抗氧化作用可能是人参皂甙Rg1抗细胞凋亡的机制

目的 :探讨人参皂甙Rg1对MPP+诱导SHSY5Y细胞凋亡的保护作用及其可能的机制。方法 :用吖啶橙溴化乙锭染色观察SHSY5Y细胞凋亡率 ,流式细胞仪检测线粒体跨膜电位及细胞内活性氧 (ROS)水平 ,WesternBlot法检测bcl 2、bax蛋白表达水平。结果 :经 10 μmol·L- 1Rg1预处理后 ,MPP+诱导的SHSY5Y细胞凋亡受到明显的抑制 ,虽然线粒体跨膜电位无明显改变 ,但细胞内ROS下降 ,而bcl 2蛋白表达水平增加 ,bax蛋白表达水平减少。结论 :Rg1可抑制MPP+诱导的SHSY5Y细胞凋亡 ,其作用机制可能是通过清除ROS、调节bcl 2、bax蛋白表达水平起作用。     

松果菊苷对TNFα诱导的SH-SY5Y细胞凋亡的保护作用

目的　探讨肉苁蓉提取物松果菊苷对TNFα诱导的SH SY5Y细胞凋亡的保护作用。方法　用MTT法检测细胞存活率,DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生,以激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生和线粒体膜电位的变化,并用荧光酶标仪测定caspase 3的活性。结果　100μg·L-1 TNFα处理细胞 36h显著降低细胞的存活率;诱导细胞发生凋亡,凋亡率达37%;细胞内活性氧水平及caspase 3的活性升高;而线粒体膜电位却明显降低,红 /绿荧光强度的比值由正常的 5 97降低为 0 35左右。而预先给予 1, 10或者 100mg·L-1浓度的松果菊苷处理细胞 2h,可提高细胞存活率;并可有效抑制DNAladder的发生;流式细胞仪检测凋亡率分别降低到25 9%, 18 3% 和 8 2%;激光共聚焦显微镜结果显示松果菊苷可明显抑制细胞内活性氧产生;并可逐渐恢复线粒体的高能量状态;caspase 3的活性不断降低,并呈现了一定的剂量依赖性。结论　松果菊苷能抑制TNFα诱导的SH SY5Y细胞凋亡,其神经细胞保护作用可能与降低细胞内活性氧水平,抑制caspase 3的活性和维持线粒体膜电位的高能状态有关。     

瓜子金皂苷丙对MPP~+诱导PC12细胞凋亡的抑制作用

目的观察瓜子金皂苷丙对MPP+诱导的PC12细胞凋亡的影响,并且探讨其作用机制。方法采用MTT法检测细胞存活率,碘化丙啶染色流式细胞术(FCM)检测PC12细胞凋亡,Western blotting检测Bax和Bcl-2蛋白的表达,罗丹明123染色FCM检测细胞线粒体膜电位(ΔΨm),荧光酶标仪检测细胞内活性氧(R0S)的含量。结果不同浓度MPP+作用PC12细胞24 h后,细胞存活率显著下降(P<0.01),细胞凋亡明显,凋亡相关蛋白Bcl-2/Bax比之下降,线粒体膜电位降低,细胞内ROS显著增加。与MPP+处理组相比,瓜子金皂苷丙10μmol·L-1组,细胞存活率显著升高(P<0.01);细胞凋亡率下降(P<0.01);Bcl-2/Bax比率增加(P<0.01),线粒体膜电位上升(P<0.01),ROS含量减少。结论瓜子金皂苷丙可以抑制MPP+诱导的PC12细胞凋亡,其作用机理可能与上调Bcl-2和下调Bax蛋白的表达,维持线粒体正常膜电位,稳定线粒体功能,清除R0S有关。     

人参皂苷Rg1可能通过丝裂素活化蛋白激酶途径抑制细胞凋亡

目的探讨人参皂苷Rg1对MPP+诱导细胞凋亡保护作用的可能信号传导途径.方法用吖啶橙-溴化乙锭染色观察SHSY5Y细胞凋亡率,流式细胞仪检测细胞内活性氧ROS水平,Western Blotting法检测JNK(c-jun NH2-terminal kinase)激酶活性,免疫细胞化学染色法检测裂解的Caspase-3阳性细胞的表达率.结果经10 μmol*L-1 Rg1或2.5 mmol*L-1 N-乙酰半胱氨酸预处理后,MPP+诱导的SHSY5Y细胞凋亡受到明显抑制,同时细胞内ROS下降,JNK激酶的活性减弱,裂解的Caspase-3阳性细胞表达率下降.结论 Rg1可抑制MPP+诱导的SHSY5Y细胞凋亡,其作用机制可能是通过清除ROS、减弱JNK激酶的活性,从而减少Caspase-3的激活.     

