柯里拉京对氧化应激损伤SH-SY5Y和N9细胞的保护作用及其机制研究
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摘要
近年来,氧化应激被认为是引起衰老和神经退行性疾病的主要原因,其中活性氧在神经退行性疾病的发病过程中扮演着举足轻重的角色:脑组织的氧化代谢率很高,神经元膜上含有大量的多不饱和脂肪酸等氧化底物;过氧化氢酶活性低,铁离子结合力低,比其他部位更容易受到氧化作用的影响,最易受到活性氧侵袭损害。
研究表明,炎症也可以引发神经细胞死亡,从而形成恶性循环,加速病情的恶化。参与炎症介导的神经退行性病变的几种细胞中,巨噬细胞样细胞-小胶质细胞是最重要的一种。氧化应激是导致神经退行性疾病的主要原因,而小胶质细胞正是产生氧化应激的最主要来源;越来越多的证据表明氧化应激诱导小胶质细胞的损伤优先激发神经元的损伤。小胶质细胞过度激活所释放的大量NO、TNF-α等促炎症细胞因子和ROS等神经毒性因子,会对神经元产生毒性作用,导致神经元的变性和死亡,进而导致AD、PD等神经退行性疾病的发生。
因此,通过增加细胞抗氧化能力、阻断或抑制氧化应激引起的神经元和神经胶质细胞损伤以及抑制小胶质细胞异常激活都可能成为神经退行性疾病的防治策略。致力于寻找新的抗氧化、抗炎药物以减轻氧化损伤和炎症反应,延缓病情发展将成为神经退行性疾病治疗的新的着眼点。
研究发现许多化学合成物质如多酚类,具有很强的清除自由基能力,但常有一些严重副作用,因此找寻可以清除自由基和对细胞氧化损伤有保护作用,具有神经保护潜能的天然物质也已成为学者新近关注的热点。
柯里拉京,又称云实精,是一种天然多酚类单宁酸化合物,易溶于MeOH、EtOH、Aceton、DMSO,在冷水中溶解度小,是叶下珠、老鹳草、蜜柑草、白三草等植物中的有效成分,也存在于余甘子、龙眼、橄榄等具有抗氧化、抗衰老功效的植物果实中。柯里拉京具有抗氧化、抗动脉粥样硬化、抗纤溶、降血压、抗肿瘤、抑制病毒、抗菌、抗炎等广泛的生物学活性。但迄今为止,国内外对其的药理作用研究多集中在其抗肿瘤、抗菌和心血管保护等方面,且多为提取组份,而对神经保护作用方面却未见报道,值得我们进行探讨。
1目的
本课题应用人神经母细胞瘤株(SH-SY5Y细胞)和鼠小胶质细胞(N9细胞),建立氧化应激损伤的体外细胞模型,探讨柯里拉京单体的神经保护作用潜能。以叔丁基过氧化氢诱导损伤细胞,研究柯里拉京对这两种细胞的保护作用,观察其对细胞抗氧化功能和自由基的影响;观察柯里拉京对叔丁基过氧化氢诱导SH-SY5Y和N9细胞凋亡的抑制作用;观察柯里拉京对N9细胞过度激活引起的炎症抑制作用;并对相关作用机制进行探讨,为神经退行性病变的防治和新药开发提供可靠的实验依据。
2方法
本课题首先应用SRB法检测柯里拉京本身的细胞毒性;通过MTT法检测细胞活性建立了叔丁基过氧化氢诱导细胞氧化应激损伤的体外模型;通过观察细胞形态学的变化以及MTT法和LDH释放法检测细胞损伤程度考察了柯里拉京非药物毒性剂量下预处理对体外细胞氧化应激损伤的保护作用。
通过荧光探针标记,以荧光显微镜观察细胞内活性氧变化,以激光共聚焦和流式细胞技术观察细胞线粒体膜电位变化;通过生化比色法检测细胞总抗氧化能力T-AOC、抗氧化酶SOD活力,脂质过氧化等,探讨柯里拉京抗氧化特性在对叔丁基过氧化氢诱导细胞氧化应激损伤保护作用中的作用。
通过AO/EB染色法观察细胞形态、琼脂糖凝胶电泳分析细胞DNA片段化和流式细胞术分析细胞凋亡峰;通过酶活性检测、免疫印迹法等分析细胞凋亡相关信号分子的表达,探讨柯里拉京抗细胞凋亡作用及其可能机制。
通过Griess法和比色法分别检测细胞NO的分泌和NOS活力;以ELISA法检测炎症相关因子TNF-α;实时荧光PCR法相对定量分析细胞iNOS mRNA基因的表达;以荧光免疫法细胞原位检测NF-κB激活核转录等,探讨柯里拉京的抗炎特性及其在抗N9细胞过度激活引起炎性反应的抑制作用。
课题定量结果以均值±标准差( x±s)表示;组间均数比较采用单因素方差分析和t-检验法,p <0.05为差异显著。
3结果
(1)保护作用柯里拉京在高浓度范围对SH-SY5Y和N9细胞具有一定的细胞毒作用,24 h、48 h IC50分别为:SH-SY5Y,389.64±29.83、202.80±17.64 (μM);N9,179.72±46.54、61.64±6.80 (μM);但低浓度对细胞无毒性作用。TBHP可对SH-SY5Y和N9细胞产生毒性,使其形态学发生明显改变,大部分SH-SY5Y细胞胞体皱缩,突起缩短、断裂或消失,细胞变圆;N9细胞出现皱缩变形并有泡状突起,大小不一;细胞稀疏重叠,可见细胞残骸。细胞代谢活力也明显降低,呈现一定的时间和剂量依赖性。其作用24 h抑制率接近50%时TBHP分别为40和600μM。较之损伤模型组,药物预处理组细胞形态明显改善,代谢活力明显增强,LDH释放显著降低,且呈剂量依赖性。结果提示柯里拉京对TBHP诱导的氧化应激损伤具有一定的保护作用,作用机制可能涉及线粒体途径。
(2)抗氧化作用TBHP模型组SH-SY5Y和N9细胞的DCF荧光强度增大,Rh123荧光强度减弱,提示TBHP诱导损伤线粒体膜,线粒体膜电位降低,胞内自由基增加;较之损伤模型组,柯里拉京预孵育细胞DCF荧光强度减弱,Rh123荧光强度增加,且呈现一定的剂量依赖性。提示柯里拉京可通过清除自由基、阻滞线粒体膜电位的降低保护神经细胞。
