【作者】 王晨静；

【导师】 李元建；

【作者基本信息】 中南大学 ， 药理学， 2010， 博士

【摘要】 研究背景动脉粥样硬化(atherosclerosis, AS)是一种慢性、进行性、多发性血管内膜疾病,严重危害人类的健康。AS发病原因及机制十分复杂,目前认为与血脂、吸烟、高血压、糖尿病、肥胖、感染等危险因素密切相关。大量研究证明,同型半胱氨酸(homocysteine, Hcy)也是促进AS形成的重要因素。Hcy能促进动脉粥样斑块形成,增加冠心病患者的病死率,被认为是AS的独立危险因素。衰老是AS发生发展的重要危险因素之一。年龄相关的血管疾病的很多改变都与衰老血管细胞的特征性改变相一致。内皮功能紊乱引起血管收缩异常、血小板粘附聚集以及动脉中膜平滑肌增生,被认为是AS发病的始发事件。因此,保护血管内皮细胞,拮抗内皮细胞衰老是防治心血管病特别是动脉粥样硬化的重要研究方向。大量研究表明与衰老密切相关的Sirt1基因在维持血管内皮功能方面发挥重要作用。Sirt1是近几年发现的抗衰老长寿基因,参与氧化应激诱导的内皮细胞衰老。研究显示,Hcy致AS作用与其诱导内皮细胞衰老有关。卷柏属于蕨类卷柏科卷柏属植物,具有降血压、降血糖、增强人体免疫功能和抑制氧化应激等作用。Selaginellin是从卷柏中提取的一种具有全新结构的单体化合物,体外抗氧化实验结果显示,selaginellin具有较强的抗氧化作用。基于以上研究背景,本实验拟在培养的人脐静脉内皮细胞观察selaginellin对Hcy诱导的细胞衰老的影响并探讨可能涉及的机制。方法原代培养人脐静脉内皮细胞(HUVECs)。MTS法检测细胞活力；以β-半乳糖苷酶活性(染色法)、端粒酶活性(Real time PCR法)和细胞周期分布(流式)检测细胞衰老；用活性氧荧光试剂盒检测细胞内ROS的生成；实时定量PCR检测细胞内Sirt1 mRNA的表达。结果(1)Hcy(0.5 M)处理内皮细胞48 h,能显著降低细胞活性；selaginellin(10-7,3×10-7和10-6 M)预处理1 h能显著抑制Hcy诱导的细胞活性下降,且呈浓度依赖性；(2)Hcy(0.5 M)处理内皮细胞48 h,能显著增加β-半乳糖苷酶活性,降低细胞端粒酶活性,增加Gl期细胞比例,提示Hcy能诱导内皮细胞衰老；Selaginellin(10-7,3×10-7和10-6 M)预处理1 h能显著抑制Hcy诱导的β-半乳糖苷酶活性增加,端粒酶活性的降低及G1期细胞比例的增加,且呈浓度依赖性；(3)Hcy(0.5 M)处理内皮细胞48 h,能显著增加细胞内ROS水平；Selaginellin(10-7,3×10-7和10-6 M)预处理1 h能浓度依赖性地抑制Hcy诱导的细胞内ROS水平增加；(4)Hcy(0.5 M)处理内皮细胞48 h,能显著降低Sirt1 mRNA表达；而selaginellin本身能显著上调Sirt1 mRNA表达,且能逆转Hcy诱导的Sirt1 mRNA表达下调。结论Selaginellin能抑制Hcy诱导的内皮细胞衰老,其机制可能与抑制氧化应激和上调Sirt1表达有关。研究背景谷氨酸(Glutamate, Glu)是一种兴奋性氨基酸,对神经系统正常的功能活动起着重要的维持作用。然而,在某些病理情况下,如阿尔茨海默病、亨廷顿病及帕金森病等,谷氨酸大量堆积释放,可诱发神经细胞损伤和缺失。谷氨酸主要通过两种途径诱发神经死亡：兴奋性毒性和氧化应激。前者通过谷氨酸离子型受体的过度激活,导致胞内钙超载引发细胞死亡；后者为谷氨酸／胱氨酸转运体介导的细胞死亡途径。卷柏具有降血压、降血糖、增强人体免疫功能和抑制氧化应激等作用,其粗提取物能加速自由基的清除,激活过氧化物岐化酶。Selaginellin是我院从卷柏中提取的一种单体,具有抗氧化作用。本实验在分化的PC12细胞,研究selaginellin对Glu诱导的细胞损伤和凋亡的保护作用。依据Klotho是一个新的抗衰老基因,能延缓多种应激诱导的细胞凋亡过程,其机制与抑制氧化应激有关,因此,我们进一步探讨selaginellin的抗凋亡作用是否与上调Klotho基因有关。方法实验分设正常对照组、Glu损伤组、Glu+3个浓度的selaginellin(10-7、3×10-7和10-6 M)组和溶媒对照组。各药物处理组预先同分化的PC12细胞孵育1 h后,再用Glu(10 mM)处理24 h。以细胞形态学变化、细胞活性(MTS法)和LDH漏出(比色法)检测细胞损伤；以Hoechst染色和caspase-3活性检测细胞凋亡；用活性氧荧光试剂盒检测细胞内ROS的生成；实时定量PCR检测细胞内Klotho mRNA的表达。结果(1)PC12细胞株在含100 ng/mL NGF的培养液中孵育7 d后,细胞分化为分裂后期神经元样细胞株,生成大量树突；(2)MTS结果显示：5-20 mM Glu浓度依赖性地降低PC12细胞活性,其中,10 mM Glu可降低45%的细胞活性；(3)形态学结果显示：对照组PC12细胞边缘清晰,胞膜完整,树突完整；10 mM Glu孵育24 h后,胞体皱缩,树突断裂,细胞网状结构消失；Selaginellin预处理可浓度依赖性地减轻Glu对PC12细胞的损伤。与细胞形态学变化相一致,Glu可明显增加LDH漏出和降低细胞活性,selaginellin可浓度依赖性地逆转Glu的上述效应；(4) Hoechst 33342染色结果显示：Glu处理24 h显著增加细胞皱缩,胞核碎裂和染色质聚集,Hoechst染色阳性细胞数明显增加。与之一致的是caspase-3活性显著增加。Glu的促凋亡效应可浓度依赖性地被selaginellin所抑制；(5) Glu (10 mM)处理细胞24 h可明显增强活性氧水平；预先用selaginellin处理1 h后,能呈浓度依赖性地抑制Glu诱导的活性氧生成增加；(6)Glu可显著下调PC12细胞Klotho mRNA的表达,selaginellin预处理能显著抑制Glu所致Klotho mRNA的下调。结论Selaginellin通过降低氧化应激反应而抑制谷氨酸诱导的PC12细胞损伤和凋亡,其抗凋亡作用可能与上调Klotho基因的表达有关。研究背景阿尔茨海默病(Alzheimer’s disease, AD)是由多种病因引起的神经系统进行性退行性疾病,主要病理学特征为细胞外淀粉样物质沉积、神经纤维缠结和神经元缺失。目前AD的发病机制仍未十分明确。近年来的研究提示,AD发病可能与细胞脂质代谢异常相关。脑组织的低密度脂蛋白氧化修饰生成氧化性低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL),引起氧化应激诱导神经元凋亡。许多证据表明,神经细胞凋亡参与脑老化及AD的发生。体外实验证实,ox-LDL可诱导多种细胞的凋亡。Ox-LDL主要通过与其受体结合而发挥其生物学作用。植物凝集素样氧化性低密度脂蛋白受体-1(lectin-like oxidized low density lipoprotein receptor-1, LOX-1)是1997年由日本学者首次在牛主动脉内皮细胞发现的新型ox-LDL受体。