Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.

STUDY ON THE THERAPEUTIC EFFECTS OF GINSENOSIDE Rg-1 AND GASTRODINE ON AD MODEL RATS INDUCED BY β-AMYLOID PEPTIDE (25-35)

Objective To study the therapeutic effects of Ginsenoside Rg-1 and Gastrodine on rats model of Alzheimer's disease(AD). Methods Aggregated β-Amyloid peptide (25-35) was injected into the lateral ventricle of rats to establish AD models. Ginsenoside Rg-1, Gastrodine and Ginsenoside Rg-1+Gastrodine were intraperitoneally injected into rats of each test group(Ginsenoside Rg-1∶10mg/kg·day; Gastrodine 100mg/kg·day) for 4 weeks, the rats of control group received equal volume of saline. Passive avoidance task and Morris maze test were done to assess the ability of learning and memory. The content of superoxide dismutase (SOD), malondiadehyde (MDA), total-antioxidative capability (T-AOC), Choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) in brain tissue were measured. Results Ginsenoside Rg-1 and Gastrodine significantly improved learning and memory deficits in the rats with AD induced by β-Amyloid peptide (25-35) (P<0.05). Ginsenoside Rg-1+Gastrodine group were better than Ginsenoside Rg-1 group and Gastrodine group (P<0.05). Ginsenoside Rg-1 reduced the increase of SOD, MDA, but inhibited the decrease of T-AOC, AchE and ChAT; Gastrodine reduced the increase of SOD, MDA, while inhibited the decrease of T-AOC. Gastrodine could also prevent the activity of ChAT and AchE decline in AD rats. Conclusion Both Ginsenoside Rg-1 and Gastrodine have therapeutic effects on rats with AD; Ginsenoside Rg-1 and Gastrodine injection at the same time were better than only using one of them. Their mechanisms might different. Ginsenoside Rg-1 can not only inhibit peroxidation but also increase the activity of AchE and ChAT in brain tissue, while Gastrodine can inhibit peroxidation only, but it can't prevent the decline of ChAT and AchE activity in AD rats.

Neuroprotective Effect of Peptides Extracted from Walnut(Juglans Sigilata Dode) Proteins on Aβ25-35-induced Memory Impairment in Mice

Alzheimer＇s disease（AD） is one of the major neurodegenerative disorders of the elderly, which is characterized by the accumulation and deposition of amyloid-beta（Aβ） peptide in human brains. Oxidative stress and neuroinflammation induced by Aβ in brain are increasingly considered to be responsible for the pathogenesis of AD. The present study aimed to determine the protective effects of walnut peptides against the neurotoxicity induced by Aβ25-35 in vivo. Briefly, the AD model was induced by injecting Aβ25-35 into bilateral hippocampi of mice. The animals were treated with distilled water or walnut peptides（200, 400 and 800 mg/kg, p.o.） for five consecutive weeks. Spatial learning and memory abilities of mice were investigated by Morris water maze test and step-down avoidance test. To further explore the underlying mechanisms of the neuroprotectivity of walnut peptides, the activities of superoxide dismutase（SOD）, glutathione（GSH）, acetylcholine esterase（ACh E）, and the content of malondialdehyde（MDA） as well as the level of nitric oxide（NO） in the hippocampus of mice were measured by spectrophotometric method. In addition, the levels of 8-hydroxy-2＇-deoxyguanosine（8-OHd G）, tumor necrosis factor-α（TNF-α）, interleukin 1β（IL-1β） and IL-6 in the samples were determined using ELISA. The hippocampal expressions of inducible nitric oxide synthase（i NOS） and nuclear factor κB（NF-κB） were evaluated by Western blot analysis. The results showed that walnut peptides supplementation effectively ameliorated the cognitive deficits and memory impairment of mice. Meanwhile, our study also revealed effective restoration of levels of antioxidant enzymes as well as inflammatory mediators with supplementation of walnut peptides（400 or 800 mg/kg）. All the above findings suggested that walnut peptides may have a protective effect on AD by reducing inflammatory responses and modulating antioxidant system.

Regulation of adenosine triphosphate-sensitive potassium channels suppresses the toxic effects of amyloid-beta peptide(25-35)

In this study, we treated PC12 cells with 0-20 μM amyloid-β peptide （25-35） for 24 hours to induce cytotoxicity, and found that 5-20 μM amyloid-β peptide （25-35） decreased PC12 cell viability, but adenosine triphosphate-sensitive potassium channel activator diazoxide suppressed the decrease in PC12 cell viability induced by amyloid-β peptide （25-35）. Diazoxide protected PC12 cells against amyloid-β peptide （25-35）-induced increases in mitochondrial membrane potential and intracellular reactive oxygen species levels. These protective effects were reversed by the selective mitochondrial adenosine triphosphate-sensitive potassium channel blocker 5-hydroxydecanoate. An inducible nitric oxide synthase inhibitor, Nw-nitro-L-arginine, also protected PC12 cells from amyloid-β peptide （25-35）-induced increases in both mitochondrial membrane potential and intracellular reactive oxygen species levels. However, the H202-degrading enzyme catalase could not reverse the amyloid-β peptide （25-35）-induced increase in intracellular reactive oxygen species. A 24-hour exposure to amyloid-13 peptide （25-35） did not result in apoptosis or necrosis, suggesting that the increases in both mitochondrial membrane potential and reactive oxygen species levels preceded cell death. The data suggest that amyloid-β peptide （25-35） cytotoxicity is associated with adenosine triphosphate-sensitive potassium channels and nitric oxide. Regulation of adenosine triphosphate-sensitive potassium channels suppresses PC12 cell cytotoxicity induced by amyloid-β peptide （25-35）.

