维生素E对鼠海马神经元细胞的抗氧化损伤作用

目的 观察维生素E对老年痴呆(Alzheimer’s disease)的治疗作用，研究氧化损伤对鼠海马神经元细胞的损伤作用及维生素E对其氧化损伤的保护作用。方法 用氧化(H202)、维生素E处理后氧化的方法对鼠海马神经细胞(HT-22)进行分组处理。用二维电泳法、蛋白银染法及特殊氧化蛋白免疫染色法探测被氧化的蛋白。结果 氧化处理12h后出现细胞生存率明显下降31．19％；经维生素E处理后再氧化处理的细胞生存率几乎达到正常组水平。经H202氧化处理的鼠海马神经元细胞的氧化蛋白数目增加了，而维生素E提前处理过的那组没有增加。结论 维生素E对鼠神经元细胞的氧化损伤起保护作用，是有效的抗氧化治疗剂。     

黄芩茎叶总黄酮对过氧化氢诱导人脐静脉内皮细胞氧化损伤的保护作用及其机制

目的探讨黄芩茎叶总黄酮(SSTF)对过氧化氢(H2O2)所致人脐静脉内皮细胞(HUVEC)氧化损伤的保护作用及其可能机制。方法在由H2O2制备血管内皮细胞氧化损伤模型的基础上,采用MTT法测定各组细胞活力;生化比色法测定各组细胞培养上清液中乳酸脱氢酶(LDH)的活性,丙二醛(MDA)的含量及超氧化物歧化酶(SOD)的活性;流式细胞术检测各组细胞的凋亡率以及western-blot法测定各组细胞凋亡抑制蛋白Bcl-2的表达情况。结果 H2O2处理HUVEC后其存活率较正常HUVEC下降,细胞培养上清液中LDH活性和MDA含量明显增加,SOD活性较正常HUVEC降低;HUVEC凋亡率明显升高(P<0.05或P<0.01);而SSTF(100~400mg/L)各剂量都能提高H2O2损伤的HUVCE的细胞活力,明显降低LDH活性,显著减少MDA的含量,提高SOD的活性,明显降低HUVEC凋亡率,明显提高Bcl-2蛋白表达水平(P<0.05或P<0.01)。结论 SSTF能减轻H2O2对HUVEC的氧化损伤作用,降低H2O2所致HUVEC的凋亡率,其机制可能与上调凋亡抑制蛋白Bcl-2的表达水平有关。     

抗衰老酶1-p53信号通路在吸入麻醉致原代海马神经元细胞凋亡中的神经保护作用

目的:探讨抗衰老酶1(sirtuin 1,SIRT1)在七氟醚(sevoflurane,Sev)联合一氧化二氮(N_2O)吸入麻醉导致的原代海马神经元细胞凋亡中的作用。方法:原代海马神经元予以1.3%Sev联合50%N_2O吸入麻醉(对照组给予50%O_2)处理2 h,或吸入麻醉前分别予以SIRT1拮抗剂sirtinol(50μmol/L)、salermide(50μmol/L)或激动剂resveratrol(100μmol/L)处理24 h。24 h后收集细胞,采用蛋白质印迹法检测Bax、剪切型半胱氨酸蛋白酶3(cleaved caspase-3,c-caspase-3)、剪切型聚腺苷酸二磷酸核糖转移酶[cleaved poly (ADP-ribose) polymerase,c-PARP]、磷酸化组蛋白H2A家族成员X(phosphorylated H2A histone family member X,γ-H2AX)、SIRT1和乙酰化p53[acetylated p53(lysine 381),acetyl-p53]蛋白水平,采用免疫荧光染色法检测c-caspase-3、SIRT1和acetyl-p53蛋白的分布及表达情况。结果:Sev联合N_2O吸入麻醉显著上调原代海马神经元中促凋亡蛋白Bax、c-caspase-3、c-PARP及DNA损伤相关指标γ-H2AX的表达水平(P0.05);同时麻醉组SIRT1蛋白表达水平也显著升高,而acetyl-p53表达水平显著降低(P0.05)。吸入麻醉处理前给予SIRT1拮抗剂sirtinol或salermide预处理可抑制吸入麻醉引起的SIRT1蛋白表达上调,但增加了促凋亡蛋白Bax、c-caspase-3、c-PARP及DNA损伤指标γ-H2AX的表达(P0.05);SIRT1激动剂预处理则呈现相反的结果(P0.05)。结论:Sev联合N_2O吸入麻醉引起的SIRT1-p53信号通路激活可抑制其所致的原代海马神经元细胞凋亡。     

