黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

当药黄素对H_2O_2诱导PC12细胞损伤的保护作用

研究当药黄素在H_2O_2诱导PC12细胞损伤中的作用。建立H_2O_2诱导的PC12细胞损伤模型,采用MTT法测定细胞活力,比色法测定细胞MDA和上清液LDH含量,以及SOD、CAT和GSH-Px酶活力,采用DCFH-DA荧光染色测定细胞ROS含量,JC-1染色测定细胞线粒体膜电位。当药黄素能够提高H_2O_2诱导损伤的PC12细胞的细胞活力,增加细胞内SOD、CAT和GSH-Px活力,降低MDA、LDH含量,抑制细胞内ROS增加,稳定细胞线粒体膜电位。当药黄素对H_2O_2诱导的PC12细胞损伤有保护作用。     

菟丝子提取物对活性氧引起已分化的PC12细胞损伤的保护作用

以常用的神经嗜铬细胞瘤PC12细胞株为实验模型,通过比较活性氧(ROS)作用细胞后的细胞活力、凋亡相关蛋白(p53、Bax)水平以及细胞中SOD、GSH、MDA的差异,发现菟丝子提取物不仅能提高ROS损伤的已分化PC12细胞活力,调节细胞中凋亡相关基因的表达,而且还能提高细胞中SOD和GSH的含量,降低MDA水平。由此表明,菟丝子提取物对ROS造成的PC12细胞损伤有一定的保护作用。     

异槲皮苷对Aβ25-35导致的PC12细胞损伤的保护作用及机制研究

探讨异槲皮苷对β-淀粉样蛋白(Aβ25-35)导致的PC12细胞氧化损伤的保护作用.首先通过分子对接技术分析异槲皮苷与AMPK的结合情况.采用Aβ25-35(20 μmol/L)损伤PC12细胞建立细胞氧化损伤模型,采用甲基噻唑蓝(MTT)法检测细胞活力,通过试剂盒检测乳酸脱氢酶(LDH)漏出量、活性氧(ROS)含量、...     

山楂叶金丝桃苷对高糖诱导SH-SY5Y细胞损伤的保护作用及机制

为研究金丝桃苷对高糖诱导的人神经母细胞瘤(SH-SY5Y)细胞氧化损伤的保护作用及机制,用含100mmo L/L葡萄糖和分别为20、50、100μmo L/L金丝桃苷的培养基共同孵育SH-SY5Y细胞36 h,检测细胞活力、细胞培养液中乳酸脱氢酶(LDH)水平及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性,细胞内活性氧(ROS)水平、丙二醛(MDA)、还原型谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性及SIRT1和NF-кB基因的mRNA水平和蛋白含量。结果显示金丝桃苷可提高高糖诱导后SH-SY5Y细胞的存活率,抑制细胞LDH释放,清除ROS,降低MDA含量与caspase-3活性,增强SOD、CAT活性和GSH含量;同时,金丝桃苷还能提高SIRT1基因的mR-NA表达及蛋白含量,降低NF-кB基因的mRNA水平和蛋白含量。结果表明金丝桃苷能通过激活SIRT1基因,抑制NF-кB基因保护高糖所致SH-SY5Y细胞的氧化损伤。     

