低氧预适应大鼠脑脊液减轻氧糖剥夺对培养新生鼠海马神经元的损伤及可能的机制

本文旨在观察低氧预适应(hypoxic preconditioning,HPC) Wistar大鼠脑脊液对新生鼠海马神经元氧糖剥夺(oxygen glucose deprivation,OGD)损伤的影响及机制.原代培养大鼠新生鼠(出生12 h)海马神经元,随机分为正常对照组、OGD组(OGD培养1.5 h)、正常脑脊...     

缺糖缺氧对体外培养海马神经元的影响

目的：观察缺糖缺氧诱导的培养海马神经元损伤。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液在常氧条件下继续培养24h。用0．4％台盼蓝染色,检测神经元坏死,并用TUNEL法检测神经元凋亡,计算存活、坏死和凋亡神经元所占百分率。同时用图像分析仪测定存活、坏死和凋亡神经元的胞体面积、周长和等园直径。结果：培养的海马神经元急性缺糖缺氧后0．5～4h,随缺糖缺氧时间的延长,坏死神经元逐渐增多,缺糖缺氧后0．5～2h再恢复糖和氧供应后24h,凋亡神经元明显增多。图像分析的结果表明,坏死神经元的胞体面积、周长和等园直径均明显大于凋亡神经元。结论：缺糖缺氧可引起海马神经元严重损伤,在急性缺糖缺氧后0．5～4h引起的神经元死亡以坏死为多见,但在缺糖缺氧后0．5～2h再恢复糖和氧供应后24,神经元死亡则以凋亡为多见。     

白花丹参的核型分析及小孢子发生过程观察

对白花丹参(Salvia miltiorrhiza f.alba)进行了核型分析及小孢子发生过程观察.结果表明,白花丹参染色体数目为2n=16,核型公式为2n=16=8m+8sm,其中2条染色体具有随体,没有观察到多倍体细胞和B染色体;16条染色体在减数分裂中期I正常配对形成8个二价体,减数分裂的胞质分裂属于同时型;在白花丹参的减数分裂前期I观察到频率较高的细胞融合现象.白花丹参的核型为2B型,其花粉母细胞中的细胞融合现象为首次报道.     

Protective effects of Lycium barbarum polysaccharide on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion

This study investigated the protective effects of Lycium barbarum polysaccharide (LBP) on alleviating injury from oxygen-glucose deprivation/reperfusion (OGD/RP) in primary cultured rat hippocampal neurons. Cultured hippocampal neurons were exposed to oxygen-glucose deprivation (OGD) for 2?h followed by a 24?h re-oxygenation. The MTT assay and the lactate dehydrogenase (LDH) release were used to determine the neuron viability. Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX), malondialdehyde (MDA) were determined by spectrophotometry using commercial kits. Mitochondrial membrane potential (MMP) and the intracellular free calcium concentration ([Ca²⁺]_i) in hippocampal neurons were measured using the confocal laser scanning microscope (CLSM). Treatment with LBP (10–40?mg/l) significantly attenuated neuronal damage and inhibited LDH release in a dose-dependent manner. Furthermore, LBP enhanced activities of SOD and GSH-PX but it decreased their MDA content, inhibited [Ca²⁺]_i elevation and decrease of MMP in ischemia–reperfusion treated hippocampal neurons. These findings suggested that LBP may be a potential neuroprotective agent for cerebral?ischemia–reperfusion injury.     

白花丹参与丹参活性成分积累比较研究

研究了山东产丹参（Salvia miltiorrhiza Bge．）、白花丹参（Salvia miltiorrhiza Bge．f．alba）不同生育阶段脂溶性与水溶性成分含量的动态变化,对活性成分积累过程进行数学模拟。结果表明：白花丹参脂溶性成分积累有两个高峰期,分别在7月底和10月底,丹参脂溶性成分的积累高峰在7月底;白花丹参和丹参水溶性成分的积累高峰期均出现在6—7月份。两年生白花丹参活性成分高于丹参;两种丹参的迷迭香酸含量均以地上部为高,其他活性成分均以地下部为高;白花丹参与丹参根中丹酚酸B的含量积累方程分别为y=-8．105X＋81．047和y=-13．777X＋117．917,即随着植株的不断发育,丹参根部丹酚酸B的含量呈下降趋势。该研究结果可为丹参栽培及其质量控制提供理论依据。     