三七总皂苷对L6大鼠成肌细胞抗化学损伤模型的保护作用

背景:三七总皂苷具有活血祛瘀,通脉活络等多重药理作用,但其具体机制尚不明确。目的:观察三七总皂苷对L6大鼠成肌细胞H2O2损伤的保护作用及其对Bax和Bcl-2蛋白表达的影响。方法:建立L6大鼠成肌细胞H2O2损伤模型,应用MTT法、DAPI化学荧光法和流式细胞仪分析检测各种剂量三七总皂苷(0.5,0.1,0.02g/L)对L6大鼠成肌细胞存活率的影响;通过免疫荧光抗体细胞化学法研究各种剂量三七总皂苷(0.5,0.1,0.02g/L)对L6大鼠成肌细胞Bax和Bcl-2蛋白表达的影响。结果与结论:L6大鼠成肌细胞经H2O2损伤后细胞存活率降低、凋亡率增加,三七总皂苷能明显提高经H2O2损伤后细胞的存活率,降低凋亡率;免疫荧光抗体细胞化学法表明三七总皂苷能促进Bcl-2表达,抑制Bax表达。结果可见三七总皂苷对L6大鼠成肌细胞H2O2损伤具有保护作用,依次为0.1g/L>0.02g/L>0.5g/L,其作用可能与上调Bcl-2蛋白的表达、抑制Bax蛋白的表达有关。     

维生素C保护视网膜色素上皮细胞的相关机制研究

目的研究氧化应激状态下维生素C作为抗氧化剂对视网膜色素上皮细胞的保护作用以及维生素C和SIRT1之间的调节机制。方法以人视网膜色素上皮细胞-19(ARPE-19)细胞为研究对象,分为空白对照组,无维生素C组(0μmol),低浓度维生素C组(20μmol),中等浓度维生素C组(100μmol)和高浓度维生素C组(500μmol)。培养过程中添加不同浓度维生素C之后对细胞加以H2O2(100μmol)处理12 h或24 h建立氧化应激模型。利用MTT法检测ARPE-19细胞存活率,膜联蛋白V-FITC凋亡检测试剂盒检测细胞凋亡以及活性氧(ROS)试剂盒测定细胞内活性氧的改变。使用SIRT1靶向siRNA进行SIRT1敲除,细胞使用预定浓度的维生素C进行孵育,并分为空白对照组,阴性对照组,siRNA干扰组。先用10 m M SIRT1激动剂白藜芦醇(RSV)和5 m M SIRT1抑制剂烟酰胺(NA)对细胞进行孵育,然后加入H2O2(100μmol)处理12 h或24 h,RT-PCR及Western blot方法检测SIRT1、p53和Foxo3基因表达水平。结果经过H2O212 h处理后,较高浓度的维生素C(500μmol)与较低浓度(20μmol)的维生素C无明显保护作用,而中等浓度的维生素C(100μmol)能够显著提高细胞的生存能力(P0.05),减少ARPE-19细胞的凋亡数量(P0.05),降低细胞内ROS水平(P0.05),差异有统计学意义。维生素C的作用可上调H2O2作用后SIRT1转录因子和应激反应因子(p53和Foxo3)的表达。RSV及NA可分别上调及下调维生素C对H2O2刺激的ARPE-19细胞存活力,细胞凋亡和细胞内ROS水平的影响。RT-PCR及Western blot结果表明:维生素C浓度增加时,SIRT1表达增加,p53和Foxo3基因表达水平升高。敲除或上调SIRT1表达,可相应明显地增加或减少p53和Foxo3转录和蛋白水平的表达。结论维生素C可降低细胞内ROS水平,减少ARPE-19细胞凋亡,起到抗氧化损伤保护作用,有望成为年龄相关性黄斑变性的有效治疗方法。     

