慢性睡眠剥夺对颞下颌关节微结构的影响

目的:研究慢性睡眠障碍对大鼠颞下颌关节微结构的影响。方法:采用改良多平台法(MMPM)建立睡眠剥夺模型,将90只Wistar大鼠随机分为3组(n=30),分别为小平台组、网格组和对照组。小平台组和网格组大鼠接受每天18 h的睡眠剥夺和6 h间歇期(10:00—16:00),间歇期大鼠正常笼养。实验第7、14和21天时分别行动物行为学观察、旷场试验和动物血浆检测,并通过HE染色和扫描电镜观察颞下颌关节微结构的变化。结果:与对照组和网格组相比,小平台组大鼠血清促肾上腺激素(ACTH)和皮质醇(CORT)水平均增高(P<0.05),髁突软骨HE染色显示软骨细胞层次及厚度改变;扫描电镜结果显示关节盘表面纤维排列松散。结论:慢性睡眠障碍可能导致颞下颌关节微结构发生病理性改变。     

MMP-1、MMP-3 和MMP-13在慢性睡眠剥夺所致颞下颌关节损伤中的变化

目的：观察MMP-1、MMP-3 和MMP-13 在慢性睡眠剥夺所致颞下颌关节损伤中表达的变化,探讨慢性睡眠剥夺所致颞下颌 关节损伤的可能机制。方法：采用改良多平台(MMPM)建立大鼠慢性睡眠剥夺模型,将90 只成年雄性Wastar 大鼠随机分为小平 台组、网格组和对照组。小平台组和网格组大鼠接受每天18 h的睡眠剥夺和6 h间歇期(10:00-16:00),间歇期大鼠正常笼养。实验 第7、14 和21 d时分别观察动物的行为学观察、检测动物血浆皮质醇(CORT)和促肾上腺皮质激素(ACTH)水平检测,通过免疫印 迹法和实时定量聚合酶链反应(PRC)检测颞下颌关节软骨中MMP-1、MMP-3 和MMP-13 的蛋白和mRNA表达,并通过HE 染色 法观察颞下颌关节结构的变化。结果：与对照组和网格组大鼠相比,小平台组大鼠第14 d和21 d 时髁突软骨中间部位表面纤维 在出现明显的炎症、松解及脱落现象;第21天时的血浆ACTH 和CORT 水平均显著高于网格组和对照组,差异有统计学意义 (P<0.05);第7、14、21 d时关节软骨MMP-1 和MMP-13 蛋白和mRNA 的表达水平均显著上调(P<0.05)。结论：慢性睡眠剥夺所致 的颞下颌关节损伤可能与关节软骨中MMP-1、MMP-3 和MMP-13 的表达上调有关。     

Smad7基因对胞外信号调节激酶磷酸化水平的调控

研究在BEP2D细胞中,作为Smads蛋白家族的抑制分子,Smad7对胞外信号调节的蛋白激酶(ERK1／2或p44／42)磷酸化水平的调控。将Smad7真核表达载体或人工合成的Smad7-siRNA转染BEP2D细胞,TGF—β刺激,通过Western印迹检测Smad7对p44／42蛋白磷酸化的影响。结果在永生化BEP2D细胞中,TGF-β1刺激后5min开始,可以检测到磷酸化的p44／42;到60min达到高峰,之后逐渐降低。细胞转染Smad7,TGF-β作用60rain后,p44／42磷酸化水平明显增高;而转染Smad7-SiRNA,TGF-β作用60min后,p44／42磷酸化水平显降低。p44／42蛋白水平基本上不受TGF—β1刺激及Smad7表达水平的影响。以上结果说明,在BEP2D细胞中,Smad7可参与TGF—β对ERK／MAPK通路的活化作用。     

The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.     

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2?/?Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2?/?Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.     