细胞抗氧化能力和脂质过氧化测定结果显示:较之正常细胞,TBHP模型组SH-SY5Y、N9细胞的MDA明显增高,T-AOC、GSH和SOD值明显下降;与模型组比较,SH-SY5Y细胞药物预处理组MDA值显著下降,T-AOC、GSH和SOD值显著增加,其中MDA和T-AOC的改变呈现剂量依赖性;与模型组比较,N9细胞MDA显著下降、SOD显著升高,中、高剂量组T-AOC显著升高,GSH增加,但无统计意义。提示,柯里拉京可上调抗氧化酶活性,减少脂质过氧化、提高细胞抗氧化能力。
(3)抗凋亡AO/EB双染、DNA Ladder分析和流式细胞术对细胞亚二倍体峰的检测结果均表明TBHP可诱导SH-SY5Y和N9细胞产生凋亡;柯里拉京预处理可抑制TBHP诱导的细胞凋亡。较之正常细胞,模型组SH-SY5Y细胞的Caspase-3活性显著增强,柯里拉京可降低Caspase-3活性;免疫印迹检测结果显示,损伤模型组Caspase-3,Caspase-9,Bax表达增加,Bcl-2表达下降,药物预处理组细胞Caspase-3,Caspase-9下调,Bcl-2的表达上调;对Bax未见明显影响。提示,柯里拉京可下调Caspase-9和Caspase-3的表达及活性,阻止其诱导的凋亡级联反应;上调Bcl-2的表达,对氧化应激诱导的细胞凋亡产生抑制作用。
(4)抗炎作用TBHP模型组N9细胞NO含量、iNOS活性、TNF-a水平均显著高于正常细胞;柯里拉京本身具有体外清除NO作用,还可降低TBHP诱导的N9细胞NO含量,减少TNF-a的分泌,但对iNOS的活性无明显影响;实时RT-PCR检测结果显示,柯里拉京可抑制TBHP诱导的N9细胞iNOS mRNA表达;细胞原位免疫荧光标记显示,TBHP模型组细胞NF-κB激活转运入核明显增加,药物预处理减弱了NF-κB的激活转位。结果提示,除了自身清除NO外,柯里拉京可下调iNOS mRNA基因表达,抑制NO生成和TNF-a等炎性因子的分泌;机制可能涉及NF-κB通路。
4结论
柯里拉京对叔丁基过氧化氢诱导的SH-SY5Y细胞和N9细胞氧化应激损伤具有一定的保护作用。该保护作用主要与其抗氧化、抗凋亡和抗炎特性相关:可清除细胞自由基、活性氧,增加细胞抗氧化能力;通过线粒体途径抑制氧化应激诱导的细胞凋亡;可抑制氧化应激引起小胶质细胞过度激活的炎性损伤作用。
由此可见,柯里拉京对神经元和神经胶质细胞的氧化应激损伤有显著的保护作用,显示出一定的神经保护潜能,可能成为NDD防治的有利选择。
Introduction
Oxidative stress plays important roles in aging and neurodegenerative disorders. With a high content of oxidizable substrates such as polyunsaturated fatty acids, poor catalase activity and low iron-binding capacity, brain tissue may be prone to ROS damage. It has been found that inflammation the most important cause.
It has been proven that inflammation induced the death of neuron, which accelerated the inflammation in turn. Microglia, the macrophage-like cells, has been implicated as mediators of inflammation in the central nervous system. ROS-mediated stress in microglia could result in cellular injuries and evoke neuronal injury preferentially. Activated microglia produce inflammatory mediators including nitric oxide, reactive oxygen species (ROS), and cytokines, such as tumor necrosis factor-a, which are associated with several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis.
Therefore, scavenging free radicals, preventing ROS induced toxicities and inhibiting the microglia over-activation might be some reasonable therapeutic strategies for the treatment of neurodegenerative diseases. Phenolic compounds have strong radical scavenging abilities, but they usually have some severe adverse effects. So, finding natural substances with neuroprotective potential that can protect cells from oxidative damage has drawn considerable attention.
Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose), a novel member of the tannin family which has been discovered in many medicinal plants such as Phyllanthus species etc., has been proven to possess a multiformity of pharmacological activity, including antitumor, antioxidative, antiatherogenic, thrombolytic, antihypertensive, hepatoprotective, antiviral, antibacterial and anti-inflammatory effects. Although its antioxidant and anti-inflammatory properties were confirmed, there are no reports concerning the neuroprotective effects available. In addition, reports were almost focusing on the extract containing corilagin. Therefore corilagin, especially the purified substance, deserves an in-depth and further exploitation.
Objective
The present study was aimed to investigate the potential protective effects of corilagin on cell oxidative stress models of human neuroblastoma SH-SY5Y cells and N9 murine microglia cells which induced by tert-butyl hydroperoxide (TBHP) and elucidate the related mechanisms, which were undertaken to supply the experimental foundations for the potential clinical application of corilagin as a kinds of antioxidative and anti-inflammatory drugs in treatment of neurodegenerative diseases.
Methods
First of all, cytotoxicities of corilagin on SH-SY5Y and N9 cells determined by SRB assay and oxidative stress models were induced by TBHP in SH-SY5Y and N9 cells. With the MTT and LDH release tests and observation of the morphological changes, the protective effects of corilagin were evaluated.
Secondly, the ROS levels and mitochondrial membrane potential were analyzed using the fluorescent probe DCFH-DA and Rh-123, respectively. In addition, the levels of MDA, T-AOC, T-SOD and GSH were measured by biochemistry assay to evaluate the influence of antioxidative abilities of corilagin on SH-SY5Y and N9 cells.
Thirdly, with AO/EB fluorescent staining used to observe apoptosis, agarose gel electrophoresis to detect DNA fragmentation and flow cytometry (FCM) to detect the cell sub-G1 peaks, the influence of corilagin on cell apoptosis induced by TBHP were observed. In order to make clear the relative mechanisms of antiapoptosic effects of corilagin, chromometry was used to measure the activation of Caspase-3 and western blotting analysis used to observe the effects of corilagin on the signaling molecules Caspase-3, Caspase-9, Bcl-2 and Bax, which are relevant with cell apoptosis.