脑复聪对PC12细胞拟缺血损伤保护作用的血清学研究

目的观察中药复方脑复聪颗粒对H2 O2 损伤的大鼠肾上腺髓质嗜铬瘤分化细胞株 (PC12细胞 )的影响。方法选用SD雄性大鼠 ,分别灌胃脑复聪和蒸馏水后制备血清。培养的PC12细胞加入不同血清孵育后 ,再加入不同浓度的超氧化合物H2 O2造成拟缺血模型 ,采用MTT法观察细胞生存率。结果对受不同剂量H2 O2 损伤的PC12细胞 ,脑复聪含药血清可明显提高其生存率 (P <0 0 5— 0 0 1) ;加入脑复聪血清后的神经细胞可明显抵抗H2 O2 所致的损伤 ,细胞形态基本恢复正常。结论脑复聪颗粒对H2 O2 损伤的PC12细胞有明显保护作用。     

病毒硫氧还蛋白基因在Neur02A细胞中的重组与表达

背景：目前对抗氧化基因硫氧还蛋白的研究逐步受到重视，但从基因治疗的角度对其研究较少。目的：采用硫氧还蛋白转染Neuro-2A细胞，观察细胞内表达相应蛋白因子对细胞的具体保护效果，并分析其发挥保护作用的可能机制。方法：以质粒PIRES2-EGFP-TRX转染Neuro-2A细胞，经RT-PCR鉴定，建立能够稳定表达硫氧还蛋白的细胞株。利用不同浓度的H202处理正常细胞及转染细胞，建立氧化应激模型。观察受到氧化损伤后两组细胞的形态学、存活率、还原型谷胱甘肽的浓度及细胞内DNA链断裂情况。结果与结论：经H2O2作用后，两组细胞形态均出现损伤，但转染组细胞比正常组细胞损伤减轻、细胞存活率升高、细胞内还原型谷胱甘肽水平增高、DNA链断裂程度减轻。说明人类硫氧还蛋白基因可以在Neuro-2A细胞中被重组并顺利表达，对细胞具有一定的保护作用；这一作用可能是通过清除氧自由基，维持细胞内还原型谷胱甘肽水平，从而保护细胞DNA免受氧化性损伤来实现的。     

抑制STAT3表达增加H_2O_2诱导人胶质瘤U251细胞损伤

目的探讨RNA干涉技术抑制STAT3基因表达对过氧化氢(H2O2)复制的人胶质瘤U251细胞损伤模型的影响。方法转染pSilencer1.0-U6-SiRNA-STAT3重组质粒到U251细胞;Western blot方法观察转染重组质粒对STAT3蛋白表达的影响;MTT法检测细胞生存率,TUNEL法检测细胞凋亡,Western blot检测凋亡相关蛋白BCL-2、BAX表达。结果转染pSilencer1.0-U6-SiRNA-STAT3重组质粒能有效抑制U251细胞STAT3蛋白表达(抑制率50%);抑制STAT3表达能促进H2O2诱导U251细胞生存率下降,增加细胞凋亡率(P0.05)。STAT3抑制能促进H2O2作用下U251细胞BCL-2表达降低,BAX表达增加(P0.05)。结论pSilencer1.0-U6-SiRNA-STAT3可有效抑制U251细胞STAT3的表达,并促进H2O2诱导U251细胞凋亡。     

高压氧对脑缺血再灌注海马神经元Bcl-2和Bax蛋白表达的影响

目的观察高压氧(HBO)作用下脑缺血再灌注海马CA1区神经元Bcl-2和Bax蛋白表达的变化情况,进一步研讨高压氧治疗脑缺血再灌注损伤、减轻神经元凋亡从而发挥保护作用的机制。方法沙土鼠20只,采用随机数字法将实验动物分为正常对照组、缺血组、0.15MPaHBO治疗组、0.25MPaHBO治疗组,0.25MPa压力空气(hyperbaricair,HBA)对照组,每组4只动物。采用“双侧颈总动脉阻断法”前脑缺血模型,缺血20min后再灌注3d,并用0.15MPa和0.25MPa压力的高压氧治疗(60min/d,连续3d)后,应用免疫组化LSAB方法,观察高压氧对海马CA1区神经元凋亡相关基因Bcl-2和Bax的蛋白表达的影响。结果沙土鼠脑缺血再灌注3d组海马CA1区大量神经元表达Bax蛋白,并且神经元发生凋亡,未见神经元表达Bcl-2蛋白;高压氧治疗组则大量神经元表达Bcl-2蛋白,并且0.25MPa高压氧治疗组比0.15MPa高压氧治疗组变化更显著,而各组表达Bax蛋白的神经元数目无明显变化,但高压氧治疗组Bax蛋白阳性的神经元形态正常。结论HBO暴露可诱导大量神经元表达Bcl-2蛋白,对Bax蛋白表达则无明显作用,使Bcl-2和Bax蛋白表达的比值增高,从而起到保护神经元的作用,这可视为HBO治疗脑缺血性损伤减少神经元凋亡的机制之一。     

