Dual inhibition of protein phosphatase-1/2A and calpain rescues nerve growth factor-differentiated PC12 cells from oxygen-glucose deprivation-induced cell death

In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC12 cells.     

Immature rat brain slices exposed to oxygen-glucose deprivation as an in vitro model of neonatal hypoxic-ischemic encephalopathy

To analyze whether exposure to oxygen-glucose deprivation (OGD) of immature rat brain slices might reproduce the main pathophysiologic events leading to neuronal death in neonatal hypoxic-ischemic encephalopathy (NHIE), 500 microm-thick brain slices were obtained from 7-day-old Wistar rats, and incubated in oxygenated physiological solution. In OGD group, oxygen and glucose were removed from the medium for 10-30 min (n = 25); then, slices were re-incubated in normal medium. In control group the medium composition remained unchanged (CG, n = 30). Medium samples were obtained every 30 min for 3 h. To analyze neuronal damage, slices were stained with Nissl and CA1 area of hippocampus and cortex were observed under microscopy. In addition, neuronal death was quantified as LDH released to the medium determined by spectrophotometry. Additionally, medium glutamate (Glu) levels were determined by HPLC and those of TNFalpha by ELISA, whereas inducible nitric oxide synthase expression was determined by Western blot performed on slices homogenate. Optimal OGD time was established in 20 min. After OGD, a significant decrease in the number of neurones in hippocampus and cortex was observed. LDH release was maximal at 30 min, when it was five-fold greater than in CG. Furthermore, medium Glu concentrations were 200 times greater than CG levels at the end of OGD period. A linear relationship between Glu and LDH release was demonstrated. Finally, 3 h after OGD a significant induction of iNOS as well as an increase in TNFalpha release were observed. In conclusion, OGD appears as a feasible and reproducible in vitro model, leading to a neuronal damage, which is physiopathologically similar to that found in NHIE.     

Acute and repeated restraint stress influences cellular damage in rat hippocampal slices exposed to oxygen and glucose deprivation

Several studies have shown that high corticosteroid hormone levels increase neuronal vulnerability. Here we evaluate the consequences of in vivo acute or repeated restraint stress on cellular viability in rat hippocampal slices suffering an in vitro model of ischemia. Cellular injury was quantified by measuring lactate dehydrogenase (LDH) and neuron-specific enolase released into the medium. Acute stress did not affect cellular death when oxygen and glucose deprivation (OGD) was applied both immediately or 24h after restraint. The exposure to OGD, followed by reoxygenation, resulted in increased LDH in the medium. Repeated stress potentiated the effect of OGD both, on LDH and neuron-specific enolase released to the medium. There was no effect of repeated stress on the release of S100B, an astrocytic protein. Additionally, no effect of repeated stress was observed on glutamate uptake by the tissue. These results suggest that repeated stress increases the vulnerability of hippocampal cells to an in vitro model of ischemia, potentiating cellular damage, and that the cells damaged by the exposure to repeated stress+OGD are mostly neurons. The uptake of glutamate was not observed to participate in the mechanisms responsible for rendering the neurons more susceptible to ischemic damage after repeated stress.     

亚低温对体外缺氧缺糖损伤后神经元的保护作用及其对谷氨酸转运体、亲代谢型谷氨酸受体表达的影响

目的通过研究亚低温对体外缺氧缺糖损伤的神经细胞谷氨酸转运体(GLT-1)、亲代谢型谷氨酸受体(mGluR2/3)表达的影响,探讨亚低温的神经保护途径。方法分组培养大鼠大脑皮层细胞,亚低温组在33 C条件、常温组在37 C条件,缺氧缺糖培养2h后,复氧复糖37 C培养,经时(6h、12h、24h、3d)检测LDH释放量、GLT-1和mGluR2/3蛋白表达量。结果在复氧复糖后,两组LDH释放均呈现上升趋势,其中24h和3d亚低温组的LDH上升水平显著降低(P<0.05);两组GLT-1表达均呈现先下降后上升的趋势,其中6h和12h亚低温组的GLT-1下降水平显著降低(P<0.05);两组mGluR2/3表达均呈现上升趋势,其中12h和24h亚低温组的mGluR2/3升高水平显著增加(P<0.05)。结论亚低温能够在体外水平,通过抑制GLT-1蛋白的表达下调,促进mGluR2/3的表达上调,抑制神经元兴奋性损伤。     

