Protective Effects Of Paeonol On Rat Cultured Hippocampale Neurons Subjected To Oxygen-Glucose Deprivation And Reperfusion

Background:Ischemic cerebrovascular disease (ICVD) is an usually pathologic process and influence human health and life badly. At present cerebrovascular disease have become one of these disease that can cause the seriouse complication, lingering effects and so much as cause death all over the word. It is very important to research the mechanism of cerebrovascular disease and look for the efficacious drugs actively. As we know, today the therapy of thrombolysis and brain protection for ischemic cerebrovascular disease is epidemic trend all the same . Now because of the good curative effect in the treatment of the ischemic cerebrovascular diseases, traditional Chinese medicines have been pay more and more attention to by lots of researchers. Paeonol, a major phenolic component of Moutan Cortex, as traditional Chinese medicine, has been described to possess analgesic, sedative, anti-inflammatory, anti-allergic and antimicrobial properties. Our study is to determine if paeonol can protect hippocampal neurons subjected to oxygen-glucose deprivation and reperfusion injury and elucidate the underlying mechanisms.Modern medicine researches have showed that oxygen-glucose deprivation and reperfusion injury is main pathological and physiological process of ischemic cerebrovascular disease. The mechanismes of oxygen-glucose deprivation and reperfusion refer to many theories such as free radical theory, exitotoxicity theory and calcium overload theory and so on. In 1956, American researcher Harman proposed the free radical damage theory firstly. They believed that the cells needing oxygen would produce lots of toxical superoxide free radicals during metabolic process, which resulted in creature injury and apolexis. Under the condition of ccerebral ischemia, biological oxidation function of brain cells was disordered and cells generated a great many free radicals, then free radicals entered and diffused in blood, resulting in the damage to brain tissue , cells and biomolecules. What is more, the free radicals produced wildnes lipid peroxides that had many kinds and malondialdehyde ( MDA) was the uppermost kind of that. In addition, free radicals took oxidative damage to vascular endothelial cells , capillary basement membrance and brain cells, consequently increased permeability of capillary vessel and disordered function of cells. One of the primary antioxidase was superoxide dismutase ( SOD) that can catalysed the dismictation reaction of superoxide anion radical and blocked the toxic action of free radicals. So the lipid peroxidation reaction, resulted from oxygen free radicals, was one of the main mechanisms of ischemic cerebrovascular disease. Recent study has revealed that nitrogen monoxidum ( NO ) involved in the whole pathological and physiological process of ischemic cerebrovascular disease. Under the pathological condition , NO itself was one of free radicals that caused the lipid peroxidation reaction and produce oxidation nitrous acid ion by reaction with superoxide free radical, which preoxidate lipid, DNA and protein thiol group etc., finally resulted cells death. The double biologic function of NO was decided mainly by activity of nitrioxide synthase ( NOS ). NOS was divided into three types including neuronal NOS( nNOS ), endothelial NOS( eNOS ) and induced NOS( iNOS ). In the physiological condition, nNOS and eNOS induced by calcium ion could express and had activity, while iNOS, independent of Ca²⁺, only expressed in pathologic status. During earlier period of ischemical reperfusion time, NO produced by eNOS broadened blood vessel, restrained platelet aggregation and leucocyte adherence, which showed the useful function. However, according to the lasting of reperfusion time, iNOS and nNOS began to express and produced large number of NO that resulted in damage to cells.Recently, theory about neurotoxicity cascade reaction is widely accepted for lots of researchers and excitatory amino acid ( EAA ), especial glumatic acid, have important role in ischemic cerebral vascular disease. Nowadays, theory of acute cerebral accident is transitional from the cognition of "blood suspension→no substrate→energy failure→cell death" to the theory of neurotoxicity cascade reaction, at least including four different but interconnected mechanisms: exitotoxicity, infarction surrounding depolarization, inflammation and programmed