TNF-α诱导MLO-Y4细胞发生RIP3介导的程序性坏死

目的:探讨肿瘤坏死因子α(TNF-α)能否诱导小鼠长骨骨样细胞株MLO-Y4发生程序性坏死及其发生机制。方法:将MLO-Y4细胞分为正常对照(control)组、TNF-α处理组、TNF-α+necrostatin-1(Nec-1)处理组、TNF-α+Z-VAD处理组和TNF-α+受体相互作用蛋白3(RIP3)-siRNA组。用流式细胞术检测各组细胞凋亡或坏死率,透射电镜鉴定细胞形态学变化,用Western blot法测定RIP1、RIP3和cleaved caspase-3的蛋白水平,应用激光共聚焦显微镜观察RIP1和RIP3蛋白的共表达,应用荧光标记法检测各组细胞活性氧(ROS)水平。结果:TNF-α诱导MLO-Y4细胞24 h,凋亡和坏死率明显高于control组(P0.01)。与TNF-α组相比,Nec-1、Z-VAD和RIP3-siRNA均能降低细胞的凋亡或坏死率(P0.01)。在TNF-α组可见大量坏死样细胞,在Z-VAD组仍可见到坏死样细胞,而在Nec-1和RIP3-siRNA组未见到坏死样细胞。Western blot实验结果显示Nec-1可有效抑制RIP1蛋白表达,而Z-VAD对RIP1和RIP3蛋白表达无影响,RIP3-siRNA可有效降低RIP3蛋白表达(P0.01)。与TNF-α组比较,Nec-1可有效降低RIP1-RIP3蛋白共表达阳性细胞百分率(P0.01),而Z-VAD对其无影响。与control组相比,TNF-α组的ROS水平明显增高(P0.01);与TNF-α组相比,Nec-1、Z-VAD及RIP3-siRNA均能有效抑制ROS水平(P0.01)。结论:TNF-α能诱导MLO-Y4细胞发生RIP3介导的程序性坏死;ROS可能是MLO-Y4细胞程序性坏死的执行者。     

人肾小管上皮细胞necroptosis模型的构建

 目的：以体外培养的人肾小管上皮细胞（HK-2细胞）为靶细胞,构建肾小管上皮细胞凋亡样坏死（necroptosis）的模型。方法：采用肿瘤坏死因子 α (tumor nercosis factor α, TNF-α)诱导细胞凋亡,同时采用抗霉素A (antimycin A)耗竭ATP,构建肾小管上皮细胞凋亡的模型,并以caspase-8抑制剂苄氧羰酰-缬氨酰-丙氨酰-天冬氨酰-氟甲基酮(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, zVAD-fmk) 阻断凋亡,用necroptosis的特异性抑制剂necrostatin-1（Nec-1）阻断necroptosis,观察细胞在不同的处理下形态学的变化,同时检测细胞存活率及标志物微管相关蛋白1轻链3-Ⅱ（microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ）的表达。结果：（1）在TNF-α+zVAD- fmk+antimycin A处理1 h时细胞及细胞器膨胀,电镜下细胞膜碎裂,线粒体变圆、肿胀,嵴逐渐模糊,胞浆中出现大量自噬小体,而Nec-1预处理后细胞的坏死程度较对照组明显改善。（2）在TNF-α+zVAD-fmk+antimycin A 1 h实验组,Nec-1预处理后细胞的存活率显著增加（P<0.05）。（3）TNF-α+zVAD-fmk+antimycin A干预1 h实验组在Nec-1预处理后LC3-Ⅱ的表达量明显下降（P<0.05）。结论：凋亡环境中阻断凋亡可以诱导肾小管上皮细胞necroptosis,抑制剂Nec-1能特异性阻断肾小管上皮细胞发生坏死。     

Group I metabotropic glutamate receptor inhibition selectively blocks a prolonged Ca(2+) elevation associated with age-dependent excitotoxicity

It has been recognized for some years that a prolonged Ca(2+) elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca(2+) elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity.Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca(2+) elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8-9 and 21-28 days in vitro) were exposed to a 5-min glutamate insult (400 microM in younger and 10 microM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca(2+) elevation was present in older but not most younger neurons. The N-methyl-D-aspartate receptor antagonist, MK-801, blocked the Ca(2+) elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca(2+) elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures.From these results we suggest that the post-insult Ca(2+) elevation is regulated differently from the Ca(2+) elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca(2+) elevation appears to be directly linked to an age-dependent component of vulnerability.     