栀子苷抗H_2O_2诱导人脐静脉内皮细胞凋亡的实验研究

目的探讨栀子苷对氧化应激诱导人脐静脉内皮细胞(HUVEC)凋亡的抑制作用及可能机制。方法体外培养的HUVEC细胞用不同浓度的栀子苷药物预处理24h后,加入终浓度为500μmol·L-1H2O2氧化损伤12h。荧光染色法观察细胞内DNA损伤情况,流式细胞术检测细胞凋亡率和线粒体膜电位的变化,Western blot检测Bcl-2和Bax蛋白的表达,酶免法测定caspase-3蛋白酶的活性,Real-time PCR检测超氧化物歧化酶(Mn-SOD)和谷胱甘肽过氧化物酶(GPx)的mRNA表达。结果与模型组相比,不同浓度栀子苷(12.5、25、50mg·L-1)可以明显改善H2O2对细胞内的DNA氧化损伤,降低细胞凋亡率,下调Bax蛋白表达,逐步恢复Bcl-2/Bax表达比例和线粒体膜电位的改变,降低caspase-3蛋白酶活性,上调细胞内Mn-SOD和GPx的表达,但对Bcl-2的表达无影响。结论栀子苷可以抑制H2O2诱导的HUVEC细胞凋亡,其机制可能与调节线粒体应激途径和发挥抗氧化损伤功能有关。     

瓜子金皂苷己对MPP~+诱导PC12细胞凋亡的保护作用

目的观察瓜子金皂苷己(polygalasaponin F,PS-F)对1-甲基-4-苯基-吡啶离子(1-methyl-4-phenylpyridinium,MPP+)诱导的PC12细胞损伤的影响,并且探讨其作用机制。方法 MTT法检测细胞存活率,Annexin V/PI染色流式细胞术检测PC12细胞凋亡,JC-1染色倒置显微镜检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),Western blot检测Caspase-3蛋白的水平。结果 500μmol.L-1MPP+作用PC12细胞48 h,能明显抑制细胞生长(P<0.01),诱导细胞发生凋亡,同时降低MMP,增加活性Caspase-3的蛋白水平。同时给予不同浓度PS-F处理,PC12细胞存活率增加(P<0.01);凋亡细胞量减少;MMP增高;活性Caspase-3蛋白水平降低(P<0.01)。结论 PS-F能抑制MPP+诱导的PC12细胞的凋亡,其作用机制可能与维持线粒体正常膜电位,稳定线粒体功能,降低活性Caspase-3蛋白水平有关。     

神经妥乐平对H2O2致PC12细胞损伤的保护作用

目的 探索神经妥乐平对H2O2诱导的PC12细胞氧化应激损伤的影响及其潜在机制.方法 用CCK-8法检测细胞存活率,以流式细胞术检测细胞氧化损伤的发生、细胞内活性氧的生成及线粒体膜电位的变化,荧光显微镜观察细胞内活性氧的产生,qRT-PCR测定Caspase-3、Bax和Bcl-2 mRNA的表达.结果 PC12细胞存活率随H2O2浓度的增加而逐渐下降.其中,450μM H2O2处理细胞24 h后细胞存活率、凋亡率、坏死率明显降低;细胞内活性氧水平表达明显升高;线粒体膜电位JC-1红/绿荧光比值下降;Bax和Caspase-3的mRNA表达升高,而Bcl-2的mRNA表达下降,以上指标与对照组相比,差异有统计学意义(P＜0.05).而预先给予0.01UN/ml的NTP处理细胞12h可明显提高细胞存活率,降低细胞凋亡率和坏死率,减少细胞内活性氧生成并提高线粒体膜电位,抑制Bax和Caspase-3的mRNA表达,促进Bcl-2 mRNA的表达,以上指标与H2O2组相比,差异有统计学意义(P＜0.05).结论 NTP能抑制H2O2诱导的PC12细胞损伤,其神经细胞保护作用可能与其降低细胞内活性氧水平、维持线粒体膜电位的高能状态和抑制促凋亡基因表达、促进抗凋亡基因表达有关.     