Fourthly, to investigate whether corilagin medicated in the inflammation of N9 cells activated by TBHP, Griess assay and absorption spectrometry were used for NO levels and NOS activities determination respectively; the expression of iNOS mRNA assessed by means of analysis of relative gene expression data using real-time quantitative PCR; ELISA assay used for TNF-α; In-situ immuno-flusent staining applied for the observation of the NF-KB mechanisms.
Statistic analysis
Quantitative data were presented as mean±standard deviation (S.D.). The statistical analysis between various experimental results was performed using one-way ANOVA, while those between two groups were analyzed by t-test. P<0.05 was considered statistically significant.
Results
(1) Protective Effects Corilagin exhibits cytotoxicities on SH-SY5Y and N9 at high concentrations with IC50 values of 389.64±29.83 (μM, 24 h), 202.80±17.64 (μM, 48 h) for SH-SY5Y and 179.72±46.54, 61.64±6.80 for N9 cells. Challenge with TBHP for 24 h resulted in the morphological changes: neuritis of most SH-SY5Y cells shortened, broken, or lost, cell bodies crimpled, N9 cells shrinkage, even floatation, and some of which were lysed or replaced by debris. TBHP inhibited SH-SY5Y and N9 cells viability in a time-and-dose-depended manner, the inhibition about 50% with TBHP (40μM, 24 h) for SH-SY5Y, as for N9 cells, and 600μM. Compared with the TBHP group, morphologies of cells mitigated with preincubation of corilagin 24 h prior to the exposure of TBHP, and cell viability increased, LDH leakage lowered significantly and dose-dependently. The results indicated corilagin could attenuate TBHP-induced oxidative stress in SH-SY5Y and N9 cells.
(2) Antioxidative Effects Treatment with TBHP resulted in an increase of DCF fluorescence and decrease of Rh-123 fluorescence, which suggested the accumulation of intercellular ROS, damage of michondrial membrane which resulted in the loss of MMP. Compared with TBHP group, pretreatment of corilagin decreased the DCF fluorescence and inversed the MMP reduction, which confirmed that corilagin, may protect oxidative damage through ROS scavenging and MMP stabilization. In antioxidative characteristic measurements, treatment with TBHP resulted in MDA increased, and T-AOC, SOD, GSH levels lowed. In contrast, pretreatment with corilagin ameliorate these items. Combined with the previous part, the results confirmed that corilagin can protect the oxidative damage with its antioxidative activity, and the relevant mechanisms may involve mitochondrial protection.
(3) Anti-apoptosis Apoptosis induced by TBHP was observed, which inhibited by pretreatment of corilagin. While SH-SY5Y cells preincubated with corilagin before TBHP exposure, caspase-3 enzyme activity inhibited. Expressions of caspase-3 and caspase-9 were significantly down-regulated and Bcl-2 up-regulated by corilagin without the obviously influence on Bax. The results suggest that corilagin could inhibited the apoptosis which induced by TBHP, in mitochondrial-depended pathway, with the regulation of Caspases and Bcl-2.
(4) Anti-inflammation TBHP incubation resulted in the increase of NO and TNF-αsecretion, as well as iNOS activity. With the NO scavenging activity, intervention with corilagin could decrease the secretion of NO and TNF-αsignificantly, without obviously effect on iNOS activity. Furthermore, the expression of iNOSmRNA of cells preincubated with corilagin was significantly down-regulated; NF-KB activation and translocation were attenuated. The results collectively showed that corilagin could inhibit the inflammatory factors, such as NO and TNF-α, corresponding with the down-regulation of iNOSmRNA, besides the NO scavenging itself in vitro. The mechanisms in which corilagin prevent the impairment of oxidative stress may involve NF-κB pathway.
Conclusion
Corilagin could prevent the impairment of oxidative stress challenged with TBHP on SH-SY5Y cells and N9 cells. The effects may relate to its antioxidative, antiapopototic and anti-inflammatory activities with the mechanisms involved in ROS scavenging, inhibiting apoptosis which induced in mitochondrial-dependent throughway, and anti-inflammation on the microglia activated by oxidative stress.