Ox-LDL激活LOX-1诱导细胞内ROS生成,而ROS在多种生物过程中扮演重要角色。我们推测LOX-1可能参与了ox-LDL诱导的神经细胞凋亡。NADPH氧化酶是超氧阴离子生成的重要催化酶。研究表明AD发病过程中,NADPH氧化酶参与活性氧引起的神经元损伤。卷柏具有降血压、降血糖、增强人体免疫功能和抑制氧化应激等作用,其粗提取物能加速自由基的清除,激活过氧化物岐化酶。Selaginellin是从卷柏中提取的一种单体,研究发现selaginellin有明显抗氧化的作用。本研究探讨脂质代谢异常时selaginellin对神经元NADPH氧化酶及其介导的ROS生成的影响,进一步探明其神经保护作用的机制。方法实验分设正常对照组、ox-LDL损伤组、3个浓度的selaginellin(10-7、3×10-7和10-6 M)+ox-LDL组、anti-LOX-1+ox-LDL组、selaginellin (10-6 M)组和溶媒对照组。各药物处理组预先同分化的PC12细胞孵育l h后,再用ox-LDL (100μg/mL)处理24 h。MTS法检测细胞活性；以Hoechst染色、AnnexinV-FITC/PI双染和caspase-3活性检测细胞凋亡；用活性氧荧光试剂盒检测细胞内ROS的生成；RT-PCR检测LOX-1 mRNA的表达；比色法检测NADPH氧化酶活性；实时定量PCR检测细胞内NOX-1、gp91phox及p47phox mRNA的表达。结果(1)MTS结果显示：20-100μg/mL ox-LDL浓度依赖性地降低PC12细胞活性,其中,100μg/mL ox-LDL可降低40%的细胞活性；(2)Ox-LDL处理PC12细胞24 h,能显著降低细胞活性；Selaginellin(10-3,3×10-7和10-6M)预处理1 h能显著抑制ox-LDL诱导的细胞活性,且呈浓度依赖性；(3) Hoechst 33342染色结果显示：ox-LDL处理24 h显著增加细胞皱缩,胞核碎裂和染色质聚集,Hoechst染色阳性细胞数明显增加；流式细胞仪分析结果显示,与正常对照组比较,ox-LDL组PC12细胞的凋亡比例明显升高；Selaginellin预处理组的细胞凋亡比例分别较ox-LDL组明显降低,且呈浓度依赖性；与之一致的是caspase-3活性显著增加；Ox-LDL的促凋亡效应可浓度依赖性地被selaginellin所抑制；(4) Ox-LDL (100μg/mL)处理细胞24 h可明显增强活性氧水平；预先用selaginellin处理1 h后,能呈剂量依赖性地抑制ox-LDL诱导的活性氧生成增加；Anti-LOX-1具有相似的作用；(5)Ox-LDL可显著上调PC12细胞LOX-1 mRNA的表达；Selaginellin预处理能显著抑制ox-LDL所致LOX-1 mRNA的上调；(6) NADPH氧化酶活性检测结果显示,100μg/mL的ox-LDL作用于分化的PC12细胞24 h可增加NADPH氧化酶活性；不同浓度selaginellin可剂量依赖性拮抗ox-LDL诱导的NADPH氧化酶活性升高；Real time PCR的结果显示,100μg/mL ox-LDL可显著上调NOX-1、gp91phox和p47phox mRNA的表达；Selaginellin预处理能显著抑制ox-LDL所致NOX-1、gp91phox和p47phox mRNA表达的下调,且呈浓度依赖性；Anti-LOX-1组具有相似作用。结论Selaginellin抑制氧化性低密度脂蛋白诱导的PC12细胞凋亡,其抗凋亡作用可能与抑制LOX-1/NADPH氧化酶/R0S通路有关。研究背景阿尔茨海默病(Alzheimer’s disease, AD)是中枢神经系统一种常见的退行性病变。AD病因、发病机制尚不完全清楚,目前认为与遗传因素、细胞凋亡、自由基损伤、铝中毒等因素有关。炎症反应在AD发生中的作用近来受到普遍关注。在AD发生发展过程中,伴随着炎症病理改变。AD患者脑组织中可见大量局限性和弥散性星形胶质细胞增生。在某些刺激下,星形胶质细胞可释放大量的炎症介质,参与中枢神经系统实质炎症反应的触发,加速AD进程。越来越多的证据显示,免疫反应在AD的发生和发展过程中起决定作用。Toll样受体(toll-like receptor, TLR)是介导天然免疫的重要受体,主要参与病原微生物产物的识别以及炎症信号传导,介导固有免疫和获得性免疫。研究显示,TLR4与阿尔茨海默病的发生与发展密切相关。TLR4能激活核因子-κB (nuclear factor-κB, NF-κB)信号通路并介导多种致炎作用。Selaginellin是从卷柏中提取的一种具有全新结构的单体化合物,具有较强的抗氧化作用。研究显示,selaginellin可抑制谷氨酸及氧化性低密度脂蛋白诱导的PC12细胞凋亡。因此,我们推测,selaginellin具有较强神经保护作用。本实验旨在观察selaginellin对星形胶质细胞炎症反应的影响并探讨其作用是否与抑制TLR4-NF-κB信号通路有关。方法原代培养星形胶质细胞,实验分设正常对照组、LPS损伤组、3个浓度的selaginellin(10-7、3×10-7和10-6 M)+LPS组、TLR4拮抗剂+LPS组、selaginellin(10-6 M)组和溶媒对照组。各药物处理组预先孵育星形胶质细胞1 h后,再用LPS (1μg/ml)处理24 h。MTS法检测细胞活性；实时定量PCR检测细胞TLR4、TNF-α、MCP-1、IL-6及NF-κB p65 mRNA的表达；ELISA法检测细胞培养液中TNF-α、MCP-1及IL-6的含量；电泳迁移率变更分析细胞NF-κB活性。结果(1)MTS结果显示,星形胶质细胞暴露于1μg/mL的LPS 24 h,细胞活力下降40%；不同浓度selaginellin可剂量依赖性拮抗LPS的细胞毒性作用；(2) Real time RCR结果显示,1μg/mL的LPS处理星形胶质细胞24 h,可显著上调TNF-α、MCP-1及IL-6 mRNA的表达；Selaginellin预处理能浓度依赖性地抑制LPS所致TNF-α、MCP-1及IL-6 mRNA表达的上调；TLR4拮抗剂具有相似的作用；(3) ELISA结果显示,1μg/mL的LPS孵育星形胶质细胞24 h,培养上清液中TNF-α、MCP-1及IL-6水平显著升高；预先用selaginellin孵育细胞1h后,再暴露于LPS孵育24 h,可浓度依赖性地下调培养上清液中TNF-α、MCP-1及IL-6的水平；(4)LPS处理星形胶质细胞24 h,能显著上调TLR4 mRNA表达；而selaginellin能逆转LPS诱导的TLR4 mRNA表达上调；(5) Real time PCR结果显示,1μg/mL的LPS处理星形胶质细胞24h,可显著上调NF-κB p65 mRNA的表达；Selaginellin预处理能浓度依赖性地抑制LPS所致NF-κB p65 mRNA表达上调；EMSA结果显示有明显的信号条带,LPS能显著增加NF-κB的活性；Selaginellin预孵育星形胶质细胞1 h,可浓度依赖性地降低NF-κB的活性,与TLR4拮抗剂具有相似的作用。结论Selaginellin通过减少炎症因子TNF-α、MCP-1及IL-6合成与释放而抑制LPS诱导的星形胶质细胞炎症反应,其抗炎作用可能与抑制TLR4-NF-κB途径有关。更多还原

