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。     

毛蕊花苷对MPP~+诱导的SHSY5Y细胞凋亡的保护作用

目的观察肉苁蓉提取物毛蕊花苷对MPP+诱导的SHSY5Y细胞损伤的影响。方法用MTT法检测细胞存活率,以流式细胞仪检测细胞内活性氧的产生和线粒体膜电位的变化,以及细胞凋亡的发生,并用荧光酶标仪测定caspase-3的活性,蛋白印迹测定Bcl-2的表达水平。结果200μmol·L-1MPP+处理细胞24h降低细胞的存活率;诱导细胞发生凋亡,凋亡率达38.9%;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。而预先给予0.1、1或者10mg·L-1浓度的毛蕊花苷处理细胞12h,可提高细胞存活率;流式细胞仪检测凋亡率分别降低到29.5%,15.3%和8.6%,而且细胞内活性氧的水平明显降低,并可逐渐恢复线粒体的高能量状态;caspase-3的活性不断降低,Bcl-2的表达水平增高,并呈现一定的剂量依赖性。结论毛蕊花苷能抑制MPP+诱导的SHSY5Y细胞凋亡,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caspase-3的活性有关。     

Beta-asarone attenuates beta-amyloid-induced apoptosis through the inhibition of the activation of apoptosis signal-regulating kinase 1 in SH-SY5Y cells

Beta-amyloid (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that beta-asarone improves cognitive function by suppressing neuronal apoptosis in vivo. In this study, we assessed the neuroprotective effects of beta-asarone against the toxicity of Abeta in relation to the mitochondria-mediated cell death process, and to elucidated the role of the ASK1/MKK7/JNK and mitochondrial pathways in beta-asarone-induced neuroprotection in SH-SY5Y cells. Our results show that beta-asarone afforded protection against Abeta-induced toxicity by inhibiting apoptosis in SH-SY5Y cells. This result was also confirmed by caspase-9 and caspase-3 activity assays. Expression of p-ASK1, p-MKK7, p-JNK, Bax, Bad, and cytochrome c release decreased after pretreatment with beta-asarone in SH-SY5Y cells exposed to A1-42. Interestingly, these effects of beta-asarone against Abeta1-42 insult were enhanced by ASK1 siRNA. These findings suggest that beta-asarone prevents Abeta1-42-induced neurotoxicity through attenuating neuronal apoptosis, and might be a potential preventive or therapeutic agent for AD.     

Inhibitory effects of 1,25-dihydroxyvitamin D3 and its low-calcemic analogues on staurosporine-induced apoptosis

The active form of vitamin D3 and some of its related compounds show neuroprotective effects in various models of neuronal damage, however, mechanism of their anti-apoptotic action has not been elucidated. Therefore, the present study was designed to investigate the effects of 1,25-dihydroxyvitamin D3 and its low-calcemic analogues, PRI-2191, PRI-1890 and PRI-1901 on staurosporine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Twenty-four hour incubation with staurosporine (1 microM) enhanced the caspase-3 activity, decreased mitochondrial membrane potential and increased the number of apoptotic cells as visualized by Hoechst staining. 1,25-Dihydroxyvitamin D3 and PRI-2191 attenuated the staurosporine-induced caspase-3 activity at 5, 50 and 500 nM, whereas PRI-1890 and PRI-1901 were active only at higher concentrations. Furthermore, 1,25-dihydroxyvitamin D3 (50 and 500 nM) and PRI-2191 (500 but not 50 nM) reversed the staurosporine-evoked decrease in mitochondrial membrane potential. Hoechst and calcein staining confirmed the neuroprotective effects of the secosteroids under study. Further study revealed that a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, at concentration of 100 nM antagonized the effect of 1,25-dihydroxyvitamin D3 and PRI-2191 on staurosporine-induced caspase-3 activation. These data indicate that 1,25-dihydroxyvitamin D3 and its low-calcemic analogues at nanomolar concentrations inhibited mitochondrial pathway of apoptosis in SH-SY5Y neuronal cells, though with different potency. Moreover, the activation of PI3-K/Akt signaling pathway appears to play a role in anti-apoptotic effects of the secosteroids.     

