Che-1通过抑制自噬减轻氧糖剥夺所致神经元损伤的研究

目的:研究Che-1蛋白对氧糖剥夺(Oxygen glucose deprivation, OGD)所致神经元损伤的保护作用及机制。方法:OGD处理神经元后,采用免疫荧光染色和免疫印迹法检测Che-1蛋白的表达;慢病毒转染神经元实现Che-1过表达,检测乳酸脱氢酶(Lactate dehydrogenase, LDH)释放量和流式细胞术检测神经元凋亡反映OGD所致神经元损伤程度,采用免疫荧光染色和免疫印迹法检测神经元自噬;使用自噬激动剂雷帕霉素(Rapamycin)处理神经元,并通过检测LDH释放量和流式细胞术研究自噬在Che-1保护作用中的作用。结果:免疫荧光结果显示,OGD后神经元Che-1蛋白表达明显增高;免疫印迹结果显示,OGD后6至48 h神经元Che-1蛋白表达明显增高;慢病毒转染过表达Che-1蛋白后,OGD所致神经元LDH释放量明显减低,且OGD所致神经元凋亡明显减少;过表达Che-1蛋白可显著减少OGD所致神经元Beclin1和LC3II的表达;自噬激动剂Rapamycin可逆转Che-1对OGD所致神经元损伤的保护作用。结论:过表达Che-1蛋白可通过抑制神经元自噬对OGD所致神经元损伤发挥保护作用。     

I组mGluRs反义寡核苷酸抗谷氨酸对小鼠大脑皮层神经元的兴奋毒作用

目的:研究I组代谢型谷氨酸受体(mGluRs)反义寡核苷酸对谷氨酸钠(Glu)引起的小鼠大脑皮层神经元损伤的保护作用。方法:以细胞乳酸脱氢酶(LDH)漏出、光镜下细胞形态变化为指标,观察培养液中加入Glu引起的神经元损伤及mGluRl反义寡核苷酸或mGluR5反义寡核苷酸的保护作用;用免疫细胞化学检测神经元mGlulα仪和mGluR5表达。结果:实验显示0.1mmol.L-1的谷氨酸钠可明显造成神经元损伤,使LDH漏出增加(P<0.01),mGluRl反义寡核苷酸或mGluR5反义寡核苷酸6μmol.L-1和8μmol.L-1可明显拮抗Glu引起的神经元损伤,使LDH漏出显著减少(P<0.01);免疫组化实验证实体外培养神经元mGluRlα和mGluR5阳性表达。结论:mGluRl反义寡核苷酸和mGluR5反义寡核苷酸可对抗Glu引起的皮层神经元损伤。     

自噬在高压氧预处理预防脊髓缺血再灌注损伤中 的作用机制研究*

目的:研究自噬在高压氧预处理预防脊髓缺血再灌注损伤中的机制。方法:新生大鼠脊髓神经元原代培养,分为对照组(氧糖剥夺)和高压氧(HBO)预处理组。通过应用免疫组织化学、Western blot分析两组LC3-Ⅱ与凋亡相关分子Beclin-1,Bcl-2,Casp-ase-3的表达变化。结果:发现重复高压氧预处理对氧糖剥夺诱导原代培养的脊髓神经元损伤具有明显的保护作用。免疫组化和Western blot显示与对照组相比高压氧预处理显著增加脊髓神经元细胞Bcl-2的表达,降低Beclin-1,Caspase-3以及自噬的特异性标记蛋白LC3-Ⅱ的表达。氧糖剥夺后对照组与高压氧组相比,LDH释放量明显增多(P<0.05)。结论:HBO预处理通过调节自噬减轻缺血再灌注损伤,为HBO预处理神经保护提供一条新的作用机制。     

Ⅰ组mGluRs反义寡核苷酸抗谷氨酸对小鼠大脑皮层神经元的兴奋毒作用

目的研究Ⅰ组代谢型谷氨酸受体(mGluRs)反义寡核苷酸对谷氨酸钠(Glu)引起的小鼠大脑皮层神经元损伤的保护作用.方法以细胞乳酸脱氢酶(LDH)漏出、光镜下细胞形态变化为指标,观察培养液中加入Glu引起的神经元损伤及mGluRl反义寡核苷酸或mGluR5反义寡核苷酸的保护作用;用免疫细胞化学检测神经元mGlulα仪和mGluR5表达.结果实验显示0.1 mmol·L-1的谷氨酸钠可明显造成神经元损伤,使LDH漏出增加(P＜0.01),mGluRl反义寡核苷酸或mGluR5反义寡核苷酸6 μmol·L-1和8μmol·L-1可明显拮抗Glu引起的神经元损伤,使LDH漏出显著减少(P＜0.01);免疫组化实验证实体外培养神经元mGluRlα和mGluR5阳性表达.结论mGluRl反义寡核苷酸和mGluR5反义寡核苷酸可对抗Glu引起的皮层神经元损伤.     

SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤*

目的:探讨SIRT3调控的线粒体自噬对高糖加重神经元缺氧再灌注损伤的影响及机制。方法:高糖(50 mmol/L)干预HT22细胞后,构建细胞缺氧/复氧模型,利用SIRT3抑制剂3-TYP抑制SIRT3表达。倒置显微镜观察细胞形态改变,CCK8法检测细胞存活率,流式细胞术检测细胞凋亡率,TMRE荧光试剂盒检测细胞线粒体膜电位,RT-qPCR、Western blot检测相关分子的基因和蛋白质表达。结果:高糖使神经元缺氧再灌注后的细胞碎片进一步增加,细胞存活率降低,细胞凋亡率升高(P<0.05)。此外,高糖降低了神经元缺氧再灌注后的线粒体膜电位(P<0.05)。进一步研究发现,高糖上调神经元缺氧再灌注后线粒体分裂相关蛋白DRP1的表达水平,降低了线粒体融合相关蛋白OPA1和线粒体外膜蛋白TOM20的表达;并且增加了自噬相关蛋白LC3Ⅱ、Beclin-1和线粒体自噬相关蛋白PINK1、Parkin的表达;同时,高糖升高了SIRT3的基因和蛋白质表达(P<0.05)。而SIRT3抑制剂3-TYP使神经元高糖缺氧再灌注损伤加重,同时进一步上调DRP1、LC3Ⅱ和PINK1的蛋白质表达(P<0.05)。结论:高糖可显著加重神经元缺氧再灌注损伤,破坏细胞线粒体功能,激活细胞线粒体自噬;SIRT3可抑制PINK1-Parkin通路介导的线粒体自噬并减轻神经元高糖缺氧再灌注损伤。     

SOD2在姜黄素减轻氧糖剥夺所致神经元损伤中的作用

目的:探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了姜黄素(Curcumin,Cur)对氧糖剥夺模型(Oxygen-Glucose Deprivation,OGD)损伤神经元的保护作用。方法:本研究采用HT22神经元细胞暴露于OGD环境中3 h模拟神经元缺血缺氧损伤,SOD2-si RNA抑制神经元SOD2蛋白表达后,通过噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,比色法测量培养基乳酸脱氢酶(Lactic Dehydrogenase LDH)水平,流式细胞仪计算细胞凋亡率,Western blot测定凋亡蛋白Cleaved Caspase-3表达,并观察细胞形态和线粒体功能。结果:与正常培养的Control组相比,OGD组细胞活力显著降低,LDH释放明显增加,细胞凋亡率和Cleaved Caspase-3表达显著上升,细胞形态破坏并降低线粒体膜电位(MMP)和线粒体复合物1(Mitochondrial Complex 1 Activity)的活力(P0.05),100 ng/ml的Cur可显著减轻OGD诱导的神经元细胞的上述损伤性改变(P0.05)。而SOD2-si RNA显著逆转Cur对OGD诱导的神经元细胞损伤的保护作用(P0.05),SC-si RNA则未对Cur产生的神经保护作用造成显著干扰(P0.05)。结论:Cur可能通过上调SOD2的表达,减轻OGD对神经元细胞的损伤。     

SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义

目的:通过建立体外脑缺血模型,探讨沉默信息因子3(SIRT3)在小鼠皮层神经元氧糖剥夺再灌注(OGD/R)损伤后的表达和意义。方法:C57BL/6J小鼠皮层神经元原代培养7天后,以氧糖剥夺不同时长(2 h、4 h、6 h、8 h)再灌注24 h作为观察时间点,利用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)检测细胞活力;小鼠乳酸脱氢酶(LDH)试剂盒检测LDH释放;蛋白印迹法(Western blot WB)观察微管相关蛋白1轻链3(LC3-Ⅱ)、活化凋亡蛋白3(Cleaved caspase-3)、以及SIRT3的表达变化;免疫荧光下进一步观察LC3-II、SIRT3表达。结果:与正常组比,随着氧糖剥夺时间的延长,LDH释放量呈台阶式升高(P0.01),而神经元活性进展性下降(P0.01);蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调,并于OGD 4h达峰值,SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状,而Cleaved caspase-3整体上调;相应的,细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达,与此同时SIRT3荧光强度亦增高。结论:神经元缺血时间越长损伤越重;LC3-Ⅱ和SIRT3表达呈现相似性;SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。     