GABA对大鼠海马脑片缺氧损伤的保护作用

目的：研究GABA对大鼠海马脑片急性缺氧损伤的保护机制。方法：采用成年大鼠离体海马脑片,用胞外记录的电生理技术,观察GABA对急性缺氧后海马脑片诱发电位的影响。结果：（1）GABA可明显延迟PV的消失,但对PS却无影响;（2）给予GABAA受体拮抗剂荷包牡丹碱（bicuculine)以及Cl^-通道阻抗剂NPPB可阻断GABA的保护作用。结论：GAB可提高海马脑片耐缺氧能力,其机制可能与GABA通过GABAA受体提高Cl^-内流有关。     

Lycopene protects against trimethyltin-induced neurotoxicity in primary cultured rat hippocampal neurons by inhibiting the mitochondrial apoptotic pathway

Lycopene is a potent free radicals scavenger with demonstrated protective efficacy in several experimental models of oxidative damage. Trimethyltin (TMT) is an organotin compound with neurotoxic effects on the hippocampus and other limbic structures and is used to model neurodegenerative diseases targeting these brain areas. Oxidative stress is widely accepted as a central pathogenic mechanism of TMT-mediated neurotoxicity. The present study investigated whether the plant carotene lycopene protects against TMT-induced neurotoxicity in primary cultured rat hippocampal neurons. Lycopene pretreatment improved cell viability in TMT-treated hippocampal neurons and inhibited neuronal apoptosis. Microfluorometric imaging revealed that lycopene inhibited the accumulation of mitochondria-derived reactive oxygen species (ROS) during TMT exposure. Moreover, lycopene ameliorated TMT-induced activation of the mitochondrial permeability transition pore (mPTP) and the concomitant depolarization of the mitochondrial membrane potential (ΔΨ_m). Consequently, cytochrome c release from the mitochondria and ensuing caspase-3 activation were markedly reduced. These findings reveal that lycopene protects against TMT-induced neurotoxicity by inhibiting the mitochondrial apoptotic pathway. The anti-apoptotic effect of lycopene on hippocampal neurons highlights the therapeutic potential of plant-derived antioxidants against neurodegenerative diseases.     

Protective effect of salidroside against H2O2-induced cell apoptosis in primary culture of rat hippocampal neurons

Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H₂O₂)-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H₂O₂-stimulated apoptotic cell death in primary culture of hippocampal neurons in a concentration-dependent manner. The measurements of caspase-3 activity, nitric oxide (NO) production, and NO synthase (NOS) activity suggest that the protection of salidroside, shown in this study, might be mediated by inhibiting caspase-3 activity, and antagonizing NO production and NOS activity during H₂O₂ stimulation. Perhaps, this study might contribute to the development of salidroside as a broad-spectrum agent for preventing and/or treating neuronal damage in neurodegenerative disorders.     

HPLC Profiles and Biomarker Contents of Australian‐Grown Salvia miltiorrhiza f. alba Roots

Salvia miltiorrhiza f. alba (Baihua Danshen) is a Chinese medicinal herb commonly used for treating cardiovascular disease. It has been grown in Australia, although the quality of its main medicinal part (dried root) has not been assessed. In this study, we investigated HPLC profiles and biomarker contents of Australian‐grown S. miltiorrhiza f. alba roots. Patterns of HPLC profiles were established in MeOH, and aqueous extracts in terms of number of common characteristic peaks and their relative retention times. The contents of three tanshinone biomarkers (cryptotanshinone ( 3 ), tanshinone I ( 1 ), and tanshinone IIA ( 2 )) were significantly higher (p<0.05) in the roots of one‐year‐old plants than those of two‐year‐old plants. In contrast, salvianolic acid B ( 4 ) content was significantly higher in the roots of two‐year‐old plants than in those of one‐year‐old plants. The findings suggest that the biomarker contents in Australian‐grown S. miltiorrhiza f. alba roots vary with the growth periods of the plants, which may be important in determining the optimal harvest time for the plant roots with targeted levels of tanshinones and salvianolic acid B ( 4 ).     