【Abstract】 Atherosclerosis (AS) is recognized as one of the major causes to threaten the life of people. It is considered that pathogenesis of AS is involved in many factors, such as blood fat, smoking, hypertension, diabetes, infection and induced pathological changes of vascular biology. Homocysteine (Hcy) can promote atherogenesis, and plasma level of Hcy are positively correlated with morbidity and mortality from coronary heart diseases, suggesting that Hcy is an independent risk factor for AS.Endothelial dysfunction is recognized as all early event in the pathogenesis of AS, which is related to senescence and damages of endothelial cells. Also, the decreased expression of anti-senescent molecules contributes to progression of cell senescence. The down-regulation of Sirtl, an enzyme to remove acetyl groups from specific proteins, has been shown to mediate oxidative stress-induced endothelial senescence. It has been reported that pro-atherogenetic properties of Hcy are related to inducing cell senescence.Selaginella Tamariscina (Beauv) spring can lower blood pressure, decrease the level of serum glucose, enhance immune function and inhibit oxidant production and so on. Selaginellin, a compound with novel chemical structure, was extracted from Selaginella Tamariscina. Our preliminary data showed that selaginellin had strong anti-oxidative activity. Based on these data mentioned above, in the present study, we investigated the effects of selaginellin on Hcy-induced senescence of endothelial cells and its underlying mechanisms.METHODSHuman umbilical vein endothelial cells (HUVECs) were cultured and used for all these studies. Cell viability was analyzed by using MTS assay. Cell senescence was evaluated by usingβ-galactosidase staining, telomerase activity detected by real time PCR and cell cycle distribution determined by staining DNA with propidium iodide. The level of intracellular reactive oxygen species (ROS) was determined by using fluorescent ROS detection kit and the level of Sirtl mRNA was detected by real time PCR.RESULTS(1) Treatment with Hcy (0.5 M) for 48 h markedly decreased the viability of endothelial cells. Pretreatment with selaginellin for 1 h prior to exposure to homocysteine concentration-dependently reversed the decrease in cell viability induced by Hcy.(2) Treatment with Hcy (0.5 M) for 48 h markedly induced the senescence of endothelial cells shown by the decrease in telomerase activity, the increase inβ-galactosidase activity and the increase in the percentage of cells in G0/G1 phage, which could be attenuated by pretreatment with selaginellin in a concentration-dependent manner.(3) Treatment with Hcy (0.5 M) significantly increased the level of intracellular ROS in endothelial cells. However, selaginellin could significantly inhibit Hcy-induced elevation of intracellualr ROS level.