The Influences Of Polydatin In ERK Pathway Of Neonatal Rats With Hypoxic-ischemic Brain Damage And The Mechanism Of Polydatin In Improving The Ability Of Learning And Memory

Objective: Hypoxic-ischemic brain damage（HIBD）in neonatal period is one ofthe most harmful diseases of the nervous system, which often causes the death ofneonates. The survival also has the varying degree of nervous system developmentaldisorders. HIBD pathogenesis is complicated. For the majority of neonatals, HIBDcomprehensive treatment alleviates the symptoms, protects brain tissue, and minimizesfurther injury of brain. But perinatal cells are easier to be damaged than in adulthood,which leads to further investigation of these therapeutic measures in the neonatals. Andwhether the effective treatment in the mature brain also take effect in the immaturebrain, whether various treatment methods has consistent protective effect for long-termcontinuous development still needs more research. Notably, during the brain damage,the body will produce corresponding protective factors against injury response.Therefore, studies of up-regulations of the protective factor level and reduction of injuryfactor level can lead to important targets in clinical treatment.Brain derived neurotrophic factor is an important regulatory factor in synapticplasticity and memory, which plays a significant role in hypoxia-ischemia brain damagerepair as a neuro-protection. While in MAPK/ERK pathway activation, BDNF cancause feedback inhibition of the neuronal excitotoxicity and plays a neuro-protectiverole. In various intracellular signaling kinase cascade chains, extracellular signaling ofERK kinase is the most possible brain-derived neurotrophic intracellular mechanism inneuroprotection. After cerebral hypoxia-ischemia, the high expression ofphosphorylated ERK may be the results of endogenous neuroprotective responses. Afterhypoxia-ischemia brain damage, the immediate early genes such as c-Jun rapidly expresses in the short term, leading to some late effect genes inhibition or activation,and ultimately necrosis and apoptosis of nerve cells. Therefore the use of drugs tomaintain protective signaling pathway activation, up-regulation of the levels of braininjury protective neurotrophic factors, while reduction of early nerve injury caused byfactors such as c-Jun, is the important strategies of clinical treatment ofhypoxic-ischemic brain damage.The existing research and our previous work demonstrated the traditional Chinesemedicine, monomer polydatin（PD）, has obvious protective effect in cardiovascular andcerebrovascular system. PD can function in the regulation of vascular tone, inhibition ofplatelet aggregation, improvement of microcirculation, protection of vascularendothelial cells and antioxidant effect. It also has the protective effect of cerebralhypoxia ischemia in acute period of brain tissue cell injury. Currently, PD achievedgood clinical results in clinical treatment of acute cerebral infarction, Parkinson’sdisease and senile dementia. These basic research and clinical practice provide a newthought to find an effective treatment for the potential drugs of neonatal HIBD. Moreimportantly, it reflects the aim of whole regulation in Chinese traditional medicinewithin the treatment of diseases. But so far, the study of the polydatin in long termlearning and memory ability of neonatal rat brains after cerebral hypoxia-ischemiainjury and its mechanism research is not well known. Based on the above research andtheoretical basis, we put forward a tentative idea about polydatin in which we appliedthe polydatin to the treatment of neonatal rat HIBD to study the effect ofhypoxic-ischemic brain damage in neonatal rats’ long term learning and memoryabilities. We further explored the possible mechanism of polydatin in HIBD neonatalrat’s long term learning and memory protection, protein levels of ERK pathway andnerve protection factor BDNF expression to clarify the mechanism of PD for HIBDneonatal rats’ learning and memory ability, and to detect the c-Jun expression to clarifythe neuroprotective mechanism of polydatin. These studies will further clarify theprotective role and mechanism of polydatin in cerebral hypoxic-ischemia injuryprotective effect, further provide the role of polydatin in clinical application andtheoretical basis of neonatal hypoxic-ischemic encephalopathy.Methods:1.7day old SD rats were randomly divided into three groups: false operationgroup (group NS), hypoxia and ischemia group (group HI), PD group (group PD).According to Rice’s methods for preparation of neonatal rat model of hypoxic-ischemic brain damage, separation and ligation of the left common carotid artery2hours afterinhalation of8%oxygen nitrogen oxygen gas mixture for2hour. In NS group, the ratswere only released left common carotid artery but were not ligated and gained anoxictreatment. In group PD, the hypoxia ischemia rats were intraperitoneal injected0.1%PD10mg/kg per day for10days. In group NS and group HI, the rats were injected theequal volume of sterile saline solution. After24hours,10days and21days hypoxiaischemia injury, the rat cortex, hippocampus brain tissue pathological structure changeswere observed by HE staining at the three times. After21days hypoxia ischemia injury,the rats were tested through Y maze and step-down test to evaluate the rats’ learningand memory function changes.2. In PD group, inject hypoxic-ischemia neonatal rats0.1%PD10mg/kgintervention therapy for1day. In group NS and HI, inject the equal volume of sterilesaline solution for1day. Use western blot to detect the expression changes of p-ERKand p-CREB of rats after24hours hypoxic-ischemia treatment. Utilizeimmunohistochemical method to detect the c-Jun expression in hippocampal CA1, CA3and DG area. Use western blot to detect the expression of c-Jun in rats after24hoursHIBD.3. In group PD, inject