羟基红花黄色素A抑制脑组织蛋白质硝基化的体外研究

目的 研究羟基红花黄色素A(HSYA)对体外亚铁血红素/亚硝酸钠/过氧化氢(heme/NaNO2/H2O2)途径引起脑组织蛋白质硝基化的抑制作用.方法 模拟体内heme/NaNO2/H2O2硝基化途径,分别以牛血清白蛋白和脑组织蛋白为硝基化底物,分为对照组及药物干预组,HSYA的终浓度依次为0.01mmol/L、0.1mmol/L、1mmol/L.以Western blotting法检测蛋白质酪氨酸硝基化水平.结果 Heme/NaNO2/H2O2途径可引起牛血清白蛋白和脑组织蛋白显著的硝基化修饰,加入不同浓度HSYA后,蛋白质硝基化水平均呈剂量依赖性地降低,其中以1 mmol/L HSYA的抑制作用最明显,与对照组相比,差异具有统计学意义(P均＜0.05).结论 HSYA可剂量依赖性地抑制heme/NaNO2/H2O2途径在体外对脑组织蛋白的硝基化修饰;提示HSYA抑制蛋白质硝基化反应可能是其对抗脑血管疾病与神经系统变性疾病的分子机制之一.     

羟基红花黄色素A对6-OHDA诱导的帕金森病小鼠模型的神经保护作用及机制研究

目的 探讨羟基红花黄色素A对6-OHDA诱导的帕金森病小鼠模型的神经保护作用及机制。方法 通过旋转实验检测羟基红花黄色素A 对6-OHDA诱导的帕金森病小鼠模型的神经保护作用; 末次行为学试验后取脑,采用高效液相色谱-荧光检测法测定5-羟色胺(5-HT)水平; 采用试剂盒测定超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶( GSH-Px)、一氧化氮合酶(NOS)水平。结果 与空白组比较,对照组的旋转圈数明显增加(P<0.05),实验组的爬杆时间明显缩短(P<0.05); 使用羟基红花黄色素A治疗后实验组的旋转圈数明显降低(P<0.05); 羟基红花黄色素A能明显提高细胞中5HT、SOD、GSH-Px的水平,降低NOS 的水平,提高抗氧化能力。结论 羟基红花黄色素A对6-OHDA诱导的帕金森病小鼠模型具有神经保护作用,其机制可能与抗氧化作用有关     

羟基脲对体外培养的脑膜瘤细胞增殖及凋亡的影响

目的观察羟基脲对体外培养的脑膜瘤细胞增殖的影响及其对细胞凋亡的诱导作用.方法采用细胞计数法测定脑膜瘤细胞的增殖情况,运用光镜、电镜、DNA电泳及流式细胞仪检测细胞的凋亡情况.结果羟基脲能明显抑制脑膜瘤细胞的增殖,电镜、DNA电泳及流式细胞仪检测有明显的凋亡改变.结论羟基脲可能通过诱导细胞产生凋亡来抑制脑膜瘤细胞的增殖.     