Early Activation of Extracellular Signal-Regulated Kinase Signaling Pathway in the Hippocampus is Required for Short-Term Memory Formation of a Fear-Motivated Learning

1. According to its duration there are, at least, two major forms of memory in mammals: short term memory (STM) which develops in a few seconds and lasts several hours and long-term memory (LTM) lasting days, weeks and even a lifetime. In contrast to LTM, very little is known about the neural, cellular and molecular requirements for mammalian STM formation.2. Here we show that early activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the hippocampus is required for the establishment of STM for a one-trial inhibitory avoidance task in the rat. Immediate posttraining infusion of U0126 (a selective inhibitor of ERK kinase) into the CA1 region of the dorsal hippocampus blocked STM formation.3. Reversible inactivation of the entorhinal cortex through muscimol infusion produced deficits in STM and a selective and rapid decrease in hippocampal ERK2 activation.4. Together with our previous findings showing a rapid decrease in ERK2 activation and impaired STM after blocking BDNF function, the present results strongly suggest that ERK2 signaling in the hippocampus is a critical step in STM processing.Lionel Muller Igaz and Milena Winograd contributed equally to this work     

Sevoflurane Postconditioning Inhibits Autophagy Through Activation of the Extracellular Signal-Regulated Kinase Cascade,Alleviating Hypoxic-Ischemic Brain Injury in Neonatal Rats

Hypoxic-ischemic brain injury (HIBI) in neonates is one of the major contributors of newborn death and cognitive impairment. Numerous animal studies have demonstrated that autophagy is substantially increased in HIBI and that sevoflurane postconditioning (SPC) can attenuate HIBI. However, if SPC-induced neuroprotection inhibits autophagy in HIBI remains unknown. To investigate if cerebral protection induced by SPC is related to decreased autophagy in the setting of HIBI. Postnatal rats at day 7 (P7) were randomly assigned to 7 different groups: Sham, HIBI, SPC–HIBI, HIBI?+?rapamycin, SPC–HIBI?+?rapamycin, HIBI?+?p-extracellular signal-regulated kinase (p-ERK) inhibitor, and SPC–HIBI?+?p-ERK inhibitor. To induce HIBI, neonatal rats underwent left common carotid artery ligation, followed by 2 h of hypoxia (8% O₂). Rats in the SPC groups were treated with 1 minimum alveolar concentration ([MAC], 2.4%) SPC for 30 min after HIBI induction. Markers of autophagy and expression of ERK cascade components were measured in the rat brains after 24 h. Spatial learning and memory function were examined 29–34 days after administration of an autophagy agonist or a p-ERK inhibitor. The expression of microtubule-associated proteins 1A/1B, light chain 3B II (LC3-II) and tuberous sclerosis complex 2 (TSC2) were decreased in the SPC–HIBI group compared to the HIBI group. Expression of the p62 sequestosome 1 (P62/SQSTM1) protein, p-ERK/ERK, phospho-mammalian target of rapamycin (p-mTOR) and phospho-p70S6 were increased in SPC–HIBI group. Rats within the SPC–HIBI groups that also received the p-ERK inhibitor or autophagy inhibitor demonstrated reduced cross platform times and increased escape latency. Approximately 30 min of 2.4% SPC treatment in the P7 rat HIBI model attenuated excessive autophagy in the brain by elevating the ERK cascade. This finding provides additional insight into HIBI and identifies new targets for therapeutic approaches to treat HIBI.

     

Distinct Changes in cAMP and Extracellular Signal-Regulated Protein Kinase Signalling in L-DOPA-Induced Dyskinesia

Background

In rodents, the development of dyskinesia produced by L-DOPA in the dopamine-depleted striatum occurs in response to increased dopamine D1 receptor-mediated activation of the cAMP - protein kinase A and of the Ras-extracellular signal-regulated kinase (ERK) signalling pathways. However, very little is known, in non-human primates, about the regulation of these signalling cascades and their association with the induction, manifestation and/or maintenance of dyskinesia.