氧化苦参碱预处理对急性心肌梗死大鼠的保护作用与机制

目的:探讨氧化苦参碱(OMT)对冠脉结扎诱导的急性心肌梗死大鼠的保护作用与机制。方法:将SD大鼠随机分为4组:假手术组、假手术+OMT组、心梗模型组,OMT预处理组(ig给予OMT 100 mg/kg)。给药12小时后,结扎冠状动脉左前降支(LAD)复制大鼠急性心肌梗死模型。8小时后,取大鼠心肌组织,通过TUNEL染色观察大鼠心肌细胞损伤及凋亡情况;收集大鼠血清,检测LDH与CK水平,过氧化氢酶(CAT)、超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH)的活力,丙二醛(MDA)含量,ELISA法分析血清中IL-1β、IL-6和TNF-α的水平。结果:与假手术组比较,模型组大鼠的凋亡心肌细胞数明显增加(P0.05),血清CK、LDH水平显著升高(P0.05);同时,血清CAT、SOD与GSH的活性明显降低(P0.001),MDA的含量、IL-1β、IL-6和TNF-α水平显著增加(P0.001)。OMT预处理明显减轻了心肌梗死大鼠心肌细胞的损伤和凋亡,降低了其血清MDA含量,IL-1β、IL-6和TNF-α水平,增加了其CAT、SOD与GSH的活性。结论:氧化苦参碱预处理能够显著减轻心肌梗死大鼠的心肌损伤,这可能与其抗炎、抗凋亡与抗氧化损伤作用有关。     

干鲜人参对PC12细胞损伤保护作用的比较研究

分别建立皮质酮、谷氨酸、过氧化氢诱导PC12细胞损伤模型,通过MTT和LDH测定,比较不同浓度的干、鲜人参水提液对于皮质酮、谷氨酸、过氧化氢诱导PC12细胞损伤的保护作用。结果表明皮质酮浓度为200μmol/L、谷氨酸浓度为30 mmol/L和过氧化氢浓度为150μmol/L时,PC12细胞的存活率分别为:53.42%、49.64%、54.27%,当PC12细胞与不同浓度人参提取液共同孵育24 h,再分别加入200μmol/L的皮质酮和150μmol/L的过氧化氢时,与模型组相比,细胞存活率明显提高,乳酸脱氢酶的释放明显减少(P0.01);并且在中、高剂量组,鲜人参组的细胞存活率明显高于干人参组(P0.01),乳酸脱氢酶释放明显低于干人参组(P0.01);但对谷氨酸诱导的PC12细胞损伤则无上述效果。干、鲜人参水提液对于皮质酮、过氧化氢诱导损伤的PC12细胞均有明显的保护作用;在中、高剂量时,鲜人参水提液对细胞保护活性明显好于干人参组,且组间表现出显著性差异(P0.01),表明鲜人参较干人参具有更好的细胞保护活性。干、鲜人参水提液对谷氨酸诱导损伤的PC12细胞却无保护活性。     

籽瓜多糖对H_2O_2致PC12细胞氧化损伤的保护作用

本文研究了籽瓜多糖(SWP)对H2O2致PC12细胞氧化应激损伤的影响及其机制。通过建立H2O2诱导PC12细胞氧化损伤模型,CCK-8法测定细胞存活率;硫辛酰胺脱氢酶催化的INT显色反应检测乳酸脱氢酶(LDH)释放量,DCFH-DA检测细胞内ROS;ELISA法检测8-OHd G;JC-1染色检测细胞线粒体膜电位;利用caspase-3可以催化底物Ac-DEVD-p NA的反应检测caspase-3活性;应用caspase-9催化特异性底物Ac-LEHDp NA检测caspase-9活性。结果显示:过氧化氢组与对照组相比,终浓度为500μmol/L H2O2作用细胞24 h后,细胞活力显著下降(P0.01);LDH释放量和细胞内ROS增加(P0.01);8-OHd G含量上升(P0.01);线粒体膜电位下降(P0.01);caspase-3和caspase-9活性增强(P0.01)。与H2O2损伤组相比,不同剂量的SWP预处理后,能显著改善H2O2引起的上述指标的变化(P0.05)。由此得出:SWP对H2O2诱导的PC12细胞的氧化损伤具有一定的保护作用。     