葛根素保护双氧水诱导的SH-SY5Y细胞凋亡

目的探讨葛根素对双氧水(H_2O_2)诱导的SH-SY5Y细胞凋亡的保护作用及其机制。方法建立体外神经元损伤模型,MTT法观察细胞存活率;Hoechst 33342染色观察细胞核改变;JC-1染色检测细胞线粒体膜电位的改变;酶活性检测线粒体caspase-3和caspase-9的变化;Western blot检测细胞中Bcl-2、Bax、p-Akt、Akt蛋白的表达。结果与H_2O_2模型组相比,葛根素预处理能明显改善H_2O_2诱导的SHSY5Y细胞存活率下降(P<0.05),缓解H_2O_2引起的线粒体膜电位的下降(P<0.01),抑制caspase-3和caspase-9的酶活性(P<0.01),减少H_2O_2诱导的细胞凋亡。此外,葛根素还促进细胞内p-Akt、Bcl-2蛋白表达,抑制Bax蛋白表达,而这种作用能被PI3K/Akt的抑制剂LY294002所抑制。结论葛根素可保护H_2O_2诱导的SH-SY5Y细胞凋亡,这种保护作用可能是通过激活PI3K/Akt信号通路实现的。     

类叶升麻苷对鱼藤酮致SH-SY5Y细胞凋亡的保护作用

目的探讨类叶升麻苷对鱼藤酮致多巴胺能神经元SH-SY5Y细胞凋亡的保护作用及其机制。方法采用MTT法检测细胞存活率,以荧光染料Hoechst33342染色分析细胞核的形态学变化,用流式细胞仪定量分析细胞凋亡峰,以2,′7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)为标记探针检测细胞内活性氧的产生。结果①0.5μmol.L-1的鱼藤酮处理SH-SY5Y细胞48 h能引起细胞存活率的显著下降;诱导细胞发生凋亡,凋亡率达47.39%;大部分细胞胞体皱缩,突起缩短消失或断裂;染色质皱缩、浓缩、断裂及形成凋亡小体;细胞内活性氧水平上升。②预先用盐生肉苁蓉提取物类叶升麻苷(10,20或40 mg.L-1)处理细胞6 h,可提高细胞存活率;明显改善鱼藤酮引起的细胞形态学变化;流式细胞仪检测凋亡率分别降低到25.87%,23.97%,10.45%;以DCFH-DA为标记探针检测到20 mg.L-1类叶升麻苷可明显抑制鱼藤酮引起的细胞内活性氧产生。结论类叶升麻苷能抑制鱼藤酮诱导的多巴胺能神经元SH-SY5Y细胞凋亡,其神经细胞保护作用可能与降低细胞内活性氧水平有关。     

新疆家蚕抗菌肽诱导人胃癌细胞AGS凋亡的研究

目的探讨新疆家蚕抗菌肽(cecropin XJ)是否通过诱导人胃癌细胞AGS凋亡产生抗肿瘤的作用。方法选择0.01~1 000 mg·L-1浓度范围内的cecropin XJ与人胃癌细胞AGS和人正常胃上皮细胞GES-1共培养24 h,采用MTT法检测cecropin XJ对AGS细胞和GES-1细胞增殖的影响;透射电镜观察细胞超微结构变化;Hoechst染色观察细胞凋亡情况;流式细胞术检测细胞内活性氧和线粒体膜电位的变化;实时荧光定量PCR(qRT-PCR)和Western blot检测Bax、Bcl-2、caspase-3以及细胞色素C mRNA和蛋白水平的表达变化。结果 Cecropin XJ在体外能明显抑制胃癌AGS细胞的增殖(P<0.05),并具有浓度依赖性,IC50值为61.19 mg·L-1,但对GES-1细胞无明显的抑制增殖作用。经cecropin XJ处理24 h后,AGS细胞核固缩,呈现典型细胞凋亡特征,同时细胞内活性氧增加,线粒体膜电位下降。qRT-PCR和Western blot结果表明,cecropin XJ能够引起Bcl-2表达下调,Bax表达上调,促进细胞色素C的释放并活化caspase-3。Cecropin XJ促进caspase-3活性呈剂量依赖,经caspase-3和caspase-9特异性抑制剂处理后可降低cecropin XJ介导的AGS细胞死亡率。结论 Cecropin XJ可通过下调Bcl-2表达,上调Bax表达和活化caspase-3诱导AGS细胞凋亡,是其抗肿瘤机制之一。     

