The Role And Mechanism Of Trichostatin A On Neonatal Rats With Hypoxia-ischaemia Brain Damage

Purpuse To explore the potential protective effect of a histone deacetylase inhibitor,trichostatin A(TSA),against hypoxic-ischemic brain damage and the epigenetic modification of the down-regulation of KCC2 in the neonatal rat model of HIBD to supply medication reference in the clinical treatment of neonatal hypoxic-ischemic brain damage.Methods 1.All 7-day-old(P7)neonatal Sprague-Dawley rats were randomly assigned into three groups: the sham group,HIBD group,and TSA group.And the hypoxicischemic brain damage(HIBD)model used in this study was induced according to the Rice model;2.Using TTC staining to investigate the cerebral infarction volume of each group of neonatal rats;3.Using locomotor activity testing to investigate the locomotor activity of each group of neonatal rats;4.Using morris water maze testing to investigate the long-term spatial learning and memory ability of each group of neonatal rats.5.Using RT-PCR to investigate the m RNA expression of KCC2 in neonatal rats at three different times post-HIBD,compare the m RNA expression of Neu N and KCC2 in neonatal rats at 3 days post-HIBD and investigate the m RNA expression of REST,Co REST,HDAC1,KCC2 in each group of neonatal rats at 3 days post-HIBD.6.Using western blot and immunofluorescence assay to investigate the protein expression of REST,Co REST,HDAC1,KCC2 in each group of neonatal rats at 3days post-HIBD.Results 1.After the ligation of the left common carotid artery,the skin color of the rats appeard pale,and returned to normal in a short time;In the process of hypoxia,the skin color of the rats deepened,accompanied by convulsions and restless;The animals rotated to the left and obtusely reacted to the outer stimulation after HIBD.2.TTC staining revealed that the HIBD group showed significant infarction in the ipsilateral hemisphere compared to the sham group,which indicated that the establishment of the HIBD model was successful.The cerebral infarction volume in the TSA group was significantly reduced compared to the HIBD group,which indicated that TSA has short-term protective effect on neonatal rats with hypoxic-ischemic brain damage.3.The locomotor activity counts of the rats in the HIBD group were significantly reduced compared to the rats in sham group.By contrast,the locomotor counts of the rats in the TSA group were higher than the rats in the HIBD group.4.Morris water maze test(1)The place navigation test showed that as time passed,the mean escape latency of each group gradually shortened,indicating that all the groups demonstrated spatial learning.However,the mean escape latency of the HIBD group was significantly longer on the third,fourth and fifth days than that in the sham group.For the rats in the TSA group,the mean escape latency was shorter than in the HIBD group.(2)The spatial probe test showed that the rats in the sham group spent more time in the goal area and crossed the platform area more often than those in the HIBD group.Improvement was observed in the rats treated with TSA.5.RT-PCR(1)we performed RT-PCR to investigate the m RNA expression of KCC2 inneonatal rats at 3 days,7 days and 14 days post-HIBD.The results demonstrated that the m RNA expression of KCC2 in the neonatal rat model of HIBD was decreased significantly at 3 days post-HIBD.Thus,we conducted later molecular experiments at 3 days post-HIBD.(2)At 3 days post-HIBD,we simultaneously investigated the m RNA expression of Neu N and KCC2 in the neonatal rat model of HIBD.The result showed that the decreased level of m RNA expression of Neu N was obviously lower than that of KCC2;that is,the decreased expression of KCC2 in the neonatal rat model of HIBD was mainly due to neuronal damage rather than death.(3)Besides that,The results respectively demonstrated that the m RNA expression of REST,Co REST,HDAC1 was increased significantly at 3 days post-HIBD compared to the sham group while the m RNA expression of KCC2 was reduced obviously.TSA could prevent the down-regulation of m RNA expression of KCC2 via the inhibition of REST,Co REST,HDAC1 in the ipsilateral hemisphere.6.Western blot:the ipsilateral hemisphere elicited a considerable increase in the REST/Co REST/ HDAC1 complex at 3 days post-HIBD compared to the sham group.In addition,a significant decrease in KCC2 was observed in the HIBD group,while the protein expression of REST,Co REST and HDAC1 was evidently reduced in the TSA group,and the protein expression of KCC2 was significantly enhanced.The results of Immunofluorescence staining were consistent with these results.Conclusion Our current study illustrated that TSA had short and long term protective effect on hypoxic-ischemic brain damage in neonatal rats.And the mechanism was that TSA could inhibit the transcription complex REST/Co REST/HDAC1 and promoted the expression of KCC2 by epigenetic regulation after cerebral ischemia and hypoxia to maintain the steady state of Cl-in neurons and play a protective role.