雌二醇对海马神经元和隔区胆碱能神经元损伤的保护作用比较

目的：研究雌二醇在体外原代培养条件下对淀粉样β25-35肽(amvloid-β25-35，Aβ25-35)诱导的海马神经元和胆碱能神经元损伤的保护作用，探讨其可能的机制。方法：实验于2002-03／2003-03在中山大学医学院解剖教研室完成。采用SD大鼠海马神经元原代培养，用终浓度为10βmol／L的AB25-35诱导体外海马和隔胆碱能神经元损伤，MTT法比色微量分析较雌二醇对这两种神经元的存活率的影响，并检测超氧化物歧化酶(superoxide dismutase，SOD)活性的变化。将两种培养细胞分为3组，分别为AB处理组、雌二醇组、空白对照组。结果：雌二醇可提高海马和胆碱能神经元存活率(与AB处理组比较，P&;lt;0．05)，经雌激素作用后，两种神经元的存活率分别为103．52%，和85．90%，它对海马神经元的保护作用优于胆碱能神经元(两者存活率比较，P&;lt;0．05)，并可诱导两种神经元Mn-SOD的活性增加(与AB处理组比较，P&;lt;0．05)，分别比AB处理组提高了63．76％和131．38%，对隔胆碱能神经元的诱导作用较强(两者活性比较，P&;lt;0．05)，对CuZn-SOD的活性无明显影响(P&;gt;O．05)。结论：雌二醇对Aβ25-35诱导毒性损伤的海马和隔胆碱能神经元有相似程度的保护作用，其机制可能与提高Mn-SOD活性，促进聚集型的Aβ溶解有关。     

Vitamin E protects the insulin sensitivity and redox balance in rat L6 muscle cells exposed to oxidative stress

BACKGROUND: The effect of reactive oxygen species (ROS) on insulin action is unclear. This study was carried out to explore the effect of prolonged low grade oxidative stress and vitamin E treatment on cultured rat L6 muscle cells. METHODS: L6 myotubes were incubated with vitamin E for 18 h and treated with H2O2 generating system for 12 h. Insulin stimulated glucose uptake, total antioxidant capacity and reduced glutathione concentration were measured. RESULTS: There was a marked decrease in insulin stimulated glucose transport in L6 cells exposed to H2O2 generating system. Pretreatment with vitamin E attenuates the effect of H2O2 on insulin action. Treatment with H2O2 caused a significant reduction in the levels of reduced glutathione and total antioxidant capacity, these alterations were reversed by vitamin E pretreatment. Vitamin E per se had no effect on insulin stimulated glucose transport in cells not exposed to oxidative stress. CONCLUSION: In conclusion, our observations indicate that vitamin E improves the free radical defense system potential and prevents oxidative stress induced insulin resistance in rat L6 muscle cells.     