hypoxic-ischemia neonatal rats with0.1%PD10mg/kgintervention therapy for10days. In group NS and HI, inject the equal volume of sterilesaline solution for10days. Utilize immunohistochemical methods to detect the BDNFexpression in left cortical, hippocampal CA1and CA3area.Results:1. Y maze experimental results show that, in the first day of the learning test andthe next day of memory retention test, compared with the NS group, the total reactiontime in HI group was significantly prolonged (P <0.01), error number increased (P<0.01), active avoidance rate reduced (P <0.01). Compared with the HI group, the totalreaction time in PD group was significantly decreased (P <0.01), error numberdecreased (P <0.01), active avoidance rate increased (P <0.01).2. The experimental results of Step-down test show that, compared with the NSgroup, in the first day learning test, in HI group, reaction time extended (P <0.01), thenumber of errors increased (P <0.01), in the next day memory retention test, in HIgroup, latency reduced (P <0.01), the number of errors increased (P <0.01).Compared with the HI group, in the first day learning test, in PD group, reaction timereduced more than in HI group (P <0.05), the number of errors was less than in HI group (P <0.01), and the number of errors compared with NS group had nodifferences (P>0.05).in the next day memory retention test, in PD group, latency islonger than in HI group (P <0.05), the number of errors in less than in HI group (P <0.01), and the number of errors compared with NS group had no differences (P>0.05).3. After24hours hypoxic-ischemia injury, the western blot results showed that rathippocampal areas in NS group had the basal expression of p-ERK, p-CREB. In HIgroup, the p-ERK, p-CREB expression in rat hippocampal area were increased whencompared with NS group (P <0.01), In PD group, rat hippocampal p-ERK, p-CREBexpression were significantly increased when compared with HI group (P <0.01).4. Immunohistochemical results showed that, after24hours hypoxic-ischemiainjury, the BDNF expression of HI rats compared with the NS group significantlyincreased in cortex contralateral hippocampal CA1and CA3area (P <0.01). After10days, it is still higher than that of group NS (P <0.01), with the prolongation of injurytime, the BDNF expression in HI group gradually fall, on the21day, there are nosignificantly different BDNF expression in HI group cortex and hippocampal CA1area,CA3area when compared with NS group (P>0.05). The PD group showed the sametrend, after24hours hypoxic-ischemia injury, the BDNF expression of PD groupsignificantly increased in cortex contralateral hippocampal CA1area, CA3areacompared with the NS group (P <0.01), while in cortical, hippocampal CA1area, therewere no significant difference (P>0.05). The expression of BDNF in CA3area ishigher than that of HI group (P <0.05). The BDNF expression in CA3area is higherthan that of HI group (P <0.05), on the21day, the BDNF expression in PD group washigher than that of group HI (P <0.05). In rats with hypoxic-ischemic hippocampal area,western blot data gain the similar results with the immunohistochemical data, afterhypoxic-ischemia, BDNF expression in HI group and PD group were increased whencompared with NS group, with the prolongation of time, the expression of BDNFgradually fall. After21days hypoxic-ischemia injury in HI group, it had fallen to thelevel of NS group, in PD group, at24hours (P <0.05),10days (P <0.01) and21days(P <0.05), BDNF expression is higher than that of HI group.5. After24hours hypoxic-ischemia injury, in HI group, in left cortex,hippocampal CA1, CA3and DG area, the immunohistochemical results showed thepositive expression of c-Jun was significantly higher than that of group NS (P <0.01),in PD group, it was significantly decreased (P <0.01). The western blot results showed that the c-Jun expression in left hippocampal in HI group was higher than that of groupNS (P <0.01), while in PD group, its expression was lower than that in the HI group (P<0.01).6. Under a light microscope, HE staining showed that the left hemisphere of thebrain organization of cortical, hippocampal CA1area in NS group displayed clear layers,normal nerve cell structure and morphology. After24hours hypoxic-ischemia injury in10days HI group, left brain tissue became thin, hippocampal pyramidal cell layerreduces, nerve cell deranged, cell swelled, dissoluted, and displayed the obviousneuronal loss and focal necrosis. After21days hypoxic-ischemia injury, cortex,hippocampus and other areas formed the glial scar. The rats in PD group at each timepoint still have various degrees of neuronal degeneration, but the majority of theneurons structure are complete, and the hippocampal pyramidal cell layer, cell shapeand arrangement are generally normal.Conclusion:1. HIBD can cause brain tissue pathological changes. The learning and memoryability of neonatal rats were reduced. Polydatin treatment can alleviate the injury ofbrain tissue of HIBD rats and improve the ability of learning and memory.2. After hypoxic-ischemic brain damage, the expression of p-ERK and p-CREBwere increased in hippocampal areas of neonatal rats, MAPK/ERK signaling pathwayis activated. Polydatin can enhance hippocampal p-ERK and p-CREB expression, whichsuggested that the learning memory protection of polydatin may be mediated by MAPK/ERK signal pathway.3. The expression of BDNF in HIBD neonatal rats is increased afterhypoxic-ischemia injury. Polydatin can up-regulate and last the expression of HIBD inbrain tissue of neonatal rats in cerebral cortex and hippocampus. This effect may beinvolved in hypoxic-ischemia neuroprotection.4. The expression of c-Jun is increased after hypoxic-ischemia injury. Polydatinreduces c-Jun expression in HIBD neonatal rats’ cerebral cortex, hippocampus, whichmay have an early protective effect in neurons after hypoxic-ischemia.