Neuroprotective Effects of Plant Polyphenols Against Oxidative Insults

Abstract
Key Personnel
Specific Objectives

Abstract
Many polyphenolic compounds from plants, including a large class of bioflavonoids, are known to offer health benefits to humans. The beneficial effects have been attributed to their anti-oxidant and anti-inflammatory properties. There is increasing evidence that besides the peripheral organs, bioflavonoids may help prevent tissue damage from oxidative stress in the brain. The brain is highly susceptible to oxidative stress and oxidative damages have been implicated in the pathology of a number of neurodegenerative diseases. Recent studies from our laboratories have provided evidence for the ability of polyphenols from grapes to ameliorate neurodegenerative changes due to chronic ethanol consumption. Individual polyphenolic compounds such as resveratrol and genistein could also protect neurons and astrocytes from oxidative damage and effects of pro-inflammatory cytokines. These studies clearly warrant further investigations to examine structural specificity, target cellular sites and molecular mechanism of action of these botanical compounds. It is equally important to determine whether these bioflavonoids may have adverse effects on specific cellular systems, since these compounds are already widely used as nutritional supplements. The focus of this research project is to use animal and cell culture models to examine whether the pholyphenolic compounds can offer neuroprotection against oxidative stress in the brain. Specific aims are: (1) Determine the ability of plant polyphenols to protect against oxidative damage in brain due to global cerebral ischemia induced to gerbils and focal cerebral ischemia induced to C57Bl mice (wild type and ERKO). Studies will include determination of neuronal cell death, infarct size as well as other biochemical endpoints, e.g., measurement of Cu/Zn SOD and Mn SOD mRNA levels. (2) Determine the ability of polyphenols to prevent neuronal damage mediated by excitatory mechanisms. These studies will include determination of mechanisms through which polyphenols may act in the cell death processes (necrotic and apoptotic) cell death processes. (3) Determine the ability of polyphenols to modulate intracellular signaling pathways in neuronal cells and astrocytes in their response to pro-inflammatory cytokines and signaling cascades leading to activation of mitogen activated protein kinase (MAPK). It is anticipated that the combined studies with animal and cell models will provide new insights regarding the mechanisms of action of the polyphenolic compounds on the nervous system. These studies will also provide new information regarding proper use of these polyphenols as supplements and/or therapeutic agents for alleviating oxidative insults and related injuries in the brain.

Key Personel
Grace Y. Sun — Principal Investigator
Albert Y. Sun — Co-investigator

Specific Objectives
Polyphenolic compounds, including a large class of flavonoids, are enriched in certain vegetables, fruits, seeds, and beverages (e.g., tea and wine). Kuhnau (1976) regarded the flavonoids as a class of semi-essential nutrients for humans. Dietary intake rich in these compounds has been suggested to improve the health of individuals and decrease the risk of cardiovascular diseases. The beneficial effects of flavonoids have been attributed to their anti-oxidant and anti-inflammatory properties (Korkina and Afanas'ev, 1997). Many of these polyphenols also possess estrogenic properties and are regarded as phytoestrogens. There is increasing evidence that flavonoids may help prevent damage in tissues that are highly susceptible to oxidative stress, including the brain. A number of neurodegenerative diseases are associated with neuronal damage from oxidative stress. Recent studies from our laboratories have provided evidence for the neuroprotective effects of polyphenols, e.g., in their ability to ameliorate neurodegenerative changes due to chronic ethanol consumption (Sun et al., 1999). In addition, individual polyphenolic compounds such as resveratrol and genistein could prevent neuronal cell death induced by oxidative stress (Chanvitayapangs, et al., 1997; Drazynska-Luziak et al., 1998; Sun et al., 1998) and inhibit cytokine-mediated induction of inducible nitric oxide synthase (iNOS) and secretory phospholipase A2 (sPLA2) (Li and Sun, 1998; Li et al., 1999). These results suggest that further studies are warranted to determine the structural specificity, cellular targets (e.g., neurons or glia) and mechanisms of actions of the polyphenolic compounds in brain. It is equally important to determine whether these polyphenols have adverse effects on specific cellular systems, since these compounds are already widely used as nutritional supplements. Establishment of a Botanical Center at the University of Missouri will provide the means to explore the neurological effects of a wide variety of bioflavonoids in animal, tissue and cell model systems.