Calcium influx through N-methyl-D-aspartate receptors triggers GABA release at interneuron-Purkinje cell synapse in rat cerebellum

Ca(2+)-dependent neurotransmitter release was originally thought to occur only following activation of presynaptic voltage-gated calcium channels after a presynaptic action potential. Recent evidence suggests that not only opening of voltage-gated but also ligand-gated ion channels, such as neurotransmitter receptors, can trigger exocytosis, as well as Ca(2+) release from intracellular Ca(2+) stores. It was shown that activation of N-methyl-d-aspartate (NMDA) receptors on presynaptic interneurons led to increases in GABA release from these neurons onto postsynaptic Purkinje cells in rat cerebellum in the presence of tetrodotoxin (TTX), suggesting a presynaptic location for the underlying NMDA receptors. However, the mechanism for the NMDA-induced increase in GABA release remained unclear. The present study addresses the question whether Ca(2+) influx through presynaptic NMDA receptors alone is sufficient to trigger presynaptic GABA release at this synapse or whether activation of presynaptic NMDA receptors leads to opening of voltage-gated Ca(2+) channels, thereby increasing exocytosis. The results suggest that the NMDA-induced increase in presynaptic GABA release neither requires activation of presynaptic voltage-gated Ca(2+) channels nor Ca(2+) release from presynaptic Ca(2+) stores. It is concluded that Ca(2+) influx through the NMDA receptor alone is sufficient to drive presynaptic GABA release at the rat interneuron-Purkinje cell synapse.     

硫化氢通过抑制坏死性凋亡对抗高糖引起的H9c2心肌细胞损伤

 目的: 探讨硫化氢(hydrogen sulfide,H₂S)能否通过调控坏死性凋亡(necroptosis)对抗高糖(HG)引起的H9c2心肌细胞损伤。方法: 应用Western blot法检测心肌细胞内能反映坏死性凋亡的RIP3蛋白和cleaved caspase-3蛋白的水平;细胞计数盒测定心肌细胞存活率;双氯荧光素染色荧光显微镜照相法检测细胞内活性氧簇(reactive oxygen species,ROS)水平;罗丹明123染色荧光显微镜照相法测定线粒体膜电位(mitochondrial membrane potential,MMP);Hoechst 33258核染色荧光显微镜照相法测定凋亡细胞的数量。结果: 应用HG(35 mmol/L葡萄糖)处理H9c2心肌细胞3 h、6 h、9 h、12 h和24 h均能明显地上调RIP3蛋白的表达水平,其中24 h时RIP3蛋白水平增加最明显。400μmol/L硫氢化钠(NaHS;为H₂S的供体)预处理或坏死性凋亡的特异性阻断剂necrostatin-1(Nec-1;100μmol/L)共处理心肌细胞均能明显地抑制HG对RIP3蛋白表达的上调作用。此外,NaHS预处理或Nec-1共处理心肌细胞均显著地抑制HG引起的心肌细胞损伤,使细胞存活率升高,ROS生成及MMP丢失减少。另一方面,400μmol/L NaHS预处理心肌细胞能使凋亡细胞数量及cleaved caspase-3表达明显减少。结论: H₂S可通过抑制坏死性凋亡保护心肌细胞,对抗高糖引起的损伤。     

N-Methyl-D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca(2+) is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca(2+). Exposure of cortical neurons to NMDA, in the absence of external Ca(2+), produced a transient rise in intracellular Ca(2+) that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca(2+) did not result from an influx of Na(+) via reversal of the mitochondrial Na(+)/Ca(2+) exchanger since it persisted in a Na(+)-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca(2+) required the presence of D-serine, was blocked by D(-)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg(2+). This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca(2+) or Na(+), or presence of Mg(2+). NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca(2+). The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2alpha subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca(2+) stores via a mechanism that is not linked to the ionic properties of NMDA receptors.     