(4) The mRNA expression of Sirt1, an important anti-senescent protein, was markedly decreased in Hcy-incubated endothelial cells. However, selaginellin could significantly upregulate the mRNA expression of Sirtl in the absence or presence of Hcy.CONCLUSIONSelaginellin attenuates Hcy-induced senescence of endothelial cells, and such effects may be related to inhibiting oxidative stress and up-regulating Sirtlexpression. L-glutamate, an excitatory amino acid, has been identified as the principal transmitter mediating excitatory synaptic responses and neuronal development via glutamate receptor activation in the central nervous system. However, excessive excitatory transmission can be transformed into an implement of neuronal destruction resulting in CNS disorder, such as cerebral ischemia, hypoxia, alcoholism, autoimmune encephalomyelitis, and Alzheimer’s, Huntington’s and Parkinson’s diseases. Glutamate can induce cell death by two different pathways: excitotoxicity and oxytosis. The excitotoxicity of glutamate is mediated through the activation of several types of excitatory amino acid receptors, and subsequently massive influx of extracellular Ca2+. The oxytosis of glutamate is mediated by competitive inhibition of cystine uptake. Selaginellin is a component extracted from S.pulvinata (Hooket Grev.) Maximo. Selaginellin contains hydroxy group, we therefore presumed that it would have the ability of binding free radicals. In the present study, we therefore studied the protective effect of selaginellin on glutamate-induced cytotoxicity and apoptosis in differentiated rat pheochromocytoma (PC 12) cells. Because Klotho, an anti-aging protein with anti-oxidative property, has been shown to have anti-apoptotic function in a variety of cell types, we also investigated whether the anti-apoptotic effect of selaginellin on PC 12 cells is related to regulation of Klotho gene expression.METHODSAfter cultured with the serum-free medium for 24 h, the differentiated PC 12 cells were pretreated with various concentrations (10-7,3×10-7 or 10-6 M) of selaginellin for 1 h prior to exposure to L-glutamate. Cell viability was analyzed by using MTS assay. Cell injury was evaluated using observation of morphologic changes, cell viability and LDH release. Cell apoptosis was determined by Hoechst staining and caspase-3 activity. The level of intracellular reactive oxygen species (ROS) was determined using fluorescent ROS detection kit and the level of Klotho mRNA was detected by real time PCR. (1) Treatment of PC 12 cells with 100 ng/mL nerve growth factor for 1 week successfully induced differentiation as shown by round cell bodies with fine dendritic networks.(2) MTS assay showed that the cell viability was inhibited by glutamate in a concentration-dependent manner. Glutamate at 10 mM led to about 45% inhibition of cell viability. We therefore chose 10 mM of glutamate for the subsequent experiments.