【作者】 陈霞；

【导师】 丁斐；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 兴奋性氨基酸尤其是谷氨酸（Glutamate, Glu）引发的神经兴奋毒性在缺血性脑损伤中占有相当重要的地位,因而减轻Glu兴奋性毒性,保护神经元成为缺血性脑损伤的重要治疗策略之一。本研究探讨了红景天苷对兴奋性毒性损伤神经元的保护作用及可能的作用机制。第一部分红景天苷对Glu损伤的保护作用及机制的研究目的:观察红景天苷对原代培养海马神经元Glu兴奋性毒性损伤的保护作用及作用机制。方法:1.通过镜下形态分析和MTT比色法,观察红景天苷对原代培养海马神经元形态和活力的影响。2.通过镜下形态观察和MTT比色法检测细胞活力,摸索诱导Glu兴奋性毒性的适当浓度及时间,建立原代培养海马神经元谷氨酸兴奋性毒性损伤模型。3.通过形态学分析、细胞活力的MTT检测、LDH释放量的检测,观察红景天苷预处理对谷氨酸损伤后海马神经元形态和细胞活力的影响。4.通过Hoechst染色、TUNEL标记和Annexin V+PI的流式分析,观察红景天苷预处理对Glu损伤的海马神经元凋亡的影响。5.通过western blotting检测,观察红景天苷预处理对Glu损伤海马神经元凋亡相关蛋白Bcl-2和Bax蛋白表达的影响。6.通过使用Caspase-3抑制剂Z-DEVD-FMK预处理,MTT检测Glu损伤后海马神经元活力,观察凋亡执行分子Caspase-3是否参与Glu诱导的兴奋性毒性。7.通过比色法检测Caspase3活性,评价Glu损伤后不同时间细胞凋亡的程度及红景天苷预处理对Glu诱导的Caspase-3活性的影响。8.钙离子荧光探针Fluo-4/AM标记海马神经元,激光共聚焦显微镜下检测红景天苷对Glu诱导的[Ca²⁺]_i变化的影响。结果:1.不同浓度的红景天苷与海马神经元共培养24h,海马神经元生长状态良好,随着红景天苷浓度的增加,神经元活力呈增加趋势,但与空白对照相比无明显统计学差异。2.细胞活力检测、Hoechst染色、TUNEL染色和caspase-3活性测定结果一致表明,Glu诱导了神经元凋亡;红景天苷预处理可抑制Glu诱导的神经元凋亡,呈现良好的量效关系。3.125μmol/LGlu损伤15min不影响Bcl-2和Bax蛋白的表达,红景天苷预处理24h也不影响这两个蛋白的表达。4.红景天苷预处理24h能显著降低Glu诱导的胞浆Ca²⁺超载。结论:红景天苷预处理24h可明显改善Glu损伤后细胞活力,减少LDH的释放,抑制Glu诱导的细胞凋亡,对Glu损伤的海马神经元有明显的保护作用,其机制与抑制Glu损伤后的caspase-3活性增加和Ca²⁺超载有关。第二部分红景天苷对NMDA损伤的保护作用及机制的研究目的:观察红景天苷对原代培养海马神经元NMDA兴奋性毒性损伤的保护作用及作用机制。方法:1.通过MTT检测,观察不同浓度NMDA对海马神经元细胞活力的影响,建立原代培养海马神经元NMDA兴奋性毒性损伤模型。2.通过形态学分析、细胞活力的MTT检测、LDH释放的检测,观察红景天苷预处理对NMDA损伤后海马神经元形态和细胞活力的影响。3.通过NF染色、TUNEL标记和Caspase-3活性的检测,观察红景天苷预处理对NMDA诱导的海马神经元凋亡的影响。4.RT-PCR检测红景天苷对海马神经元NR2A、NR2B、NR2C和NR2D mRNA表达的影响。5.Western blotting分析红景天苷对NMDA损伤海马神经元PSD-95蛋白表达的影响。6.Western blotting分析红景天苷对NMDA损伤海马神经元p38蛋白表达的影响。7.全细胞膜片钳技术检测红景天苷对NMDA诱发电流（INMDA）的影响。8.通过比色法检测培养上清中NO含量和NOS活性,探讨红景天苷对NMDA损伤海马神经元保护作用机制。9.通过MTT比色法检测细胞活力,观察红景天苷对SNP（NO供体）损伤海马神经元的保护作用。结果:1.细胞活力检测、NF染色、TUNEL染色和caspase-3活性测定结果一致表明,不同浓度NMDA均显著降低海马神经元的活力,诱导了神经元凋亡,红景天苷可抑制NMDA诱导的神经元凋亡,呈现良好的量效关系。2.红景天苷不影响NR2各亚单位的mRNA表达和PSD95蛋白的表达。3.红景天苷预处理可显著抑制NMDA诱导的NO产生和NOS活性增加。4.红景天苷预处理可显著抑制SNP（NO供体）诱导的神经元活力的下降。5.NMDA诱导的细胞凋亡性损伤与p38MAPK磷酸化相关,红景天苷可显著抑制p38MAPK蛋白的磷酸化。结论:红景天苷预处理24h可显著增加NMDA损伤的细胞活力,减少NMDA损伤导致的LDH的释放,且呈明显的剂量相关性,对NMDA损伤的海马神经元有明显的保护作用。其保护作用机制与抑制NMDA诱导的caspase-3活性的升高,NOS活性增加、NO生成和损伤以及p38MAPK蛋白的磷酸化有关。综上研究表明,红景天苷对Glu兴奋性毒性损伤的神经元有明显的保护作用,其保护作用可能与其抑制NMDA受体过度激活介导的兴奋性毒性有关(表现为抑制NMDA受体-Ca²⁺-NO路径的激活和p38MAPK蛋白的磷酸化),为其用于脑缺血等损伤的研究和应用打下了实验基础。更多还原

【Abstract】 During ischemic brain damage, excitatory amino acids, glutamate （Glu）-induced excitotoxicity in neurons play a rather important role. Thereby protecting neurons from the Glu-induced excitotoxicity in ischemic brain damage has become one of the important treatment strategies. This study aimed to evaluate the neuroprotective effects of salidroside on excitotoxicity and investigate the possible mechanism involved in the neuroprotection.Part I Protective effects and mechanism of salidroside on primary hippocampal neurons damage induced by GluObjective: To investigate the protective effects and underlying mechanism of salidroside on primary rat hippocampal