The role of RIP1 and RIP3 in the development of aplastic anemia induced by cyclophosphamide and busulphan in mice

This study aimed to investigate the role of RIP1 and RIP3 in the pathogenesis of aplastic anemia (AA) induced by cyclophosphamide and busulphan in mice. Animals were randomly divided into three groups: the control group, the AA group, and the Nec-1 group. Mouse AA model was established by intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The Nec-1 group mice received intraperitoneal injection of Nec-1 (2 mg/kg/d) for 12 days prior to intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The control mice received intraperitoneal injection of equal volume of saline. At 12 h after the last intraperitoneal injection, blood and bone marrow tissues were collected from mice. Peripheral blood cells were analyzed using hematology analyzer and the histological changes of bone marrow tissues were examined using scanning electron microscopy (SEM). The levels of RIP3 and RIP3 in bone marrow were measured using Western blot analysis and the interaction of RIP1 and RIP3 proteins was investigated on the basis of immunoprecipitation analysis. ELISA was used to measure the levels of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant. Apoptosis and necrosis of bone marrow cells were analyzed using flow cytometry. Western blot showed that the expression of RIP1 and RIP3 was significantly increases in AA mice compared to the normal controls. Immunoprecipitation detected the pro-necrotic RIP1-RIP3 complex, suggesting that RIP1 and RIP3 mediated necroptosis may involved in the damage of bone marrow cells. Compared to the AA mice, Nec-1 group mice exhibited significantly increase of peripheral blood cells and mononuclear cells in bone marrow tissues and decrease of the apoptosis/necrosis of bone marrow cells. In addition, we observed significant decrease of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant in the Nec-1 group mice compared to AA mice. Our results suggest that Nec-1 can prevent the development of AA by inhibiting bone marrow cells necrosis and the production of inflammatory mediators. RIP1 and RIP3-mediated necroptosis may involve in the pathogenesis of AA induced by cyclophosphamide and busulfan in mice.     

Metabotropic glutamate response in acutely dissociated hippocampal CA1 pyramidal neurones of the rat.

1. The metabotropic glutamate (mGlu) response was investigated in dissociated rat hippocampal CA1 pyramidal neurones using conventional and nystatin-perforated whole-cell modes of the patch recording configuration. 2. In the perforated patch recording configuration, the application of glutamate (Glu), quisqualate (QA), aspartate (Asp) and N-methyl-D-aspartate (NMDA) induced a slow outward current superimposed on a fast ionotropic inward current, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate (KA) induced only an ionotropic inward current at a holding potential (VH) of -20 mV. A specific agonist of the mGlu receptor (mGluR), trans-1-aminocyclopentane-1,3-dicarboxylate (tACPD), induced an outward current in approximately 80% of the neurones tested. Asp- and NMDA-induced outward currents were antagonized by D-2-amino-5-phosphonopentanoate (D-AP5) whereas Glu-, QA- and tACPD-induced outward currents were not antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D-AP5, indicating that the mGlu response is an outward current component. 3. L-2-Amino-3-phosphonopropionate (L-AP3) and DL-2-amino-4-phosphonobutyrate (AP4) did not block the mGlu response. 4. The relative potencies of mGlu agonists were QA > Glu > tACPD. The threshold and EC50 values of metabotropic outward currents were 10-100 times lower than those of the ionotropic inward current (iGlu response). 5. The reversal potential of the mGlu response (EmGlu) was close to EK (K+ equilibrium potential), and it shifted 59.5 mV for a tenfold change in extracellular K+ concentration. 6. In Ca(2+)-free external solution, the mGlu response was elicited by an initial application of Glu, but subsequent applications failed to induce the response. There was also an increase in the intracellular free Ca2+ concentration ([Ca2+]i) during the application of Glu and QA but not of AMPA, indicating Ca2+ release from an intracellular Ca2+ store. 7. During the activation of a Ca(2+)-dependent K+ current (IK(Ca)) by inositol trisphosphate (IP3) in the internal solution, the mGlu response was suppressed. Addition of GDP-beta-S, neomycin or heparin to the internal solution also suppressed the mGlu response, but staurosporine had no effect. The mGlu response was abolished by pretreatment with either caffeine or ryanodine, but treatment with pertussis toxin (IAP) for 6-8 h had no effect. 8. The mGlu response was suppressed by tetraethylammonium, but not by either apamin or iberiotoxin, suggesting that intermediate-conductance Ca(2+)-dependent K+ (KCa+) channels are involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMDA-evoked excitotoxicity increases tissue transglutaminase in cerebellar granule cells