Penehyclidine hydrochloride protects against oxygen and glucose deprivation injury by modulating amino acid neurotransmitters release

Abstract

Penehyclidine hydrochloride (PHC) is an anticholinergic agent, with only high degree of selectivity for M₁ and M₃ receptor subtypes. In this study, we investigated whether PHC could play a protective effect on hippocampal slice against oxygen and glucose deprivation (OGD), as well as related to the change of amino acid neurotransmitters release. Penehyclidine hydrochloride 2, 10, and 50 μM doses were adopted in the lactate dehydrogenase (LDH) leakage assay and triphenyl tetrazolium chloride (TTC) staining. The spontaneous miniature excitatory postsynaptic currents (mEPSCs) and amino acid neurotransmitters were detected by electrophysiology method and high-performance liquid chromatography (HPLC), respectively. Our study showed that PHC can lessen the LDH leakage ratio and tissue injury values according to TTC staining. Penehyclidine hydrochloride decreased the content of aspartate acid (Asp) and glutamate (Glu), and elevated the content of glycine (Gly) and gamma-aminobutyric acid (GABA). Ischemia increased the amplitude and frequency of the mEPSCs, but PHC obviously decreased the frequency and amplitude of mEPSCs. Thus, the study reveals the fact that PHC protects hippocampus slice against OGD injury by decreasing excitatory amino acids release and increasing inhibitory amino acids release.     

Apoptotic characteristics of cell death and the neuroprotective effect of homocarnosine on pheochromocytoma PC12 cells exposed to ischemia

We recently improved an in vitro ischemic model, using PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 3 hr in a special device, followed by 18 hr of reoxygenation. The cell death induced in this ischemic model was evaluated by a series of markers: lactate dehydrogenase (LDH) release, caspase-3 activation, presence of cyclin D1, cytochrome c leakage from the mitochondria, BAX cellular redistribution, cleavage of poly (ADP-ribose) polymerase (PARP) to an 85-kDa apoptotic fragment, and DNA fragmentation. The OGD insult, in the absence of reoxygenation, caused a strong activation of the mitogen-activated protein kinase (MAPK) isoforms extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and stress-activated protein kinase (SAPK), also known as p-38. The detection of apoptotic markers and activation of MAPKs during the ischemic insult strongly suggest that apoptosis plays an important role in the PC12 cell death. Homocarnosine, a neuroprotective histidine dipeptide, present in high concentrations in the brain, was found to provide neuroprotection, as expressed by a 40% reduction in LDH release and caspase-3 activity at 1 mM. Homocarnosine reduced OGD activation of ERK 1, ERK 2, JNK 1, and JNK 2 by 40%, 46%, 55%, and 30%, respectively. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that antioxidants, such as homocarnosine, may prevent OGD-induced neuronal death by inhibiting the apoptotic process and/or in relation to the differential attenuation of activity of MAPKs.     

Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia

The development of neuroprotective drugs against ischemic insults is hampered by the lack of pharmacological in vitro models. We developed an ischemic model using PC12 cell cultures exposed to oxygen-glucose-deprivation (OGD) followed by reoxygenation (18 hr) under regular atmospheric oxygen level. The toxicity induced in this model, that is partially caused by generation of reactive oxygen species (ROS), was measured morphologically as well as by the release of lactate dehydrogenase (LDH) and the prostaglandin PGE(2) from the cells. Carnosine and homocarnosine, histidine dipeptides antioxidants, found in high concentration in the brain, have been suggested to provide neuroprotection. Using the OGD model we found that 5 mM carnosine and 1 mM homocarnosine provided maximal neuroprotection of about 50% against OGD insult. This neuroprotective effect was similar to that of a known antioxidant, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol), and was not observed in a serum-deprivation toxicity model of PC12 cells, indicating that carnosine and homocarnosine may act as antioxidant-neuroprotective agents in the brain. Our ischemic model may provide a useful tool for investigating the mechanisms involved in the neuroprotection afforded by histidine dipeptides.     