cell death. Cascade reaction is initiated from exitotoxicity; that is, exitotoxicity is the most important process in the accidence of acute cerebral injury. Recently excitotoxicity has been described in virtually every region of the center nervous system ( CNS ) and is thought to stimulate a series of linked events that lead to cell death of ischemic cerebrovascular vascular disease. Recent study has revealed that N-methyl-D-aspartate (NMDA) receptors, a subclass of excitatory amino acid receptors and ligand-gated ion channels, permeable to Ca²⁺ as well as Na⁺, may play an important role in ICVD. Under ischemic conditions, the levels of glutamate in extracellular space are markedly elevated. High concentrations of extracellular space are lead to overactivation of the NMDA subtype of glutamate receptors located on neuronal plama membranes, which results in Ca²⁺ overlocated into neurons and ultimately, neuron death. The NMDA receptor is a heteromeric structure consisting of four or five subunits. Assembly of a functional receptor requires the presence of an NR1 subunit, which is derived from a single gene and exists in at least eight different splice variant forms, along with one or more NR2 subunits (NR2A, NR2B, NR2C and NR2D) and/or a NR3 subunit, all derived from separate genes. Recombination studies have shown that the combination of an NR1 subunit with a NR2 or NR3 subunit results in a functional receptor with a unique physiological and pharmacological profile. Objection:Many previous studies have shown that paeonol, as traditional Chinese medicine, could exhibit its protective effects against brain ischemia injury, resulting in reducing the infarct size, decreasing the content of inflammatory cellsand adhesion molecules and inhibiting cell apoptosis in ischemia area in vivo. In our present study, the injury model was established by oxygen-glucose deprivation in vitro, to investigate the protective effects against oxygen-glucose deprivation of paeonol and the potential neuroprotective mechanism of paeonol. Methods:1. Our study were performed using hippocampal neurons in vitro hippocampal slices.2. The cultured rat neurons were randomly divided into four groups, â‘ control group, neurons was treated with DMEM containing glucose and incubated with 5% CO₂ and 95% O₂ gas;â‘¡injury model group, model of oxygen-glucose deprivation and reperfusion injury group: neurons were washed with Earle's balanced salt solution without glucose for four times, and put into oxygen deficient box with 100μl Earle's without glucose in each dish for three hours with 100% N₂ gas. After that, the neurons were incubated in DMEM at 37 °C for 6,12, and 24 h respectively;â‘¢Paeonol treatment groups, The model of oxygen-glucose deprivation and reperfusion injury was established by changing non-sugar Hank's, then the paeonol was joined before the time of reperfusion, which included the three concentration of paeonol(5μmol/L, Pae-H; 1μmol/L, Pae-M and 0.2μmol/L, Pae-L) ;â‘£MK-801(dizolcipine maleate) group(10μmol/L). Drugs were administrated during ischemic time, acting through the processes of oxygen-glucose deprivation and reperfusion.3. Each group was observed by inverted phase contrast microscope; neuron viability was measured by the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide ( MTT ); glumatic acid was analyzed by amino acid analyzer; binding force of NMDA receptors was evaluated by liquid scintillation counting; the expression of NMDA receptors NR1 subunit mRNA was semiquantitatively determined by reverse transcription-polymerase chain reaction (RT-PCR ); the content of SOD, MDA, NO and the activity of NOS were observed by reagent box.Results:Compared with deprivation and reperfusion group at each designated period, paeonol treatment after deprivation and reperfusion obviously increased cell survival rate, reduced the binding force of NMDA receptors and the release of glutamate; down-regulating the expression of NR1 subunit, and decreased the content of SOD,MDA, NO and the activity of NOS.Conclusions:These results demonstrate that all concentration paeonol, especial high concentration paeonol, protected rat neurons from ischemia-reperfusion injury by the antioxidation of the effect on oxygen free radical and inhibiting the neurotoxicity cascade reaction resulted from the action on NMDA receptor and the decrease of the levels of extracellular glutamate direct, which resulted in alleviating the morphological damage and increasing neuron viability.