The neuroprotective activity of polyphenolic compounds isolated from Tournefortia sarmentosa Lam.

• Main Authors: Chih-Wen Chi, 紀智文
• Other Authors: Chieh-Fu Chen
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/08150010854060579346

博士 === 國立陽明大學 === 藥理學研究所 === 96 === 英文摘要 Alzheimer’s disease (AD) is the most commonly neurodegeneration disorder in aged population. ��-Amyloid peptide (A��), the component of senile plaques in AD brain, has been shown to be neurotoxic. Several mechanisms including glutamate-induced excitotoxicty have been proposed in A��-induced neuronal death. Thus, glutamate and A�� have critical roles in the pathogenesis of AD. For screening the neuroprotective components against glutamate-induced neurotoxicity, the crude extracts and its pure chemicals from Chinese herbal medicine, using cortical neurons as experimental model, were studied. The results showed that ten polyphenolic compounds ((±) catechin, procyanidin B-3, casuarinin, punicafolin, penta-o-galloyl-��-D-glucose, tournefolic acid B methyl ester (TABM), tournefolic acid B ethyl ester (TABE), isosalvianolic acid C, isosalvianolic acid C methyl ester, lithospermic acid ethyl ester), and various extracts of Sanguisorba officinalis, reduced glutamate-induced neurotoxicity. TABM was the most effective polyphenolic compound among above testing materials. Therefore the mechanisms of neuroprotective effect of TABM were investigated. Treatment of cortical neurons with glutamate 50 �嵱 for 24 h decreased cell viability by 44.8 ± 7.9% in MTT reduction assay and 6.5-fold increased in LDH releasing. TABM 50 �嵱 attenuated glutamate-induced cell death by 46.8 ± 17.8% in MTT reduction and 41.7 ± 15.2% in LDH releasing. TABM did not block the calcium influx into cytosol induced by glutamate. TABM eliminated glutamate-induced accumulation of superoxide anion and hydrogen peroxide using hydroethidine and 2’, 7’-dichlorofluorescein diacetate detecting assay. Treatment with glutamate for 12 h increased the activity of caspase 2, 3, 6, 8 and 9 by 5.5, 5.2, 4.9, 4.9 and 6.9-fold of control, respectively. TABM 40 �嵱 abrogated glutamate-mediated activation of caspase 2, 3, 6, 8, and 9 by 72.1, 68.2, 66.6, 46.2 and 69.2%, respectively. Glutamate increased endogenous antioxidant enzymes and was eliminated by TABM 50 �嵱. C-jun N-termianl kinase inhibitor (JNKI) attenuated glutamate-mediated cell death in a concentration dependent manner. Combination of TABM 20 �嵱 and JNKI 0.5 or 1 �嵱 decreased cell death by 73.1 and 91.4%, respectively. Phosphorylation of p54-JNK was activated by glutamate, and abolished by TABM 50 �嵱 in control and glutamate-treated cells. TABM attenuated glutamate-induced neurotoxicity by eliminated ROS accumulation, abrogated the activation of caspases and JNK in cortical neurons. In the meanwhile, the polyphenolic compounds were used to study the neuroprotective effect against A��25-35-induced neurotoxicity. A��25-35 5 �嵱 elicited 40.1% cell death as measured by MTT reduction. Tournefoilc acid B (TAB) inhibited the A��25-35-induced cell death in a concentration-dependent manner. A��25-35 5 and 10 �嵱 stimulated the activity of caspase 2, 3, 6, 8 and 9, and TAB abrogated caspases activation. A��25-35-induced elevation of calcium level in cytosol, mitochondria, and endoplasmic reticulum (ER) and TAB inhibited calcium elevation in cytosol and mitochondria, but not in ER. A��25-35 induced tBid increase in mitochondria and cytochrome c release to cytosol by 90.6 and 63.6%, respectively. TAB 40 �嵱 abolished cytochrome c release and tBid formation in mitochondria by 33.6 and 52.6%, respectively. Caspase 8 inhibitor protected cortical neurons from A��25-35-mediated neurotoxicity as measured by MTT reduction, and inhibited the increase of mitochondrial tBid and the release of cytochrome c. The amount of Bcl-2 agonist killer 1 (Bak) in cytosol was 27.1% increased but not in mitohondria by A���ntreatment. TAB 43.3% inhibited A��-mediated elevation of cytosolic Bak. A��25-35 decreased the level of B-cell lymphoma-2�� (Bcl-2��) by 15.3%, whereas TAB did not affect the A��-mediated decreased of mitochondria Bcl-2���nlevel�| TAB maintained the Ca2+ homeostasis and inhibited A��-mediated the activation of caspase 8-tBid-cytochrome c pathway, conferring its neuroprotective effects on A��-mediated neurotoxicity. In in vivo animal model, intraalveus injection of A�������{���� or NMDA elicits neuronal degeneration as determined by cresyl violet and fluoro-jade B staining, and TABM prevented such neuronal degeneration.