NIM811对Na2S2O4引起小鼠HT22细胞缺氧/复氧损伤的保护作用的研究

目的探讨环孢菌素衍生物NIM811对连二亚硫酸钠(Na₂S₂O₄)引起的小鼠海马神经元细胞(HT22)的缺氧/复氧损伤的保护作用及其机制。方法以小鼠HT22培养细胞制备缺氧/复氧细胞模型,实验分组为正常对照组、Na₂S₂O₄组、Na₂S₂O₄+NIM811组、NIM811组。CCK-8检测细胞生存率、流式细胞术检测细胞凋亡、JC-1试剂检测线粒体膜电位、用钙离子指示剂Rhod-2 AM观察线粒体内钙离子水平、DCFH-DA法检测细胞活性氧(ROS)水平。结果与Na₂S₂O₄组比较,给予NIM811处理后:(1)细胞活性增高38%(P<0.01);(2)细胞凋亡减少27%(P<0.01);(3)线粒体膜电位上升(P<0.01);(4)线粒体内钙离子水平下降(P<0.01);(5)活性氧(ROS)水平降低(P<0.01)。结论NIM811对Na₂S₂O₄引起小鼠海马神经元细胞缺氧/复氧损伤有保护作用,其机制可能为NIM811维持线粒体动态平衡和抑制细胞凋亡有关,NIM811对未来临床治疗缺血性脑卒中具有潜力。     

木香烃内酯通过调控LncRNA LINC01116/miR-9-5p的表达来减轻过氧化氢诱导的大鼠脑微血管内皮细胞损伤

目的 探讨木香烃内酯(Cos)对过氧化氢(H₂O₂)诱导的大鼠脑微血管内皮细胞的氧化应激、凋亡的影响及其对长链非编码RNA LINC01116(LncRNA LINC01116)/微小RNA-9-5p(miR-9-5p)的调控作用。方法 原代分离培养大鼠脑微血管内皮细胞,并随机分成Con组、H₂O₂组、Cos-L组、Cos-M组、Cos-H组、Cos-H+pcDNA组、Cos-H+pcDNA-LINC01116组; 采用化学比色法检测丙二醛(MDA)、还原型谷胱甘肽(GSH)的水平及超氧化物歧化酶(SOD)的活性; 流式细胞术检测细胞凋亡率; 实时荧光定量聚合酶链反应(qRT-PCR)检测LINC01116、miR-9-5p的表达水平; 双荧光素酶报告实验验证LINC01116与miR-9-5p的靶向关系; 蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)的表达水平。结果 H₂O₂处理后MDA的水平显著升高(P<0.05),GSH水平与SOD活性显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),Bax蛋白水平显著升高(P<0.05),Bcl-2蛋白水平显著降低(P<0.05),LINC01116的表达水平显著升高(P<0.05),miR-9-5p的表达水平显著降低(P<0.05); Cos处理后MDA的水平显著降低(P<0.05),GSH水平与SOD活性显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),Bax蛋白水平显著降低(P<0.05),Bcl-2蛋白水平显著升高(P<0.05),LINC01116的表达水平显著降低(P<0.05),miR-9-5p的表达水平显著升高(P<0.05),且Cos-L组、Cos-M组、Cos-H组上述指标的水平比较均有明显差异(P<0.05); 双荧光素酶报告实验证实LINC01116可靶向结合miR-9-5p; LINC01116过表达可减弱木香烃内酯对H₂O₂诱导的大鼠脑微血管内皮细胞凋亡及氧化应激的作用。结论 木香烃内酯可能通过抑制LINC01116的表达及促进miR-9-5p的表达来抑制H₂O₂诱导的大鼠脑微血管内皮细胞的氧化应激及细胞凋亡,从而减轻细胞损伤。     