Detailed in vitro pharmacological analysis of FK506-induced neuroprotection

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.     

Silica nanoparticles induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway

The present study was to investigate effects of Silica nanoparticles (SiNPs) on nervous system and explore potential mechanisms in human neuroblastoma cells (SH-SY5Y). Cytotoxicity was detected by cell viability and Lactate dehydrogenase (LDH) release. Flow cytometry analysis was applied to assess mitochondrial membrane potential (MMP) loss, intracellular Ca²⁺ and apoptosis. To clarify the mechanism of SiNPs-induced apoptosis, intrinsic apoptosis-related proteins were detected. Our results showed that SiNPs caused cytotoxicity, cell membrane damage and Ca²⁺ increase in a dose-dependent manner in SH-SY5Y cells. Both the mitochondrial membrane potential (MMP) loss and potential mitochondria damage resulted in Cyt C release to the cytoplasm. The elevated Cyt C and Apaf1 further triggered intrinsic apoptosis via executive molecular caspase-9 and caspase-3. The present study confirmed that SiNPs induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway and provided a better understanding of the potential toxicity induced by SiNPs on human neurocyte.     

Tubuloside B from Cistanche salsa rescues the PC12 neuronal cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis and oxidative stress

The neuroprotective effects of tubuloside B, one of the phenylethanoids isolated from the Chinese herbal medicine Cistanche salsa, on 1-methyl-4-phenylpyridinium ion (MPP +)-induced apoptosis and oxidative stress in PC12 neuronal cells were investigated. PC12 cells treated with MPP + underwent apoptotic death as determined by MTT assay, flow cytometry and DNA agarose gel electrophoresis; intracellular accumulation of reactive oxygen species (ROS) was measured by DCFH-DA staining with laser scanning confocal microscopy (LSCM). Simultaneous treatment with tubuloside B markedly attenuated MPP +-induced cytotoxicity, DNA fragmentation, and intracellular accumulation of ROS. These results strongly indicate that tubuloside B prevents MPP +-induced apoptosis and oxidative stress. Tubuloside B may be applied as an antiparkinsonian agent.     

Curcumin inhibits appoptosin-induced apoptosis via upregulating heme oxygenase-1 expression in SH-SY5Y cells

Aim:

Appoptosin (SLC25A38) is a pro-apoptotic protein, which is upregulated in Alzheimer''s disease (AD) brains and plays an important role in promoting the pathological progress of AD. The aim of this study was to investigate the effects of curcumin from the rhizome of Curcuma longa on appoptosin-induced apoptosis in SH-SY5Y cells.

Methods:

SH-SY5Y cells were pretreated with curcumin, then transfected with appoptosin or vector. The apoptotic cells were detected with Annexin V staining analysis by flow cytometry. The expression of cleaved caspase-3, appoptosin, heme oxygenase-1 (HO-1) was examined using Western blotting. Intracellular level of ROS was measured with DCFH-DA staining by flow cytometry analysis. Mitochondrial membrane potential (ΔΨm) was detected with JC-1 staining under a fluorescence microscope and quantified by fluorescence ratio detection.Overexpression of appoptosin in SH-SY5Y cells markedly increased cell apoptosis accompanied by reduced HO-1 expression, increased intracellular heme level, ROS overproduction and ΔΨm impairment. Treatment of SH-SY5Y cells with curcumin (2.5–20 μmol/L) for 24 h did not significantly affect their viability. However, pretreatment with curcumin (2.5–20 μmol/L) dose-dependently attenuated all above-mentioned pathological changes in appoptosin-transfected SH-SY5Y cells.

Results:

Overexpression of appoptosin in SH-SY5Y cells markedly increased cell apoptosis accompanied by reduced HO-1 expression, increased intracellular heme level, ROS overproduction and ΔΨm impairment. Treatment of SH-SY5Y cells with curcumin (2.5–20 μmol/L) for 24 h did not significantly affect their viability. However, pretreatment with curcumin (2.5–20 μmol/L) dose-dependently attenuated all above-mentioned pathological changes in appoptosin-transfected SH-SY5Y cells.