(3) Under a phase-contrast microscopy, normal differentiated PC 12 cells showed round cell bodies with fine dendritic networks, and the cell edges were intact and clear. In contrast, incubation of cells with 10 mM of L-glutamate for 24 h induced shrinkage of the cell bodies, disappearance in cell reticular formation and disruption of the dendritic networks. Pretreatment with selaginellin dramatically alleviated morphological manifestations of cell damage in a concentration-dependent manner. Glutamate significantly increased the release of LDH and decreased the cell viability. These effects of glutamate were reversed by selaginellin in a concentration-dependent manner.(4) Hoechst 33342 staining assay showed that treatment with glutamate (10 mM) for 24 h significantly increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. The caspase-3 activity, a marker of apoptosis, was also found to be increased in glutamate-treated cells. The number of Hoechst 33342-positive cells and the activity of caspase-3 induced by glutamate were significantly reduced by selaginellin in a concentration-dependent manner.(5) Glutamate increased intracellular ROS generation as indicated by the increase in 2’,7’-dichlorofluorescein fluorescence. Selaginellin significantly reduced the increase in ROS generation induced by glutamate in a concentration-dependent manner.(6) Incubation of PC 12 cells with glutamate (10 mM) for 24 h led to a significant decrease in Klotho mRNA expression. In contrast, the decreased mRNA expression of Klotho was reversed by pretreatment with selaginellin in a concentration-dependent manner.CONCLUSIONSelaginellin protected against L-glutamate induced cell injury and apoptosis in differentiated PC 12 cells through regulation of ROS/Klotho gene pathway. BACKGROUNDAlzheimer’s disease (AD) is a progressive neurodegenerative disorder. The major neuropathological hallmarks of AD are the formation of senile plaques following neurofibrillary tangles and neurons loss. Although the mechanisms of AD pathology are still undefined, lipoprotein dysbolism has been recently reported to be involved in the progress of AD.Low-density lipoprotein (LDL) exists within the brain and is highly vulnerable to oxidative modifications. Once formed, oxidized LDL (ox-LDL) is capable of eliciting neurons apoptosis involving in reactive oxygen species (ROS). It has been demonstrated that neurons apoptosis takes part in the progress of AD. In vitro experiments have shown that ox-LDL can induce many kinds of cell apoptosis via combining with its receptors.Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), a novel ox-LDL receptor, was firstly found to be expressed in bovine aortic endothelial cells. Ox-LDL activates LOX-1 followed by the production of intercellular reactive oxygen species (ROS), wherein ROS play an important role in a variety of biological processes. We presumed that LOX-1 might be involved in ox-LDL-induced neurons apoptosis. NADPH oxidases is the major source of ROS in diffirent cell types. Studies have shown that NADPH oxidases are involved in ROS-induced neuronal damage in the AD pathogenesis.Selaginellin, a component extracted