神经干细胞微囊泡抑制H2O2 诱导DRG 神经元氧化应激损伤机制研究

目的 探究神经干细胞微囊泡（neural stem cell microvesicles, NSC-MVs）对H2O2 诱导背根神经节（dorsal rootganglion, DRG）神经元氧化应激损伤的作用及机制。方法 超速离心提取NSC-MVs，并进行电镜和纳米颗粒示踪分析。原代培养大鼠DRG 神经元，β-tubulin Ⅲ荧光染色。建立H2O2 诱导DRG 神经元氧化应激损伤模型，确定作用浓度。经NSC-MVs 预处理，MTT( 四唑盐) 检测神经元活力，流式细胞术检测Annexin Ⅴ和PI，蛋白质印迹检测凋亡相关蛋白cleaved caspase 3，cleaved caspase 9，Bax 和Bcl-2 的表达。结果 NSC-MVs 在透射电镜下呈圆盘状，包膜完整，纳米颗粒示踪显示其粒径为50 ～ 450 nm。MTT 结果显示，与对照组相比，H2O2 组神经元活力明显抑制。当H2O2 浓度为25，50，100 和200μmol/L 时具有显著性差异，细胞活力分别为84.4 %，73.7 %，69.8 % 和49.5 %（F=127.7，P ＜ 0.01）。经100，200 和400 μg/ml 的NSC-MVs 预处理DRG 神经元，细胞活力得到明显提升，分别为51.4 %，67.4 % 和73.5 %（F=49.47，P=0.023）。流式细胞术检测结果显示，与对照组相比，H2O2 组神经元凋亡率显著上升（P ＜ 0.05），NSCMVs预处理组细胞凋亡率明显下降（P ＜ 0.05）。蛋白质印迹结果显示，与H2O2 组相比，NSC-MVs 显著抑制cleavedcaspase3，cleaved caspase 9 和Bax 蛋白表达（均P ＜ 0.05），上调Bcl-2 蛋白表达（P ＜ 0.05）。结论 NSC-MVs 能够抑制H2O2 诱导DRG 神经元氧化应激损伤，发挥神经保护作用。     

A novel neurotrophic agent, T-817MA [1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate], attenuates amyloid-beta-induced neurotoxicity and promotes neurite outgrowth in rat cultured central nervous system neurons

Progressive neuronal loss in Alzheimer's disease (AD) is considered to be a consequence of the neurotoxic properties of amyloid-beta peptides (A beta). T-817MA (1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate) was screened as a candidate therapeutic agent for the treatment of AD based on its neuroprotective potency against A beta-induced neurotoxicity and its effect of enhancing axonal regeneration in the sciatic nerve axotomy model. The neuroprotective effect of T-817MA against A beta(1-42) or oxidative stress-induced neurotoxicity was assessed using a coculture of rat cortical neurons with glia. T-817MA (0.1 and 1 microM) was strongly protective against A beta(1-42)-induced (10 microM for 48 h) or H2O2-induced (100 microM for 24 h) neuronal death. T-817MA suppressed the decrease of GSH levels induced by H2O2 exposure (30 microM for 4 h) in cortical neuron culture; therefore, T-817MA was likely to alleviate oxidative stress. Besides the neuroprotective effect, T-817MA (0.1 and 1 microM) promoted neurite outgrowth in hippocampal slice cultures and reaggregation culture of rat cortical neurons. T-817MA also increased the growth-associated protein 43 content in the reaggregation culture of cortical neurons. These findings suggest that T-817MA exerts neuroprotective effect and promotes neurite outgrowth in rat primary cultured neurons. Based on these neurotrophic features, T-817MA may have a potential for disease modification and be useful for patients with neurodegenerative diseases, such as AD.     

Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity

A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of mu-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.     

Oxidative changes and desialylation of serum proteins in hyperthyroidism

BACKGROUND: Hyperthyroidism is associated with oxidative stress. Thyroid hormones are observed to influence the metabolism of plasma proteins. The present study was carried out to explore the level of sialic acid content and the oxidative changes of serum proteins in hyperthyroid subjects and matched healthy controls. METHODS: Blood was collected from 10 hyperthyroid patients and 10 age and sex matched healthy control subjects. The extent of carbonylation and desialylation of serum protein was estimated by dinitrophenylhydrazine and thiobarbituric acid methods, respectively. The protein cleavage and cross-linking were studied by separating serum protein in SDS-PAGE. The effects of in-vitro treatment of serum proteins with H(2)O(2) on the above-mentioned parameters were observed. RESULTS: The carbonylation was significantly higher and the sialic acid content was significantly lower in serum proteins of hyperthyroid cases in comparison to that of controls. Serum proteins were found to have increased levels of cleavage and cross-linking in hyperthyroid cases. The high molecular weight proteins were mostly cleaved. In-vitro treatment of serum proteins with H(2)O(2) led to similar changes. CONCLUSIONS: The study indicated that in hyperthyroidism, serum proteins undergo increased levels of oxidative changes leading to high turnover rate of blood proteins. A significant negative correlation between carbonylation and sialic acid content of serum proteins in hyperthyroidism and enhanced desialylation and carbonylation of serum proteins by in-vitro H(2)O(2) treatment suggest that oxidative stress can cause desialylation of serum glycoproteins.     