Conclusion:

Curcumin inhibits appoptosin-induced apoptosis in SH-SY5Y cells by upregulating the expression of HO-1, reducing the production of intracellular heme and ROS, and preventing the ΔΨm loss.     

Effect of fraxetin and myricetin on rotenone-induced cytotoxicity in SH-SY5Y cells: comparison with N-acetylcysteine

The purpose of this study was to investigate the potential neuroprotective effects of myricetin (flavonoid) and fraxetin (coumarin) on rotenone-induced apoptosis in SH-SY5Y cells, and the possible signal pathway involved in a neuronal cell model of Parkinson's disease. These two compounds were compared to N-acetylcysteine. The viability of cells was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cytotoxicity was assayed by lactate dehydrogenase (LDH) released into the culture medium. Parameters related to apoptosis, such as caspase-3 activity, the cleavage of poly(ADP-ribose) polymerase and the levels of reactive oxygen species were also determined. Rotenone caused a time- and dose-dependent decrease in cell viability and the degree of LDH release was proportionally to the effects on cell viability. Cells were pretreated with fraxetin, myricetin and N-acetylcysteine at different concentrations for 30 min before exposure to rotenone. Cytotoxicity of rotenone (5 microM) for 16 h was significantly diminished as well as the release of LDH into the medium, by the effect of fraxetin, myricetin and N-acetylcysteine, with fraxetin (100 microM) and N-acetylcysteine (100 microM) being more effective than myricetin (50 microM). Rotenone-induced apoptosis in SH-SY5Y cells was detected by an increase in caspase-3 activity and in the cleavage of poly(ADP-ribose) polymerase. After exposing these cells to rotenone, a significant increase in reactive oxygen species preceded apoptotic events. Fraxetin (100 microM) and N-acetylcysteine (100 microM) not only reduced rotenone-induced reactive oxygen species formation, but also attenuated caspase-3 activity and poly(ADP-ribose) polymerase cleavage at 16 h against rotenone-induced apoptosis. The effect of fraxetin in both experiments was similar to that of N-acetylcysteine. These results demonstrated the protective action of fraxetin and suggest that it can reduce apoptosis, possibly by decreasing free radical generation in SH-SY5Y cells. Myricetin at 100 microM was without any preventive effect.     

香椿果抗补体活性多酚对补体损伤神经细胞的保护作用研究

目的评价香椿果抗补体活性多酚XCG-7对补体损伤神经细胞的保护作用。方法采用眼镜蛇毒因子特异激活血清补体,诱导人神经母细胞瘤细胞SH-SY5Y损伤。通过测定乳酸脱氢酶释放量、细胞活力和Caspase-3/7活性等指标,评价XCG-7对补体损伤神经细胞的保护作用。结果 XCG-7对SH-SY5Y细胞的生长具有促进作用,明显抑制补体损伤导致的细胞活力下降,在一定程度上减轻乳酸脱氢酶的释放,抑制细胞凋亡的发生。结论香椿果抗补体活性多酚XCG-7能促进SH-SY5Y细胞的生长,对补体损伤神经细胞具有一定的保护作用。     

Potential autophagy enhancers protect against fipronil-induced apoptosis in SH-SY5Y cells

Oxidative stress created by environmental toxicants activates several signaling pathways. Autophagy is one of the first lines of defense against oxidative stress damage. The autophagy pathway can be induced and up-regulated in response to intracellular reactive oxygen species (ROS). Recently, we reported that fipronil (FPN)-induced mitochondria-dependent apoptosis is mediated through ROS in human neuroblastoma SH-SY5Y cells. In this study, we explored the role of autophagy to prevent FPN neurotoxicity. We investigated the modulation of FPN-induced apoptosis according to autophagy regulation. FPN activated caspase-9 and caspase-3, and induced nuclear fragmentation and condensation, all of which indicate that FPN-induced cell death was due to apoptosis. In addition, we observed FPN-induced autophagic cell death by monitoring the expression of LC3-II and Beclin-1. Exposure to FPN in SH-SY5Y cells led to the production of ROS. Treatment with N-acetyl-cysteine (NAC) effectively blocked both apoptosis and autophagy. Interestingly, pretreatment with rapamycin, an autophagy inducer, significantly enhanced the viability of FPN-exposed cells; the enhancement of cell viability was partially due to alleviation of FPN-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment with 3-methyladenine (3MA) a specific inhibitor for autophagy, remarkably strengthened FPN toxicity and further induced activation of caspase-3 in these cells. Our studies suggest that FPN-induced cytotoxicity is modified by autophagy regulation and that rapamycin is neuroprotective against FPN-induced apoptosis through enhancing autophagy.     