MiR-139 protects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced nerve injury through targeting c-Jun to inhibit NLRP3 inflammasome activation

BackgroundCerebral ischemia/reperfusion (I/R) injury after ischemic stroke is usually accompanied with the activation of inflammasome which seriously impairs neurological function. MiR-139 has been reported to be associated with inflammatory regulation in multiple diseases. However, its effect and mechanism on inflammation regulation after cerebral I/R injury are still poorly understood.MethodsAn in vitro model of cerebral I/R injury was constructed with oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. TargetScan bioinformatics analysis and dual luciferase reporter assay were utilized to confirm the targeted relationship between miR-139 and c-Jun. Cell pyroptosis was verified by flow cytometry and Caspase-1 Detection Kit. qRT-PCR assay was performed to detect the expression levels of miR-139, c-Jun, NLRP3 and ASC. Western blotting was applied to measure the protein levels of c-Jun and pyroptosis-related markers NLRP3, ASC, caspase-1, GSDMD^Nterm. The ELISA assay was applied to measure the release of IL-1β, IL-18 and LDH.ResultsMiR-139 was significantly downregulated whereas c-Jun was obviously upregulated after OGD/R treatment. TargetScan analysis predicted that c-Jun was a potential target of miR-139, which was verified by the dual-luciferase reporter assay. Also, overexpression of miR-139 repressed c-Jun expression. Furthermore, miR-139 inhibited OGD/R-induced cell pyroptosis and the upregulation of NLRP3, caspase-1, ASC, GSDMD^Nterm, and the release of IL-1β, IL-18 and LDH, while miR-139 inhibition exerted the opposite effects. However, overexpression of c-Jun aggravated OGD/R-induced nerve injury and partly abolished the neuroprotective effect of miR-139.ConclusionUpregulation of miR-139 exerted neuroprotection against OGD/R-induced nerve injury by negatively regulating c-Jun/NLRP3 inflammasome signaling. This study offered insights for providing potential therapeutic targets for treating cerebral I/R injury.     

15d-PGJ_2对神经元氧糖剥夺/复氧损伤的影响

目的观察过氧化物酶体增殖物激活受体γ(PPAR-γ)激动剂15d-PGJ_2对神经元氧糖剥夺/复氧(OGD/R)损伤的影响,并探讨其可能的作用机制。方法采用原代培养的小鼠胎鼠大脑皮质神经元,建立OGD/R损伤模型。将培养的神经元细胞进行分组,Western blot检测各组神经元内LC3-1、LC3-Ⅱ、Bcl-2、Beclin1、AMPK、m TOR及p70S6K等蛋白的表达;MTT法检测细胞活性,乳酸脱氢酶(LDH)漏出率测定细胞毒性。结果 OGD/R损伤6 h、12 h及24 h后神经元LC3-Ⅱ、Beclin 1表达明显增高,而p62表达持续下降,神经元的生存率明显下降,LDH漏出率增加。15d-PGJ_2可显著降低LC3-Ⅱ和Beclin 1表达水平,改善神经元的生存率及降低LDH的漏出率。OGD/R后Bcl-2的蛋白表达水平均明显减少,而Beclin 1表达则显着增加。15d-PGJ_2预处理显著增加OGD/R 24 h的Bcl-2表达量。利用Bcl-2 siRNA或scRNA转染神经元细胞发现,Bcl-2 siRNA可消除15d-PGJ_2对OGD/R后各时间点的抑制效应。结论 15d-PGJ_2能够有效地对神经元OGD/R损伤起到保护作用,其机制可能是通过上调Bcl-2的表达进而抑制自噬的发生实现的。     

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38alpha and beta, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.     

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38α and p38β activation and confers neuroprotection in the pheochromocytoma PC12 model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38α and β, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.     