间充质干细胞外泌体对海马神经元氧化应激的调控作用

目的 探讨间充质干细胞外泌体(Mesenchymal stem cell derived-exosomes,MSC-Exo)对海马神经元氧化应激的作用。方法 首先体外培养原代小鼠海马神经元,用H₂O₂刺激建立氧化应激模型,细胞计数试剂盒(Cell counting kit-8,CCK8)筛选最佳H₂O₂水平并检测不同水平MSC-Exo对细胞活力的作用,试剂盒检测超氧化物歧化酶(Superoxide dismutase,SOD)的活性,酶联免疫吸附测定法(Enzyme linked immunosorbent assay,ELISA)检测DNA氧化损伤分子8-羟基脱氧鸟苷(8-hydroxy-2 deoxyguanosine,8-OHdG)的表达水平,然后免疫荧光和免疫印迹检测应激和损伤分子一氧化氮合成酶(Inducible nitric oxide synthase,iNOS)、高迁移率族蛋白B1(High mobility group box 1,HMGB1)的表达水平。结果 CCK8细胞活力实验显示,10 μg/mL及以上水平的MSC-Exo对海马神经元氧化损伤具有明显的保护作用; 与对照组比较,H₂O₂组SOD的活性显著下降,而在MSC-Exo+H₂O₂组有所提高,趋于正常; 与对照组比较,H₂O₂作用后iNOS,HMGB1,8-OHdG等分子的表达水平显著上调(P<0.01),MSC-Exo作用于模型后与H₂O₂组比较,这些分子的表达水平显著下调(P<0.01)。结论 MSC-Exo作用于H₂O₂诱导的海马神经元氧化应激模型后能够增加SOD的活性,抑制海马神经元iNOS,HMGB1,8-OHdG等分子的表达,表明MSC-Exo对海马神经元氧化应激有一定的调控作用。     

青蒿素通过Nrf2/HO-1通路改善Aβ_(1-42)对SH-SY5Y细胞的细胞毒性作用

目的研究青蒿素(Artemisinin)在β淀粉样蛋白1-42(amyloid-β1-42,Aβ1-42)诱导的SH-SY5Y细胞毒性作用中的保护性作用及其可能机制。方法 SH-SY5Y细胞分为control组、Aβ1-42组、control+Aβ1-42组和Artemisinin+Aβ1-42组。利用细胞增殖实验检测细胞增殖情况,乳酸脱氢酶细胞毒性检测实验检测细胞的受损情况,免疫荧光检测细胞内活性氧(reactive oxygen species,ROS)的表达水平,Western blotting检测Nrf2和HO-1蛋白的表达。结果与control组相比,Artemisinin能够改善Aβ1-42对SH-SY5Y细胞的损伤以及增殖活力的抑制。除此之外,Artemisinin还能够抑制Aβ1-42诱导的SH-SY5Y细胞内ROS的表达,上调Nrf2和HO-1蛋白的表达。结论 Artemisinin能够通过Nrf2/HO-1通路改善Aβ1-42诱导的氧化应激对SH-SY5Y细胞的毒性作用。     

CDK5在鱼藤酮诱导的多巴胺能神经元凋亡中的作用

目的探讨CDK5在鱼藤酮介导的SH-SY5Y细胞凋亡中的作用以及通过干预CDK5表达对细胞凋亡的影响。方法使用鱼藤酮处理多巴胺能神经元SH-SY5Y细胞,采用四甲基偶氮唑盐(MTT)法检测细胞活力,Annexin-V/PI染色,流式细胞仪检测不同实验组细胞凋亡率,采用Western blot法检测不同实验组CDK5的表达以及p25/p35蛋白的表达比。结果经鱼藤酮处理后细胞活力呈浓度依赖性下降,细胞凋亡率增高,且CDK5表达增高,p25/p35比值增高。CDK5抑制剂MDL28170或Olomoucine预处理细胞能够降低CDK5的表达水平,降低p25/p35蛋白的表达比,同时可以减轻鱼藤酮诱导的细胞损伤。结论 CDK5在鱼藤酮介导的SH-SY5Y细胞凋亡中可能发挥着重要作用,抑制CDK5的活性可能具有神经保护作用。     