Lonicera japonica THUNB. protects 6-hydroxydopamine-induced neurotoxicity by inhibiting activation of MAPKs, PI3K/Akt, and NF-κB in SH-SY5Y cells

In this study, we investigated the neuroprotective effects of Lonicera japonica THUNB. extract (LJ) on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. We found that LJ significantly increased cell viability decrease, lactate dehydrogenase release (LDH), morphological changes, nuclear condensation, fragmentation, and reactive oxygen species (ROS) production induced by 6-OHDA in SH-SY5Y cells. The cytoprotection afforded by pretreatment with LJ was associated with increases of the glutathione (GSH) level, superoxide dismutase (SOD) activity, and catalase (CAT) activity in 6-OHDA-induced SH-SY5Y cells. In addition, LJ strikingly inhibited 6-OHDA-induced mitochondrial dysfunctions including reduction of mitochondria membrane potential (MMP) and activation of cleaved poly-ADP-ribose polymerase (PARP), cleaved caspase-3, cleaved caspase-9, increased Bax, as well as decreased Bcl-2 and Bcl-xL. Additionally, LJ dramatically attenuated 6-OHDA-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphoinositide 3-kinase (PI3K)/Akt. Meanwhile, LJ counteracted nuclear factor-κB (NF-κB) activation by blocking its translocation to the nucleus. These findings suggest that LJ has a potent anti-parkinsonism; this effect was mediated, at least in part, by inhibition of neurotoxicity, apoptotic cascade events, and oxidative stress via activation of MAPKs, PI3K/Akt, and NF-κB.     

Neuroprotection by marine-derived compound, 11-dehydrosinulariolide, in an in vitro Parkinson’s model: a promising candidate for the treatment of Parkinson’s disease

Parkinson's disease (PD) is a neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and gait impairment. So far, very few pharmacological agents have been isolated or developed that effectively inhibit the progression of PD. However, several studies have demonstrated that inflammatory processes play critical roles in PD. Therefore, anti-inflammatory agents may suppress disease progression in PD. 11-Dehydrosinulariolide was isolated from cultured soft corals. The anti-inflammatory effect of this molecule has been observed through suppression of the expression of two main pro-inflammatory proteins: inducible nitric oxide synthase and cyclooxygenase-2, in lipopolysaccharide-stimulated macrophage cells. We also found that 11-dehydrosinulariolide significantly reduced 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and apoptosis in a human neuroblastoma cell line (SH-SY5Y). The pharmacological activity of this compound has been studied, and it is associated with the inhibition of 6-OHDA-induced activation of caspase-3 and translocation of nuclear factor kappa B. 11-Dehydrosinulariolide increased the activation of survival-signaling phospho-Akt but not phospho-ERK. The neuroprotective effect of 11-dehydrosinulariolide was assessed here using 6-OHDA-treated SH-SY5Y cells, wherein neuroprotection is mediated through regulation of phosphatidylinositol 3-kinase (PI3K). Furthermore, 11-dehydrosinulariolide caused a significant decrease in caspase-3/7 activity in comparison to the 6-OHDA-treated group, indicating that 11-dehydrosinulariolide has neuroprotective properties. We conclude that 11-dehydrosinulariolide is a promising candidate for the treatment of Parkinson's disease through its anti-apoptotic and anti-inflammatory action via PI3K signaling.     