羟基红花黄色素A对谷氨酸诱导损伤神经元过氧化物酶体增殖物激活受体γ表达的影响

目的探讨羟基红花黄色素A(HSYA)拮抗谷氨酸诱导神经元损伤的保护机制。方法采集经体外原代培养第7天的Sprague Dawley胎鼠皮质神经元,通过形态学观察、噻唑蓝法、Westernblotting法分别检测过氧化物酶体增殖物激活受体γ(PPARγ)和磷酸化PPARγ(pPPARγ,即PPARγ的失活形式)表达水平,以探讨HSYA抗谷氨酸诱导神经元损伤的保护机制。结果经谷氨酸处理后神经元发生肿胀、部分崩解、死亡。与正常对照组相比,谷氨酸不同剂量组(1.00、5.00和10.00mmol/L组)的神经元存活率(66.60%、33.40%和21.60%)和PPARγ蛋白表达水平显著降低,对照组灰度值:1.06±0.18,谷氨酸5.00和10.00mmol/L组灰度值分别为:0.32±0.09、0.28±0.07(均P=0.000),pPPARγ蛋白表达水平明显升高,灰度值分别为:0.37±0.05、1.83±0.17和1.75±0.21(均P=0.000)。HSYA与谷氨酸共同处理后神经元形态明显改善、细胞相对存活率提高,谷氨酸5.00mmol/L,HSYA0.01、0.10和1.00mmol/L组分别为33.40%、35.30%、51.00%和72.50%(P=0.742、0.033、0.002),PPARγ蛋白表达水平呈现逐渐升高趋势,对照组,谷氨酸5.00mmol/L,HSYA0.01、0.10和1.00mmol/L组灰度值分别为:1.20±0.16、0.25±0.06、0.39±0.10、0.41±0.12、0.37±0.08,但差异未达到统计学意义(均P>0.05),pPPARγ蛋白表达水平降低,对照组,谷氨酸5.00mmol/L,HSYA0.01、0.10和1.00mmol/L组灰度值分别为:0.51±0.14、1.91±0.25、1.70±0.26、1.25±0.23、0.85±0.19,(P=0.022、0.004、0.000)。结论 HSYA对由谷氨酸诱导的神经元损伤具有保护作用,其机制与抑制PPARγ蛋白磷酸化即抑制PPARγ活性有关。     

纳米羟基磷灰石颗粒诱导对大鼠巨噬细胞凋亡影响的初步研究

由于其尺寸效应,纳米颗粒（NPs）可以结合细胞内大分子,从而引起细胞坏死或凋亡。而目前针对纳米颗粒的生物安全性,缺乏对颗粒致细胞凋亡的机理研究。目的：从细胞及分子水平上探讨纳米颗粒致细胞凋亡的机理,为纳米颗粒的安全应用提供科学依据。设计、时间及地点：2007年1月至2008年12月,在上海生物材料研究测试中心完成以下相关实验。材料：原代培养大鼠巨噬细胞,并在300W/40KHZ的超声条件下制备纳米羟基磷灰石颗粒（HAP NPs）的悬浮液（30-80nm）。方法：为探讨纳米陶瓷颗粒对细胞的损伤作用及凋亡机制,本研究应用透射电镜（TEM）观察细胞凋亡的超微结构表征;AnnexinV-EGFP/PI双染检测凋亡结果;Western Blot方法检测凋亡调控相关基因P53的表达变化。主要观察指标：凋亡率及P53的蛋白表达水平。结果：TEM 观察单核巨噬细胞经HAP NPs作用后具有典型的凋亡形态学特征;并且细胞的凋亡与NPs的浓度呈正相关;Western Blot结果显示,100 µg/ml HAP NPs引起P53表达显著上调。 结论：本研究结果提示HAP NPs颗粒能够通过蛋白磷酸化上调P53蛋白,从而能够启动下游相关基因,最终导致细胞凋亡。同时也可以认为,P53是用来进行纳米陶瓷颗粒安全性评价较为理想的指标。     

亚低温治疗对重型颅脑损伤患者预后及其脑脊液Aβ水平的影响

目的 探讨亚低温治疗对重型颅脑损伤患者预后及其脑脊液Aβ水平的影响.方法 选取2009-10-2011-10于我院接受治疗的重型颅脑损伤患者72例,随机分为观察组与对照组,每组36例,观察亚低温治疗对重型颅脑损伤患者预后及其脑脊液Aβ水平的影响.结果 6个月后观察组患者预后情况良好的占44.44％,明显高于对照组的16.67％ (P＜0.05),同时,观察组患者植物生存和死亡的病例明显低于对照组(P＜0.05).对照组患者术后脑脊液Aβ水平呈逐步上升的趋势,治疗后5d、7d脑脊液Aβ水平明显高于治疗前(P＜0.05);观察组患者术后脑脊液Aβ水平则呈逐步下降的趋势,其治疗后3d、5d、7d脑脊液Aβ水平明显低于治疗前(P＜0.05),而且观察组治疗后3d、5d、7d脑脊液Aβ水平明显低于对照组(P＜0.05).结论 亚低温治疗用于重型颅脑损伤患者效果显著,值得推广.     

