Studies On Effects Of Ginseng Saponin On Human Microglial Functions

The aims of this study is to investigate the modulating effects of Chinese medicine herb Ginseng water-soluble extracts(GS) on human microglial functionns and to develop GMSE to become an uesful therapeutic medicine of central nerve system(CNS) neurodegenerative disease,injury and ischemia.Therefore,our study is divided into six parts:isolation, culturation and identification of human microglia from fetal brain tissues;modulation of GS on activation of microglial cells;influence of GS on phagocytosis of microglia;regulation of GS on NO synthesis of microglia;modulation of GS on production of ROS in microglia;impact of GS on lysophosphlipids-induced calcium influx in microglia.First,we isolated and purified human microglial cells from artificial abortive fetal brain tissue.After purification,we used morphological and immuno-fluorescence methods to identify the purity of microglia we got.We found that more than 98%of cells were microglia. On this condition,we made researches on the following parts.When microglial cells exert functions,they must alter their state from resting to activated. The activated microglia can elaborate both neurotrophic and neurotoxic factors,while its morphological change is membrane ruffling.So our results showed:both ATP and GS could make microglia activated.As the main innate immue cells in CNS,microglia has many important functions,like phagocytosis.Therefore,we tried to find out the effects of GS on microglial phagocytosis.GS obviously enhanced microglia engulfing fluorescent spheres.NO is another functional molecule that microglia produces.Meanwhile,if too much NO synthesized by over-activated microglia,it can cause neurotoxin.In the thesis,we performed experiments to evaluate the impacts of GS on NO production by microglia.Our results showed:GS could increase NO basic-level synthesis;however,it could suppress NO production stimulated by LPS to some extent.The physiological or toxic effects of ROS is depended on its intracellular level.Low-level ROS is a basic element that microglia needs to function normally;midi-level can activate multiple kinases and factors,such as JNK,p38 NF-κB and API,and so on;high-level may induce apoptosis or necrosis of microglia and neurons.So we examined the influence of GS on ROS production by microglia.We found that GS inhibited ROS synthesis.Bioactive lysophophlipids are a group of natural lipids,they can modulate several cellular function,like proliferation and injury-healing.Lysophophatidycholine(LPC),which is the representative member of lysophophlipids,is an important modulator of immune cells.It also correlates tightly with inflammation.We explored the effects of GS on LPC-induced Ca²⁺ influx of microglia.As the results showed,LPC stimulated Ca²⁺ influx instantaneously,but later on,microglia had no response to calcium ionophore.When microglias were pre-incubated with GS,and then added LPC into supernatant,persistent but not instantaneous Ca²⁺ influx occurred;furthermore,those cells still responded to calcium ionophore efficiently.In summary,at a series of definitive concentrations,GS enhanced microglial phagocytosis;GS stimulated microglial activation;GS promoted or suppressed NO synthesis depending on microglial states;GS prevented ROS production by over-activated microglia; GS protected microglia from anergy to calcium ionophore caused by LPC.Consequently,GS could be developed to be a useful medicine used to prevent and therapy neurodegenerative diseases.