In a word, corilagin presents the potential protection on neuron and microglia cell. Hence, it would be considered to be a potential choice of prevention and cure to NDD.
研究表明,炎症也可以引发神经细胞死亡,从而形成恶性循环,加速病情的恶化。参与炎症介导的神经退行性病变的几种细胞中,巨噬细胞样细胞-小胶质细胞是最重要的一种。氧化应激是导致神经退行性疾病的主要原因,而小胶质细胞正是产生氧化应激的最主要来源;越来越多的证据表明氧化应激诱导小胶质细胞的损伤优先激发神经元的损伤。小胶质细胞过度激活所释放的大量NO、TNF-α等促炎症细胞因子和ROS等神经毒性因子,会对神经元产生毒性作用,导致神经元的变性和死亡,进而导致AD、PD等神经退行性疾病的发生。
因此,通过增加细胞抗氧化能力、阻断或抑制氧化应激引起的神经元和神经胶质细胞损伤以及抑制小胶质细胞异常激活都可能成为神经退行性疾病的防治策略。致力于寻找新的抗氧化、抗炎药物以减轻氧化损伤和炎症反应,延缓病情发展将成为神经退行性疾病治疗的新的着眼点。
研究发现许多化学合成物质如多酚类,具有很强的清除自由基能力,但常有一些严重副作用,因此找寻可以清除自由基和对细胞氧化损伤有保护作用,具有神经保护潜能的天然物质也已成为学者新近关注的热点。
柯里拉京,又称云实精,是一种天然多酚类单宁酸化合物,易溶于MeOH、EtOH、Aceton、DMSO,在冷水中溶解度小,是叶下珠、老鹳草、蜜柑草、白三草等植物中的有效成分,也存在于余甘子、龙眼、橄榄等具有抗氧化、抗衰老功效的植物果实中。柯里拉京具有抗氧化、抗动脉粥样硬化、抗纤溶、降血压、抗肿瘤、抑制病毒、抗菌、抗炎等广泛的生物学活性。但迄今为止,国内外对其的药理作用研究多集中在其抗肿瘤、抗菌和心血管保护等方面,且多为提取组份,而对神经保护作用方面却未见报道,值得我们进行探讨。
1目的
本课题应用人神经母细胞瘤株(SH-SY5Y细胞)和鼠小胶质细胞(N9细胞),建立氧化应激损伤的体外细胞模型,探讨柯里拉京单体的神经保护作用潜能。以叔丁基过氧化氢诱导损伤细胞,研究柯里拉京对这两种细胞的保护作用,观察其对细胞抗氧化功能和自由基的影响;观察柯里拉京对叔丁基过氧化氢诱导SH-SY5Y和N9细胞凋亡的抑制作用;观察柯里拉京对N9细胞过度激活引起的炎症抑制作用;并对相关作用机制进行探讨,为神经退行性病变的防治和新药开发提供可靠的实验依据。
2方法
本课题首先应用SRB法检测柯里拉京本身的细胞毒性;通过MTT法检测细胞活性建立了叔丁基过氧化氢诱导细胞氧化应激损伤的体外模型;通过观察细胞形态学的变化以及MTT法和LDH释放法检测细胞损伤程度考察了柯里拉京非药物毒性剂量下预处理对体外细胞氧化应激损伤的保护作用。
通过荧光探针标记,以荧光显微镜观察细胞内活性氧变化,以激光共聚焦和流式细胞技术观察细胞线粒体膜电位变化;通过生化比色法检测细胞总抗氧化能力T-AOC、抗氧化酶SOD活力,脂质过氧化等,探讨柯里拉京抗氧化特性在对叔丁基过氧化氢诱导细胞氧化应激损伤保护作用中的作用。
通过AO/EB染色法观察细胞形态、琼脂糖凝胶电泳分析细胞DNA片段化和流式细胞术分析细胞凋亡峰;通过酶活性检测、免疫印迹法等分析细胞凋亡相关信号分子的表达,探讨柯里拉京抗细胞凋亡作用及其可能机制。
通过Griess法和比色法分别检测细胞NO的分泌和NOS活力;以ELISA法检测炎症相关因子TNF-α;实时荧光PCR法相对定量分析细胞iNOS mRNA基因的表达;以荧光免疫法细胞原位检测NF-κB激活核转录等,探讨柯里拉京的抗炎特性及其在抗N9细胞过度激活引起炎性反应的抑制作用。
课题定量结果以均值±标准差( x±s)表示;组间均数比较采用单因素方差分析和t-检验法,p <0.05为差异显著。
3结果