Neuroprotection afforded by prior citicoline administration in experimental brain ischemia: effects on glutamate transport

BACKGROUND AND PURPOSE: Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine, has shown beneficial effects in a number of CNS injury models including cerebral ischemia. Citicoline is the only neuroprotectant that has proved efficacy in patients with moderate to severe stroke. However, the precise mechanism by which citicoline is neuroprotective is not fully known. The present study was designed to search for mechanisms of citicoline neuroprotective properties using in vivo and in vitro models of brain ischemia. METHODS: Focal brain ischemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Brain glutamate levels were determined at fixed intervals after occlusion. Animals were then sacrificed, and infarct volume and brain ATP levels were measured. As in vitro model of ischemia, rat cultured cortical neurones or astrocytes, isolated or in co-culture, were exposed to oxygen-glucose deprivation (OGD) either in the absence or in the presence of citicoline (1-100 microM). Viability was studied by measuring LDH release. Glutamate release and uptake, and ATP levels were also determined. RESULTS: Citicoline (0.5, 1 and 2 g/kg i.p. administered 1 h before the occlusion) produced a reduction of the infarct size measured at striatum (18, 27 and 42% inhibition, respectively, n = 8, P < 0.05 vs. ischemia), effect that correlated with the inhibition caused by citicoline on ischemia-induced increase in glutamate concentrations after the onset of the ischemia. Citicoline also inhibited ischemia-induced decrease in cortical and striatal ATP levels. Incubation of cultured rat cortical neurones with citicoline (10 and 100 microM) prevented OGD-induced LDH and glutamate release and caused a recovery in ATP levels after OGD, confirming our previous results. In addition, citicoline (100 microM) caused an increase in glutamate uptake and in EAAT2 glutamate transporter membrane expression in cultured rat astrocytes. CONCLUSIONS: Our present findings show novel mechanisms for the neuroprotective effects of citicoline, which cooperate to decrease brain glutamate release after ischemia.     

Quercetin protects oligodendrocyte precursor cells from oxygen/glucose deprivation injury in vitro via the activation of the PI3K/Akt signaling pathway

The aim of this study was to investigate the protection of quercetin (QUE) on oligodendrocyte precursor cells (OPCs) from oxygen/glucose deprivation (OGD)-induced injury in vitro and explore whether the PI3K/Akt signaling pathway contributed to the protection provided by quercetin. The OGD condition was induced by including 2mM sodium dithionite (Na(2)S(2)O(4)) in glucose-free DMEM medium. The concentration of QUE in this study ranged from 3μM to 81μM. OPCs were identified by immunocytochemical staining. Cell viability was analyzed using the water soluble tetrazolium salt-8 (WST-8) and lactate dehydrogenase assay (LDH). The morphological changes of the nucleus were measured using Hoechst 33258 nuclear staining, and the ratio of apoptotic cells was determined by FITC annexin V- and propidium iodide (PI) flow cytometry assay kit. In addition, the levels of pro-apoptotic proteins such as cleaved-caspase-3 and Bax and the anti-apoptotic proteins p-Akt and Bcl-2 were quantified using western blotting. The results showed that the OPC cell survival rate was significantly increased by incubation in conditioned medium supplemented with QUE as measured by the WST-8 assay, while the LDH release rate was significantly decreased as analyzed by the LDH assay. Furthermore, apoptosis assay showed that the apoptosis ratio of OPCs was also dramatically reduced by QUE. Western blotting showed that the expression levels of Bax and cleaved-caspase-3 proteins were down-regulated, while Bcl-2 and p-Akt were up-regulated. Further study showed that the increase in p-Akt by QUE was reduced by the PI3K inhibitor LY294002. These results indicated that QUE effectively protected OPCs from OGD-induced injury and that the mechanism might be related to the activation of the PI3K/Akt signaling pathway.     

The relationship between exercise participation and well-being of the retired elderly

The objective of this study is to identify the relationship between physical exercise and the feelings of well-being of the retired elderly. Face-to-face questionnaire survey was adopted, and quota sampling was chosen to select the respondents. A total of 352 valid questionnaires were collected in selected parks in Taipei. The survey found that the Taiwanese retired elderly participated in a wide range of sports, from light exercise such as walking to vigorous exercise such as tennis and aerobics, and their participation frequency was very high. Most of the respondents (87.2%) were defined as having positive well-being. The results showed that exercise frequency and well-being were positively correlated, but a negative correlation was found between exercise intensity and well-being. The survey found that the intensity of exercise was self-evaluated by as being low to moderate, but most of the activities were in the categories of moderate or vigorous intensity according to the metabolic equivalents suggested by American College of Sports Medicine. The study suggest that the elderly felt more comfortable and gained more pleasure psychologically while participating in exercises less intensive. As a result, the retired elderly are recommended to take exercise as frequently as possible. As to exercise intensity, self-evaluated low-to-moderate intensity exercise might be better for the psychological well-being of the elderly.     