在低氧损伤中牛磺酸对视网膜神经节转化细胞RGC-5抗氧化作用的研究

目的:观察低氧(Hypoxia,Hyp)对大鼠视网膜神经节转化细胞(retina ganglion cell-5,RGC-5)氧化应激损伤的影响及牛磺酸(Taurine,Tau)的防护效应.方法:将RGC-5置于低氧条件(5%O2,5%CO2,90%N2),加入不同浓度的牛磺酸(0.05mM、0.1mM、0.5mM、1mM)预处理后培养12h,24h和48h,使用MTT法检测细胞活力,并通过对NO、GSH、MDA等指标的检测,观察牛磺酸对RGC-5的保护效应.结果:低氧处理后RGC-5细胞活力明显降低(P＜0.05),牛磺酸处理组细胞活力明显高于低氧组,其中0.1mM牛磺酸组作用最为显著(P＜0.05);低氧组与常氧组比较,RGC-5的NO、GSH含量明显降低(P＜0.05),而MDA含量显著升高(P＜0.05);牛磺酸处理组与低氧组比较,RGC-5细胞GSH,NO的含量显著升高(P＜0.05),而MDA的含量显著降低(P＜0.05).结论:牛磺酸能有效增强低氧损伤中RGC-5细胞的活力,其机制可能与牛磺酸可以提高其抗氧化能力有关.     

延龄草苷对过氧化氢诱导的PC12细胞氧化损伤和炎症因子表达的影响

探讨延龄草苷对过氧化氢(H_2O_2)诱导的PC12细胞氧化损伤和炎症因子表达的影响。采用MTT法和LDH活性测定观察延龄草苷对PC12细胞模型的影响,采用相关试剂盒检测活性氧(ROS)和丙二醛(MDA)含量,超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,Western blot检测Sirt1、NF-κB和TNF-α的蛋白表达。延龄草苷(5~20μM)能够显著提高H_2O_2损伤的PC12细胞的活力,提高细胞抗氧化能力,并上调Sirt1的表达,下调NF-κB和TNF-α的表达,其保护作用可能与提高抗氧化能力,降低炎症因子损伤,调控Sirt1/NF-κB信号通路有关。     

Antioxidant activity of phenylethanoid glycosides on glutamate-induced neurotoxicity

ABSTRACT

Exposure of PC12 cells to 10 mM glutamate caused significant viability loss, cell apoptosis, decreased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as increased levels of malondialdehyde (MDA). In parallel, glutamate significantly increased the intracellular levels of ROS and intracellular calcium. However, pretreatment of the cells with acteoside and isoacteoside significantly suppressed glutamate-induced cellular events. Moreover, acteoside and isoacteoside reduced the glutamate-induced increase of caspase-3 activity and also ameliorated the glutamate-induced Bcl-2/Bax ratio reduction in PC12 cells. Furthermore, acteoside and isoacteoside significantly inhibited glutamate-induced DNA damage. In the mouse model, acteoside significantly attenuated cognitive deficits in the Y maze test and attenuated neuronal damage of the hippocampal CA1 regions induced by glutamate. These data indicated that acteoside and isoacteoside play neuroprotective effects through anti-oxidative stress, anti-apoptosis, and maintenance of steady intracellular calcium.     

Protective effect of sesaminol from Sesamum indicum Linn. against oxidative damage in PC12 cells

Sesaminol is one component of sesame oil and has been widely used as the stabilizer to extend the storage period of food oil in China. In this study, we tried to investigate the antioxidant activity of sesaminol on rat pheochromocytoma (PC12) cells oxidative damaged by H₂O₂. Cell viability, LDH level and apoptosis of the PC12 cells were assayed after treatment with sesaminol for 3 h and exposure to H₂O₂. Furthermore, superoxide (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px) and intracellular ROS were assayed after exposure of the PC12 cells to H₂O₂. The results showed that pre‐treatment with sesaminol prior to H₂O₂ exposure significantly elevated cell survival rate and SOD, CAT and GSH‐Px activity. Meanwhile, sesaminol declined the secreted LDH level, apoptosis rate and ROS level of H₂O₂ exposed cells. Thus, sesaminol may protect PC12 against oxidative injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Ginsenoside Rg1 protects rat cardiomyocyte from hypoxia/reoxygenation oxidative injury via antioxidant and intracellular calcium homeostasis