RAPD分析用白花丹参叶片DNA的提取方法

本研究采用改良CTAB法和SDS法提取新鲜白花丹参叶片的基因组DNA,初步筛选RAPD扩增引物。结果表明改良CTAB法提取的DNA较SDS法质量好,随机引物p2扩增条带相对较为清晰。再利用p2随机引物对2种方法提取的DNA进行RAPD检测比较,结果显示仅改良CTAB法能扩增出有效条带,说明改良CTAB法更适合用于白花丹参基因组DNA的RAPD检测分析。本文将为白花丹参叶片DNA的提取提供方法学参考。     

Protective effect of apelin on cultured rat bone marrow mesenchymal stem cells against apoptosis

Bone marrow-derived mesenchymal stem cells (BMSCs) have shown great promise for ischemic tissue repair. However, poor viability of transplanted BMSCs within ischemic tissues has limited their therapeutic potential. Apelin, an endogenous peptide, whose level is elevated following ischemia, has been shown to enhance survival of cardiomyocytes and neuronal cells during ischemia. We hypothesized that apelin-13 protects BMSCs from apoptotic death. In this paper we determined the potential mechanism of apelin-13 effects using cultured BMSCs from adult rats. Apoptosis was induced by the specific apoptotic insult serum deprivation (SD) for up to 36 h. Apoptotic cell death was measured using immunostaining and Western blotting in the presence and absence of apelin-13 (0.1 to 5.0 nM) co-applied during SD exposure. SD-induced apoptosis was significantly reduced by apelin-13 in a concentration-dependent manner. SD-induced mitochondrial depolarization, cytochrome c release, and caspase-3 activation were largely prevented by apelin-13. The apelin-13 anti-apoptotic effects were blocked by inhibiting the MAPK/ERK1/2 and PI3K/Akt signaling pathways. Taken together, our findings indicate that apelin-13 is a survival factor for BMSCs and its anti-apoptotic property may prove to be of therapeutic significance in terms of exploiting BMSC-based transplantation therapy.     

Protection by 3'-methoxypuerarin of rat hippocampal neurons against ischemia/reperfusion injury

3'-Methoxypuerarin (3'-MOP) is an isoflavone extracted from radix puerariae. The aim of this study was to investigate the role and the mechanism of 3'-MOP in the protection of hippocampal neurons against cerebral ischemia/reperfusion (I/R) injury in rats. I/R injury was induced by a modified four-vessel occlusion model. Rats were randomly divided into an I/R group, an I/R + 3'-MOP group and a control group. Histological changes in the neurons of the hippocampal CA1 region were observed with hematoxylin and eosine (H&E) staining. The apoptotic neurons in the hippocampal CA1 area were counted with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The results showed that compared with the I/R group, 3'-MOP increased the number of surviving neurons in the hippocampal CA1 region (P < 0.001) and markedly reduced the number of apoptotic pyramidal neurons (P < 0.001) after I/R injury. In conclusion, 3'-MOP can protect hippocampal neurons against I/R injury by inhibiting apoptosis.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons

The loss of ATP, which is needed for ionic homeostasis, is an early event in the neurotoxicity of glutamate and beta-amyloid (A(beta)). We hypothesize that cells supplemented with the precursor creatine make more phosphocreatine (PCr) and create larger energy reserves with consequent neuroprotection against stressors. In serum-free cultures, glutamate at 0.5-1 mM was toxic to embryonic hippocampal neurons. Creatine at >0.1 mM greatly reduced glutamate toxicity. Creatine (1 mM) could be added as late as 2 h after glutamate to achieve protection at 24 h. In association with neurotoxic protection by creatine during the first 4 h, PCr levels remained constant, and PCr/ATP ratios increased. Morphologically, creatine protected against glutamate-induced dendritic pruning. Toxicity in embryonic neurons exposed to A(beta) (25-35) for 48 h was partially prevented by creatine as well. During the first 6 h of treatment with A(beta) plus creatine, the molar ratio of PCr/ATP in neurons increased from 15 to 60. Neurons from adult rats were also partially protected from a 24-h exposure to A(beta) (25-35) by creatine, but protection was reduced in neurons from old animals. These results suggest that fortified energy reserves are able to protect neurons against important cytotoxic agents. The oral availability of creatine may benefit patients with neurodegenerative diseases.     