维生素A对体外培养小鼠精原干细胞生长增殖的影响*

背景：维生素A在体内对精原干细胞生长具有重要作用,目前还没有发现在体外培养过程中能够很好促进精原干细胞生长与分化的诱导物质。 目的：探讨维生素A对体外培养小鼠精原干细胞生长增殖的影响。 方法：无菌收集5~7 d龄昆明雄性小鼠双侧睾丸,采用差速贴壁联合非连续性Percoll密度梯度离心法分离纯化精原干细胞。无菌取出12~15 d龄昆明雄性小鼠双侧睾丸,酶消化法分离纯化Sertoli细胞,贴壁并极化后作为饲养层,将精原干细胞接种在单层Sertoli细胞上。设立2组,实验组向DMEM/F12培养液中加入1 g/L维生素A,对照组不添加维生素A。采用酶联仪测定精原干细胞生长增殖情况,流式细胞仪检测精原干细胞生长周期。 结果与结论：共培养6,9,12,15 d时,实验组精原干细胞吸光度值明显高于对照组(P < 0.05或0.01)。随共培养时间的延长,实验组精原干细胞S期染色体含量逐渐增多,然后又逐渐下降,开始另一个分裂周期;与实验组比较,对照组精原干细胞S期染色体含量增长缓慢(P < 0.05)。小鼠精原干细胞在体外培养过程中,维生素A可促进其增殖分化。     

XIAP过度表达对未成熟脑急性放射损伤后脑组织硝基酪氨酸和4-羟基壬烯醛表达的影响

目的探讨急性放射损伤对未成熟脑高增殖细胞的影响以及X-染色体连锁的凋亡抑制剂(XIAP)过度表达对放射后氧化应激产物硝基酪氨酸(NT)和4-羟基壬烯醛(4-HNE)形成的影响。方法新生10日龄XIAP过度表达转基因C57BL/6小鼠(XIAP组)及同期野生型10日龄C57BL/6小鼠在8Gy单一剂量放射线照射后6h或7d处死取脑,进行脑组织硝基酪氨酸和4-羟基壬烯醛免疫组化染色,以及海马齿状回、侧脑室下区面积测量。结果照射后7d海马齿状回、侧脑室下区面积较对照组明显下降,照射后脑组织硝基酪氨酸和4-羟基壬烯醛免疫活性明显增加,照射后6hXIAP过度表达组大脑海马齿状回、侧脑室下区硝基酪氨酸和4-HNE的阳性细胞数明显低于野生组(P<0.05,P<0.001)。结论放射线照射可导致活性氧、活性氮基团过度表达,进而引起高增殖细胞损伤;XIAP过度表达可能抑制照射后硝基酪氨酸和4-HNE的形成。     

氯硝西泮预处理对癫痫大鼠海马区γ-氨基丁酸A受体γ2亚单位表达的影响

目的 探讨氯硝西泮预处理对癫痫大鼠海马区γ-氨基丁酸A受体γ2亚单位(GABAARγ2)表达的影响.方法 60只健康雄性SD大鼠随机分为假手术组、癫痫组和氯硝西泮预处理组;癫痫组再分为6h、12 h、1 d、3d、7d、15 d和30 d7个亚组;药物预处理组再分为假预处理组、预处理6h、12h和ld亚组.药物预处理组给予氯硝西泮6 mg/(kg·d)分2次灌胃,连续5d;然后癫痫组和预处理组通过向大鼠海马C3区注射海人酸建立颞叶癫痫模型;采用免疫组化法在相应时点检测各组大鼠海马区GABAARγ2的表达.结果 与假手术组比较,癫痫组CA1区癫痫发作ld后、CA3区癫痫发作后各时间点GABAARγ2表达明显下降(P＜0.05 ～0.01).与癫痫组相应亚组比较,预处理6h、12 h亚组海马CA3区及预处理ld亚组海马CA1区及CA3区GABAARγ2的表达明显增高(P＜0.05～0.01).结论 氯硝西泮预处理可上调癫痫大鼠海马区GABAARγ2的表达.     