Ginsenoside Rg1 is a major active ingredient of Panax notoginseng radix which has demonstrated a number of pharmacological actions including a cardioprotective effect in vivo. This study investigated the protective effect and mechanism of ginsenoside Rg1 in cardiomyocytes hypoxia/reoxygenation (H/R) model. Pretreatment with ginsenoside Rg1 (60–120 µM) reduced lactate dehydrogenase release and increased cell viability in a dose‐dependent manner. Fluorescence analysis demonstrated ginsenoside Rg1 reduced intracellular ROS and suppressed the intracellular [Ca²⁺] level. Cell lysate detected an increase of T‐SOD, CAT, and GSH levels. The myocardial protection of ginsenoside Rg1 during H/R is partially due to its antioxidative effect and intracellular calcium homeostasis. J. Cell. Biochem. 108: 117–124, 2009. © 2009 Wiley‐Liss, Inc.     

Protective Effects of Ginsenoside Rg1 Against Colistin Sulfate-Induced Neurotoxicity in PC12 Cells

The present study aimed to examine the protective effect of ginsenoside Rg1 against colistin-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Ginsenoside Rg1 was shown to elevate cell viability, decrease levels of malondialdehyde and intracellular reactive oxygen species, enhance activity of superoxide dismutase and glutathione, and decrease the release of cytochrome-c, formation of DNA fragmentation in colistin-treated PC12 cells. Ginsenoside Rg1 also reversed the increased caspase-9 and -3 mRNA levels caused by colistin in PC12 cells. These results suggest that ginsenoside Rg1 exerts a neuroprotective effect on colistin-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress, prevention of apoptosis mediated via mitochondria pathway. Co-administration of ginsenoside Rg1 highlights the potential to increase the therapeutic index of colistin.     

Protective Effects of YC-1 Against Glutamate Induced PC12 Cell Apoptosis

Glutamate, one of the major neurotransmitters in the central nervous system, is released into the synaptic spaces and bound to the glutamate receptors which facilitate normal synaptic transmission, synaptic plasticity, and brain development. Past studies have shown that glutamate with high concentration is a potent neurotoxin capable of destroying neurons through many signal pathways. In this research, our main purpose was to determine whether the specific soluble guanylyl cyclase activator YC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole) had effect on glutamate-induced apoptosis in cultured PC12 cells. The differentiated PC12 cells impaired by glutamate were used as the cell model of excitability, and were exposed to YC-1 or/and ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) with gradient concentrations for 24 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay was used to detect the cellular viability. Radioimmunoassay (RIA) was used to detect the cGMP (cyclic guanosine monophosphate) concentrations in PC12 cells. Hoechst 33258 staining and flow cytometric analysis were used to detect the cell apoptosis. The cellular viability was decreased and the apoptotic rate was increased when PC12 cells were treated with glutamate. Cells treated with YC-1 or/and ODQ showed no significant differences in the cell viability and intracellular cGMP levels compared with those of control group. The specific soluble guanylyl cyclase (sGC) inhibitor ODQ showed an inhibitory effect on cGMP level and aggravated the apoptosis of PC12 cells induced by glutamate. YC-1 elevated cGMP level thus decreased PC12 cell apoptosis induced by glutamate, but this effect could be reversed by ODQ. These results revealed that YC-1 might attenuate glutamate-induced PC12 cell apoptosis via a sGC–cGMP involved pathway.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

