The Research Of PKCγ/NMDAR1 In Mechanism Of Nitroglycerin Induced Migraine Model

BackgroundsMigraine is an incapacitating neurovascular disodrer,which badly affects the patient' s life and work quality.At present the etiopathogenisis of migraine remains elusive.With the development of neurobiology, molecular pharmacology and brain-imaging technology,central hypersensitivity in the great role of migraine attack has been recognized by people.At present many studies show central hypersensitivity in peripheral inflammatory pain and central neuropathic pain is mediated by PKC-NMDAR loop in the spinal cord level.Protein kinase C(PKC) is a member of serine / threonine kinase family,which is a kind of Ca2+ and phospholipid-dependent phosphorylase.As an intracellular second messengers,PKC plays an important role in neuronal excitability modulation,signal transduction, the release of neurotransmitters and synaptic plasticity.PKCγis unique to the brain and spinal cord,which plays an important role in signal processing in pain and the induction and maintenance of central sensitization.N-methyl-D-asparate receptor(NMDAR) is a type of excitatory neurotransmitter glutamate receptors in central nervous system,which is a ionotropic receptor.NMDAR includes NR1,NR2 and NR3 three subunits and is a polymer composed by NR1 and NR2 or NR3.NR1 is an essential component,and NR2 or NR3 modify the functional property of the receptor.It involves many complex physiological and pathological processes in vivo,including wind-up,central sensitization,long-term potentiation,peripheral sensitization,visceral pain,cell necrosis and apoptosis.In addition,it also take part in the emergence and maintenance of hyperalgesia. Some researches show,when excitatory impulses come, presynaptic membrane releases a large number of excitatory amino acids (EAAs) and make postsynaptic membrane produce a fast EPSP,leading to the removal of Mg2+ which blocks Ca2+ influx through the NMDAR channel.So extracellular Ca2+ can be inflow,and activate intracellular Ca2+-dependent PKC and induce the transposition of PKC, phosphorylating ion channels associated with NMDAR in cell membrane, reducing the Mg2+ block of NMDAR channels,which ultimately enhances NMDAR activity and the inflow of more extracellular Ca2+ and further enhances PKC activity and its effect on the NMDAR channel. The PKC-NMDAR positive feedback loop plays an important role in central sensitization.Flunarizine,a calcium channel antagonist,is effective in preventing migraine attacks.Double-blind trials reveal that flunarizine is effective in limiting frequency of the attacks and pain severity.But its concrete mechanism is undefined.ObjectiveThe study will engage in PKCγ,NMDAR1 and phosphorylated NMDAR1,build up Nitroglycerin induced experimental migraine model and simultaneously set up NS control group and Flunarizine group in order to investigate the possible mechanism of the PKC-NMDAR positive feedback loop in the procession of migraine.Methods1.Grouping and building model:30 Sprague-Dawley(SD) rattus were divided into three groups randomly.Model group:according to Tassorelli,NTG was injected subcutaneouly by 10mg/kg,1 time every week for 5 weeks,at the same time NS was given by intragastric administration by 10ml/kg each day;NS control group:NS was injected subcutaneouly by 10mg/kg,1 time every week for 5 weeks;Flunarizine group:NTG was injected subcutaneouly by 10mg/kg,1 time every week for 5 weeks,at the same time flunarizine was given by intragastric administration by 5mg/kg/d by each day.2.At 2 hour or on the 4^th day after NTG was injected subcutaneously for the 5^th,the brain stem was taken out from the rats and stored in liquid nitrogen.3.The expressions of PKCγand NMDAR1 mRNA in the brain stem was detected by RT-PCR.4.The expressions of PKCγ,NMDAR1 and phosphorylated NMDAR1 protein in the brain stem was detected by Western-blotting.Results1.Compared with NS control group,regardless of in period of onset and intermission of migraine,there is no statistical difference in the expression of brainstem PKCγmRNA and protein in the model group and flunarizine group(p＞0.05);2.Compared with NS control group,regardless of in period of onset and intermission of migraine,there is no statistical difference in the expression of brainstem NMDAR1 mRNA and nonphosphorylated protein in the model group and flunarizine group(p＞0.05);3.Compared with NS control group,the expression of phosphorylated NMDAR1 protein in model group was significantly upregulated(p＜0.05),but there is no statistical significance between flunarizine group and NS control group(p＞0.05);between in period of onset and in period of intermission in model group and between in period of onset and in period of intermission in flunarizine group,the expression of phosphorylated NMDAR1 protein did not change significantly (p＞0.05).Conclusions1,The upregulated expression of PKCγ-dependent phosphorylated NMDAR1 protein may have a correlation with occurrence and development of migraine,nor do PKCγmRNA and protein and NMDAR1 mRNA and protein(nonphosphorylated).2,One of the mechanisms of flunarizine to prevent migraine attacks may be interrupt infernal circle of the PKC-NMDAR positive feedback loop.