花生四烯酰多巴胺对H2O2诱导SH-SY5Y细胞氧化应激损伤的保护作用

目的 探讨花生四烯酰多巴胺(N-arachidonoyl dopamine,NADA)对H₂O₂诱导的人神经母细胞瘤SH-SY5Y细胞氧化损伤的保护作用。方法 H₂O₂处理SH-SY5Y细胞制作细胞氧化损伤模型,通过CCK8法检测细胞活力,明确不同水平的NADA对细胞存活的影响,分析NADA对SH-SY5Y细胞的保护作用; 利用生化方法检测丙二醛(MDA)和乳酸脱氢酶(LDH)的水平; 运用DCFH-DA荧光探针检测细胞内氧自由基(ROS)水平; Hoechst33342/PI双染法检测NADA对细胞氧化损伤的保护作用; 蛋白免疫印迹实验检测抗凋亡蛋白bcl-2的表达水平。结果 H₂O₂处理SH-SY5Y细胞可导致细胞活力降低,增加MDA水平和LDH活力,促进ROS的产生,降低抗凋亡蛋白bcl-2的表达,加剧细胞凋亡。NADA可剂量依赖性提高H₂O₂诱导的SH-SY5Y细胞的存活率、降低MDA水平和LDH活力以及ROS的产生,促进抗凋亡蛋白bcl-2的表达,降低细胞凋亡。结论 NADA对H₂O₂诱导的细胞氧化损伤模型具有保护作用,其机制可能是通过抑制胞内氧化应激,促进抗凋亡蛋白bcl-2的表达,从而减少神经细胞凋亡。     

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。     

地黄多糖调控环状RNA0010729/miR-326通路对海马神经元缺氧复氧损伤的影响

目的 探讨地黄多糖对海马神经元缺氧复氧损伤的影响及其保护机制是否与调控环状RNA 0010729(circ_0010729)和微小RNA-326(microRNA-326,miR-326)表达有关。方法 体外培养大鼠海马神经元建立缺氧复氧损伤模型; 将大鼠海马神经元分为对照组、模型组、模型+地黄多糖低剂量组、模型+地黄多糖中剂量组、模型+地黄多糖高剂量组、模型+小干扰RNA阴性对照(Small interfering RNA negative control,si-NC)组、模型+si-circ_0010729组、模型+地黄多糖高剂量+空载质粒(Empty plasmid,pcDNA)组、模型+地黄多糖高剂量+pcDNA-circ_0010729组; 流式细胞术检测细胞凋亡; 试剂盒检测丙二醛(Malonaldehyde,MDA)水平和超氧化物歧化酶(Superoxide dismutase,SOD)活性; 实时定量聚合酶链式反应(Polymerase chain reaction,PCR)检测circ_0010729,miR-326表达水平; 荧光素酶报告实验确定circ_0010729和miR-326靶向关系。结果 与对照组比较,模型组细胞凋亡率、MDA水平、circ_0010729表达水平升高(P<0.05),SOD活性、miR-326表达水平降低(P<0.05); 与模型组比较,模型+地黄多糖低剂量组、模型+地黄多糖中剂量组、模型+地黄多糖高剂量组细胞凋亡率、MDA水平、circ_0010729表达水平降低(P<0.05),SOD活性、miR-326表达水平升高(P<0.05); 与模型+si-NC组比较,模型+si-circ_0010729组细胞凋亡率、MDA水平降低(P<0.05),SOD活性升高(P<0.05); 与模型+地黄多糖高剂量+pcDNA组比较,模型+地黄多糖高剂量+pcDNA-circ_0010729组细胞凋亡率、MDA水平升高(P<0.05),SOD活性降低(P<0.05)。结论 地黄多糖能够有效抑制缺氧复氧诱导的大鼠海马神经元凋亡和氧化损伤,其机制可能与抑制circ_0010729/miR-326通路有关。     