Differential vulnerability of cortical and cerebellar neurons in primary culture to oxygen glucose deprivation followed by reoxygenation

The effects of glucose and O2 deprivation (OGD) on the survival of cortical and cerebellar neurons were examined to characterize the biochemical mechanisms involved in OGD and OGD followed by reoxygenation. To this aim, neurons were kept for different time periods in a hypoxic chamber with a controlled atmosphere of 95% N(2) and 5% CO2 in a glucose-free medium. After OGD, reoxygenation was achieved by exposing the cells to normal O2 and glucose levels. Neither MTT, an index of mitochondrial oxidative phosphorylation, nor malondialdehyde (MDA) production, a parameter measuring lipid peroxidation, were affected by 1 hr of OGD in cortical neurons. When OGD was followed by 24 hr of reoxygenation, MTT levels were reduced by 40% and MDA was significantly increased, whereas cellular ATP content did not change. Cerebellar granule cells, on the other hand, did not show any reduction of mitochondrial activity after exposure to 1 hr OGD or to 1 hr OGD plus 24 hr of reoxygenation. When OGD was prolonged for 2 hr, a significant reduction of the mitochondrial activity and of cellular ATP content occurred, coupled to a significant MDA increase in cerebellar granule cells, whereas in cortical neurons a reduction of MTT levels after 2 hr OGD was not accompanied by a decrease of cellular ATP content nor by an increase of MDA production. Moreover, 24 hr of reoxygenation further reinforced lipid peroxidation, LDH release, propidium iodide positive neurons and the reduction of ATP content in cerebellar granule cells. The results of the present study collectively show that cortical and cerebellar neurons display different levels of vulnerability to reoxygenation followed by OGD. Furthermore, the impairment of mitochondrial activity and the consequent overproduction of free radicals in neurons were observed for the first time occurring not only during the reoxygenation phase, but already beginning during the OGD phase.     

Neuroprotective effects of the beta-catenin stabilization in an oxygen- and glucose-deprived human neural progenitor cell culture system

β-Catenin stabilization achieved either via GSK-3β specific inhibition or involving canonical Wnt signalling pathway, contributes to neuroprotection in an oxygen-glucose deprivation (4 h OGD) in vitro hypoxia model performed on human cortical neural progenitor cells previously differentiated into neurons and glia. Neuroprotection mechanisms include both acquiring tolerance to injury throughout preconditioning (72 h prior to OGD) or being pro-survival during 24 h reoxygenation after the insult. Four hours of OGD induced apoptotic cell death elevation to 73 ± 1% vs. 12% measured in control and the LDH level, indicative of necrotic cell injury, elevation by 67 ± 7% (set to 100%). A significant reduction in apoptosis occurred at 24 h reoxygenation with indirubin supplement which was 49 ± 6% at 2.5 μM BIO while LDH level was only 47 ± 5% of OGD. Kenpaullone was efficient in reducing both cell deaths at 5 μM (apoptosis 38 ± 1% and necrosis 33 ± 3% less than in OGD). Wnt agonist reduced apoptosis to 45 ± 3% at 0.01 μM, while LDH value was decreased to a level of 53 ± 5% of control. Our findings suggest that GSK-3beta inhibitors/β-catenin stabilizers may ultimately be useful drugs in neuroprotection and neuroregeneration therapies in vivo.     