Tricin 7-glucoside protects against experimental cerebral ischemia by reduction of NF-κB and HMGB1 expression

There have been several studies of nuclear factor-κB (NF-κB) and high-mobility group box1 (HMGB1) associated with the pathophysiology of cerebral ischemia. Tricin 7-glucoside, a major bioactive compound extracted from Sedum sarmentosum Bunge. The objectives of this study were to determine the effects of Tricin 7-glucoside on a cultured neuronal cell line, SH-SY5Y in vitro and experimental ischemic stroke in vivo. For oxygen-glucose deprivation (OGD) and tumor necrosis factor-α (TNF-α) stimulated SH-SY5Y cell line in vitro, SH-SY5Y cells were incubated with Tricin 7-glucoside. For in vivo experiment, rats were subjected to middle cerebral artery occlusion (MACO) for 1h, then followed by reperfusion for 23 h. Treatment of SH-SY5Y cells with Tricin 7-glucoside reduced the OGD-induced apoptosis and cytotoxicity, blocked TNF-α-induced NF-κB and IκB-α phosphorylation, and decreased HMGB1 expression. At doses higher than 50mg/kg, Tricin 7-glucoside produced a significant neuroprotective potential in rats with ischemia and reperfusion (I/R). Tricin 7-glucoside (100mg/kg) demonstrated significant neuroprotective activity even after delayed administration at 2h and 4h after I/R. Tricin 7-glucoside 100mg/kg attenuated histopathological damage, decreased brain edema, inhibited NF-κB activation and reduced HMGB1 expression. These data show that Tricin 7-glucoside protects brain against I/R injury with a favorable therapeutic time-window by alleviating cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and its protective effects may involve HMGB1 and NF-κB signaling pathway.     

Diclofenac, a non-steroidal anti-inflammatory drug, suppresses apoptosis induced by endoplasmic reticulum stresses by inhibiting caspase signaling

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in the treatment of inflammation and pain. In many reports, NSAIDs have induced apoptosis in a variety of cell lines such as colon cancer cells. On the other hand, more recently a few reports have found that NSAIDs protect against apoptosis. Here we investigate endoplasmic reticulum (ER)-stress-induced apoptosis of neuronal cells. The aim of this study is to examine the involvement of NSAIDs, in particular diclofenac, on ER-stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. Diclofenac significantly suppressed SH-SY5Y cell death induced by two types of ER-stress-inducing agents: thapsigargin, an inhibitor of Ca2+-ATPase on the endoplasmic reticulum membrane, and tunicamycin, a glycosylation blocker. Other NSAIDs, such as indomethacin, ibuprofen, aspirin, and ketoprofen, also suppressed ER-stress-induced SH-SY5Y cell death. The dose-dependent anti-apoptotic effect of diclofenac did not correlate with the reduction of prostaglandin release. Administration of prostaglandin E2, which was a primary product of arachidonic metabolism, showed no effects against anti-apoptotic effects produced by diclofenac. Thapsigargin and tunicamycin each significantly activated caspase-3, -9, and -2 in the intrinsic apoptotic pathway in SH-SY5Y cells. Diclofenac suppressed the activation of caspases induced by both ER stresses. Thapsigargin and tunicamycin decreased the mitochondrial membrane potential in SH-SY5Y cells. Diclofenac suppressed the mitochondrial depolarization induced by both ER stresses. Diclofenac inhibited ER-stress-induced apoptosis of SH-SY5Y cells by suppressing the activation of caspases in the intrinsic apoptotic pathway. This is the first report to find that diclofenac has protective effects against ER-stress-induced apoptosis.     

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。     

Ara-C induces apoptosis in monkey fibroblast cells.