Methodology/Results

We here studied, in the gold-standard non-human primate model of Parkinson''s disease, the changes in PKA-dependent phosphorylation of DARPP-32 and GluR1 AMPA receptor, as well as in ERK and ribosomal protein S6 (S6) phosphorylation, associated to acute and chronic administration of L-DOPA. Increased phosphorylation of DARPP-32 and GluR1 was observed in both L-DOPA first-ever exposed and chronically-treated dyskinetic parkinsonian monkeys. In contrast, phosphorylation of ERK and S6 was enhanced preferentially after acute L-DOPA administration and decreased during the course of chronic treatment.

Conclusion

Dysregulation of cAMP signalling is maintained during the course of chronic L-DOPA administration, while abnormal ERK signalling peaks during the initial phase of L-DOPA treatment and decreases following prolonged exposure. While cAMP signalling enhancement is associated with dyskinesia, abnormal ERK signalling is associated with priming.     

Tumor Promoter Arsenite Activates Extracellular Signal-Regulated Kinase through a Signaling Pathway Mediated by Epidermal Growth Factor Receptor and Shc

Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.     

Tyrosine Phosphorylation of Extracellular Signal-Regulated Protein Kinase 4 in Response to Growth Factors

Abstract: Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.     

Effects of Deprivation of the Quick-Wave Sleep in Rats with Inherited Predisposition to Audiogenic Convulsions

In experiments on rats of Krushinskii–Molodkina line (KM) with genetic predisposition to audiogenic convulsions, effects of the 3- and 6-h periods of the absence of the quick-wave sleep (QS) were studied in animals under natural conditions as well as of selective deprivations of QS on EEG spectral characteristics in the wakening–sleep cycle, on organization of the cycle, and on intensity of convulsive symptoms. The QS deprivation for 3, 6, 9, and 12 h was produced by the classic methods of small platforms or of soft awakening. The data are presented about changes of the cycle parameters in the course of natural and experimental deprivations as well as about the dynamics of restoration of the cycle structure for 12 h of the post-deprivation period. It was established that during and after the QS deprivations (by any duration), in EEG of the hippocampus, caudate nucleus, medial central nucleus of thalamus, somato-sensory, visual and auditory cortex of the KM rat brain, no appearance of the paroxysmal fires was revealed in any of the states of the wakening–sleep cycle. It was also found that the selective QS deprivation did not affect duration of the latent period and parameters of the generalized tonic-clonic audiogenic convulsions. It is stated that in rats of the KM line that have the hidden convulsive syndrome, the used kinds and methods of QS deprivation fail to activate the epileptiform manifestations.     

Sleep Deprivation Impairs Consolidation of Cued Fear Memory in Rats

Post-learning sleep facilitates negative memory consolidation and also helps preserve it over several years. It is believed, therefore, that sleep deprivation may help prevent consolidation of fearful memory. Its effect, however, on consolidation of negative/frightening memories is not known. Cued fear-conditioning (CuFC) is a widely used model to understand the neural basis of negative memory associated with anxiety disorders. In this study, we first determined the suitable circadian timing for consolidation of CuFC memory and changes in sleep architecture after CuFC. Thereafter, we studied the effect of sleep deprivation on CuFC memory consolidation. Three sets of experiments were performed in male Wistar rat (n = 51). In experiment-I, animals were conditioned to cued-fear by presenting ten tone-shock paired stimuli during lights-on (7 AM) (n = 9) and lights-off (7 PM) (n = 9) periods. In experiment-II, animals were prepared for polysomnographic recording (n = 8) and changes in sleep architecture after CuFC was determined. Further in experiment-III, animals were cued fear-conditioned during the lights-off period and were randomly divided into four groups: Sleep-Deprived (SD) (n = 9), Non-Sleep Deprived (NSD) (n = 9), Stress Control (SC) (n = 9) and Tone Control (n = 7). Percent freezing amount, a hallmark of fear, was compared statistically in these groups. Rats trained during the lights-off period exhibited significantly more freezing compared to lights-on period. In CuFC trained animals, total sleep amount did not change, however, REM sleep decreased significantly. Further, out of total sleep time, animals spent proportionately more time in NREM sleep. Nevertheless, SD animals exhibited significantly less freezing compared to NSD and SC groups. These data suggest that sleep plays an important role in the consolidation of cued fear-conditioned memory.     

