Screening And Mechanism Studies Of The Natural Compounds On The Inhibition Of Activated Microglia

Dementia is a syndrome of neurodysfunction, which results in the development of multiple cognitive deficits, with memory impairment being the principal. Alzheimer's disease (AD) is the most common type of dementia. AD is neuropathologically characterized by the amyloid and neurofibrillary tangle depositions, basal forebrain cholinergic deficit, and extensive neuronal loss and synaptic changes in the cortex and hippocampus. Recent researches suggest that the appearance of activated-microglia is another characteristic of AD. Microglia, the main immune cell of the central nervous system (CNS), plays critical role as resident immunocompetent and phagocytic cells in CNS. During the inflammatory processes in CNS, activated-microglia can be induced by many factors. Activation of microglia induces the release of some neurotoxic substances such as nitric oxide (NO) and TNF-alpha (TNF-α). Researches suggest that inhibition of the activation of microglia may be an important strategy of preventing AD.The present dissertation evaluated the effect of some compounds from Chinese medicinal herbs on the NO production in LPS-activated N9 microglial cells, as well as the mechanisms of action of the active compounds.Firstly, the effects of 16 compounds on the NO production in LPS-activated N9 microglial cells were assessed by Griess reaction. Among the 16 compounds tested, only Rg1, Re, RT5 and resveratrol could obviously inhibit the production of NO in LPS-activated N9 microglial cells. The results were confirmed by using primary rat microglial cells. Then, the mechanisms of action of these active compounds were investigated in further studies. The results showed that all studied compounds, ginsenoside-Rb2, -Rd, -Rgl and -Re; PF₁₁and RT5, and resveratrol could strongly suppress TNF-αproduction in LPS-activated N9 cells. However, only ginsenoside-Rgl, -Re, ocotillol type saponins RT5 and resveratrol, but not ginsenoside-Rb2, Rd and ocotillol type saponins PF₁₁, inhibited the production of NO in LPS-activated N9 cells.Western blot analysis was performed as described previously in the present study. The expression of iNOS protein and the degradation of iκBαprotein induced by LPS in N9 cells were studied. Mitogen-activated protein kinases (MAPKs) pathways related proteins mediating the synthesis and the release of neurotoxic substances in activated microglia, p38 and ERK MAPKs, were also examined in this experiment. The results showed that Rg1, Re, RT5 and resveratrol significantly inhibited the degradation of iκBα, and the iNOS/NO synthesis in LPS-activated N9 microglial cells. They also decreased the expression of phosphated level of p38, p42/44 MAPKs. However, Rd, Rb2 inhibited the activation of p42/44MAPKs and the degradation of iκBα, but not iNOS/NO synthesis and the activation of p38 MAPKs in LPS -activated N9 microglial cells. The most interesting is PF₁₁, which inhibited the activation of p38, p42/44MAPKs, and the degradation of iκBα, but not iNOS/NO synthesis in LPS-activated N9 microglial cells. The above results suggested that the mechanism of inhibition on the production of NO and TNF-αby LPS-activated N9 microglia of seven compounds may be through the inhibition of the LPS-induced degradation of iκBαand the phosphorylation of p38, p42/44 MAPKs. Especially, p38 MAPKs may the action target of compounds.Studies suggest that neurotoxic substances secreted by stimulated microglia were toxic to neuronal cells, which could cause neuronal cell death directly. In order to further investigate the protective effects on the neuronal cell of the tested compounds, which inhibited the activation of microglia, in the present study, the effect of seven compounds on cytoplasmic calcium induced by condition medium of LPS-activated N9 microglial cells (CM-LPS) in PC12 cells was studied. The direct action of seven compounds on resting cytoplasmic calcium level in PC12 cells and the influence of CM-LPS on the cell viability on PC12 cells were also investigated. The results showed that CM-LPS did not affect PC12 cell viability, but significantly increased the levels of intracellular free calcium [Ca²⁺]i in PC12 cells. Among of seven compounds, Rb2, Rg1 and resveratrol could decrease the level of resting [Ca²⁺]i, however, PF₁₁, RT5, Re and Rd did not influence the level of resting [Ca²⁺]i. PF₁₁, RT5, Re and Rg1 also inhibited the elevation of cytoplasmic calcium induced by CM-LPS. These results show that seven compounds may play important neuroprotective roles in protecting the neurons from the toxic influence by the activated microglia.In conclusion, the effects on the production of NO and TNF-αby LPS-activated N9 microglia and the action mechanisms of seven compounds were studied in the present study, the results suggested that seven compounds could inhibit the activation of microglia, and have protective effect on the neurons from the toxic influence by the activated microglia.