人参皂苷Rg1可能通过丝裂素活化蛋白激酶途径抑制细胞凋亡人参皂苷Rg1可能通过丝裂素活化蛋白激酶途径抑制细胞凋亡

目的探讨人参皂苷Rg1对MPP⁺诱导细胞凋亡保护作用的可能信号传导途径。方法用吖啶橙-溴化乙锭染色观察SHSY5Y细胞凋亡率,流式细胞仪检测细胞内活性氧ROS水平,Western Blotting法检测JNK(c-jun NH₂-terminal kinase)激酶活性,免疫细胞化学染色法检测裂解的Caspase-3阳性细胞的表达率。结果经10 μmol·L^-1 Rg1或2.5 mmol·L^-1 N-乙酰半胱氨酸预处理后,MPP⁺诱导的SHSY5Y细胞凋亡受到明显抑制,同时细胞内ROS下降,JNK激酶的活性减弱,裂解的Caspase-3阳性细胞表达率下降。结论Rg1可抑制MPP⁺诱导的SHSY5Y细胞凋亡,其作用机制可能是通过清除ROS、减弱JNK激酶的活性,从而减少Caspase-3的激活     

Protective effect of verbascoside on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells

The neuroprotective effects of verbascoside, one of phenylpropanoid glucoside isolated from the Chinese herbal medicine Buddleja officinalis Maxim, on 1-methyl-4-phenylpyridinium ion (MPP(+)) induced apoptosis and oxidative stress in PC12 neuronal cells were investigated. Treatment of PC12 cells with MPP(+) for 48 h induced apoptotic death as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, the activation of caspase-3 measured by the caspase-3 activity assay kit, the reduction in mitochondrial membrane potential with laser scanning confocal microscopy and the increase in the extracellular hydrogen peroxide level. Simultaneous treatment with verbascoside markedly attenuated MPP(+)-induced apoptotic death, increased extracellular hydrogen peroxide level, the activation of caspase-3 and the collapse of mitochondrial membrane potential. These results strongly indicate that verbascoside may provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.     

Tetrahydroxystilbene glucoside protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity

1-methyl-4-phenylpyridinium (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin for inducing a cell model of Parkinson's disease. This study aimed to evaluate the effects of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from Polygonum multiflorum, on MPP+-induced cytotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. The results from the MTT and lactate dehydrogenase (LDH) assays showed that incubating cells with 500 μM MPP+ for 24 h decreased cell viability and increased LDH leakage, whereas preincubating cells with 3.125 to 50 μM TSG for 24 h protected the cells against MPP+-induced cell damage. Using 2',7'-dichlorofluorescin diacetate (DCFH-DA) and rhodamine 123, respectively, we found that TSG inhibited both the elevation of intracellular reactive oxygen species and the disruption of mitochondrial membrane potential induced by MPP+. In addition, TSG suppressed both the upregulation of the ratio of Bax to Bcl-2 and the activation of caspase-3 induced by MPP+, and TSG inhibited apoptosis as detected by flow cytometric analysis using Annexin-V and propidium (PI) label. These results suggest that TSG may protect neurons against MPP+-induced cell death through improving mitochondrial function, decreasing oxidative stress and inhibiting apoptosis, and this may provide a potentially new strategy for preventing and treating neurodegenerative disorders such as Parkinson's disease.     

Protocatechuic acid suppresses MPP+ -induced mitochondrial dysfunction and apoptotic cell death in PC12 cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, showed antioxidant neuroprotective effect in our previous study. Here, we investigated the effect of PCA on the MPP(+)-induced mitochondrial dysfunction and apoptotic cell death in PC12 cells. The apoptosis in MPP(+)-induced PC12 cells was associated with loss of mitochondrial membrane potential, the formation of reactive oxygen species (ROS), GSH depletion, activation of caspase-3 and down-regulation of Bcl-2. In contrast, treatment of PC12 cells with PCA significantly prevented the above-mentioned mitochondrial dysfunction. Our data pointed to the potential clinical application/use of PCA to overcome neurodegenerative diseases such as Parkinson's disease.     

Protective effect of tubuloside B on TNFalpha-induced apoptosis in neuronal cells.

