Neuroprotective Effects Of 9 Natural Compounds On The Exitotoxicity Damage Of Rat Cortical Neuron Induced By Activated Microglia

Alzheimer’s disease(AD)is the most common type of primary demenia.Recent studies suggest that the appearance of activated-microglia is another characteristic of AD.Microglia, the main immune cell of the central nervous system(CNS),plays the critical role as resident immunocompetent and phagocytic cells in CNS.During the inflammatory processes in CNS, activated microlia can directly and indirectly induce the dysfunction and death of neurons through the oxidative stress,exitotoxicity,cytokine and so on.Thus,inhibition of microglia activation will be an effective therapeutic approach to alleviate the progression of diseases such as AD.According to the present study,models of the release of excitatory amino acids and the DNA damage of rat cortical neurons induced by lipopolysaccharides(LPS)-activated microglia were established.Then,we evaluated the effects of the 9 natural compounds which were extracted from ginseng or grape seed on the models,as well as the mechanisms of action of the active compounds.Firstly,the conditioned medium collected from normal or LPS-activated microglia(C-CM and L-CM,respectively)was used to estimate whether the release of excitatory amino acids (EAA)was related to the damage of rat cortical neurons.The concentration of EAA was determined by HPLC with fluorescence detector.The time-effect curve of the release of glutamate indicated that primary microglia treated with 1μg/ml LPS for 24 h increased the release of glutamate significantly.The release of glutamate from malactivated microglia was mainly conveyed by the Xc exchange sysrem.LPS-induced glutamate synthesis in microglia was primarily through the glutaminase pathway.In our study,the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine(DON)and the Xc exchange system inhibitor L-2-aminoadipic acid (AAA)suppressed LPS-induced glutamate release significantly. We estimated the effect of minocycline and 9 natural compounds on the model of EAA release.Compared with the LPS-treated group,minocycline,ginsenosides Rd,Rg1,PF11 attenuated microglial glutamate release and had no effect on the concentration of aspartate and glutamine.Ginsenoside RT5 depressed the aspartate and glutamate release and increased the content of glutamine.Ginsenosides Re,grape extracts resveratrol,procyanidine and polyphenol depressed the glutamate release,increased the content of glutamine and had no effect on the concentration of aspartate;Ginsenoside Rb2 increased the concentration of glutamine and had no effect on the aspartate and glutamate release.Then,we investigated the DNA damage of rat cortical neuron induced by malactivated microglia using the single cell gel electrophoresis(Comet assay),a simple and sensitive technique for genotoxicity studies.First,the LPS treated primary microglia-conditioned medium was collected with or without AAA,DON and natural compounds for 48 h,which was referred to as "NC-CM".Compared with the neurons treated with L-CM,the microglia-conditioned medium with AAA and DON depressed the DNA damage of rat cortical neuron.Meanwhile, MK-801,an antagonist of NMDA receptor,depressed the DNA damage of rat cortical neuron induced by L-CM.The result indicated that neuronal damage by LPS-activated microglia was likely to be excitoneurotoxicity through NMDA receptor signaling.We estimated the neuroprotective role of the 9 natural compounds on the DNA damage of rat cortical neuron induced by malactivated microglia.The results showed that the neuronal injury by L-CM can be protected by treating with the NC-CM of minocycline and 9 natural compounds.In addition,minocycline,ginsenosides Rd,Rb2,Re,RT5,PF11 and grape exteacts resveratrol,polyphenol,procyanidine had the protection on the DNA level of primary rat cortical neuron.Next,We detected the 1,1-diphenyl-2-picrylhydrazyl(DPPH-)radical and MDA scavenging effect of minocycline and 9 natural compounds.The result showed that minocycline, grape exteacts resveratrol,polyphenol and procyanidine had strong radical scavenging activity towards DPPH.Ginsenosides Rb2,Rg1,RT5,PF11,grape exteacts resveratrol,polyphenol and procyanidine had a certain lipid peroxidation scavenging activity towards MDA.In conclusion,the release of glutamate from malactivated microglia involved in the DNA damage of primary rat cortical neurons.The neuroprotective effects of Minocycline and 9 natural compounds against neuronal DNA damage induced by the LPS-activated microglia were confirmed.Both the glutaminase inhibitor and Xc exchange system inhibitors might be a novel effective strategy for the treatment of AD with minimum adverse side effects.