Discovery of novel hippocampal neurogenic agents by using an in vivo stable isotope labeling technique

Neurogenesis occurs in discrete regions of adult mammalian brain, including the subgranular zone of the hippocampus. Hippocampal neurogenesis is enhanced by different classes of antidepressants, but screening for neurogenic actions of novel antidepressants has been inefficient because of limitations of 5-bromo-2'-deoxyuridine labeling techniques. We describe an efficient in vivo method for measuring hippocampal neurogenesis involving incorporation of the stable isotope, (2)H, into genomic DNA during labeling with (2)H(2)O (heavy water). Male rodents received 8 to 10% (2)H(2)O in drinking water; DNA was isolated from hippocampal progenitor cells or neurons. Label incorporation into progenitor cells of Swiss-Webster mice revealed subpopulation kinetics: 16% divided with t(1/2) of 2.7 weeks; the remainder did not divide over 1 year. Progenitor cell proliferation rates in mice were strain-dependent. Chronic antidepressant treatment for 3 weeks, with (2)H(2)O administered during the final week, increased progenitor cell proliferation across all the strains tested. Fluoxetine treatment increased (2)H incorporation into DNA of gradient-enriched neurons or flow-sorted neuronal nuclei 4 weeks after (2)H(2)O labeling, representing the survival and differentiation of newly divided cells into neurons. By screening 11 approved drugs for effects on progenitor cell proliferation, we detected previously unrecognized, dose-dependent enhancement of hippocampal progenitor cell proliferation by two statins and the anticonvulsant topiramate. We also confirmed stimulatory activity of other anticonvulsants and showed inhibition of progenitor cell proliferation by isotretinoin and prednisolone. In conclusion, stable isotope labeling is an efficient, high-throughput in vivo method for measuring hippocampal progenitor cell proliferation that can be used to screen for novel neurogenic drugs.     

Protective effect of mesenchymal stem cell-derived exosomal treatment of hippocampal neurons against oxygen-glucose deprivation/reperfusion-induced injury

BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are more stable and highly conserved than MSCs.This study was conducted to investigate the therapeutic effects of MSC-derived exosomes(MSC-Exo)on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R),and to explore the underlying mechanisms.METHODS:Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment,with or without MSC-Exo treatment.Exosomal integration,cell viability,mitochondrial membrane potential,and generation of reactive oxygen species(ROS)were examined.Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nickend labeling(TUNEL)staining was performed to detect neuronal apoptosis.Moreover,mitochondrial function-associated gene expression,Nrf2 translocation,and expression of downstream antioxidant proteins were determined.RESULTS:MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation(P<0.05).The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus(2.14±0.65 vs.5.48±1.09,P<0.01)and increased the intracellular expression of antioxidative proteins,including superoxide dismutase and glutathione peroxidase(17.18±0.97 vs.14.40±0.62,and 20.65±2.23 vs.16.44±2.05,respectively;P<0.05 for both).OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial functionassociated genes,such as PINK,DJ1,LRRK2,Mfn-1,Mfn-2,and OPA1.The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons.CONCLUSIONS:MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons.Therefore,MSCExo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.     

Oxidative stress modulates membrane bound ATPases in brain regions of PCB (Aroclor 1254) exposed rats: protective role of alpha-tocopherol.

