| Objective:Microglia are immunocompetent cells in the CNS. A variety of pathological signals can activate microglia. Activated microglia not only have the effects of neural protection, but secrete a wide array of toxical substance, such as superoxide anion, NO, arachidonic acid and excitatory amino acid. A lot of studies have found that extracellular adenosine-5'-triphosphate (ATP) acting as neurotransmitters plays an important role during neurotransmission, synaptic plasticity and neutrotrophy following the activation of P2 receptors. Almost all CNS injury such as inflammatory, ischemia and degenerative disease cause nerve cell death and dramatic release of ATP, leading to excessive activation of P2 receptor and aggravating neuron death and microglial activation.P2 purinoceptor can be divided into two families, ionotropic receptors (P2X) and metaotorpic receptors (P2Y). Functional studies have shown that both primary mouse microglial cells and the BV-2 cell line can express purinergic receptors for extracellular ATP of both the P2X and P2Y subtypes. Compared with other P2X subtypes, P2X7 receptor has caused great concern for its distinguished structure and functional properties. P2X7 receptor are widely expressed in microglia, and play an important role in immunoregulation and inflammatory reaction. P2X7 subtype appeared involve in the mechanisms of cellular damage. In many cell types, the activation of the P2X7 receptor leads to rapid cytoskeletal rearrangements, membrane blebbing and cell lysis. Activation of P2 receptors by ATP mediates cytotoxic injury and apoptosic for microglia, and the principal roles of the P2X7 subtype in this process in BV-2 cell line is less reported. The main aim of this study is to investigate the cytotoxic and apoptosic effects of ATP, P2X7 receptor agonist BzATP, P2X antagonist PPADS and P2X7 receptor antagonist BBG on cultured BV-2 cell line in vitro.Methods:The BV-2 microglia were treated with ATP, BzATP, PPADS and BBG in vitro. MTT assay were used to study cell survival after treatment. Phase-contrast microscope and Hoechst 33342 nuclei-staining were used to observe ATP, PPADS, BzATP and BBG mediated injury in BV-2 cells. Flow cytometry were used to detect apoptosis effects of ATP, PPADS, BzATP and BBG on BV-2 cells.Results:1. Compared to the control group, lower concentration of ATP (10μM, 30μM,50μM and 70μM) had no cytotoxic effects. The cell survival rate demonstrated by MTT assay was 98.0±3.7%,97.4±1.7%,95.5±1.8%, 93.5±1.9% respectively, P>0.05, which presents no statistical significance. The concentration of≥100μM ATP (100μM,300μM,500μM,700μM and 1mM) could mediate BV-2 cells damage as following ATP dose-dependent. The higher concentration of ATP (3mM,5mM and 7mM) could mediate severe damage, while cell survival rate decreased to 30% or lower. BzATP also could mediate cell damage. PPADS and BBG could block the cytotoxic effects of ATP, and increased cell survival rate markedly.2. Phase-contrast microscope showed that ATP mediated the morphological changes, such as normal cells changed to ameboid, cellular members shrinking, nuclei fragmentation and vacuole denaturation. PPADS and BBG could block the changes before-mentioned efficiently.3. Hoechst33342 nuclei-staining and flow cytometry showed that 3mMATP and 500μMBzATP could mediate cell apoptosis, and PPADS and BBG could inhibit apoptosis obviously.Conclusions:1. Lower concentration of ATP(<100μM) had no toxic effects on the cultured BV-2 microglia,100μM concentration of ATP could mediate marked neural damage and higher concentration of ATP(≥3mM) could mediate cell apoptosis.2. P2 receptor antagonist PPADS could attenuate the BV-2 cell toxic damage and apoptosis, and had neuroprotective effects.3. P2X7 receptor agonist BzATP had obvious cytotoxicity and its antagonist BBG blocked ATP mediated damage and apoptosis of BV-2 cells obviously.4. P2X7 receptor might be the major subtype of P2X receptor by higher concentration of ATP mediated BV-2 cytotoxic damage and apoptosis. |
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