(1)保护作用柯里拉京在高浓度范围对SH-SY5Y和N9细胞具有一定的细胞毒作用,24 h、48 h IC50分别为:SH-SY5Y,389.64±29.83、202.80±17.64 (μM);N9,179.72±46.54、61.64±6.80 (μM);但低浓度对细胞无毒性作用。TBHP可对SH-SY5Y和N9细胞产生毒性,使其形态学发生明显改变,大部分SH-SY5Y细胞胞体皱缩,突起缩短、断裂或消失,细胞变圆;N9细胞出现皱缩变形并有泡状突起,大小不一;细胞稀疏重叠,可见细胞残骸。细胞代谢活力也明显降低,呈现一定的时间和剂量依赖性。其作用24 h抑制率接近50%时TBHP分别为40和600μM。较之损伤模型组,药物预处理组细胞形态明显改善,代谢活力明显增强,LDH释放显著降低,且呈剂量依赖性。结果提示柯里拉京对TBHP诱导的氧化应激损伤具有一定的保护作用,作用机制可能涉及线粒体途径。
(2)抗氧化作用TBHP模型组SH-SY5Y和N9细胞的DCF荧光强度增大,Rh123荧光强度减弱,提示TBHP诱导损伤线粒体膜,线粒体膜电位降低,胞内自由基增加;较之损伤模型组,柯里拉京预孵育细胞DCF荧光强度减弱,Rh123荧光强度增加,且呈现一定的剂量依赖性。提示柯里拉京可通过清除自由基、阻滞线粒体膜电位的降低保护神经细胞。
细胞抗氧化能力和脂质过氧化测定结果显示:较之正常细胞,TBHP模型组SH-SY5Y、N9细胞的MDA明显增高,T-AOC、GSH和SOD值明显下降;与模型组比较,SH-SY5Y细胞药物预处理组MDA值显著下降,T-AOC、GSH和SOD值显著增加,其中MDA和T-AOC的改变呈现剂量依赖性;与模型组比较,N9细胞MDA显著下降、SOD显著升高,中、高剂量组T-AOC显著升高,GSH增加,但无统计意义。提示,柯里拉京可上调抗氧化酶活性,减少脂质过氧化、提高细胞抗氧化能力。
(3)抗凋亡AO/EB双染、DNA Ladder分析和流式细胞术对细胞亚二倍体峰的检测结果均表明TBHP可诱导SH-SY5Y和N9细胞产生凋亡;柯里拉京预处理可抑制TBHP诱导的细胞凋亡。较之正常细胞,模型组SH-SY5Y细胞的Caspase-3活性显著增强,柯里拉京可降低Caspase-3活性;免疫印迹检测结果显示,损伤模型组Caspase-3,Caspase-9,Bax表达增加,Bcl-2表达下降,药物预处理组细胞Caspase-3,Caspase-9下调,Bcl-2的表达上调;对Bax未见明显影响。提示,柯里拉京可下调Caspase-9和Caspase-3的表达及活性,阻止其诱导的凋亡级联反应;上调Bcl-2的表达,对氧化应激诱导的细胞凋亡产生抑制作用。
(4)抗炎作用TBHP模型组N9细胞NO含量、iNOS活性、TNF-a水平均显著高于正常细胞;柯里拉京本身具有体外清除NO作用,还可降低TBHP诱导的N9细胞NO含量,减少TNF-a的分泌,但对iNOS的活性无明显影响;实时RT-PCR检测结果显示,柯里拉京可抑制TBHP诱导的N9细胞iNOS mRNA表达;细胞原位免疫荧光标记显示,TBHP模型组细胞NF-κB激活转运入核明显增加,药物预处理减弱了NF-κB的激活转位。结果提示,除了自身清除NO外,柯里拉京可下调iNOS mRNA基因表达,抑制NO生成和TNF-a等炎性因子的分泌;机制可能涉及NF-κB通路。
4结论
柯里拉京对叔丁基过氧化氢诱导的SH-SY5Y细胞和N9细胞氧化应激损伤具有一定的保护作用。该保护作用主要与其抗氧化、抗凋亡和抗炎特性相关:可清除细胞自由基、活性氧,增加细胞抗氧化能力;通过线粒体途径抑制氧化应激诱导的细胞凋亡;可抑制氧化应激引起小胶质细胞过度激活的炎性损伤作用。
由此可见,柯里拉京对神经元和神经胶质细胞的氧化应激损伤有显著的保护作用,显示出一定的神经保护潜能,可能成为NDD防治的有利选择。
Introduction
Oxidative stress plays important roles in aging and neurodegenerative disorders. With a high content of oxidizable substrates such as polyunsaturated fatty acids, poor catalase activity and low iron-binding capacity, brain tissue may be prone to ROS damage. It has been found that inflammation the most important cause.
