLK3 and GluK2. In addition, inhibition of kainate-evoked GluK2 endocytosis decreased the activation of MLK3-JNK3 signaling and the binding of MLK3-GluK2 in cultured cortical neurons. These results suggest that the internalization of GluK2 following SUMO modification promotes its binding with MLK3, thereby activating the MLK3-JNK3 pathway, which may be responsible for ischemic neuronal cell death.     

Oncogenic role of mortalin contributes to ovarian tumorigenesis by activating the MAPK–ERK pathway

Mortalin is frequently overexpressed in human malignancies. Previous studies have suggested that mortalin contributes to ovarian cancer development and progression, but further investigation is warranted. The aim of this study is to elucidate the mechanism of mortalin in ovarian cancer development and progression. In this study, lentivirus‐delivered mortalin short hairpin RNA (shRNA) was used to knockdown mortalin expression in A2780 and A2780/cis ovarian cancer cell lines, and lentiviral mortalin‐pLVX‐AcGFP was used to generate mortalin‐overexpressing cell lines. The results demonstrated that decreased mortalin expression reduced ovarian cancer cell proliferation, colony formation, migration and invasion by Cell Counting Kit‐8 assay, colony formation assay, wounding healing assay and Transwell cell invasion assay, respectively. Flow cytometry results suggested that mortalin promotes the G1 transition, leading to faster restoration of a normal cell‐cycle distribution. Cell‐cycle proteins, including C‐myc and Cyclin‐D1, significantly increased, and Cyclin‐B1 remarkably decreased upon mortalin down‐regulation. Western blot analysis showed that mortalin knockdown significantly decreased p‐c‐Raf and phospho‐extracellular–regulated protein kinases (p‐ERK1/2) pathways but not the Jun N‐terminal kinase pathway, whereas mortalin overexpression had the opposite effect. Taken together, these results indicate that mortalin is an oncogenic factor, and mitogen‐activated protein kinase‐ERK signalling pathway activation by mortalin may contribute to ovarian cancer development and progression.     

Cell activation in the hypothalamus after exposure to an antigen (based on c-fos gene expression)

Increase of the c-fos mRNA positive cells number was insignificant in 30 min. following activation of the rat hypothalamic structures with the tetanus toxoid (TT). Elevation of the c-fos mRNA positive cells number occurred in the hypothalamic' posterior (PHA), lateral (LHA), anterior (AHA), areas dorsomedial (DMH), and ventromedial (VMH) nuclei within 2 hours of the TT administration. In 6 hours the c-fos mRNA positive cells number decreased in PHA, LHA, DMH. The c-fos mRNA expression was stable in arquate and supraoptic hypothalamic nuclei following either the TT or saline administration.     

The expression and activation of ERK/MAPK pathway in human esophageal cancer cell line EC9706

While there have been more and more studies concerning mitogen-activated protein kinases (MAPKs) signaling pathways, which control many cellular complex programmes, such as cell proliferation, differentiation, cell death and embryogenesis. However, few studies are carried out about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) in human esophageal cancer cell line. Therefore, in the present study, we investigated the expression and activation of ERK1/2 in human esophageal cancer cell line EC9706 and human normal esophageal epithelial cell line Heepic, which is as control. This study showed that ERK1/2 was transiently phosphorylated both in EC9706 and Heepic, the kinetics of which were slightly different. To further study the ERK/MAPK signaling pathway in EC9706 and Heepic cell line, U0126 a kind of specific inhibitor of MEK was used. This study showed that U0126 can block the phosphorylation of ERK1/2 in a short time, the complete inhibition concentration for EC9706 and Heepic cell line is 50 and 20 ??M, respectively. Incidentally, to further investigate the different roles of ERK1 and ERK2, vector-based short hairpin interference vectors targeted on ERK1/2 was constructed. Moreover, the effective interference target sequence was screened out in a transient transfection manner. MTT experiment showed that ERK2 is more important than ERK1 in the proliferation of EC9706 cells.     

Sevoflurane suppresses noxious stimulus-evoked expression of Fos-like immunoreactivity in the rat spinal cord via activation of endogenous opioid systems

We investigated the antagonism of sevoflurane antinociception by opioid antagonists in the rat formalin test. Formalin injection into the hindpaw of the rat induces the nocifensive flinching behavior and the expression of Fos-like immunoreactivity (Fos-LI) in the spinal cord. Sevoflurane significantly suppressed the flinching behavior and decreased the number of Fos-LI neurons in the dorsal horn of spinal cord compared with the control group. Moreover, pretreatment with intraperitoneal naloxone plus naltrexone antagonized the suppression of flinching behavior and the decrease of the number of Fos-LI neurons produced by 3% sevoflurane. Intraperitoneal opioid antagonists themselves had no effects on both the behavior response and the expression of Fos-LI induced by formalin injection. This study supports the hypothesis that sevoflurane suppresses the nociceptive response, at least in part, by activating endogenous opioid systems.