AIM: To investigate the neuroprotective effect of tubuloside B, one of the phenylethanoids isolated from the stems of Cistanche salsa, on tumor necrosis factor-alpha (TNFalpha)-induced apoptosis in SH-SY5Y neuronal cells. METHODS: Cell viability was analyzed using MTT assay. Apoptotic cells were detected using Hoechst33342 staining, and confirmed by DNA fragmentation and flow cytometric analysis. The activity of caspase-3 was measured with special assay kit. The concentration of free intracellular calcium was determined with the probe Indo-1 by spectrometer. The level of intracellular reactive oxygen species and the potential of mitochondrial membrane were determined by laser scanning confocal microscopy (LSCM) combined with fluorescence probe H2DCFDA or JC-1 respectively. RESULTS: SH-SY5Y cells treated with TNFalpha 100 microg/L for 36 h showed typical morphological changes of apoptosis. DNA ladder could be observed by agarose gel electrophoresis. The highest percentage of apoptotic cells accumulated to 37.5 %. Following 36 h treatment with TNFalpha, accumulation of intracellular ROS and [Ca2+]i and decrease in mitochondrial membrane potential were observed, and caspase-3 activity increased by about five-fold compared with controls. However, pretreatment with tubuloside B (1, 10, or 100 mg/L) for 2 h attenuated the TNFalpha-mediated apoptosis. The antiapoptotic action of tubuloside B was partially dependent on an anti-oxidative stress effects, maintain of mitochondria function, decrease of concentration of free intracellular calcium and inhibition of caspase-3 activity. CONCLUSION: Tubuloside B has the neuroprotective capacity to antagonize TNFalpha-induced apoptosis in SH-SY5Y cells and may be useful in treating some neurodegenerative diseases.     

Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro

Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. We have investigated the role of AST in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra (SN) neurons in the mouse model of Parkinson’s disease (PD) and 1-methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity of SH-SY5Y human neuroblastoma cells. In in vitro study, AST inhibits MPP+-induced production of intracellular reactive oxygen species (ROS) and cytotoxicity in SH-SY5Y human neuroblastoma cells. Preincubation of AST (50 μM) significantly attenuates MPP+-induced oxidative damage. Furthermore, AST is able to enhance the expression of Bcl-2 protein but reduce the expression of α-synuclein and Bax, and suppress the cleavage of caspase-3. Our results suggest that the protective effects of AST on MPP+-induced apoptosis may be due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of superoxide dismutase (SOD) and catalase and regulating the expression of Bcl-2 and Bax. Pretreatment with AST (30 mg/kg) markedly increases tyrosine hydroxylase (TH)-positive neurons and decreases the argyrophilic neurons compared with the MPTP model group. In summary, AST shows protection from MPP+/MPTP-induced apoptosis in the SH-SY5Y cells and PD model mouse SN neurons, and this effect may be attributable to upregulation of the expression of Bcl-2 protein, downregulation of the expression of Bax and α-synuclein, and inhibition of the activation of caspase-3. These data indicate that AST may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson’s disease.     

脂质立方液晶设计制备与表征评价研究进展

目的：研究伊拉地平（ ISR ）对1-甲基-4-苯基吡啶离子（ MPP＋）损伤的PC12细胞的保护作用及可能机制。方法MPP ＋处理PC12细胞建立帕金森病细胞模型;4-甲基偶氮唑蓝（ MTT）比色法检测细胞存活率;双氯荧光黄乙酸乙酯（ DCFH-DA）染色流式细胞术检测细胞内活性氧（ ROS）的生成;JC-1染色流式细胞术检测细胞线粒体膜电位（ MMP）。结果1 mmol · L－1MPP＋处理PC12细胞24 h后能明显抑制细胞生长（P＜0．01）;降低线粒体膜电位;ROS含量增加。2μmol· L－1伊拉地平预处理后, PC12细胞存活率显著增加（ P＜0．01）;线粒体膜电位升高;ROS生成减少。结论伊拉地平对MPP＋损伤的PC12细胞具有保护作用,其作用机制可能与维持线粒体正常膜电位,稳定线粒体功能,阻止线粒体氧化应激发生有关。     

Protocatechuic acid inhibits apoptosis by mitochondrial dysfunction in rotenone-induced PC12 cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, showed the significant neuroprotective effects on hydrogen peroxide (H2O2) or MPP+-induced apoptosis in cultured PC12 cells. However, the mechanism how PCA suppresses rotenone-induced neurotoxicity in cultured PC12 cells remains unclear. In this study, we investigated the protective effects of PCA in PC12 cells exposed to rotenone as an in vitro model of mitochondrial dysfunction and apoptotic cell death. The apoptosis in rotenone-induced PC12 cells was accompanied by the loss of mitochondrial membrane potential, the formation of reactive oxygen species (ROS), the total glutathione depletion, activation of caspase-3 and down-regulation of Bcl-2. In contrast, PCA markedly attenuated the above-mentioned mitochondrial dysfunction in a dose-dependent manner. Taken together, these results suggest that treatment of PC12 cells with PCA can block rotenone-induced apoptosis via ameliorating the mitochondrial dysfunction.