In neuronal cells, excessive activation of glutamate receptors causes excitotoxic damage culminating in apoptotic and necrotic cell death. The molecular mechanism of excitotoxicity has been associated with excessive Ca(2+) influx and overload, triggering biochemical events that lead to cell death and tissue degeneration. Following mild insults via NMDA-receptor activation, central neurons undergo several biochemical modifications recognizable as early events in apoptotic machinery.Tissue transglutaminase, the most ubiquitous among cell transglutaminases, catalyzes the Ca(2+)-dependent protein cross-linking probably associated with morphological changes in several neurodegenerative disorders. The possible involvement of this enzyme in excitotoxicity-mediated events was investigated in primary cultures of cerebellar granule cells exposed for 30 min to NMDA (100 microM) in Locke's buffer. Under these conditions time-dependent increases in transglutaminase activity were observed. Tissue transglutaminase expression reached the highest levels within 3-4 h of NMDA exposure. Similarly, high levels of incorporation of fluorescent substrates were observed in living cells. Confocal laser microscopy analysis showed that fluorescein-labelled structures were distributed within the cytoplasm and close to the membranes of NMDA-exposed cells.These effects were dependent on the Ca(2+) influx triggered by the excitotoxic stimulus. Morphological changes in NMDA-treated cells gave evidence of significant cell damage which appeared within 5-6 h of NMDA exposure.These results suggest that increases in tissue transglutaminase may be associated to the effects of NMDA-induced excitotoxicity. Therefore, it is reasonable to hypothesize that if tissue transglutaminase levels and activity are up-regulated under such conditions, the protein cross-linking could be likely involved in excitotoxic response.     

Ca2+ flux and signaling implications by nicotinic acetylcholine receptors in rat medial habenula

The fraction of inward current carried by Ca(2+) (FCa(2+)) through nicotinic acetylcholine receptors (nAChRs) on acutely isolated rat medial habenula (MHb) neurons was calculated from experiments that simultaneously monitored agonist-induced membrane currents and intracellular [Ca(2+)], measured with patch-clamp and indo-1 fluorescence, respectively. In physiological concentrations of extracellular Ca(2+) (2 mM) at -50 mV, the percentage of current carried by Ca(2+) was determined to be roughly 3-4%, which is in close agreement with measurements from other heteromeric nicotinic receptors expressed in peripheral tissue. Among factors that may have affected this measurement, such as Ca(2+) influx through voltage-gated Ca(2+) channels, the concentration of intracellular Ca(2+) buffer, and Ca(2+) sequestration and release from intracellular stores, only Ca(2+) uptake by mitochondria was shown to confound the analysis. Furthermore, we find that because of the high density of nAChRs on MHb cells, low concentrations of ACh (10 microM) and its hydrolysis product, choline (1 mM), can significantly elevate intracellular Ca(2+). Moreover, during persistent activation of nAChRs, the level of intracellular Ca(2+) is proportional to its extracellular concentration in the physiological range. Together, these findings support the suggestion that nAChRs may be capable of sensing low concentrations of diffusely released neurotransmitter and, in addition, transfer information about ongoing local synaptic activity by changes in extracellular Ca(2+).     

Multiple P2 receptors contribute to a transient increase in intracellular Ca2+ concentration in ATP-stimulated rat brown adipocytes

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca(2+) concentration ([Ca(2+)](i)), which arises primarily from a release of Ca(2+) from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca(2+)](i) elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 microM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca(2+)](i) elevation mediated through store-operated Ca(2+) entry. The inhibitory effect of ATP was antagonized by PPADS with IC(50) of 0.7 microM. In the presence of PPADS at concentrations of more than 5 microM, the ATP-induced [Ca(2+)](i) elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca(2+)](i) elevation was reduced in a concentration-dependent manner by PPADS with an IC(50) of 200 microM. In contrast, the ATP-induced [Ca(2+)](i) elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca(2+)](i) responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca(2+) stores, which is probably followed by the activation of store-operated Ca(2+) entry. Extracellular ATP, however, would inhibit this Ca(2+) entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca(2+)](i) elevation in response to extracellular ATP.     