醒脑静对Aβ诱导的SH-SY5Y细胞凋亡的保护作用及机制

目的探讨醒脑静对Aβ25~35诱导的人神经母细胞瘤SH-SY5Y细胞凋亡的保护作用及其机制。方法建立Aβ25~35诱导的SH-SY5Y细胞凋亡模型;MTT法检测不同浓度醒脑静对Aβ25~35诱导的SH-SY5Y细胞增殖的影响;Annexin-V/PI流式细胞分析法检测不同浓度醒脑静对Aβ25~35诱导的SH-SY5Y细胞凋亡的影响;Western blot法检测细胞中线粒体凋亡相关蛋白Bcl-2及Bax表达水平变化情况。结果 SH-SY5Y细胞经过Aβ25~35造模处理后,细胞形态由梭形变为方形,细胞大小固缩,数量减少,凋亡小体形成,凋亡模型构建成功;醒脑静可以促进Aβ诱导的SH-SY5Y细胞增殖;醒脑静可以抑制Aβ25~35诱导的SH-SY5Y细胞凋亡、下调Bax蛋白表达及上调Bcl-2蛋白表达抑制Aβ25~35诱导的SH-SY5Y细胞凋亡。结论醒脑静对Aβ25~35诱导的SHSY5Y细胞凋亡具有一定的保护作用,其机制可能是通过调节Bcl-2及Bax水平抑制细胞凋亡的发生。     

EGCG对H2O2诱导BV2细胞氧化损伤保护作用机制的研究

目的探讨EGCG对H2O2诱导BV2细胞氧化损伤的保护作用机制。方法以200μmol/L H2O2制备BV2细胞氧化应激损伤模型,用CCK-8法检测不同浓度EGCG(0、1、5、10、20、40、80μmol/L)的保护作用,Ho-echst33258染色法检测细胞的凋亡,Western bloting法检测caspase-9蛋白的表达。结果 10及20μmol/L的EGCG组保护效果较明显;EGCG保护组的凋亡细胞显著少于无保护组;20μmol/L EGCG保护组的caspase-9蛋白表达较无保护组明显下调(P=0.03<0.05)。结论 10及20μmol/L浓度的EGCG对H2O2诱导的BV2细胞损伤模型有保护作用,其机制可能是通过减少caspase-9蛋白的表达,进而部分阻断caspase-9所介导的caspases级联反应,减少BV2细胞的凋亡。     

大蒜素对6-羟多巴胺所致PC12细胞损伤的保护作用及机制

目的研究大蒜素对6-羟多巴胺(6-hydroxydopamine,6-OHDA)引起的PC12细胞损害的保护作用和相关机制。方法使用6-OHDA在PC12细胞建立损伤模型,细胞活力、乳酸脱氢酶(lactate dehydrogenase,LDH)释放和细胞凋亡检测被用于观察大蒜素的保护作用,氧自由基(reactive oxygen species,ROS)和内源性抗氧化酶活性被用于检测其抗氧化活性,蛋白免疫印迹用于检测大电导、钙离子激活的钾离子通道(Large-conductance Ca2+-activated K+channels,BK)蛋白表达并研究可能的相关机制。结果 1大蒜素显著减轻6-OHDA所致的细胞活力下降和LDH释放,并抑制细胞凋亡。2大蒜素显著抑制6-OHDA所致的ROS产生和丙二醛(malondialdehyde,MDA)升高,并增加内源性抗氧化酶的活性。3大蒜素显著增加BK蛋白表达,使用BK阻滞剂paxilline可部分逆转大蒜素的保护作用。结论大蒜素通过激活BK和抗氧化机制对6-OHDA损伤的PC12细胞发挥保护作用。     

LINC00612靶向结合Bcl-2抑制Aβ1-42孵育的神经元凋亡

目的探讨LINC00612对β-淀粉样蛋白(amyloid protein β,Aβ) 1-42孵育的SH-SY5Y细胞凋亡的影响。方法利用Aβ1-42孵育的SH-SY5Y细胞建立阿尔茨海默病神经元损伤模型。应用qRT-PCR实验检测Aβ1-42孵育的SH-SY5Y细胞中LINC00612的表达。将LINC00612过表达质粒转染至Aβ1-42孵育的SH-SY5Y细胞,Western blot实验检测Bcl-2的表达,Annexin V-FITC/PI染色法检测细胞凋亡。RNA-pulldown和RIP实验检测LINC00612与Bcl-2的结合作用。结果 LINC00612在Aβ1-42孵育的SH-SY5Y细胞中低表达。过表达LINC00612显著增加Bcl-2表达量,降低细胞凋亡。RNA-pulldown和RIP实验结果显示LINC00612能够与直接Bcl-2结合。结论过表达LINC00612能够上调Bcl-2表达量,抑制Aβ1-42孵育的SH-SY5Y细胞凋亡。     

Inhibition of hydrogen peroxide-induced apoptosis but not arachidonic acid release in GH3 cell by EGF