Protective Effect of Ecdysterone on PC12Cells Cytotoxicity Induced by Beta-amyloid_(25-35)

Objective： To examine the protective effect of ecdysterone （ECR） against beta-amyloid peptide fragment25-35 （Aβ25-35）-induced PC12 cells cytotoxicity, and to further explore its mechanism. Methods： Experimental PC12 cells were divided into the Aβ group （treated by Aβ25-35 100μmol/L）, the blank group （untreated）, the positive control group （treated by Vit E 100 μmol/L after induction） and the ECR treated groups （treated by ECR with different concentrations of 1, 50 and 100 μmol/L）. The damaged and survival condition of PC12 cells in various groups was monitored by lactate dehydrogenase （LDH） release and MTT assay. The content of malondialdehyde （MDA） was measured by fluorometric assay to indicate the lipid peroxidation. And the antioxidant enzymes activities in PC12 cells, including superoxide dismutases（SOD）, catalase （CAT） and glutathione peroxidase（GSH-Px）, were detected respectively. Results： After PC12 cells were treated with Aβ25-35 （100 μmol/L） for 24 hrs, they revealed a great decrease in MTT absorbance and activity of antioxidant enzymes, including SOD, CAT and GSH-Px as well as a significant increase of LDH activity and MDA content in PC12 cells （P〈0.01）. When the cells was pretreated with 1-100 μmol/L ECR for 24 hrs before Aβ25-35 treatment, the above-mentioned cytotoxic effect of Aβ25-35 could be significantly attenuated dose-dependently, for ECR 50 μmol/L, P〈0.05 and for ECR 100 μmol/L, P〈0.01. Moreover, ECR also showed significant inhibition on the Aβ25-35 induced decrease of SOD and GSH-Px activity, but not on that of CAT. Conclusion： ECR could protect PC12 cells from cytotoxicity of Aβ25-35, and the protective mechanism might be related to the increase of SOD and GSH-Px activities and the decrease of MDA resulting from the ECR-pretreatment.

Cyclophilin A affects Bcl-2 and Bax expression following beta-amyloid fragment 25-35-induced injury to PC12 cells

BACKGROUND： Cyclophilin A can protect neurons against oxidative stress. OBJECTIVE： To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochro-mocytoma （PC12） cells treated with beta-amyloid fragment 25-35 （Aβ25-35）, and to verify the protection pathway of cyclophilin A. DESIGN, TIME AND SETTING： The initial experiment was performed at the Laboratory of Department of Neurology, First Clinical College, China Medical University from November 2006 to July 2007. MATERIALS： PC12 cells were cultured at the Cell Center of Peking Union Medical College. Aβ25-35 （Sigma, USA）, antibodies of Bcl-2 and Bax （Wuhan Boster, China）, and recombinant human cyclophilin A （Biomol, USA） were used in this study. METHODS： PC12 cells were divided into three groups. Cells in the control group were incubated in culture medium. Cells in the Aβ25-35 injury group were incubated in medium containing a final concentration of 10 μmol/L of Aβ25-35. Cells in the cyclophilin A group were incubated in medium containing a final con-centration of 10 nmol/L of cyclophilin A for 30 minutes, and then treated with 10 μmol/L Aβ25-35. MAIN OUTCOME MEASURES： After 24 hours of culture, immunohistochemistry was used to detect Bcl-2 and Bax expression in PC12 cells. Annexin-V flow cytometry was employed to measure the apoptosis rate of PC12 cells. The MTT method was applied to examine the survival rate of PC12 cells. RESULTS： Bcl-2 expression decreased, whereas Bax expression increased in PC12 cells treated with Aβ25-35 （t = 2.277, 5.957, P 〈 0.05）. However, in PC12 cells treated with Aβ25-35 and cyclophilin A, Bcl-2 expression increased and Bax expression decreased （t = 4.497, 2.531, P 〈 0.05）. The survival rate of PC12 cells significantly decreased and the apoptosis rate increased （t=8.509, 22.886, P 〈 0.05） following Aβ25-35 treatment. Cyclophilin A enhanced the survival rate of PC12 cells to Aβ25-35-induced apoptosis （t = 4.895, 10.042, P 〈 0.05）. CONCLUSION： Cyclophilin A can increase Bcl-2 expression and decrease Bax expression in PC12 cells treated with Aβ25-35, which indicates that cyclophilin A has a protective effect on Aβ25-35-induced injury to PC12 cells.

Fructus Broussonetae extract improves cognitive function and endoplasmic reticulum stress in Alzheimer's disease models

This study investigated the effects and possible targets of Fructus Broussonetiae extract, a traditional Chinese medicinal herb, on a model of Alzheimer＇s disease induced by beta-amyloid peptide 25 35 and D-galactose. The results revealed that intragastric administration of Fructus Broussonetiae significantly increased the expression of immunoglobulin-binding protein, a key factor in the endoplasmic reticulum stress-signaling pathway in rat hippocampus. In contrast, the treatment significantly decreased expression levels of PKR-like endoplasmic reticulum kinase and C/EBP homologous protein, and substantially improved learning, memory and spatial recognition dysfunction in rats. This evidence indicates that Fructus Broussonetiae extract improves spatial learning and memory abilities in rats by affecting the regulation of hippocampal endoplasmic reticulum stress and activation of the apoptosis pathway.