The effect of cytosine arabinoside (Ara-C) on cell viability has been studied in African green monkey kidney fibroblasts (CV1-P). It has been shown previously that Ara-C- induced cell death in neurons is mediated by apoptosis. We investigated whether Ara-C can induce apoptosis also in CV1-P cells, and if the apoptosis is p53-associated. For comparison, human neuroblastoma cells (SH-SY5Y) were studied as a model of human neuronal cells. SYTO13/propidium iodide staining revealed condensed and fragmented nuclei in both cell lines. Ara-C treatment for 48 h induced approximately 24% apoptosis in CV1-P cells whereas approximately 55% of SH-SY5Y cells were apoptotic. Ara-C increased the level of p53 in both CV1-P and SH-SY5Y cells compared to control. The maximum level of p53 in SH-SY5Y cells was reached at 12 h and this then rapidly faded whereas CV1-P cells p53 levels remained elevated after reaching their maximum. Caspase-3 activity was 5-fold higher in human neuroblastoma cells than in monkey fibroblasts, this reflected the decreased cell viability. Our results prove that Ara-C- induced apoptosis in CV1-P cells is associated with an increase of p53 and activation of caspase-3. Ara-C-induced toxicity in CV1-P cells is modest compared to that seen in neuronal cells.     

DCMQA,a caffeoylquinic acid derivative alleviates NMDA-induced neurotoxicity via modulating GluN2A and GluN2B-containing NMDA receptors in vitro

Compound DCMQA (4, 5-O-dicaffeoyl-1-O-[4-malic acid methyl ester]-quinic acid) isa natural caffeoylquinic acid derivative isolated from Arctium lappa L. roots. Caffeoylquinic acid derivatives have been reported to possess neuroprotective effects through inhibiting oxidative stress and apoptosis in vitro. However, whether DCMQA exerts protective effects on N-methyl-D-aspartate (NMDA)-induced neurotoxicity and the underlying mechanism has not been elucidated. In this study, the results indicated that pretreatment of DCMQA prevented the loss of cell viability and attenuated the LDH leakage in SH-SY5Y cells exposed to NMDA. Hoechst 33342 staining and Annexin V-PI double staining illustrated that DCMQA suppressed NMDA-induced morphological damage and neuronal apoptosis. Moreover, DCMQA inhibited NMDA-mediated Ca²⁺ influx, excessive intracellular ROS generation and loss of mitochondrial membrane potential (MMP). Western blot analysis showed that DCMQA attenuated the Bax/Bcl-2 ratio, release of cytochrome c as well as expression of caspase-9 and caspase-3. Besides, DCMQA down-regulated GluN2B-containing NMDA receptors (NMDARs) and up-regulated GluN2A-containing NMDARs, promoted the disruption of nNOS and PSD95 as well as activation of CaMK II-α. Furthermore, computational docking study indicated that DCMQA possessed a good affinity for NMDARs. These results indicated that DCMQA protects SH-SY5Y cells against NMDA-induced neuronal damage. In addition, the underlying mechanisms of DCMQA-mediated neuroprotection are associated with modulating NMDARs and disruption of nNOS-PSD95 as well as the activation of CaMK II-α.     

9-Benzylidene-naphtho[2,3-b]thiophen-4-ones and benzylidene-9(10H)-anthracenones as novel tubulin interacting agents with high apoptosis-inducing activity

Tubulin-binding 9-benzylidene-naphtho[2,3-b]thiophen-4-ones 1a and 1b and benzylidene-9(10H)-anthracenone 2 were evaluated for their ability to induce cell death. We examined the effect of the molecules on cell cycle progression, organization of microtubule networks, and apoptosis induction. As determined by flow cytometry, cancer cells were predominantly arrested in metaphase with 4N DNA before cell death occurred. By using indirect immunofluorescence techniques we visualized microtubule depolymerization recognizable by short microtubule fragments scattered around the nucleus. The incubation with 1a and 2 resulted in chromatin condensation, nuclear fragmentation, and cell shrinkage, which are, among others, typical features of apoptotic cell death. Furthermore, time- and dose-dependent induction of apoptosis in SH-SY5Y cells was detected via cleavage of Ac-DEVD-AMC, a fluorigenic substrate for caspase-3. We observed a lower apoptotic activity in neuroblastoma cells overexpressing Bcl-xL, suggesting activation of the mitochondrial apoptosis pathway. Western blot analysis demonstrated that caspase-3, an apoptosis mediator, was activated in a time-dependent manner after exposure of SH-SY5Y cells to drugs 1a and 2. Taken together, the agents investigated in the present study display strong apoptosis-inducing activity and therefore show promise for the development of novel chemotherapeutics.