Hydroxysafflor yellow A improves learning and memory in a rat model of vascular dementia by increasing VEGF and NR1 in the hippocampus

Hydroxysafflor yellow A (HSYA) has angiogenesisregulating and neuro-protective effects, but its effects on vascular dementia (VaD) are unknown. In this study, 30 adult Sprague-Dawley rats were randomly allocated to five groups: normal, sham-operation, VaD alone (bilateral carotid artery occlusion), VaD plus saline (control), and VaD plus HSYA. One week after operation, the HSYA group received one daily tail-vein injection of 0.6 mg/100 g HSYA for two weeks. Five weeks after operation, the spatial memory of all five groups was evaluated by the water maze task, and synaptic plasticity in the hippocampus was assessed by the long-term potentiation (LTP) method. Vascular endothelial growth factor (VEGF) and N-methyl-Daspartic acid receptor 1 (NR1) expression in the hippocampus was detected via Western blot. We found that, compared with the group with VaD alone, the group with HSYA had a reduced escape latency in the water maze (P < 0.05), and the LTP at CA3-CA1 synapses in the hippocampus was enhanced (P < 0.05). Western blot in the late-phase VaD group showed slight up-regulation of VEGF and downregulation of NR1 in the hippocampus, while HSYA significantly up-regulated both VEGF and NR1. These results suggested that HSYA promotes angiogenesis and increases synaptic plasticity, thus improving spatial learning and memory in the rat model of VaD.     

Elevated protein carbonylation in the brain white matter and gray matter of patients with multiple sclerosis

Oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS). Increased levels of reactive oxygen species (ROS) derived from infiltrating macrophages and microglial cells have been shown to reduce the levels of endogenous antioxidants and to cause the oxidation of various substrates within the MS plaque. To determine whether oxidative damage takes place beyond visible MS plaques, the occurrence of total carbonyls (TCOs) and protein carbonyls (PCOs) in the normal-appearing white matter (NAWM) and gray matter (NAGM) of eight MS brains was assessed and compared with those of four control brains. The data show that most (7/8) of the MS-WM samples contain increased amounts of PCOs as determined by reaction with 2,4-dinitrophenylhydrazine and Western blot analysis. These samples also have high levels of glial fibrilary acidic protein (GFAP), suggesting that oxidative damage is related to the presence of small lesions. In contrast, we detected no evidence of protein thiolation (glutathionylation and cysteinylation) in the diseased tissue. To our surprise, MS-NAGM specimens with high GFAP content also showed three times the concentration of TCOs and PCOs as the controls. The increase in PCOs is likely to be a consequence of reduced levels of antioxidants, in that the concentration of nonprotein thiols in both MS-WM and -GM decreased by 30%. Overall, our data support the current view that both NAWM and -GM from MS brains contain considerable biochemical alterations. The involvement of GM in MS was also supported by the decrease in the levels of neurofilament light protein in all the specimens analyzed. To the best of our knowledge, this is the first study demonstrating the presence of increased protein carbonylation in post-mortem WM and GM tissue of MS patients.     

Neuroprotective role of prostaglandin PGE2 EP2 receptor in hemin-mediated toxicity

Heme (Fe²⁺ protoporphyrin IX) and hemin (Fe³⁺), the prosthetic group of hemoprotein, are cytotoxic due to their ability to contribute to the production of reactive oxygen species, increased intracellular calcium levels, and stimulate glutamate-mediated excitotoxicity. Previous work by our group showed that blockade of the prostaglandin E₂ (PGE₂)-EP1 receptor reduced hemin-induced cytotoxicity in primary cortical neuronal cultures. However, the role of the prostaglandin E2 (PGE2)-EP2 receptor in hemin neurotoxicity remains unclear. Activation of the EP2 receptor in neurons results in increased cyclic AMP (cAMP) and protein kinase A signaling; therefore, we hypothesized that the activation of the EP2 receptor decreases hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wildtype-control (WT) and EP2^−/− mice, we investigated the role of the EP2 receptor in hemin neurotoxicity by monitoring cell survival with the Calcein-AM live-cell and lactate dehydrogenase assays. MitoTracker staining was also performed to determine how mitochondria were affected by hemin. Hemin neurotoxicity in EP2^−/− neurons was 37.2 ± 17.0% greater compared to WT neurons. Of interest, cotreatment with the EP2 receptor agonist, butaprost (1 and 10 μM), significantly attenuated hemin neurotoxicity by 55.7 ± 21.1% and 60.1 ± 14.8%, respectively. To further investigate signaling mechanisms related to EP2 receptor mediating cytoprotection, neurons were cotreated with hemin and activators/inhibitors of both the cAMP-protein kinase A/exchange protein directly activated by cAMP (Epac) pathways. Forskolin, a cAMP activator, and 8-pCPT-cAMP, an Epac activator, both attenuated hemin neurotoxicity by 78.8 ± 22.2% and 58.4 ± 9.8%, respectively, as measured using the lactate dehydrogenase assay. Together, the results reveal that activation of the EP2 receptor is protective against hemin neurotoxicity in vitro and these findings suggest that neuroprotection occurs through the cAMP-Epac pathway in neuronal cultures. Therefore, activation of the EP2 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.     