瑞香素通过抑制TXNIP/NLRP3信号通路来减轻OGD/R诱导的海马神经元炎症反应和凋亡

目的 探讨瑞香素通过抑制硫氧还蛋白结合蛋白(Thioredoxin-interacting protein,TXNIP)/核苷酸结合寡聚化结构域样受体蛋白3(Nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)信号通路来对氧糖剥夺/复氧(Oxygen-glucose deprivation/Reoxygenation,OOGD/R)诱导的海马神经元炎症反应和凋亡的影响。方法 对体外培养的小鼠海马神经元HT-22做OGD/R处理诱导建立细胞损伤模型,采用细胞计数试剂盒-8(Cell counting Kit-8,CCK-8)实验检测0、5、10、20、40、80 μmol/L的瑞香素对其细胞活力的影响,筛选出最佳作用水平; 将体外培养的HT-22细胞随机分为对照组、模型组、瑞香素(40 μmol/L)组、TXNIP空载(转染TXNIP空载质粒)组、TXNIP过表达(转染TXNIP过表达质粒)组、瑞香素(40 μmol/L)+TXNIP过表达组,以瑞香素和质粒提前处理12 h后进行OGD/R诱导建立细胞损伤模型; 采用二苯甲亚胺、三氯化氢2'-(4-羟基苯基)-5-(4-甲基-1-哌嗪基)-2,5'-二-1H-苯并咪唑(2'-(4-Hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole,trihydrochloride,Hoechst)33258染色及流式细胞实验检测各组HT-22细胞凋亡情况; 采用试剂盒检测各组HT-22细胞线粒体膜电位; 应用酶标仪测量各组HT-22细胞肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素(Interleukin,IL)-6、IL-1β、IL-18、超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)、活性氧(Reactive oxygen species,ROS)及乳酸脱氢酶(Lactate dehydrogenase,LDH)释放水平; 采用免疫印迹实验检测各组HT-22细胞TXNIP/NLRP3通路蛋白表达水平。结果 选择40μmol/L瑞香素处理OGD/R诱导HT-22细胞进行后续实验; 与对照组比较,模型组HT-22细胞凋亡率、细胞TNF-α,IL-6,IL-1β,IL-18及ROS水平、细胞LDH释放水平、细胞TXNIP,NLRP3及天冬氨酸特异性半胱氨酸蛋白酶1(Cysteine-containing aspartate-specific proteases-1,Caspase-1)蛋白表达水平明显升高(P<0.05),线粒体膜电位、细胞SOD及CAT水平明显降低(P<0.05)。分别与模型组、瑞香素+TXNIP过表达组比较,瑞香素组HT-22细胞凋亡率、细胞TNF-α,IL-6,IL-1β,IL-18及ROS水平、细胞LDH释放水平、细胞TXNIP,NLRP3及Caspase-1蛋白表达水平均降低(P<0.05),线粒体膜电位、细胞SOD及CAT水平均升高(P<0.05); TXNIP过表达组HT-22细胞凋亡率、细胞TNF-α,IL-6,IL-1β,IL-18及ROS水平、细胞LDH释放水平、细胞TXNIP,NLRP3及Caspase-1蛋白表达水平均升高(P<0.05),线粒体膜电位、细胞SOD及CAT水平均降低(P<0.05); TXNIP空载组HT-22细胞各指标水平均无明显变化(P>0.05)。结论 瑞香素可通过抑制TXNIP/NLRP3信号通路来降低OGD/R诱导的ROS和炎性因子过量表达,抑制炎症和氧化应激反应,缓解海马神经元线粒体损伤,减轻神经元凋亡。     

The patterns of exercise-induced β-endorphin expression in the central nervous system of rats