Necrostatin-1 Protects Against <Emphasis Type="SmallCaps">d</Emphasis>-Galactosamine and Lipopolysaccharide-Induced Hepatic Injury by Preventing TLR4 and RAGE Signaling

Fulminant hepatic failure (FHF) is a life-threatening clinical syndrome results in massive inflammation and hepatocyte death. Necroptosis is a regulated form of necrotic cell death that is emerging as a crucial control point for inflammatory diseases. The kinases receptor interacting protein (RIP) 1 and RIP3 are known as key modulators of necroptosis. In this study, we investigated the impact of necroptosis in the pathogenesis of FHF and molecular mechanisms, particularly its linkage to damage-associated molecular pattern (DAMP)-mediated pattern recognition receptor (PRR) signaling pathways. Male C57BL/6 mice were given an intraperitoneal injection of necrostatin-1 (Nec-1, RIP1 inhibitor; 1.8 mg/kg; dissolved in 2% dimethyl sulfoxide in phosphate-buffered saline) 1 h before receiving d-galactosamine (GalN; 800 mg/kg)/lipopolysaccharide (LPS; 40 μg/kg). Hepatic RIP1, RIP3 protein expression, their phosphorylation, and RIP1/RIP3 complex formation upregulated in the GalN/LPS group were attenuated by Nec-1. Nec-1 markedly reduced the increases in mortality and serum alanine aminotransferase activity induced by GalN/LPS. Increased serum high mobility group box 1 (HMGB1) and interleukin (IL)-33 release, HMGB1-toll-like receptor 4 and HMGB1-receptor for advanced glycation end products (RAGE) interaction, and nuclear protein expressions of NF-κB and early growth response protein-1 (egr-1) were attenuated by Nec-1. Our finding suggests that necroptosis is responsible for GalN/LPS-induced liver injury through DAMP-activated PRR signaling.     

Oxygen sensitivity of NMDA receptors: relationship to NR2 subunit composition and hypoxia tolerance of neonatal neurons

Neonatal rats survive and avoid brain injury during periods of anoxia 25 times longer than adults. We hypothesized that oxygen activates and hypoxia suppresses NMDA receptor (NMDAR) responses in neonatal rat neurons, explaining the innate hypoxia tolerance of these cells. In CA1 neurons isolated from neonatal rat hippocampus (mean postnatal age [P] 5.8 days), hypoxia (PO(2) 10 mm Hg) reduced NMDA receptor-channel open-time percentage and NMDA-induced increase in [Ca(2+)](i) (NMDA DeltaCa(2+)) by 38 and 68% (P<0.01), respectively. In P20 neurons the reductions were not significant. In P3-10 CA1 neurons within intact hippocampal slices, hypoxia reduced NMDA DeltaCa(2+) by 52% (P=0.002) and decreased NMDA-induced death by 45% (P=0.004). Phalloidin, a microtubule stabilizer, prevented hypoxia-induced inhibition of NMDA DeltaCa(2+) in P3-10 neurons. To test whether NMDARs prevalent in neonates (NR1 plus NR2B or NR2D subunits) are inhibited by hypoxia compared with those in mature neurons (NR2A and NR2C), we expressed these receptors in Xenopus oocytes. Compared with responses in 21% O(2), hypoxia (PO(2) 17 mm Hg) reduced currents from neonatal type NR1/NR2D receptors by 25%, increased currents from NR1/NR2C by 18%, and had no effect on NR1/NR2A or NR1/NR2B. Modulation of NMDARs by hypoxia may play an important role in the hypoxia tolerance of the mammalian neonate. In addition, oxygen sensing by NMDARs could play a significant role in postnatal brain development.     

Calcium transients in the garter snake vomeronasal organ

The signaling cascade involved in chemosensory transduction in the VN organ is incompletely understood. In snakes, the response to nonvolatile prey chemicals is mediated by the vomeronasal (VN) system. Using optical techniques and fluorescent Ca(2+) indicators, we found that prey-derived chemoattractants produce initially a transient cytosolic accumulation of [Ca(2+)](i) in the dendritic regions of VN neurons via two pathways: Ca(2+) release from IP(3)-sensitive intracellular stores and, to a lesser extent, Ca(2+) influx through the plasma membrane. Both components seem to be dependent on IP(3) production. Chemoattractants evoke a short-latency Ca(2+) elevation even in the absence of extracellular Ca(2+), suggesting that in snake VN neurons, Ca(2+) release from intracellular stores is independent of a preceding Ca(2+) influx, and both components are activated in parallel during early stages of chemosensory transduction. Once the response develops in apical dendritic segments, other mechanisms can also contribute to the amplification and modulation of these chemoattractant-mediated cytosolic Ca(2+) transients. In regions close to the cell bodies of the VN neurons, the activation of voltage-sensitive Ca(2+) channels and a Ca(2+)-induced Ca(2+) release from intracellular ryanodine-sensitive stores secondarily boost initial cytosolic Ca(2+) elevations increasing their magnitude and durations. Return of intracellular Ca(2+) to prestimulation levels appears to involve a Ca(2+) extrusion mediated by a Na(+)/Ca(2+) exchanger mechanism that probably plays an important role in limiting the magnitude and duration of the stimulation-induced Ca(2+) transients.     