Exendin-4对甲基乙二醛诱导PC12细胞氧化应激的影响

目的：探讨肠促胰岛素类似物（Ex-4）对甲基乙二醛（MG）诱导PC12细胞氧化应激的影响及其机制。方法：传代培养PC12细胞,不同浓度MG（0、0.25、0.50、0.75、1.0、2.0 mmol/l）处理PC12细胞12~48 h,或用不同浓度Ex-4（25、50、100、200 nmol/L）预处理24 h后加用MG（0.75 mmol/L）干预24 h后,MTT比色法检测细胞存活率;荧光探针法检测活性氧（ROS）含量,黄嘌呤氧化酶法检测超氧化物歧化酶（SOD）活力。Ex-4（100nmol/L）预处理PC12细胞24h加用MG（0.75 mmol/L）干预1 h后,Western blot检测蛋白P-IκB-α、IκB-α表达情况。结果：随着MG浓度的增加和作用时间的延长,PC12细胞存活率逐渐降低;加用不同浓度Ex-4预处理后,PC12细胞存活率较单独MG处理组逐渐升高。100 nmol/L的Ex-4预处理PC12细胞后,ROS表达量较MG单独处理组下降65.30%（P<0.01）; NAC预处理组（阳性对照）ROS表达量下降107.40%（P<0.01）;Ex-4预处理组SOD活力增加5.30 U/mg prot（P<0.01）;NAC预处理组SOD活力增加8.53 U/mg prot（P<0.01）。Ex-4预处理组P-IκB-α/IκB-α表达比例下降25.50%（P<0.01）; NAC预处理组P-IκB-α/IκB-α表达比例下降35.14%（P<0.01）。结论：Ex-4浓度依赖性地增加MG诱导的PC12细胞的存活率。Ex-4能够减轻MG诱导的PC12细胞的氧化应激,其机制可能涉及抑制蛋白IκB-α的活化。     

Neuroprotective effect of individual ginsenosides on astrocytes primary culture

Most of the known pharmacological effects of Panax ginseng on the central nervous system are due to its major components - ginsenosides. Although the antioxidant ability of ginseng root has already been established, this activity has never been evaluated for isolated ginsenosides on astrocytes. The activity of protopanaxadiols Rb(1), Rb(2), Rc and Rd, and protopanaxatriols Re and Rg(1) was evaluated in vitro on astrocytes primary culture by means of an oxidative stress model with H(2)O(2). The viability of astrocytes was determined by the MTT reduction assay and by the LDH release into the incubation medium. The effects on the antioxidant enzymes catalase, superoxide dismutase (SOD), glutathione peroxidases (GPx) and glutathione reductase (GR) and on the intracellular reactive oxygen species (ROS) formation were also investigated. Exposure of astrocytes to H(2)O(2) decreased cell viability as well as the antioxidant enzymes activity and increased ROS formation. Oxidative stress produced significant cell death that was reduced by previous treatment with the tested ginsenosides. Ginsenosides Rb(1), Rb(2), Re and Rg(1) were effective in reducing astrocytic death, while Rb(1), Rb(2), Rd, Re and Rg(1) decreased ROS formation, ginsenoside Re being the most active. Ginsenosides from P. ginseng induce neuroprotection mainly through activation of antioxidant enzymes.     

Neuroprotective Effects of Butanol Fraction of Cordyceps cicadae on Glutamate‐Induced Damage in PC12 Cells Involving Oxidative Toxicity

The current study was aimed at investigating the neuroprotective effects of the butanol fraction from Cordyceps cicadae (C_BU), which was responsible for the anti‐aging effect of this medicine. Glutamate‐induced PC12 cells were used as a model to determine the neuroprotective effect against oxidative cell death. Cell viability, cytotoxicity, flow cytometry, mitochondrial transmembrane potential (MMP), reactive oxygen species (ROS), glutathione peroxidase (GSH‐Px), and superoxide dismutase (SOD) levels were analyzed to assess neuronal cell survival or death. The results obtained from the above evaluations showed that C_BU was the most effective fraction and even better than pure compounds present in C. cicadae in terms of suppressing glutamate‐induced damage in PC12 cells, increasing cell viability, decreasing lactase dehydrogenase (LDH) release, and reduction of apoptosis induced by exposure to glutamate. Furthermore, C_BU protected cells against mitochondrial dysfunction and oxidative stress as indicated by the suppression of ROS accumulation and up regulation of the levels of GSH‐Px and SOD. In summary, the above results showed that C_BU exerted neuroprotective effect against oxidative damage, and this activity could be partly due to the action of nucleosides present in the C_BU.