Exercise at different intensities is able to induce different physical and psychological statuses of the subjects. The β-endorphin (β-EP) in central nervous system is thought to play an important role in physical exercise. However, its expression patterns and physiological effects in the central nuclei under different exercise states are not well understood. Five-week old male Sprague-Dawley rats were randomly divided into two groups of 21 each: Control and Exercise. Control rats were sedentary while Exercise rats were arranged to run on a treadmill (5-week adapting or moderate exercise and 2-week high-intensity exercise). Seven rats were taken from each group at day33, day42 and day49 for examination of blood biochemical parameters (lactate, Lac; blood urea nitrogen, BUN; glucose) and for detection of nuclei β-EP level with immunohistochemistry. The results showed that Lac and BUN levels were significant increased after the high intensity exercise. The five-week exercise caused a significantly increased β-EP in caudate putamen (CPu), amygdala, paraventricular thalamic nucleus (PVT), ventromedial hypothalamus nucleus (VMH) and gigantocellular reticular nucleus (Gi). The high intensity exercise induced an elevated β-EP in CPu and nucleus of the solitary tract (Sol), but a decreased β-EP in globus pallidus (GP). Compared with Control, exercise rats showed an elevated β-EP in CPu, PVT, VMH, accumbens nucleus, Gi and Sol, and a decreased β-EP in GP at day49. The β-EP levels in acurate nucleus, periadueductal gray and parabrachial nucleus were not changed at day33, 42 and 49. In conclusion, β-EP levels in different nuclei changed under the moderate and high intensity exercises, which may contribute to modifying exercise-produced psychological and physiological effects.     

MiR-29a-3p Enhances the Viability of Rat Neuronal Cells that Injured by Oxygen-Glucose Deprivation/Reoxygenation Treatment Through Targeting TNFRSF1A and Regulating NF-κB Signaling Pathway

ObjectiveWe attempt to investigate the role of TNFRSF1A and its underlying mechanism in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in rat pheochromocytoma PC12 cells.MethodsPublic datasets GSE61616 and GSE106680 were downloaded from GEO database. PC12 cells were used to construct OGD/R models. QRT-PCR and western blot were implemented to test the relative mRNA and protein levels, respectively. The miRNA online prediction website TargetScan was used to predict TNFRSF1A upstream regulated miRNAs, which were then confirmed by luciferase reporter assay. The changes in cell viability and apoptosis were evaluated using cell counting kit 8 (CCK-8), lactose dehydrogenase (LDH), and flow cytometry assays.ResultsBioinformatics analysis demonstrated that the expression of TNFRSF1A was upregulated in CI/RI and middle cerebral artery occlusion models compared with control, respectively. And a significant upregulation was also observed in OGD/R-damaged PC12 cells. Depletion of TNFRSF1A can notably enhance the cells proliferation after OGD/R treatment, while enlargement of TNFRSF1A presented the opposite outcomes. Moreover, miR-29a-3p was shown to be the upstream regulatory miRNA of TNFRSF1A. The levels of TNFRSF1A were inversely mediated by miR-29a-3p. Overexpression of miR-29a-3p can raise the cell viability, decrease the LDH activity, and reduce the apoptotic ratio in OGD/R-treated cells. Besides, TNFRSF1A can attenuate the protective effect of miR-29a-3p on OGD/R-treated cells. Furthermore, miR-29a-3p mimic inhibited, while overexpression of TNFRSF1A promoted the activation of NF-κB signaling pathway, and TNFRSF1A can attenuate the suppressive effect of miR-29a-3p on the NF-κB pathway.ConclusionOur research illustrated that the potential regulatory role of miR-29a-3p/TNFRSF1A axis in neurons cells suffered from OGD/R, and their effects on NF-κB signaling pathway, providing a possible bio-target for protecting cells from OGD/R damage .     

Activation of CysLT receptors induces astrocyte proliferation and death after oxygen-glucose deprivation

We recently found that 5-lipoxygenase (5-LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT(1) and CysLT(2) receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5-LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen-glucose deprivation (OGD) followed by recovery to induce ischemic-like injury in the cultured rat astrocytes. We found that 1-h OGD did not injure astrocytes (sub-lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4-h OGD moderately injured the cells (moderate OGD) and led to death 24-72 h after recovery. Inhibition of phospholipase A(2) and 5-LOX attenuated both the proliferation and death. Sub-lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5-LOX inhibitors. Sub-lethal OGD increased the expressions of CysLT(1) receptor mRNA and protein, while moderate OGD induced the expression of CysLT(2) receptor mRNA. Exogenously applied leukotriene D(4) (LTD(4)) induced astrocyte proliferation at 1-10 nM and astrocyte death at 100-1,000 nM. The CysLT(1) receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT(2) receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD(4) (100 nM) increased the expression of CysLT(2) receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT(1) receptor-mediated proliferation and CysLT(2) receptor-mediated death.