| Background Hypoxic-ischemic encephalopathy(HIE) is the main causes of neonatal mortality and nerve system dysfunction of infants. The treatment of the acute phase of HIE emphasized the supportive therapy and positive symptomatic treatment to save the lives of most infants, but the corresponding sequela incidence such as cerebral palsy, epilepsy, mental retardation, learning difficulties, audio-visual obstacles increased significantly,and recovery is not ideal, so to explore the clinically safe and effective treatment is the hot spot of peripheral medical research at home and abroad. In recent years, Studies have found that cytokine plays a very important role in hypoxic-ischemic encephalopathy. Some cytokines such as interleukin-1(IL-1), interleukin-6(IL-6), inter-leukin-8(IL-8) and tumor necrosis factor-alpha (TNF-alpha) associated with hypoxia ischemic injury, and some cytokines such as transforming growth factor-beta1(TGF-β1) is associated with the repair of hypoxic ischemic encephalopathy.Recombinant human erythropoietin(rhEPO), as a acid glycoprotein, had been regard-ed as a body fluid factor concerning promoting proliferation anddifferentiation of the blood ancestral cell. People have studied more about the relationship between EPO and the cyto-kines such as IL-1, IL-6and TNF-a,the study about the relationship between rhEPO and TGF-β1was less, moreover there is no related reports about the hypoxic ischemic enceph-alopathy in newborns and infants. Through the experiment we would observe the protect-ive effect of rhEPO on the hypoxic-ischemic brain tissue and the influence on TGF-β1pro-duction which protect brain tissue. Objective Seven-day-old newborn health Sprague-Dawley(SD)rats, established animal model by hypoxia-ischemia, rats were instantly given single does of recombinant human erythropoietin(rhEPO) by intraperitoneal injection following the hypoxic-ischemic brain damage, authenticated the neuropertective role of rhEPO by observe the effect of rhEPO on the body weight of newborn rats, pathological changes of brain tissue, cell apoptosis of the injured side tissue, the learning and memory abilities of pups; To investigate the expression of TGF-β1in the brain tissue of newborn HIE rats, then observe the effectiveness of rhEPO on the expression of TGF-β1after hypoxic-ischemic in rat brain,and explore the possible mechanism of the neuroprotective effect of rhEPO.Methods1.220Seven-day-old newborn health SD rats,were randomly divided into sham group, HIE model group, low-does of rhEPO therapy group and high-does of rhEPO therapy group, each group had55rats. In sham-operated group, rats were performed operation by separated the left common carotid artery,ligation and hypoxic-ischemic were not conducted. In HIE group, the left common carotid artery of rats were separated and double ligated. Then the rats were returned to their dam and allowed to recuperate for2hours. Rats were then placed in a2000ml normal temperature and pressure hypoxic chamber that contained8%oxygen balanced with92%nitrogen for2hours, at the flow speed of2L/min. Sham operation and HIE groups after modeling, the rats were injected intraperitoneally0.5ml saline; In low-does of rhEPO therapy group, rats were instantly given single does of recombinant human erythropoietin (1000IU/Kg) by intraperitoneal injection following the hypoxic-ischemic brain damage; In high-does of rhEPO therapy group, rats were instantly given single does of recombinant human erythropoietin (5000IU/Kg) by intraperitoneal injection following the hypoxic-ischemic brain damage. Both does of rhEPO were diluted with saline to0.5ml.2.Observe the behavior changes of the rats in every group during the preparation of model. Every group of rats were wighted at6h,24h,72h,7d after recovery from hypoxic-ischemia. Taken one of the72h point rats of every group then sacrificed them to make2,3,5-triphenyltertrazolium chloride(TTC) staining of brain tissue, and then six rats of every group at every time point were sacrificed, brain tissue of them were made into paraffin section, which was for hematoxylin-eosin(HE) staining to observe pathological changes of the brain tissue, to measure cell apoptosis by Tunel and to analyze the expression of TGF-β1protein of cortex and hippocampus in the damage side; Six rats in every subgroup were randomly sacrificed at6h,24h,72h,7d after hypoxia-ischemia, the brain tissue from cortex and hippocampus of the left side were saved in liquid nitrogen tank, using western-blot method to analyze the expression of TGF-β1protein; The other part of the rats were used to conduct morris water-maze test when they were twenty-eight days old, every group had6rats.SPSS16.0software was used to analyze the date, a=0.05was considered the level of significance.Result1.The HIE model、low-does of rhEPO therapy group and high-does of rhEPO therapy group rats showed various abnormal behaviors after hypoxic, while there was no behavior changes in sham-operation rats; The gross specimen and TTC staining of brain tissue showed that the side of the injury brain tissue of HIE model、low-does of rhEPO therapy group and high-does of rhEPO therapy group rats appeared white color,which was the feature of cerebral ischemia; the brain tissue of the sham operation had no such phenomenon; Compared with sham group, the body weight of the rats in model group, high-dose rhEPO and low-dose rhEPO at24h,72h,7d grew slower (P<0.05), apoptosis percentage was higher(P<0.05), the pathological changes of the damaged brain tissue was more seriouser, the average escape latency of water maze test was longer(P<0.05), the differences were statistically significant.2. The body weight of the newborn rats with high-dose of rhEPO treatment and low-dose of rhEPO therapy groups was heaver than HIE model group in every time point(P<0.05); Pathological changes of the ipsilateral brain tissue was lighter compared with HIE model group,significantly decreased apoptosis(P<0.05), the time of the water maze test from one end to swim to the other end of the platform was significantly shortened (P<0.05),there were significant differences. High-dose of rhEPO therapy group compared with low-dose of rhEPO treatment group, the body weight was heavier at72h,7d time point(P<0.05), relieved pathological changes of brain tissue, reduce apoptosis (P<0.05), the time of the water maze test from one end to swim to the other end of the platform was significantly shortened(P<0.05), that the differences were statistically significant.3. Immunohistochemical staining showed that TGF-β1mainly expressed in the cortex and hippocampal of the damaged side after hypoxic-ischemic injury,and mainly expressed in cytoplasm of the neurons, glial cells and vascular endothelial cell. In sham group, Immunohistochemistry and western-blot analysis showed TGF-β1protein was less expressed, the changes at every time point had no statistical difference. The expression of TGF-β1in HIE model group, high-dose of rhEPO and low-dose of rhEPO therapy group appear at6h after hypoxic-ischemic, increased at24h, cytoplasm staining and particles enlarged,the express peaked at72h then reduced at7d; The expression of TGF-β1increased first and then decreased, peaked at72h; There had statistical difference compared with sham group(P<0.05). There were significant differences about the mean gray value of the expression of TGF-β1in high-dose and low-dose rhEPO therapy at every time point compared with HIE model group(P<0.05); There had significant difference about the mean gray value of the expression of TGF-β1in high-dose rhEPO therapy at every time point compared with low-dose rhEPO therapy(P<0.05).Conclusion1.rhEPO plays a neuroprotective effect in HIE, and high-dose of rhEPO has better neuroprotective effect than low-dose rhEPO.2. rhEPO can increase the expression of TGF-β1in damaged brain after hypoxia-ischemia. Presumably, by upregulating expression of TGF-β1maybe a mechanism of rhEPO in neuroprotective effect. |
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