Polychlorinated biphenyls (PCBs) are a class of widely dispersed and environmentally persistent organic compounds. PCBs exhibit a wide range of toxicological effects including neurotoxicity. Vitamin E (alpha-tocopherol) is an important lipid soluble antioxidant placed in a special region of membranes. Large amounts of energy are required to maintain the signaling activities of the cells in the central nervous system (CNS). Membrane proteins that control ion gradients across organellar and plasma membranes appear to be particularly susceptible to oxidation-induced changes. The aim of this study was to determine the protective role of vitamin E on Aroclor 1254 induced modulation in membrane bound ATPases in brain regions of rats. One group of rats received corn oil as vehicle for 30days as control. The other group of rats were administered Aroclor 1254 at a dose of 2mgkg(-1) bwday(-1) intraperitoneally for 30days. One group of rats received vitamin E (50mgkg(-1) bwday(-1)) orally simultaneously with Aroclor 1254 for 30days. After 30days, the animals were euthanized and the brain was dissected to hypothalamus and hippocampus to determine the following parameters. Hydrogen peroxide (H2O2), Lipid peroxidation (LPO) and the activities of Na+K+-ATPase, Ca2+-ATPase and Mg2+-ATPase were determined. Reduced glutathione (GSH) level was also determined. Activities of all the enzymes were decreased while an increase in H2O2 and LPO were observed in selected brain regions of PCB treated animals. Simultaneous vitamin E treatment in PCB exposed animals restored all the parameters significantly. These results suggest that oxidative stress is involved in the inhibitory effect of PCB (Aroclor 1254) on membrane bound ATPases in selected brain regions. alpha-tocopherol acts against PCB induced neurotoxicity by decreasing oxidative stress.     

Superoxide anion overproduction in sepsis: effects of vitamin e and simvastatin

Oxidative stress during sepsis induces tissue damage, leading to organ dysfunction and high mortality. The antioxidant effects of vitamin E have been reported in several diseases, but not in sepsis. Statins have cholesterol-independent anti-inflammatory effects that are related to a decrease of isoprenoid proteins and oxidative stress. Therefore, we evaluated superoxide anion (O2- degree) production and ex vivo effects of vitamin E and simvastatin in sepsis. Fourteen healthy volunteers, 14 intensive care unit (ICU) nonseptic, and 14 ICU patients with sepsis were included in this prospective study. Plasma cholesterol, triglyceride, and vitamin E levels were determined by routine laboratory tests. Superoxide anion production was measured in the venous blood by chemiluminescence technique after phorbol myristate acetate stimulation. Effects of vitamin E and simvastatin on O2- degree production was investigated ex vivo. Luminescence was indexed to the leukocyte count. We also investigated the in vitro effect of simvastatin on translocation of NADPH oxidase p21 Rac2 subunit in THP-1 monocytic cell line. The ratio of vitamin E/cholesterol + triglycerides was significantly decreased in septic as compared with nonseptic patients and volunteers. The O2- degree production was significantly higher in the group of septic patients than in the others, regardless of the polymorphonuclear leukocyte count. Vitamin E and simvastatin induced ex vivo an inhibition of O2- degree production of 20% and 40% respectively. In vitro, simvastatin inhibited phorbol myristate acetate-induced- O2- degree production by monocytes through NADPH oxidase inactivation. We conclude that sepsis is associated with a significant decrease in vitamin E and an overproduction of O2- degree. Vitamin E and simvastatin lessen this phenomenon through NADPH oxidase inactivation.     

应激状态下肠上皮细胞凋亡水平的变化及其机制

目的探讨在氧化应激状态下肠上皮细胞凋亡水平的变化以及凋亡异常发生的分子机制。方法使用过氧化氢(H2O2)处理培养的HT-29细胞模拟机体活性氧(ROS)损伤肠上皮细胞的体内状况,采用四甲基偶氮唑盐(MTT)微量酶反应比色法进行细胞生存力的检测;采用流式细胞术进行细胞凋亡的检测;采用蛋白质免疫印迹法(Westernblot)检测凋亡相关蛋白的表达。结果H2O2可降低HT-29细胞生存率,且呈现剂量依赖性和时间依赖性(P均<0.05);与空白对照组相比,随着H2O2浓度的增高,细胞凋亡率增加(P均<0.05),随着作用时间的延长,细胞凋亡率也增加(P<0.05);以不同浓度H2O2刺激HT-29细胞24h后发现,与空白对照组相比,Bax的表达随着H2O2浓度的增高而增加,Bcl-2的表达随着H2O2浓度的增高而降低。以500μmol/L,浓度的H2O2刺激HT-29细胞发现,Bax表达随着H2O2作用时间延长而增加,Bcl-2表达随着H2O2作用时间延长而降低。结论应激状态下,肠上皮细胞氧化应激水平与其凋亡程度相关,凋亡调控蛋白Bcl-2/Bax比值失调可能是肠上皮细胞凋亡过度的机制之一。