neurons damage induced by Glu.Methods:1. Phase contrast microscope and MTT assay were used to observe the effects of salidroside on morphology and viability of primary cultured hippocampal neurons.2. To build an excitatory neurotoxic model of hippocampal neurons, MTT colorimetric assay and LDH （in medium） release assay were applied to detect cell viability after incubation with different concentrations of Glu for different periods.3. Phase contrast microscope observation and MTT assay were used to identify the effect of salidroside against Glu-induced cell damage.4. Hoechst staining, TUNEL assay and flow cytometry analysis, were applied to detect the effect of salidroside against Glu-induced apoptosis of hippocampal neurons.5. Western blotting analysis were used to observe the effect of salidroside on the total amount of anti-apoptotic protein Bcl-2 or pro-apoptotic protein Bax in cultured hippocampal neurons after exposure to Glu.6. To investigate whether caspase-3-like enzymes involved in Glu-induced excitotoxicity, we detect cell viability of Glu-injuried hippocampal neurons after preincubation with Z-DEVD-FMK, a cell-permeable irreversible inhibitor of caspase-3-like enzymes.7. Caspase-3 activity assay were performed to examine the effect of salidroside on Caspase-3 activity after exposure to Glu.8. Cultured hippocampal neurons labeled with fluo-4/AM were collected and quantified by confocal laserscanning microscope to determine whether NRF could protect the neurons against Glu-induced excitotoxicity by attenuating Glu-induced rise in [Ca²⁺]_i.Results:1. Co-incubition with different concentrations of salidroside resulted in neither cell viability loss nor morphological alteration of hippocampal neurons.2. The results of MTT, Hoechst staining, TUNEL assay, flow cytometry analysis and detection of caspase-3 activity indicated that Glu could evoke neurotoxicity in cultured hippocampal neurons, with the concentrations of Glu and the incubation time increased, the neurotoxicity augmented. Salidroside could significantly prevent cultured hippocampal neurons from Glu-induced neuronal apoptosis, showing an excellent dose-effect relationship.3. Neither treatment with 125μmol/L Glu for 15min nor pretreatment with salidroside affected the expressions of Bcl-2 and Bax protein.4. Salidroside could significantly inhibit the up-regulation of [Ca²⁺]_i induced by Glu. Conclusion: salidroside efficiently protects hippocampal neurons against Glu-induced apoptosis. The protective effects are mediated by inhibiting the increased caspase-3-like activity and excessive Ca²⁺ influx triggered by Glu. Further studies need to be done to explore the underlying mechanisms.Part II Protective effects and mechanism of salidroside on primary hippocampal neurons damage induced by NMDAObjective: To investigate the protective effects and underlying mechanism of salidroside on primary rat