Neuroprotection of N-benzylcinnamide on scopolamine-induced cholinergic dysfunction in human SH-SY5Y neuroblastoma cells

Alzheimer's disease, a progressive neurodegenerative disease, affects learning and memory resulting from cholinergic dysfunction. Scopolamine has been employed to induce Alzheimer's disease-like pathology in vivo and in vitro through alteration of cholinergic system. N-benzylcinnamide (PT-3), purified from Piper submultinerve, has been shown to exhibit neuroprotective properties against amyloid-β-induced neuronal toxicity in rat cortical primary cell culture and to improve spatial learning and memory of aged rats through alleviating oxidative stress. We proposed a hypothesis that PT3 has a neuroprotective effect against scopolamine-induced cholinergic dysfunction. PT-3 (125–200 nM) pretreatment was performed in human neuroblastoma SH-SY5Y cell line following scopolamine induction. PT-3 (125–200 nM) inhibited scopolamine (2 mM)-induced generation of reactive oxygen species, cellular apoptosis, upregulation of acetylcholinesterase activity, downregulation of choline acetyltransferase level, and activation of p38 and JNK signalling pathways. These findings revealed the underlying mechanisms of scopolamine-induced Alzheimer's disease-like cellular dysfunctions, which provide evidence for developing drugs for the treatment of this debilitating disease

Curcumin Attenuates Hydrogen Peroxide-Induced Cytotoxicity in Human Neuroblastoma SK-N-SH Cells

Objective: Cellular damage induced by oxidative stress has been involved in the development of neurodegeneration. Curcumin, a dietary polyphenol found in the rhizome of Curcuma longa, has been shown, both in vitro and in vivo, to be an effective reactive oxygen scavenging molecule. We investigated an anti-oxidative effect of curcumin against H2 O2 -induced toxicity in human neuroblastoma cell line SK-N-SH. Methods: The SK-N-SH cells were pre-treated with curcumin 2 hours prior to H2 O2 treatment. We measured cell viability, intracellular reactive oxygen species (ROS) levels, expression of apoptotic-related proteins and caspase-3 activity 24 hours post H2 O2 -induced cytoxicity. Results: Treatment with curcumin at concentrations ranging from 5 to 50 μg/mL was not cytotoxic. Pre-treatment with curcumin at the concentrations of 5 to 50 μg/mL prior to H2 O2 exposure caused a significant decrease in intracellular ROS levels and a significant increase in cell viability in a dose-dependent manner. Expression of activated form of caspase-3 and BAX, a pro-apoptotic protein, measured by Western blotting were reduced when the SK-N- SH cell line was pre-incubated with curcumin. The curcumin pre-treated cells also exhibited less caspase-3 activity. Conclusion: Curcumin has protective effects against H2 O2 -induced toxicity in a dose-dependent manner through its anti-apoptotic and anti-oxidative properties in an in vitro H2 O2 -treated SK-N-SH model

Derivation of an induced pluripotent stem cell line (MUSIi004-A) from dermal fibroblasts of a 48-year-old spinocerebellar ataxia type 3 patient

Dermal fibroblasts were obtained from a 48-year-old female patient with spinocerebellar ataxia type 3 (SCA3). Fibroblasts were reprogrammed by nucleofection with episomal plasmids, carrying L-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1 and shRNA against p53. The SCA3 patient-specific iPSC line, MUSIi004-A, was characterized by immunofluorescence staining to verify the expression of pluripotent markers. The iPSC line exhibited an ability to differentiate into three germ layers by embryoid body (EB) formation. Karyotypic analysis of the MUSIi004-A line was normal. The mutant allele was still present in the iPSC line. This iPSC line represents a useful tool for studying neurodegeneration in SCA3

Effects of ME on reactive oxygen species (ROS) level and caspase-3 activity in mouse brain homogenates.

<p>ROS levels and caspase-3 activity were determined in brain homogenates of water/scopolamine-treated mice with/without prior ME treatment for 14 days. Data were presented as percentage compared to untreated cells and reported as Mean ± SEM (n=6). * <i>P</i> < 0.05 compared to the control; ^#<i>P</i> < 0.05 compared to scopolamine- treated group. </p

Effects of ME on oxidative stress and apoptosis <i>in</i><i>vitro</i>.