Sleep Deprivation Induces Changes in Immunity in Trichinella spiralis-Infected Rats

Sleep is considered an important predictor of immunity. A lack of sleep may reduce immunity, which increases susceptibility to any type of infection. Moreover, sleep deprivation in humans produces changes in both, the percent of circulating immune cells (T cells and NK cells) and cytokine levels (IL-1, IFNγ, TNΦ-αα, IL-6 and IL-17). The aim of our study was to investigate whether sleep deprivation produces deregulation on immune variables during the immune response generated against the helminth parasite Trichinella spiralis. Because sleep deprivation is stressful per se, we designed another experiments to compared stress alone (consisting in movement restriction and single housing) with sleep deprivation, in both control (uninfected) and experimental (infected) rats. Our results demonstrate that the sleep deprivation and stress have a differential effect in mesenteric lymph nodes (MLN) and spleen. In uninfected rats sleep deprivation alone produces an increase in natural killer cells (NK+) and B cells (CD45+), accompanied by a decrease in cytotoxic T cells (CD3+CD8+) in spleen; while, in MLN, produces only an increase in natural killer cells (NK+). Both, SD and stress, produce an increased percentage of total T cells (CD3+) in spleen. In the MLN both are also associated to an increase in cytotoxic T cells (CD3+CD8+) and B cells (CD45+). In the spleens of parasitized rats, cell populations did not change. In spleens of both, sleep-deprived and stressed infected rats, we observed an increase in B cells (CD45+). In infected rats, sleep deprivation alone produced an increase in NK cells (NK+). In mesenteric node cell populations of parasitized rats, we observed a decrease in NK cells and an increase in T helper (CD4+) cells in both SD and stressed rats. Rats that were only subjected to stress showed a decrease in B cells (CD45+). These findings suggest that the immune response generated against infection caused by T. spiralis is affected when the sleep pattern is disrupted. These results support the notion that sleep is a fundamental process for an adequate and strong immune response generated against this parasite.     

Activation of Extracellular Signal-Regulated Kinase1/2 in the Medial Prefrontal Cortex Contributes to Stress-Induced Hyperalgesia

Stressful stimuli can exacerbate persistent pain disorder. However, the underlying mechanism is still unknown. Here, to reveal the underlying mechanism for stressful stimuli-induced hyperalgesia in chronic pain, we investigated the effect of extracellular signal-regulated kinase1/2 (ERK1/2) activation on pain hypersensitivity using single-prolonged stress (SPS) model, complete Freund’s adjuvant (CFA) model and SPS?+?CFA model. The experimental results revealed significantly reduced paw withdrawal threshold in the SPS, CFA, and SPS?+?CFA group compared with the control group. However, the increased phosphorylation of ERK1/2 in the medial prefrontal cortex (mPFC) was observed in the SPS- or SPS?+?CFA-exposed group but not the CFA group compared with control group. There was also a significant increase in mPFC ERK1/2 phosphorylation and mechanical allodynia after SPS?+?CFA treatment compared to SPS or CFA treatment alone. Furthermore, inhibiting ERK1/2 phosphorylation by microinjection of U0126, a MAPK kinase (MEK) inhibitor, into the mPFC attenuated SPS?+?CFA- and SPS- but not CFA-induced mechanical allodynia, anxiety-like behavior, and cognitive impairments. These results suggest that the activation of ERK1/2 in the mPFC may contribute to the process of stress-induced cognitive and emotional disorders, leading to an increase in pain sensitivity.