from S.pulvinata (Hooket Grev.) Maximo. has the ability of binding free radicals. In the present study, we therefore studied the protective effect of selaginellin on ox-LDL-induced apoptosis in differentiated rat pheochromocytoma (PC 12) cells. We also investigated whether the anti-apoptotic effect of selaginellin on PC 12 cells is related to regulation of LOX-1/NADPH oxidase/ROS pathway.METHODSAfter cultured with the serum-free medium for 24 h, the differentiated PC 12 cells were pretreated with various concentrations (10-7,3×10-7 or 10-6 M) of selaginellin for 1 h prior to exposure to ox-LDL. Cell viability was analyzed by using MTS assay. Cell apoptosis was determined by Hoechst staining, flow cytometry with annexin V-FITC/PI double staining and caspase-3 activity. The level of intracellular reactive oxygen species (ROS) was determined using fluorescent ROS detection kit. The level of LOX-1 mRNA was detected by RT-PCR. The activity of NADPH oxidase was measured using cell NADPH oxidase detection kit and the expression of NOX-1, gp91phox and p47phox mRNA was detected by real time PCR. RESULTS(1) MTS assay showed that the cell viability was inhibited by ox-LDL in a concentration-dependent manner. Ox-LDL at 100μg/mL led to about 40% inhibition of cell viability. We therefore chose 100μg/mL of ox-LDL for the subsequent experiments.(2) Ox-LDL significantly decreased the cell viability. These effects of ox-LDL were reversed by selaginellin in a concentration-dependent manner.(3) Hoechst 33342 and annexin V-FITC/PI staining assay showed that treatment with ox-LDL (100μg/mL) for 24 h significantly increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. The caspase-3 activity was also found to be increased in ox-LDL-treated cells. The number of Hoechst 33342-positive cells and annexin V-FITC/PI staining cells as well as the activity of caspase-3 induced by ox-LDL were significantly reduced by selaginellin in a concentration-dependent manner.(4) Ox-LDL increased intracellular ROS generation. Selaginellin significantly reduced the increase in ROS generation induced by ox-LDL in a concentration-dependent manner.(5) Incubation of PC 12 cells with ox-LDL (100μg/mL) for 24 h led to a significant increase in LOX-1 mRNA expression. In contrast, the decreased mRNA expression of LOX-1 was reversed by pretreatment with selaginellin in a concentration-dependent manner.(6) Ox-LDL activated NADPH oxidase, which was reversed by selaginellin. Also, as shown by the results of real time PCR, incubation of PC 12 with ox-LDL for 24 h led to a significant increase in NOX-1, gp91phox and p47phox mRNA expression. In contrast, the increased mRNA expression of NOX-1, gp91phox and p47phox was reversed by pretreatment with selaginellin. Co-incubation with anti-LOX-1 could completely block ox-LDL-induced the activation of NADPH oxidase.CONCLUSIONSelaginellin protected against ox-LDL apoptosis in differentiated PC 12 cells through regulation of LOX-1/NADPH oxidase/ROS pathway. BACKGROUNDAlzheimer’s disease (AD) is