抑制SHARPIN激活Rip1促进LNCaP-AI细胞坏死性凋亡

目的:探讨SHARPIN对去势抵抗性前列腺癌细胞LNCaP-AI坏死性凋亡关键因子Rip1的调控作用,以及对细胞坏死性凋亡的影响。方法:将LNCaP-AI细胞分为TNF-α+Z-VAD(caspase抑制剂)处理组与TNF-α+Z-VAD+Nec-1(Rip1抑制剂)处理组,应用MTS检测各组细胞的活力,研究坏死性凋亡机制在诱导LNCaP-AI细胞死亡中的作用。将LNCaP-AI细胞分为阴性对照组和SHARPIN干扰(si-SHARPIN)组,RT-qPCR验证抑制效率,通过免疫荧光等技术进一步探讨SHARPIN调控坏死性凋亡的具体分子机制。结果:与对照组相比,TNF-α+Z-VAD处理组的LNCaP-AI细胞活力下降28%(P0.05),而TNF-α+Z-VAD+Nec-1处理组的细胞在Rip1被抑制后,细胞活力无明显改变。在LNCaP-AI细胞中,通过siRNA抑制SHARPIN表达后,Rip1表达水平上调,同时,LNCaP-AI细胞坏死性凋亡比例升高。结论:LNCaP-AI细胞可通过坏死性凋亡机制诱导死亡,下调SHARPIN可能通过激活Rip1增强LNCaP-AI细胞坏死性凋亡。     

Electrophysiological mechanisms of delayed excitotoxicity: positive feedback loop between NMDA receptor current and depolarization-mediated glutamate release

Delayed excitotoxic neuronal death after insult from exposure to high glutamate concentrations appears important in several CNS disorders. Although delayed excitotoxicity is known to depend on NMDA receptor (NMDAR) activity and Ca(2+) elevation, the electrophysiological mechanisms underlying postinsult persistence of NMDAR activation are not well understood. Membrane depolarization and nonspecific cationic current in the postinsult period were reported previously, but were not sensitive to NMDAR antagonists. Here, we analyzed mechanisms of the postinsult period using parallel current- and voltage-clamp recording and Ca(2+) imaging in primary hippocampal cultured neurons. We also compared more vulnerable older neurons [about 22 days in vitro (DIV)] to more resistant younger (about 15 DIV) neurons, to identify processes selectively associated with cell death in older neurons. During exposure to a modest glutamate insult (20 microM, 5 min), similar degrees of Ca(2+) elevation, membrane depolarization, action potential block, and increased inward current occurred in younger and older neurons. However, after glutamate withdrawal, these processes recovered rapidly in younger but not in older neurons. The latter also exhibited a concurrent postinsult increase in spontaneous miniature excitatory postsynaptic currents, reflecting glutamate release. Importantly, postinsult NMDAR antagonist administration reversed all of these persisting responses in older cells. Conversely, repolarization of the membrane by voltage clamp immediately after glutamate exposure reversed the NMDAR-dependent Ca(2+) elevation. Together, these data suggest that, in vulnerable neurons, excitotoxic insult induces a sustained positive feedback loop between NMDAR-dependent current and depolarization-mediated glutamate release, which persists after withdrawal of exogenous glutamate and drives Ca(2+) elevation and delayed excitotoxicity.     