锌离子调节皮质酮对原代培养大鼠海马神经元的损伤

探讨皮质酮对原代培养大鼠海马神经元的损伤效应及锌的调节作用。用原位染色和RT-PCR方法,分别检测神经元的损伤情况及NMDA受体三种亚基(NRl、NR2A、NR2B)mRNA的表达。皮质酮(5μmol／L)作用2,4h可明显降低海马神经元的存活率,导致神经元凋亡,并随着作用时间的延长而加重;锌离子明显影响皮质酮对海马神经元的损伤效应：同时加入皮质酮和低、中浓度Zn^2 (10、100μmol／L),可明显降低神经元凋亡率,而加入高浓度Zn^2 (250μmol／L)则加重神经元损伤。皮质酮作用24h后,海马神经元NRl、NR2BmRNA的表达水平增高,而同时加入低、中浓度Zn^2 (10、100μmol／L)的海马神经元NRl、NR2BmRNA表达水平与对照组接近;NR2AmRNA表达无明显变化。这些结果表明,锌对皮质酮所致应激损伤的调节具有双向性;NMDA受体亚基水平的变化可能是其中重要环节之一。     

七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用:线粒体KATP通道的作用

利用离体海马脑片缺氧无糖(oxygen-glucose deprivation,OGD)损伤模型,探讨七氟醚预处理对神经细胞的保护作用及该作用与线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoKATPchannels)的关系,随机将脑片用2%、4%、6%七氟醚,以及6%七氟醚复合mitoKATP通道阻滞剂5-羟基奎酸盐(5-hydroxydecanoic acid,5-HD)预处理30 min,观察OGD损伤14 min复氧1 h期间顺向群峰电位(orthodromic population spike,OPS)的变化,并应用透射电镜观察细胞超微结构的改变.结果表明,与单纯OGD组相比,七氟醚预处理可使海马脑片OPS消失时间明显延长(P＜0.01),使OPS明显恢复,其中4%、6%七氟醚组的恢复率均为71.4%(P＜0.05 vs OGD),相应恢复程度为(61.0±42.3)%和(78.7±21.1)%(P＜0.01),而且6%七氟醚的保护作用可被5-HD取消.OGD组的海马CA1区锥体细胞明显水肿,核膜皱缩、破裂,染色质聚集,线粒体肿胀畸形,嵴断裂或消失,而4%和6%七氟醚组仅见海马CA1区锥体细胞轻度水肿,核膜皱缩不明显,染色质均匀,线粒体轻度肿胀.结果提示,七氟醚预处理对大鼠海马脑片OGD损伤有一定的保护作用,且七氟醚对神经细胞的保护作用与激活mitoKATP通道有关.     

Single-channel currents activated by low intracellular pH in cultured hippocampal neurons of rat.

Patch clamp technique was applied to the plasma membrane of cultured hippocampal neurons of rat. Elementary currents of a cation-selective channel were elicited by low intracellular pH (pHi 3.5-4.5). Channel activity starts with 1-2 min delay from the application of low pHi, and persists upon restoration of physiological pH conditions. The channel has a conductance of approx. 110 pS in symmetrical 300 mM NaCl, and is strongly selective for cations over anions. The channel is active over the whole voltage range tested (from +75 mV to -75 mV). Mean open time is function of voltage, increasing with depolarization. Low pH applied extracellularly did not activate the channel.     

Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: Involvement of PI3K/Akt and GSK-3 beta pathway

Erythropoietin (EPO) prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the neuroprotective effect of EPO in ketamine-induced neurotoxicity in primary cortical neurons. EPO in combination with ketamine greatly increased the cell viability and reduced the number of TUNEL-positive cells. To elucidate a possible mechanism by which EPO exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase pathway using LY294002. The neuroprotection of EPO was prevented by LY294002. Immunoblotting revealed that EPO induced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3β). Moreover, the caspase-3-like activity was increased by addition of ketamine, and decreased by administration of ketamine with EPO. Decreased caspase-3-like activity by administration of ketamine with EPO was restored by LY294002. Our results suggest that PI3K/Akt and GSK-3β pathway are involved in the neuroprotective effect of EPO. You Shang and Yan Wu have contributed equally to this work.