Reactive oxygen species (ROS) and arachidonic acid (AA) can both function as extra- and intra-cellular messengers to regulate various cell functions including cell death. The effect of ROS on phospholipase A₂ (PLA₂) activity and/or AA release has not been extensively studied in neuronal cells. In this study, we investigated the effects of H₂O₂ on AA release and apoptosis in GH3 cells, a clonal strain from rat anterior pituitary. Incubation with H₂O₂ for 1 h stimulated [³H]AA release in a concentration-dependent manner from prelabeled GH3 cells. [³H]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA₂, and cytosolic PLA₂ protein with a molecular mass of 100 kDa was detected by immunoblotting. Culture with 0.2 mM H₂O₂ and 30 μM AA for 24 h induced lactate dehydrogenase (LDH) leakage, DNA laddering and DNA fragmentation in GH3 cells. In GH3 cells pretreated with EGF (50 ng/ml) for 24 h, LDH leakage and DNA fragmentation by H₂O₂ and AA were inhibited, although H₂O₂-induced [³H]AA release was not modified. Mastoparan, a wasp venom peptide, induced [³H]AA release and cell death in GH3 cells. Neither effect of mastoparan was inhibited by EGF treatment. These findings suggest that (1) H₂O₂ stimulates AA release via activation of cytosolic PLA₂, (2) H₂O₂ and AA induce apoptotic death of GH3 cells and (3) treatment with EGF protects H₂O₂- and AA-, but not mastoparan-, induced GH3 cell death.     

Effects of lidocaine reversible inactivation of the median raphe nucleus on long-term potentiation and recurrent inhibition in the dentate gyrus of rat hippocampus

Hydrogen peroxide (H₂O₂) causes oxidative stress and is considered a mediator of cell death in various organisms. Our previous studies showed that prolonged (>6 h) treatment of Aplysia sensory neurons with 1 mM H₂O₂ produced hyperpolarization of the resting membrane potential, followed by apoptotic morphological changes. In this study, we examined the effect of H₂O₂ on the membrane conductance of Aplysia sensory neurons. Hyperpolarization was induced by 10 mM H₂O₂ within 1 h, and this was attributed to increased membrane conductance. In addition, treatment with 10 mM H₂O₂ for 3 min produced immediate depolarization, which was due to decreased membrane conductance. The H₂O₂-induced hyperpolarization and depolarization were completely blocked by dithiothreitol, a disulfide-reducing agent. The later increase of membrane conductance induced by H₂O₂ was completely blocked by 100 mM TEA, a K⁺ channel blocker, suggesting that H₂O₂-induced hyperpolarization is due to the activation of K⁺ conductance. However, the inhibition of K⁺ efflux by TEA did not protect against H₂O₂-induced cell death in cultured Aplysia sensory neurons, which indicates that the signal pathway leading to H₂O₂-induced cell death is more complicated than expected.     

Overexpression of superoxide dismutase and catalase in immortalized neural cells: toxic effects of hydrogen peroxide

Hydrogen peroxide (H₂O₂) is a known toxicant which causes its damage via the production of hydroxyl radicals. It has been reported to cause both necrotic and apoptotic cell death. The present study was undertaken to evaluate the mode of H₂O₂-induced cell death and to assess if overexpression of catalase could protect against its toxicity. H₂O₂ causes cell death of immortalized CSM 14.1 neural cells in a dose-dependent manner. H₂O₂-induced death was associated with DNA laddering as shown by agarose gel electrophoresis. Stable overexpression of catalase by transfection of a vector containing human cDNA into these cells markedly attenuated H₂O₂-induced toxic effects. Transfection of a vector containing a SOD cDNA afforded no protection. These results indicate that H₂O₂ can lead to the activation of endonuclease enzyme that breaks DNA into oligosomes. These cells which overexpress catalase or SOD will help to determine the specific role of H₂O₂ or O₂^−. in the deleterious effects of a number of toxins.     