半胱氨酰白三烯受体拮抗剂通过下调自噬减轻长爪沙鼠的全脑缺血再灌注损伤

目的 探讨半胱氨酰白三烯受体拮抗剂(普鲁司特、HAMI 3379)对长爪沙鼠全脑缺血再灌注损伤的保护作用及其作用机制。方法 采用结扎双侧颈总动脉缺血10 min再灌注24 h,建立长爪沙鼠全脑缺血再灌注损伤模型,随机分为假手术组、模型组、普鲁司特、HAMI 3379组,每组20只,术前3 d开始腹腔注射给药,1次/日,术前30 min给药1次。观察再灌注24 h神经症状及功能;尼氏染色法观察皮层及海马区神经元;免疫印迹法检测皮层、海马中自噬相关蛋白beclin-1及LC3的表达情况;电镜观察海马区自噬小体。结果与模型组比较,普鲁司特、HAMI 3379组可提高神经症状评分,减少神经功能损伤,减轻皮层及海马区神经元损伤及丢失,减少自噬相关蛋白beclin-1及LC3的表达及海马区自噬小体的数量。结论 半胱氨酰白三烯受体拮抗剂通过下调大脑皮层、海马区的自噬减轻长爪沙鼠全脑缺血再灌注损伤。     

人参皂甙Rd预处理可减轻谷氨酸所致的PC12细胞损伤

目的:研究人参皂甙Rd(Ginsenoside Rd)预处理对谷氨酸所致PC12细胞损伤的影响。方法:将体外培养的PC12细胞分为3组,分别为对照组(Control)、谷氨酸损伤组(Glu)和人参皂甙Rd预处理组(Rd)。Control组细胞正常培养;Glu组细胞暴露于含10mM谷氨酸的DMEM培养基中损伤24 h;Rd组细胞经50μM的人参皂甙Rd预处理30 min后,在谷氨酸浓度为10 mM的DMEM培养基中损伤24 h。采用MTT检测细胞活力和乳酸脱氢酶(LDH)检测试剂盒检测LDH释放量;流式细胞仪检测胞内活性氧(ROS)水平;Western blot检测还原型谷胱甘肽蛋白(GSH)表达;专用试剂盒检测细胞内过氧化氢酶(CAT)和超氧化物歧化酶(SOD)含量,相差显微镜观测细胞形态。结果:50μM的人参皂甙Rd预处理30 min,可明显提高谷氨酸诱导的PC12细胞的活力,降低其LDH释放量、胞内ROS含量,并提高胞内GSH蛋白表达,增加CAT、SOD含量并改善细胞形态。结论:人参皂甙Rd预处理可减轻谷氨酸引起的PC12细胞损伤。     

脑益康药物血清对谷氨酸诱导海马神经元损伤的保护作用

目的:探讨脑益康药物血清对谷氨酸(Glu)诱导的海马神经元损伤的保护作用。方法:大鼠海马神经元培养后,采用形态学观察、MTT法及DAPI染色法检测脑益康药物血清对Glu损伤细胞活力的影响,采用RT-PCR和免疫组化方法检测脑益康药物血清对Glu损伤细胞PTEN表达的影响。结果:脑益康药物血清可明显提高Glu损伤的海马神经元的细胞活力,减少PTEN的表达。结论:脑益康药物血清对Glu诱导的海马神经元损伤有保护作用,其机制可能与减少PTEN表达,抑制神经元凋亡有关。     

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia-reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3,?-8, and?-9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50?μg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and?-8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.     

The modulation of the pulse activity of neocortical neurons during a conditioned reflex]

Spontaneous and evoked activity of neurons in the sensorimotor cortex was recorded in cats with learned conditioned placing reaction before, during, and after the iontophoretic application of synaptically active substances. It was shown that apart from direct excitatory effect on the cortical neurons during its application, glutamate (Glu) exerted some modulatory influence on unit activity in subsequent 20 min. Noradrenaline suppressed the background and evoked activity through beta 1 adrenoreceptors. Activation of beta 2 adrenoreceptors by metaproterenol was accompanied by facilitation of the background and evoked activity during application and 10-20 min after. The joint application of Glu and metaproterenol improved facilitation of neuronal responses evoked by conditioned stimuli. Application of levodopa, like Glu, increased the background and evoked activity of many sensorimotor cortical neurons. The joint effect of Glu and levodopa was not substantially more intensive than the changes produced by the isolated application of any of these substances. A nonselective blocker of DA1 and DA2 receptors haloperidol either increased or did not change the background and evoked activity of some cortical neurons. In contrast to isolated application of Glu, simultaneous application of Glu and haloperidol to neocortex suppressed the neuronal responses associated with conditioned movements. The results suggest that the Glu-induced potentiation is substantially realized through molecular mechanisms common for Glu and dopamine, probably, through Gi-proteins. The conclusion is drawn that the adrenergic and dopaminergic inputs to neocortical neurons are involved in the Glu-mediated plastic changes in the cortex during conditioning.     

Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos,necroptosis, and apoptosis,but not autophagy

Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.     

Peroxisome Proliferator-Activated Receptor-γ Agonist 15d-Prostaglandin J2 Mediates Neuronal Autophagy after Cerebral Ischemia-Reperfusion Injury

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has recently emerged as potential therapeutic agents for cerebral ischemia-reperfusion (I/R) injury because of anti-neuronal apoptotic actions. However, whether PPAR-γ activation mediates neuronal autophagy in such conditions remains unclear. Therefore, in this study, we investigated the role of PPAR-γ agonist 15-PGJ₂ on neuronal autophagy induced by I/R. The expression of autophagic-related protein in ischemic cortex such as LC3-II, Beclin 1, cathepsin-B and LAMP1 increased significantly after cerebral I/R injury. Furthermore, increased punctate LC3 labeling and cathepsin-B staining occurred in neurons. Treatment with PPAR-γ agonist 15d-PGJ₂ decreased not only autophagic-related protein expression in ischemic cortex, but also immunoreactivity of LC3 and cathepsin-B in neurons. Autophagic inhibitor 3-methyladenine (3-MA) decreased LC3-II levels, reduced the infarct volume, and mimicked some protective effect of 15d-PGJ₂ against cerebral I/R injury. These results indicate that PPAR-γ agonist 15d-PGJ₂ exerts neuroprotection by inhibiting neuronal autophagy after cerebral I/R injury. Although the molecular mechanisms underlying PPAR-γ agonist in mediating neuronal autophagy remain to be determined, neuronal autophagy may be a new target for PPAR-γ agonist treatment in cerebral I/R injury.     

ACEA改善线粒体复合体活性诱导神经保护作用研究

目的:探讨线粒体复合体活性对大麻素CB1受体选择性激动剂ACEA神经保护作用的影响。方法:将原代大鼠皮层神经元分为4组:对照组(Control)、氧糖剥夺组(OGD)、ACEA+OGD组和溶剂(Vehicle)+OGD组,分别检测各组神经元损伤程度和线粒体复合体Ⅰ、Ⅱ和Ⅳ的活性。为进一步证实线粒体复合体活性对ACEA神经保护的影响,将原代大鼠皮层神经元分为5组:对照组(Control)、氧糖剥夺组(OGD)、ACEA+OGD组、线粒体复合体Ⅰ抑制剂(rotenone)+ACEA+OGD组和线粒体复合体Ⅱ抑制剂(TTFA)+ACEA+OGD组,检测和比较各组神经元细胞的损伤情况。结果:在OGD后24小时,ACEA明显增加神经元活性,减少LDH释放,降低神经元凋亡率(P0.05),改善OGD损伤后线粒体复合体Ⅰ和Ⅳ的活性(P0.05),而对复合体Ⅱ的活性没有影响;rotenone可以部分逆转ACEA的神经保护作用(P0.05),但TTFA却没有这一作用。结论:ACEA可以诱导神经保护作用,其机制是与改善线粒体呼吸链复合体活性有关。     

Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy

Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl₂ to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl₂ induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl₂-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl₂-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.     

Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia

Recent reports indicate that autophagy serves as a stress response and may participate in pathophysiology of cerebral ischemia. Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), the rate-limiting enzyme in mammalian NAD⁺ biosynthesis, protects against ischemic stroke through inhibiting neuronal apoptosis and necrosis. This study was taken to determine the involvement of autophagy in neuroprotection of Nampt in cerebral ischemia. Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in cultured cortical neurons were performed. Nampt was overexpressed or knocked-down using lentivirus-mediated gene transfer in vivo and in vitro. Immunochemistry (LC3-II), electron microscope and immunoblotting assays (LC3-II, beclin-1, mammalian target of rapamycin [mTOR], S6K1 and tuberous sclerosis complex-2 [TSC2]) were performed to assess autophagy. We found that overexpression of Nampt increased autophagy (LC3 puncta immunochemistry staining, LC3-II/beclin-1 expression and autophagosomes number) both in vivo and in vitro at 2 hours after MCAO. At the early stage of OGD, autophagy inducer rapamycin protected against neuronal injury induced by Nampt knockdown, whereas autophagy inhibitor 3-methyladenine abolished the neuroprotective effect of Nampt partly. Overexpression or knockdown of Nampt regulated the phosphorylation of mTOR and S6K1 signaling pathway upon OGD stress through enhancing phosphorylation of TSC2 at Ser1387 but not Thr1462 site. Furthermore, in cultured SIRT1-knockout neurons, the regulation of Nampt on autophagic proteins LC3-II and beclin-1 was abolished. Our results demonstrate that Nampt promotes neuronal survival through inducing autophagy via regulating TSC2-mTOR-S6K1 signaling pathway in a SIRT1-dependent manner during cerebral ischemia.