hippocampal neurons damage induced by NMDA.Methods:1. To build an excitatory neurotoxic model of hippocampal neurons, MTT colorimetric assay and LDH （in medium） release assay were applied to detect cell viability after incubation with different concentrations of NMDA. 2. Phase contrast microscope observation and MTT assay were used to identify the effect of salidroside against NMDA-induced injury.3. NF staining and TUNEL assay were applied to detect the effect of salidroside against NMDA-induced apoptosis of hippocampal neurons.4. RT-PCR were used to observe the effect of salidroside on mRNA expressions of NR2A、NR2B、NR2C and NR2D.5. Western blotting analysis was used to observe the effect of salidroside on protein expression of PSD-95 in cultured hippocampal neurons after exposure to NMDA.6. Western blotting analysis were used to observe the effect of salidroside on protein expressions of phosphorylated p38 MAPK and p38 MAPK.7. Whole-cell patch-clamp technique was applied to detect the effect of salidroside on NMDA-induced current （INMDA）.8. NO production and NOS activity were measured by the NO2/3 assay kit and the NOS assay kit according to the instructions provided with.9. MTT colorimetric assay cell viability was used to observe the effect of salidroside on SNP （NO donor）-induced cell viability in hippocampal neurons.Results:1. MTT and LDH assays, together with NF staining and TUNEL assay, flow cytometric analysis and dictation of caspase-3 activity indicated that NMDA induced the dose-dependent excitotoxicity in cultured hippocampal neurons. Salidroside pretreatment attenuated NMDA-induced apoptosis in primary cultured hippocampal neurons, showing a dose-dependent pattern.2. Salidroside pretreatment does not affect the mRNA expression of NR2 subunit and protein expression of PSD95.3. Salidroside pretreatment significantly inhibited the NMDA-induced NO production and NOS activity.4. Salidroside pretreatment significantly inhibited the SNP-induced increase of cell viability.5. NMDA-induced apoptosis is associated with p38MAPK phosphorylation. Salidroside significantly inhibited p38MAPK protein phosphorylation.Conclusion: Salidroside efficiently protected hippocampal neurons against NMDA-induced apoptotic cell death. Its protective mechanism related to the inhibition of NMDA-induced caspase-3 activity and NOS activity increased NO production and NO-induced damage, and expression of phosphorylation of p38MAPK protein.Togather, salidroside could efficiently protect hippocampal neurons against Glu-induced excitotoxicity. The protective effects may be related to the inhibition of NMDA receptor mediated excitotoxicity which is related to the activiation of the NMDA receptor-Ca²⁺-NO pathway and phosphorylation of p38MAPK protein. Its neuroprotection may be used to develop a potential therapeutic approach for preventing and/or treating ischemic brain damage.更多还原