Bcl-2反义核酸对SK-N-SH细胞增殖和凋亡的影响

目的研究Bcl-2反义核酸对人神经母细胞瘤细胞株SK-N-SH的增殖和凋亡的作用。方法设Bcl-2反义核酸ASODN组、正义核酸SODN组、脂质体组及空白对照组。MTT法检测Bcl-2反义核酸对SK-N-SH细胞增殖的抑制作用;流式细胞术检测细胞周期及细胞凋亡情况。结果 ASODN组72h细胞增殖抑制率达(68.24±2.41)%,较空白对照组差异显著(P<0.01);ASODN可使SK-N-SH细胞周期阻滞于G2/M期,72h细胞凋亡率达(23.25±2.34)%。结论 Bcl-2反义核酸可抑制SK-N-SH细胞增殖并诱导其发生凋亡。     

CircTLK1通过miR-424-5p/FBXO3轴对氧糖剥夺/复氧诱导的神经细胞损伤的影响

目的 探讨环状RNA TLK1(CircRNA TLK1,CircTLK1)对氧糖剥夺/复氧(Oxygen glucose deprivation/Reoxygenation,OGD/R)诱导的神经元HT22损伤的影响以及对微小RNA(microRNA,miR)-424-5p/F-box蛋白3(F-box protein 3,FBXO3)的调控作用。方法 将HT22细胞分为对照组、OGD/R组、sh-NC组、沉默环状RNA TLK1(Silencing circular RNA tlk1,sh-circTLK1)组、sh-circTLK1+抑制剂NC组、sh-circTLK1+miR-424-5p抑制剂组、sh-circTLK1+miR-424-5p抑制剂+sh NC组、sh-circTLK1+miR-424-5p抑制剂+sh FBXO3组,除对照组外其余各组细胞均行OGD/R操作,细胞计数试剂盒8(Cell counting Kit 8,CCK-8)法测定HT22细胞活力; 脂连蛋白V-异硫氰酸荧光素(Adiponectin V-fluorescein isothiocyanate,Annexin V-FITC)细胞凋亡试剂盒测定HT22细胞凋亡; 测定HT22细胞乳酸脱氢酶(Lactate dehydrogenase,LDH)漏出率; 实时荧光定量聚合酶链反应(Real time fluorescent quantitative polymerase chain reaction,RT-qRCR)法测定HT22细胞miR-424-5p,FBXO3 mRNA水平; 蛋白免疫印记法(Western Blot)检测B淋巴细胞瘤-2关联基因X(B lymphoma-2 gene association X,Bax)、活化半胱天冬酶-3(Cleaved caspase-3)、FBXO3水平; 双荧光素酶测定CircTLK1与miR-424-5p以及miR-424-5p与FBXO3靶向关系,并使用RNA下拉实验验证CircTLK1与miR-424-5p关系。结果 与对照组比较,OGD/R组miR-424-5p,HT22细胞活力降低(P<0.05),circTLK1,FBXO3 mRNA水平,HT22细胞凋亡率、Bax,Cleaved caspase-3水平、LDH漏出率升高(P<0.05); 与OGD/R组比较,sh-circTLK1组HT22细胞活力增加(P<0.05),HT22细胞凋亡率、Bax,Cleaved caspase-3水平,LDH漏出率降低(P<0.05); 与sh-circTLK1组比较,sh-circTLK1+miR-424-5p抑制剂组细胞活力降低(P<0.05),HT22细胞凋亡率、Bax,Cleaved caspase-3水平、LDH漏出率升高(P<0.05); 与sh-circTLK1+miR-424-5p抑制剂组比较,sh-circTLK1+miR-424-5p抑制剂+sh FBXO3组细胞活力升高(P<0.05),HT22细胞凋亡率、Bax,Cleaved caspase-3水平、LDH漏出率降低(P<0.05); CircTLK1与miR-424-5p以及miR-424-5p与FBXO3均存在靶向关系。结论 CircTLK1沉默可能通过调控miR-424-5p/FBXO3对OGD/R诱导的HT22细胞损伤来发挥保护作用。     