<p>(<b>A</b>, <b>B</b>) Intracellular levels of ROS in SK-N-SH cells treated with H₂O₂ (<b>A</b>) or PCB-52 (<b>B</b>) for 24 h with or without prior ME incubation. ROS levels were determined using DCFH-DA assay. The fluorescence was measured and expressed as percentage compared to untreated cells (n=3 independent experiments for each bar). (<b>C</b>, <b>D</b>) Caspase-3 activity in SK-N-SH cells treated with H₂O₂ (<b>C</b>) or PCB-52 (<b>D</b>) with or without ME preincubation. Caspase-3 activity was measured using colorimetric assay. Data were expressed as percentage of the activity compared to untreated cells (n=5 independent experiments for each bar). *<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared to untreated control; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 compared to cells treated with corresponding chemicals concentration with no ME preincubation.</p

Western blot analysis of karyopherin β1 (KPNB1) in mouse brain homogenates.

<p>Scopolamine-treated mice with or without prior ME treatment were obtained from passive avoidance test. At the end of the test, whole brain from the mice was subjected to protein extraction. A total 30 µg proteins derived from brain extracts (n=3) were resolved by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking non-specific bindings, the membrane was incubated with mouse monoclonal anti-KPNB1 (1:1,000 in 1% skim milk/PBS) and then incubated with rabbit anti-mouse IgG conjugated with horseradish peroxidase (1:2,000 in 1% skim milk/PBS). β-actin served as the loading control. The immunoreactive bands were visualized by chemiluminescence and autoradiography. Data was presented as KPNB1 band intensity relative to β-actin band. KPNB1 (97 kDa) level was significantly decreased after treatment with scopolamine but could be successfully preserved at its basal level by mangosteen extract. Data were reported as Mean ± SEM (n=3). * <i>P</i> < 0.05 compared to the control. </p

Cytotoxicity screening of mangosteen extract in SK-N-SH cells.

<p>SK-N-SH cells were treated with mangosteen extract at the concentrations of 25-800 µg/ml. After 24-hour incubation, cytotoxicity was determined by MTT assay. Data were presented as mean ± SEM (n=8 for each group). ***<i>P</i> < 0.001 compared to control.</p

Effects of ME on chemical-induced cytotoxicity.

<p>(<b>A</b>) Cytotoxic effects of H₂O₂ on SK-N-SH cells after 24-h incubation. (<b>B</b>) Effects of ME on H₂O₂-induced cytotoxicity. (<b>C</b>) Cytotoxic effects of PCB-52 on SK-N-SH cells after 24-h incubation. (<b>D</b>) Effects of ME on PCB-52-induced cytotoxicity. SK-N-SH cells were preincubated with ME for 3 h before the treatment of H₂O₂ and PCB-52 for 24 h. Cell viability was determined by MTT assay and expressed as percentage compared to untreated cells. Data were reported as mean ± SEM (n=10 independent experiments for H₂O₂). *<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared to untreated control; ^#<i>P</i> < 0.05, ^###<i>P</i> < 0.001 compared to cells treated with corresponding chemicals concentration with no ME preincubation.</p

Protective Effects of Mangosteen Extract on H₂O₂-Induced Cytotoxicity in SK-N-SH Cells and Scopolamine-Induced Memory Impairment in Mice

<div><p>Mangosteen extracts (ME) contain high levels of polyphenolic compounds and antioxidant activity. Protective effects of ME against β-amyloid peptide (Aβ), induced cytotoxicity have been reported. Here, we further studied the protective effects of ME against oxidative stress induced by hydrogen peroxide (H₂O₂) and polychlorinated biphenyls (PCBs), and demonstrated the protection against memory impairment in mice. The cytoprotective effects of ME were measured as cell viability and the reduction in ROS activity. In SK-N-SH cell cultures, 200 μg/ml ME could partially antagonize the effects of 150 or 300 µM H₂O₂ on cell viability, ROS level and caspase-3 activity. At 200, 400 or 800 µg/ml, ME reduced AChE activity of SK-N-SH cells to about 60% of the control. <i>In vivo</i> study, Morris water maze and passive avoidance tests were used to assess the memory of the animals. ME, especially at 100 mg/kg body weight, could improve the animal’s memory and also antagonize the effect of scopolamine on memory. The increase in ROS level and caspase-3 activity in the brain of scopolamine-treated mice were antagonized by the ME treatment. The study demonstrated cytoprotective effects of ME against H₂O₂ and PCB-52 toxicity and having AChE inhibitory effect in cell culture. ME treatment in mice could attenuate scopolamine-induced memory deficit and oxidative stress in brain. </p> </div