In line with the central focus of the Botanical Center in University of Missouri, this research project is aimed at examining whether plant polyphenols can offer neuroprotection against oxidative stress induced by cerebral ischemia. It is well recognized that cerebral ischemia leads to the rapid release of excitatory amino acids and calcium influx. In turn, this leads to an increased production of reactive oxygen species and ultimately results in cell death. In some instances, cerebral ischemia causes the activation of astrocytes and the recruitment of microglia. These events are associated with increased release of pro-inflammatory cytokines and stimulation of signaling pathways involving protein kinase C, tyrosine kinases and mitogen activated protein (MAP) kinases. The possibility that specific plant polyphenols can ameliorate these ischemia-mediated damages has not been examined in detail. The major objective of this project is to use animal and cell models to examine the ability of polyphenolic compounds from plants to protect against oxidative damage in neuronal cells. The use of neuronal and glial cell culture models will be help to understand molecular mechanisms whereby these compounds prevent cellular damage due to inflammatory and mitogenic insults.

Objective 1
Determine the ability of plant polyphenols to protect against neuronal damage due to cerebral ischemia. Two models of cerebral ischemia will be tested: (a) the gerbil model in which transient global cerebral ischemia is induced by occlusion of both common carotid arteries (CCA), and (b) the mouse model in which focal cerebral ischemia is induced by occlusion of the middle carotid artery (MCA) in wild type and estrogen recptor knockout (ERKO) C57Bl mice. Initial studies will examine the effects of two widely used plant extracts, i.e., soy isoflavones and grape polyphenols. Subsequently, studies will use purified polyphenolic compounds, e.g., genistein, daidzien, resveratrol, catechin, and quercetin as well as newly identified compounds from the Plant and Analytical Cores. Plant extracts will be administered through supplementation of a well-defined diet (AIN 76) and specific polyphenolic compounds will be administered orally or systematically prior to or after the ischemic insult. Assessments of ischemic damage will include measurements of neuronal cell death in the hippocampal CA1 area (gerbil model), infarct volume (mouse model) and the levels of mRNA and protein to Cu/Zn SOD, Mn SOD, IP3R, SERCA-IIB, iNOS and nNOS.

Objective 2
Determine the ability of polyphenols to prevent neuronal damage mediated by excitotoxic mechanisms. Using primary neurons in culture, studies will be carried out to test the ability of plant polyphenols to prevent neuronal cell death due to action of excitatory amino acids, namely, glutamate. Experiments include treatment of neuronal cells with varying doses of the polyphenols and determination of the extent of necrotic and/or apoptotic cell death at multiple times after excitotoxic insult. Cell death will be detected by several procedures including measurements of LDH release, PARP hydrolysis, cell staining with acridine orange, DNA laddering, and the MTT and TUNEL assays. Compounds capable of preventing neuronal cell death due to excitotoxic insults will be further studied to elucidate their mode of action at the cellular levels.

Objective 3
Determine the ability of polyphenols to modulate intracellular signaling pathways in glial cells. Primary or immortalized (DITNC) astrocytes will be used to examine the effect of polyphenols on cellular pathways that have been previously associated with cerebral ischemia including (a) the induction of iNOS and sPLA2 by the endotoxin (LPS) and the pro-inflammatory cytokines, e.g., TNFa, IL-1b, and IFNg, and (b) the activation of signaling cascades involving mitogen activated protein (MAP) kinases. Upon identification of active polyphenols, studies will elucidate whether the effects are mediated through transcriptional or post-translational mechanisms.

This project will focus on establishing cell and animal models for assessing the neuroprotective effects of polyphenols from plants. Studies with animal (gerbils and mice) models will give information regarding the types of plant extracts and purified compounds that protect against oxidative insults and ischemia-induced neuronal cell death. Studies with the estrogen receptor knock out (ERKO) mice (in collaboration with Research Project #1) will provide additional information on the phytoestrogen properties of these compounds on the brain, since phytoestrogens have been implicated to offer protective effects to the brain. Studies with cell culture model (neurons and glia) will provide a better understanding of the target site and biochemical actions of specific polyphenolic compounds. In fact, the combined cell and animal studies can be used to test any compounds that may act as anti-oxidants, or anti-inflammatory and anti-mitogenic agents in the brain. It is anticipated that results from this project will aid in identification of new plant species and botanical compounds and their possible use as therapeutic agents to alleviate oxidative stress and related injuries in the brain.