丹红注射液对自然衰老大鼠心脏、大脑组织CAT及H2O2的影响

丹红注射液是采用丹参、红花配制成的中药制剂,主要成分有丹参酚酸、丹参酮等,具有活血化瘀和通脉舒络的作用,对心血管、脑血管、呼吸等系统均具有一定作用[1]。衰老是一种普遍存在于生物界的自然生理现象,是随着年龄的增长机体内的器官功能逐渐减弱或消失的过程[2]。作者利用     

miR-125a-5p靶向MEIS2基因对垂体瘤AtT20细胞增殖、凋亡的影响

目的探讨微小RNA-125a-5p(miR-125a-5p)是否可靶向调控MEIS2基因表达并分析其对垂体瘤AtT20细胞增殖及凋亡的影响。方法采用实时荧光定量PCR(qRT-PCR)检测AtT20细胞及小鼠正常垂体细胞(NPC)中miR-125a-5p及MEIS2 mRNA表达量。采用瞬时转染技术分别将miR-125a-5p mimic质粒、anti-miR-125a-5p抑制剂及si-MEIS2质粒转染至AtT20细胞。双荧光素酶报告基因检测miR-125a-5p与MEIS2的靶基因关系,同时共转染miR-125a-5p mimic与pcDNA-MEIS2验证miR-125a-5p是否通过靶向MEIS2表达发挥作用。MTT法检测各组细胞增殖能力;流式细胞术检测各组细胞凋亡率;蛋白免疫印迹(Western blotting)检测各组细胞MEIS2蛋白表达。结果 qRT-PCR检测结果显示,与NPC比较,miR-125a-5p在AtT20细胞中表达水平显著降低,MEIS2 mRNA表达水平显著升高;与miR-NC组比较,miR-125a-5p组miR-125a-5p表达水平显著升高。W...     

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。     

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。     

Immunosuppressive and non-immunosuppressive immunophilin ligands improve H2O2-induced cell damage by increasing glutathione levels in NG108-15 cells

We examined the effects of FK506 and its non-immunosuppressive derivative, GPI1046, on H₂O₂-induced oxidative cell damage in NG 108-15 cells. Our results suggest that the protective properties of GPI1046 are equipotent with those of FK506 and may be mediated by increased intracellular concentrations of glutathione (GSH). Thus, non-immunosuppressive immunophilin ligands such as GPI1046 might be potentially for neurodegenerative diseases, particularly since they do not have serious side effects such as immune deficiency.     

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。     

Pretreatment of MQA,a caffeoylquinic acid derivative compound,protects against H2O2-induced oxidative stress in SH-SY5Y cells

Objectives: Compound MQA (1,5-O-dicaffeoyl-3-O-[4-malic acid methyl ester]-quinic acid) is a natural caffeoylquinic acid derivative isolated from Arctium lappa L. roots. This study aims to explore the neuroprotective effects of MQA against hydrogen peroxide (H₂O₂)-induced oxidative stress in SH-SY5Y neuroblastoma cells.

Methods: The SH-SY5Y cells were divided into four groups, including control, 20 μM MQA, 200 μM H2O2, 200 μM H2O2 + 20 μM MQA groups. The effects of MQA on H₂O₂-induced cell death were measured by MTT and LDH assays. Hoechst 33342 and Annexin V-PI double staining were used to observed H2O2-induced apoptosis. Also, the effects of MQA on antioxidant system and mitochondrial pathway were explored. Further, steady-state phosphorylation levels of ERK1/2, Akt and GSK-3β were examined by Western blot analysis.

Results: Pretreatment with MQA prevented cell death in SH-SY5Y cells exposed to 200 μM H2O2 for 3 h. Meanwhile, Hoechst 33342 and Annexin V-PI double staining showed that MQA attenuated H₂O₂-induced apoptosis. These changes are related to elevation in SOD activity, reduction in MDA production and ROS formation, and increases in mitochondrial membrane potential (MMP). In addition, the potential mechanisms of MQA against H₂O₂-induced apoptosis are associated with increases in the Bcl-2/Bax ratio, decreases in cytochrome c release, caspase-3 and caspase-9 expressions, phosphorylation of ERK1/2, and dephosphorylation of AKT and GSK-3β.