It has been proven that inflammation induced the death of neuron, which accelerated the inflammation in turn. Microglia, the macrophage-like cells, has been implicated as mediators of inflammation in the central nervous system. ROS-mediated stress in microglia could result in cellular injuries and evoke neuronal injury preferentially. Activated microglia produce inflammatory mediators including nitric oxide, reactive oxygen species (ROS), and cytokines, such as tumor necrosis factor-a, which are associated with several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis.
Therefore, scavenging free radicals, preventing ROS induced toxicities and inhibiting the microglia over-activation might be some reasonable therapeutic strategies for the treatment of neurodegenerative diseases. Phenolic compounds have strong radical scavenging abilities, but they usually have some severe adverse effects. So, finding natural substances with neuroprotective potential that can protect cells from oxidative damage has drawn considerable attention.
Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose), a novel member of the tannin family which has been discovered in many medicinal plants such as Phyllanthus species etc., has been proven to possess a multiformity of pharmacological activity, including antitumor, antioxidative, antiatherogenic, thrombolytic, antihypertensive, hepatoprotective, antiviral, antibacterial and anti-inflammatory effects. Although its antioxidant and anti-inflammatory properties were confirmed, there are no reports concerning the neuroprotective effects available. In addition, reports were almost focusing on the extract containing corilagin. Therefore corilagin, especially the purified substance, deserves an in-depth and further exploitation.
Objective
The present study was aimed to investigate the potential protective effects of corilagin on cell oxidative stress models of human neuroblastoma SH-SY5Y cells and N9 murine microglia cells which induced by tert-butyl hydroperoxide (TBHP) and elucidate the related mechanisms, which were undertaken to supply the experimental foundations for the potential clinical application of corilagin as a kinds of antioxidative and anti-inflammatory drugs in treatment of neurodegenerative diseases.
Methods
First of all, cytotoxicities of corilagin on SH-SY5Y and N9 cells determined by SRB assay and oxidative stress models were induced by TBHP in SH-SY5Y and N9 cells. With the MTT and LDH release tests and observation of the morphological changes, the protective effects of corilagin were evaluated.
Secondly, the ROS levels and mitochondrial membrane potential were analyzed using the fluorescent probe DCFH-DA and Rh-123, respectively. In addition, the levels of MDA, T-AOC, T-SOD and GSH were measured by biochemistry assay to evaluate the influence of antioxidative abilities of corilagin on SH-SY5Y and N9 cells.
Thirdly, with AO/EB fluorescent staining used to observe apoptosis, agarose gel electrophoresis to detect DNA fragmentation and flow cytometry (FCM) to detect the cell sub-G1 peaks, the influence of corilagin on cell apoptosis induced by TBHP were observed. In order to make clear the relative mechanisms of antiapoptosic effects of corilagin, chromometry was used to measure the activation of Caspase-3 and western blotting analysis used to observe the effects of corilagin on the signaling molecules Caspase-3, Caspase-9, Bcl-2 and Bax, which are relevant with cell apoptosis.