醒脑静对Aβ25-35诱导的SH-SY5Y细胞线粒体损伤的保护作用

目的 观察醒脑静注射液对Aβ_25-35诱导的SH-SY5Y细胞线粒体损伤的保护作用。方法 将培养的SH-SY5Y细胞随机分为5组,即(1)正常对照组:细胞正常培养27 h;(2)AD细胞模型组:细胞正常培养3 h,随后用老化的Aβ_25-35(25μmol/L)处理24 h;(3)醒脑静低浓度组:醒脑静注射液(5 μl/mL)预处理细胞3 h,随后用老化的Aβ_25-35(25 μmol/L)处理24 h;(4)醒脑静中浓度组:醒脑静注射液(10 μl/mL)预处理细胞3 h,随后用老化的Aβ_25-35(25μmol/L)处理24 h;(5)醒脑静高浓度组:醒脑静注射液(20 μl/mL)预处理细胞3 h,随后用老化的Aβ_25-35(25 μmol/L)处理24 h; 处理后各组细胞利用MTT法检测细胞存活率,紫外分光光度法检测细胞内ATP含量,流式细胞仪检测线粒体膜电位水平。结果 AD细胞模型组细胞存活率较正常对照组降低(P<0.05),醒脑静不同浓度组细胞存活率较AD细胞模型组均明显升高(P<0.05); AD细胞模型组细胞内ATP含量、线粒体膜电位水平较正常对照组下降(P<0.05),醒脑静不同浓度组细胞内ATP含量、线粒体膜电位水平较AD细胞模型组显著升高(P<0.05)。结论 醒脑静预处理可抑制Aβ_25-35诱导的SH-SY5Y细胞凋亡,提高细胞存活率,其机制可能与醒脑静提高细胞内ATP含量及线粒体膜电位水平而发挥线粒体保护作用有关。     

Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC 12 cells via inhibition of ROS generation and Ca(2+) influx

Glutamate is considered to be responsible for the pathogenesis of cerebral ischemia disease. [Ca²⁺]_i influx and reactive oxygen species (ROS) production are considered to be involved in glutamate-induced apoptosis process. In this study, we investigated the neuroprotective effects of ginkgolide K in the glutamate-induced rat's adrenal pheochromocytoma cell line (PC 12 cells) and the possible mechanism. Glutamate cytotoxicity in PC 12 cells was accompanied by an increment of malondialdehyde (MDA) content and lactate dehydrogenase (LDH) release, as well as Ca²⁺ influx, bax/bcl-2 ratio, cytochrome c release, caspase-3 protein and ROS generation, and reduction of cell viability and mitochondrial membrane potential (MMP). Moreover, treatment with glutamate alone resulted in decrease activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity. However, pretreatment with ginkgolide K significantly reduced MDA content, LDH release, as well as Ca²⁺ influx, cytochrome c release, bax/bcl-2 ratio, caspase-3 protein and ROS production, and attenuated the decrease of cells viability and MMP. In addition, ginkgolide K remarkedly up-regulated SOD and GSH-PX activities. All these findings indicated that ginkgolide K protected PC12 cells against glutamate-induced apoptosis by inhibiting Ca²⁺ influx and ROS production. Therefore, the present study supports the notion that ginkgolide K may be a promising neuroprotective agent for the treatment of cerebral ischemia disease.     

褪黑素通过下调SLC7A11表达来诱导胶质瘤细胞SH-SYSY铁死亡的机制研究

目的 探讨褪黑素(Melatonin, MT)通过下调溶质载体家族7成员11(Solute carrier family 7member 11,SLC7A11)表达对胶质瘤细胞(Glioma cell, SH-SYSY)铁死亡的影响。方法 使用不同水平的MT处理人胶质瘤SH-SY5Y细胞,采用细胞计数试剂盒-8(Cell counting kit-8,CCK-8)法检测细胞活性,进而选择合适的MT水平;实验分组为二甲基亚砜(Dimethyl sulfoxide, DMSO)组(加入常规培养基)、MT组(加入1 mmol/L的MT)、凋亡抑制剂氟甲基酮(Z-VAD-FMK)+MT组(加入1 mmol/L的MT和20μmol/L的凋亡抑制剂Z-VAD-FMK)、坏死性凋亡抑制剂Necrosulfonamide(Nec-1)+MT组(加入1 mmol/L的MT和1μmol/L的坏死性凋亡抑制剂Nec-1)、自噬抑制剂Chloroquine+MT组(加入1 mmol/L的MT和5 mmol/L的自噬抑制剂Chloroquine)、特异性铁死亡抑制剂Deferoxamine(DFO)对照组(加入...