血管紧张素-(1-7)通过抑制TLR4激活和坏死性凋亡的相互作用对抗高糖引起的H9c2心肌细胞损伤

目的:研究血管紧张素-(1-7)[Ang-(1-7)]能否通过抑制Toll样受体4(TLR4)激活和坏死性凋亡的相互作用对抗高糖(HG)引起的H9c2心肌细胞损伤。方法:应用Western blot检测心肌细胞受体相互作用蛋白3(RIP3;反映坏死性凋亡的指标)和TLR4的表达水平;CCK-8法测定心肌细胞存活率;用试剂盒检测细胞培养液中乳酸脱氢酶(LDH)的活性;ELISA检测细胞培养液中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的分泌水平;双氯荧光素染色法测定细胞内活性氧簇(ROS)水平;罗丹明123染色法测定线粒体膜电位(MMP)。结果:HG(35 mmol/L葡萄糖)作用H9c2心肌细胞24 h可使RIP3的表达水平明显升高,应用30μmol/L TAK-242(TLR4抑制剂)和HG共处理心肌细胞24 h可抑制HG对RIP3的上调;另一方面,HG可上调TLR4的表达,100μmol/L坏死性凋亡的特异性抑制剂necrostatin-1(Nec-1)和HG共处理心肌细胞24 h可抑制HG对TLR4的上调;而1μmol/L Ang-(1-7)和HG共处理心肌细胞24 h能同时抑制HG对RIP3和TLR4的上调。此外,1μmol/L Ang-(1-7)、30μmol/L TAK-242或100μmol/L Nec-1与HG共处理心肌细胞均能对抗HG引起的心肌细胞损伤,细胞存活率升高,LDH活性降低,ROS生成和MMP丢失减少,同时IL-1β和TNF-α的分泌减少。结论:Ang-(1-7)通过抑制TLR4激活和坏死性凋亡的相互作用对抗高糖引起的H9c2心肌细胞损伤。     

Motor imagery in blind subjects: the influence of the previous visual experience

Interleukin-1beta (IL-1beta) has been found to play an important role in various diseases in the central nervous system (CNS) and exhibit neuroprotective effects in some conditions. The transmitter release in brain is controlled by voltage-gated Ca(2+) channels (VGCCs), predominantly N-type Ca(2+) channels (NCCs). Although both IL-1beta and NCCs are implicated regulating excitotoxicity and Ca(2+) homeostasis, it is not known whether IL-1beta modulates NCCs directly. In present study, we examined the effects of IL-1beta treatment (10 ng/ml, 24 h) on NCCs in cultured cortical neurons using patch-clamp recording and immunoblot assay. Our results showed that IL-1beta decreased NCC currents by approximately 50%, which made up 40% of the whole-cell Ca(2+) current demonstrated by omega-conotoxin-GVIA, and also significantly downregulated the expression of NCC protein. The residual Ca(2+) currents except L-type Ca(2+) channel currents and NCC currents were not affected by IL-1beta. Our finding, IL-1beta inhibits the activity of NCC via suppressing NCC protein expression provides new insight into the neuroprotective role of IL-1beta in CNS.     

自噬对肾大部切除大鼠肾小管上皮细胞程序性坏死的影响

目的:探讨自噬是否参与肾大部切除(SNx)大鼠肾小管上皮细胞的过度死亡,及其与程序性坏死的关系。方法:48只雄性SD大鼠随机分为control组(6只)和SNx组(42只),分别行假手术和SNx。将24只SNx大鼠分为0、4、8和12周组;其余SNx大鼠分为SNx+vehicle组、SNx+necrostatin-1(Nec-1)组和SNx+3-甲基腺嘌呤(3-MA)组,每组6只。检测0、4、8和12周组大鼠肾组织RIP1、RIP3、LC3和beclin-1的mRNA和蛋白表达水平;用Nec-1和3-MA干预SNx大鼠,Western blot法检测LC3-Ⅰ、LC3-Ⅱ和beclin-1的蛋白水平,透射电镜和TUNEL染色判定Nec-1和3-MA对SNx大鼠肾小管上皮细胞死亡的影响,并观察Nec-1和3-MA对SNx大鼠肾组织的病理变化、活性氧簇(ROS)、血尿素氮(BUN)和血肌酐(SCr)含量的影响。结果:SNx术后8周大鼠肾组织RIP1、RIP3、LC3和beclin-1的mRNA和蛋白水平达最高值(P0.01);Nec-1和3-MA干预SNx大鼠的LC3-Ⅱ/Ⅰ和beclin-1蛋白水平、发生程序性坏死的肾小管上皮细胞及TUNEL阳性细胞数量均显著降低(P0.01)。另外,Nec-1减低SNx大鼠肾组织的ROS含量,但3-MA无此作用。结论:自噬参与了SNx大鼠肾小管上皮细胞过度死亡;抑制自噬可减轻SNx大鼠肾小管上皮细胞程序性坏死及其肾损伤。