The Effects Of P2Y₁ Receptor On Reactive Astrogliosis, GDNF Secretion Of Astrocytes And The Related Signal Pathways

Reactive astrogliosis is a repair process after various insults to Central Nervous System.Astrogliosis manifests hypertrophy with increase of its GFAP contents as well as hyperplasia of astrocyte.The relevance of astrogliosis remains controversial,especially with respect to the beneficial or detrimental influence of reactive astrocytes on CNS recovery. The reactive astrocytes can secret neurotrophic factors and confine the spread of inflamation and toxic chemicals.At the same time,The formation of a glial scar may interfere with neuronal repair or axonal regeneration in the CNS.Therefore controllable astrogliosis might be an admirable goal:to enhance its beneficial effects of gliosis and to avoid its detrimental effects.Extracellular ATP and its derivants have been considered as transmitters for years. "Purinergic" receptors were first formally recognized and classified as P1 and P2 subtypes by Bumstock in 1978.The widespread and abundant distribution of the P2Y1 receptor within the brain arouses much interests.P2 receptors can activate STAT3 serine 727 phosphorylation in astrocytes and activation of STAT3 maybe play a key role in astrogliosis.Extracellular signal-regulated kinase(ERK) attributes to CREB phosphorylation that leads to GDNF production in cultured astrocytes.The roles of P2Y1 receptor in astrogliosis, STAT3 phosphorylation and GDNF secretion and the related signal pathways under ischemic brain injury remain elucidation.Our study is targeting on the roles of P2Y1 receptor during the process of astrogliosis and GDNF secretion with the temporal right middle cerebral artery occlusion (MCAO) in vivo,and Oxygen-nurturer deprivation of cultured astrocytes in vitro as the model of temporal ischemic injury.The related signal pathways would be investigated.We hope our work will help us to deepen our understanding of related pathophysiological processes and might arouse new therapeutic approach.Part One The effect of P2Y1 receptor on gliosis and GDNF secretion of rat brain astrocytes after temporal ischemiaObjective:The study was aimed to investigate the effects of P2Y1 receptor on the gliosis and GDNF secretion of astrocytes after temporal ischemia in vivo and in vitroMethods:MCAO in vivo and Oxygen-nurturer deprivation in vitro were used as the model of brain temporal ischemic injury.The expression of GFAP as the biomarker of astrogliosis.MRS2179 was used as selective P2Y1 receptor antagonist. The location of P2Y1 receptor and GDNF was observed by double immunofluorescence labeling.Real time RT-PCR and Western blotting were used to measure the mRNA and protein levels of GFAP.Elisa was used to measure the GDNF expression of damaged tissue and the supernate of cultured cells.Results:Inhibition of P2Y1 receptor could reduce the production of GFAP (P＜0.05) and increase the GDNF secretion of astrocytes after the temporal ischemic injury.Summary:Inhibition of P2Y1 receptor could interfere in the astrogliosis and increase the GDNF secretion of astrocytes after the temporal ischemic injury.Part Two The signaling pathways during astrogliosis and GDNF secretion of astrocytes after temporal ischemic injury in vitro Objectives:The purpose is to investigate the signaling pathways during astrogliosis and GDNF secretion of astrocytes after temporal ischemic injuryMethods:Measuring of the level of phosphorylation of signaling molecules involving PI3-K/AKT/CREB and JAK2/STAT3 pathways in the cultured cells after inhibition of the P2Y1 receptor and temporal Oxygen-nurturer deprivation.Summary:Under ischemic injury and blocking the P2Y1 receptor the phosphorylation of AKT and CREB was increased and the phosphorylation of STAT3 was decreased.AG490 could reduce the expression of pSTAT3 and GFAP.LY294002 could reduce the expression of pAKT and pCREB.U0126 could reduce the expression of pAKT,pCREB,pJAK2,and pSTAT3.Conclussions:The study showed that blockage of P2Y1 receptor might decrease the contents of GFAP,while increase the secretion of GDNF of astrocyte.It indicated that synthesis of GFAP and GDNF,influenced by P2Y1 receptor,showed negative correlation.P2Y1 receptor on astrocytic membrane affected GFAP synthesis through JAK2/STAT3 pathway,and influenced GDNF secretion via PI3-K/AKT/CREB pathway respectively.Some molecules,such as MEK1/2 and ERK1/2 in RAS/ERK pathway might be upstream signal molecules of JAK2/STAT3 and PI3-K/AKT/CREB.Crosstalk existed between signal pathways of JAK/STAT3 and PI3-K/AKT/CREB.