a progressive neurodegenerative disorder. It is considered that pathogenesis of AD is involved in many factors, such as genetic factor, cell apoptosis, oxidation and aluminium poisoning. Inflammation has been recently reported to be involved in the progress of AD. Astrocytes express an array of inflammatory mediators that are involved in the initiation of inflammatory reactions within CNS parenchyma. It is documented that toll-like receptor 4 (TLR4) plays a key role in the program of AD pathology. TLR4 can activate nuclear factor-κB (NF-κB) signal pathway, which mediate the pro-inflammatory effects of many kinds of inflamatory cytokines. Selaginellin, a compound with novel chemical structure, was extracted from Selaginella Tamariscina. Our preliminary data showed that selaginellin had strong anti-oxidative activity, which inhibited L-glutamate-and ox-LDL-induced apoptosis of differentiated PC 12 cell. Therefore, we presumed that selaginellin had neuronal protective effects. The aim of the present study was to investigate the effect of selaginellin on inflammation of astrocytes and whether anti-inflammatory effect of selaginellin was related to TLR4-NF-κB pathway. METHODSCell viability was analyzed by using MTS assay. The expression of TLR4, TNF-α, MCP-1, IL-6 and NF-κB p65 mRNA was detected by real time PCR. The levels of TNF-α, MCP-1 and IL-6 in culture medium were assayed by enzyme linked immunosorbent assay (ELISA). NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA).RESULTS(1) Treatment with LPS (1μg/mL) for 24 h markedly decreased the activity of astrocytes. Pretreatment with selaginellin for 1 h prior to exposure to LPS concentration-dependently reversed the decrease in cell viability induced by LPS.(2) In cultured astrocytes, incubation with LPS significantly up-regulated the mRNA expression of inflammatory factors tumor necrosis factor-α(TNF-α), MCP-1 and IL-6, which could be attenuated by pretreatment with selaginellin in a concentration-dependent manner.(3) The levels of TNF-α, MCP-1 and IL-6 in cultured medium were increased after incubated with LPS for 24 h. In contrast, selaginellin decreased the levels of TNF-α, MCP-1 and IL-6 in a concentration-dependent manner. (4) The mRNA expression of TLR4 was markedly increased in LPS-incubated astrocytes. In contrast, selaginellin could significantly downregulate the mRNA expression of TLR4 in the presence of LPS.(5) Incubation of astrocytes with LPS for 24 h led to a significant increase in NF-κB p65 mRNA expression. In contrast, the increased mRNA expression of NF-κB p65 was reversed by pretreatment with selaginellin in a concentration-dependent manner. Also, as shown by the results of EMSA, LPS activated NF-κB, which was reversed by selaginellin. Co-incubation with TLR4 antagonist could completely block LPS-induced the activation of NF-κB.CONCLUSIONSelaginellin can decrease inflammatory cytokines TNF-a, MCP-1 and IL-6 synthesis and release in astrocytes induced by LPS via inhibiting TLR4-NF-κB signaling pathway.更多还原