Conclusion: These findings suggest that protective effects of MQA against H₂O₂-induced apoptosis might be associated with mitochondrial apoptosis, ERK1/2 and AKT/GSK-3β pathway.     

In cortical cultures of trisomy 16 mouse brain the upregulated metallothionein-I/II fails to respond to H2O2 exposure or glutamate receptor stimulation

To assess whether a defective oxidative defense may contribute to Down's syndrome, we studied the regulation of the metallothionein(MT)-I/II isoforms in primary cultures of cerebral cortex from fetal trisomy 16 mice and their euploid littermates. Western blot analysis showed that MT-I/II was upregulated and the protein carbonyl content was higher in trisomy 16 compared with euploid cultures. Addition of N-acetyl-

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-cysteine to the culture medium reduced the increment of MT-I/II in trisomy 16 cortical cells. In euploid, but not trisomic cortical cultures, kainic acid, trans-(±)-ACPD, or H₂O₂ exposure elicited a dose-dependent increase of the MT-I/II immunoblots. In trisomic cells, the MT-I/II immunoblot densities were not increased beyond their elevated basal levels. In contrast, 25 μM Pb induced MT-I/II, to a similar extent, in cortical cultures from euploid and trisomy 16 mice. This suggests that the antioxidant—but not the metal—response element of the MT-I/II promoter was altered by increased oxidative stress. Our data suggest that, in the trisomy 16 mouse, the effects of increased production of reactive oxygen species, due to the increased SOD-1, GluR5, or amyloid precursor protein gene dosage, is exacerbated by an insufficient or missing antioxidant response.     

The effect of dopamine depletion on the H2O2 production in the rat striatum following transient middle cerebral artery occlusion

The changes in the extracellular concentrations of rat striatal H₂O₂, dopamine (DA) and its metabolites during middle cerebral artery (MCA) occlusion and reperfusion were simultaneously examined by microdialysis, and the relationship between the ischemia-induced release of DA and the generation of H₂O₂ was estimated by assessing the effect of the lesion of the substantia nigra (SN). In the rats without SN lesions, a significant increase in the striatal H₂O₂ level was observed during the ischemia and reperfusion phases. In the rats with SN lesions, the ischemia-induced H₂O₂ production was not attenuated. These results suggest that DA is not an important source of H₂O₂ in cerebral ischemia and reperfusion.     

Involvement of oxidative stress in di-2-ethylhexyl phthalate (DEHP)-induced apoptosis of mouse NE-4C neural stem cells

Di-2-ethylhexyl phthalate (DEHP) has been widely used as a plasticizer in industry and can cause neurotoxicity; however, the underlying mechanism remains unclear. In the study, we found that DEHP significantly inhibited viability of mouse NE-4C neural stem cells and caused lactate dehydrogenase (LDH) release from the cells. DEHP dramatically increased the levels of apoptosis-related proteins such as cleaved Caspase-8, cleaved Caspase-3 and Bax, as well as decreased Bcl-2 protein level. DEHP could also significantly increase the total numbers of AnnexinV-positive/PI-negative and AnnexinV-positive/PI-positive staining cells. Hoechst 33342 staining showed that marked DNA condensation and apoptotic bodies could be found in the ZnO NPs-treated cells. These results indicated that DEHP could induce apoptosis of NE-4C cells. Meanwhile, DEHP could significantly increase malondialdehyde (MDA) level, and decrease the content of glutathione (GSH) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), respectively, implying that DEHP could induce oxidative stress of NE-4C cells. Furthermore, N-Acetyl-l-cysteine (NAC), an inhibitor of oxidative stress, could rescue the inhibition of cell viability and induction of apoptosis by DEHP. Taken together, our results showed that oxidative stress was involved in DEHP-induced apoptosis of mouse NE-4C cells.