抑胶素对C6胶质瘤细胞p16蛋白表达的影响

目的探讨不同浓度的抑胶素对C6胶质瘤细胞p16蛋白表达的影响。方法体外培养C6胶质瘤细胞,应用顺铂作为对照,通过免疫细胞化学染色法检测不同浓度的抑胶素作用后细胞周期负性调控基因p16在不同时问点的蛋白表达。结果 C6胶质瘤细胞经过抑胶素处理后,随着抑胶素浓度的增加,p16蛋白的表达也明显增强。结论抑胶素能够抑制C6胶质瘤细胞的增殖,并且具有改变肿瘤细胞增殖周期的作用。     

Differential involvement of spinal protein kinase C and protein kinase A in neuropathic and inflammatory pain in mice

In the present study, we demonstrated the differential role of spinal protein kinases in neuropathic and inflammatory pain. Mice with sciatic nerve ligation exhibited a spinal protein kinase C (PKC)-dependent neuropathic pain-like state. In contrast, an intraplanter injection of inflammatory agent caused a protein kinase A (PKA)-related thermal hyperalgesia. These findings suggest that the substantial activation of spinal PKC and PKA may differentially contribute to the development of respective chronic pain-like state in mice.     

Zinc binds to and directly inhibits protein phosphatase 2A in vitro

Zinc induces protein phosphatase 2A(PP2A) inactivation and tau hyperphosphorylation through PP2A(tyrosine 307) phosphorylation in cells and the brain, but whether Zn2+ has a direct inhibitory effect on PP2 A is not clear. Here we explored the effect of Zn2+ on PP2 A and their direct interaction in vitro. The results showed that Zn2+ mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2 a cell lysates. PP2 A activity assays indicated that a low concentration(10 μmol/L) of Zn2+ inhibited PP2 A directly. Further Zn2+-IDA-agarose affinity binding assays showed that Zn2+ bound to and inhibited PP2Ac(51-270) but not PP2Ac(1-50) or PP2Ac(271-309). Taken together, Zn2+ inhibits PP2 A directly through binding to PP2Ac(51-270) in vitro.     

Interactive effects of a protein kinase AII inhibitor and testosterone on spatial learning in the Morris water maze

Neurohormones such as testosterone (TE) are important in modulation of learning and memory. In the present study, we investigated the interactive effects of pre-training bilateral intra-hippocampal infusions of testosterone and H-89, a selective PKAII inhibitor, on spatial acquisition in the Morris water maze (MWM). Different doses of TE (20, 40 and 80 μg/side) and H-89 (5 and 10 μM/side) were administered 30 min before start of the training each day. Control animals received bilateral intra-hippocampal infusions of DMSO as vehicle for TE and H-89. Animals were trained for 4 days and each day included one block of four trials. The results of this study showed that bilateral infusion of TE (40 and 80 μg/side) or H-89 (10 μM/side) impaired spatial learning as indicated by significant increases in escape latency and traveled distance compared to the control group. Although pre-training bilateral infusions of a low concentration of either TE (20 μg/side) or H-89 (5 μM/side) into the CA1 region of the hippocampus did not affect learning capabilities, but the combination of the low doses of the drugs led to significant deficits in spatial acquisition. Overall, our data suggest that spatial acquisition was affected by PKAII inhibition or TE administration. Moreover, when co-administered, these drugs had a negative synergistic impact on acquisition.