The Role And Mechanism Of Melatonin On Hypoxic-ischemic Brain Damage In Neonatal Rats

Research background and purpose:Neonatal hypoxic-ischemic brain damage(HIBD)is the main cause of neurological sequelae in children,such as learning disabilities,epilepsy and even cerebral palsy.Neuron death is an important mechanism of HIBD that it is difficult to recover from neurological sequelae.Therefore,the main strategies for the prevention and treatment of neurological sequelae of HIBD are protecting neurons damage and reducing their death.Ferroptosis is a newly discovered mechanism of cell death,which depends on lipid peroxidation and plays a vital role in a variety of nervous system diseases.In recent years,studies have shown that melatonin(Mel)has the advantages of high-fat solubility,low toxicity,and strong antioxidant capacity.Mel combined with mild hypothermia treatment can reduce acute death and improve prognosis in hypoxic-ischemic encephalopathy,but its mechanism is still unclear.This study intends to investigate the potential mechanism by which Mel can improve the prognosis of HIBD through HIBD rat model,and to provide reliable experimental and theoretical basis for clinical application of Mel for the treatment of HIBD.Materials and Methods:The neonatal rat model of HIBD was established according to Rice.The expression of Gpx4,the key regulatory enzyme of ferroptosis in the ipsilateral hippocampus,was detected by Western blot at different time points(3h,6h,12h,24h,48h,and 72h)after hypoxia-ischemia,and the time point of Gpx4 expression valley value was selected as the sample havest time.After intraperitoneal injection of low,middle and high doses of Mel,the learning and memory function of rats were evaluated by Morris water maze test,and then the most effective dose of Mel was selected.Firstly,rats were randomly divided into Sham group and HIBD group.The morphology changes of hippocampal tissue were detected by HE staining;NeuN positive(NeuN~+)neurons were measured by immunohistochemical staining;the protein levels of ferroptosis markers(Gpx4 and4-HNE)were evaluated by Western blot;the content of glutathione(GSH)was detected by specific kit,and the pathological changes of neuronal mitochondria was detected by Electron microscopic.The above tests were applied to show the role of ferroptosis in the pathogenesis of HIBD.Secondly,the rats were randomly divided into four groups,including Sham group,HIBD group,control(DMSO)group,and Mel treated(Mel)group.The pathological changes of hippocampal tissue,NeuN~+neurons,ferroptosis markers(Gpx4?4-HNE and GSH),and neuronal mitochondria were detected by the previously described methods to study the effect of Mel on neurons ferroptosis in HIBD rats.Finally,the rats were randomly divided into Sham group,HIBD group,DMSO group,Mel group,PI3K/Akt inhibitor LY294002 intervention Mel group(LY294002),Nrf2 inhibitor ML385 intervention Mel group(ML385)and Gpx4 inhibitor RSL3intervention Mel(RSL3)group.The protein levels of Akt,p-Akt,Nrf2,Gpx4 and4-HNE were detected by Western blot,the content of GSH was detected by specific kit,and the learning and memory function of rats was evaluated by Morris water maze test,which to explore the mechanism of Mel on improving HIBD.Result:(1)The role of neurons ferroptosis in HIBDThe expression of Gpx4 decreased significantly at 6 h,12 h,24 h,48 h,and 72 h after HI,and reached the trough at 48 h.Compared with the Sham group,the cells in the hippocampal CA1 region of HIBD rats were disorganized,and the nucleus was small,darkened,or even fragmented.In addition,the number of NeuN~+neurons and the expression of ferroptosis markers Gpx4 and GSH decreased post HIBD.Moreover,the expression of 4-HNE increased,and the neuron mitochondria shrunk and the mitochondrial membrane density increased in HIBD rats.(2)Effect of Mel on neurons ferroptosis in HIBD ratsThe results of Morris water maze test showed that compared with Sham group,the escape latency of HIBD group was significantly increased and the number of times of crossing the platform was significantly decreased.Compared with DMSO group,the escape latency of rats in Mel(10mg/kg)and Mel(20mg/kg)groups was relatively decreased,and the number of times of crossing the platform was markedly increased,especially in Mel(10mg/kg)group.Compared with DMSO group,The cells in the hippocampal CA1 area of the Mel(10mg/kg)group were relatively regular,with larger nuclei and larger circles.Meanwhile,the number of NeuN~+neurons and the expression of ferroptosis markers Gpx4 and GSH in hippocampus were increased in the Mel(10mg/kg)group.Furthermore,the expression of 4-HNE was decreased,and the shrinkage of neuron mitochondria as well as the increase of mitochondrial membrane density were relatively alleviated in the Mel(10mg/kg)group compared with DMSO group.(3)Effect of the Akt/Nrf2/Gpx4 signaling pathway on the protective effect of Mel in HIBDCompared with Mel(10mg/kg)group,the cells in CA1 region of hippocampus in LY294002 group,ML385 group and RSL3 group were relatively scattered,the nucleus was small and dark,the number of NeuN~+neurons and the expression of ferroptosis markers Gpx4 and GSH decreased,the expression of 4-HNE increased,the neuron mitochondrial shrinkage and mitochondrial membrane density increased significantly,and the escape latency increased significantly and the number of crossing the platform decreased.Conclusion:Neurons ferroptosis is an important mechanism of learning and memory impairment in HIBD,and Mel can inhibit neurons ferroptosis through Akt/Nrf2/Gpx4signal pathway,which can improve the learning and memory function recovery post HIBD.