Fourthly, to investigate whether corilagin medicated in the inflammation of N9 cells activated by TBHP, Griess assay and absorption spectrometry were used for NO levels and NOS activities determination respectively; the expression of iNOS mRNA assessed by means of analysis of relative gene expression data using real-time quantitative PCR; ELISA assay used for TNF-α; In-situ immuno-flusent staining applied for the observation of the NF-KB mechanisms.
Statistic analysis
Quantitative data were presented as mean±standard deviation (S.D.). The statistical analysis between various experimental results was performed using one-way ANOVA, while those between two groups were analyzed by t-test. P<0.05 was considered statistically significant.
Results
(1) Protective Effects Corilagin exhibits cytotoxicities on SH-SY5Y and N9 at high concentrations with IC50 values of 389.64±29.83 (μM, 24 h), 202.80±17.64 (μM, 48 h) for SH-SY5Y and 179.72±46.54, 61.64±6.80 for N9 cells. Challenge with TBHP for 24 h resulted in the morphological changes: neuritis of most SH-SY5Y cells shortened, broken, or lost, cell bodies crimpled, N9 cells shrinkage, even floatation, and some of which were lysed or replaced by debris. TBHP inhibited SH-SY5Y and N9 cells viability in a time-and-dose-depended manner, the inhibition about 50% with TBHP (40μM, 24 h) for SH-SY5Y, as for N9 cells, and 600μM. Compared with the TBHP group, morphologies of cells mitigated with preincubation of corilagin 24 h prior to the exposure of TBHP, and cell viability increased, LDH leakage lowered significantly and dose-dependently. The results indicated corilagin could attenuate TBHP-induced oxidative stress in SH-SY5Y and N9 cells.
(2) Antioxidative Effects Treatment with TBHP resulted in an increase of DCF fluorescence and decrease of Rh-123 fluorescence, which suggested the accumulation of intercellular ROS, damage of michondrial membrane which resulted in the loss of MMP. Compared with TBHP group, pretreatment of corilagin decreased the DCF fluorescence and inversed the MMP reduction, which confirmed that corilagin, may protect oxidative damage through ROS scavenging and MMP stabilization. In antioxidative characteristic measurements, treatment with TBHP resulted in MDA increased, and T-AOC, SOD, GSH levels lowed. In contrast, pretreatment with corilagin ameliorate these items. Combined with the previous part, the results confirmed that corilagin can protect the oxidative damage with its antioxidative activity, and the relevant mechanisms may involve mitochondrial protection.
(3) Anti-apoptosis Apoptosis induced by TBHP was observed, which inhibited by pretreatment of corilagin. While SH-SY5Y cells preincubated with corilagin before TBHP exposure, caspase-3 enzyme activity inhibited. Expressions of caspase-3 and caspase-9 were significantly down-regulated and Bcl-2 up-regulated by corilagin without the obviously influence on Bax. The results suggest that corilagin could inhibited the apoptosis which induced by TBHP, in mitochondrial-depended pathway, with the regulation of Caspases and Bcl-2.
(4) Anti-inflammation TBHP incubation resulted in the increase of NO and TNF-αsecretion, as well as iNOS activity. With the NO scavenging activity, intervention with corilagin could decrease the secretion of NO and TNF-αsignificantly, without obviously effect on iNOS activity. Furthermore, the expression of iNOSmRNA of cells preincubated with corilagin was significantly down-regulated; NF-KB activation and translocation were attenuated. The results collectively showed that corilagin could inhibit the inflammatory factors, such as NO and TNF-α, corresponding with the down-regulation of iNOSmRNA, besides the NO scavenging itself in vitro. The mechanisms in which corilagin prevent the impairment of oxidative stress may involve NF-κB pathway.
Conclusion
Corilagin could prevent the impairment of oxidative stress challenged with TBHP on SH-SY5Y cells and N9 cells. The effects may relate to its antioxidative, antiapopototic and anti-inflammatory activities with the mechanisms involved in ROS scavenging, inhibiting apoptosis which induced in mitochondrial-dependent throughway, and anti-inflammation on the microglia activated by oxidative stress.
In a word, corilagin presents the potential protection on neuron and microglia cell. Hence, it would be considered to be a potential choice of prevention and cure to NDD.
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