Ramelteon Protects Against Ischemic Brain Injury In Mice

Objective:Melatonin receptors is a potential target for ischemic brain injury therapy.Ramelteon which is used for insomnia treatment is a more potent and selective melatonin receptor agonist than endogenous melatonin.We aimed to identify that whether Ramelteon has anti-ischemic brain injury in mice and further explore the mechanisms.Methods:C57BL/6 mice were subjected for permanent middle cerebral artery occlusion(pMCAO)to establish an animal model of acute cerebral ischemia.At the completion of pMCAO surgery,mice were intragastric administrated ramelteon 0.33,1,3 mg/kg,and infarct volumes and neurological deficit scores were measured 24 hours after occlusion;Ramelteon 3 mg/kg,melatonin receptor antagonist 4-P-PDOT 3 mg/kg or both two drugs were intraperitoneally injected respectively at the same time to investigate whether the effect of ramelteon was antagonized by melatonin receptor antagonists;Edaravone of 3,10,30 mg/kg(acute cerebral infarction treatment drug)was intraperitoneally injected to determine the best dose in this experimental model.At the onset(0 h)or 4 h or 6 h after pMCAO onset,ramelteon 3 mg/kg or edaravone 10 mg/kg was intragastric administrated to investigate the time window of ramelteon and also compared the curative effect of ramelteon to edaravone against cerebral ischemic injury.C57B/6 mice were subjected for photochemical embolization to establish an animal model of chronic cerebral ischemia.On the 7th day after thrombosis,ramelteon 3 mg/kg or 4-P-PDOT 3mg/kg was intragastric administrated respectively;cylinder task and grid-walking task were performed every other day to detect the motor function of mice,and detect the mice's fine motor coordination ability respectively;the cerebral infarction volume of mice was detected by toluidine blue staining after 15 days of ischemia.After pMCAO onset,mice were adminitrated ramelteon 3 mg/kg,4-P-PDOT 3 mg/kg or both,then we detected the expression of the autophagy marker proteins(SQSTM1,LC3-?)and the autophagy signaling pathway proteins(AMPK,p-AMPK,p-mTOR,p-p70S6)after 24 h.Ramelteon 3 mg/kg or autophagy activator rapamycin 20 pmol was administrated to observe the effect of ramelteon by autophagy activator at the onset of pMCAO.Seven days after photothrombosis,mice were intragastrically administered with ramelteon 3 mg/kg or 4-P-PDOT 3 mg/kg at the same time.The expressions of cell autophagy marker proteins and autophagy signaling pathway proteins were detected 15 days later.Result:After pMCAO onset,ramelteon 3 mg/kg could significantly reduce the brain infarct volume and neurological deficit scores,and the protective effect can be extended to 4 h after pMCAO.Administration of melatonin receptor antagonist 4-P-PDOT 3 mg/kg can reverse the protective effect of ramelteon.We found that the volume of brain infarct and neurological deficit scores were significantly ameliorated when edaravone was administered at 10 mg/kg.Compared with ramelteon 3 mg/kg and edaravone 10 mg/kg,there was no statistical difference with brain infarct volume and neurological deficit scores,and it showed similar effective time window.Ramelteon 3 mg/kg significantly reduced brain infarct volume in mice after photochemical embolization of cerebral ischemia after 15 days,and significantly reduced the footfault index and the asymmetry index.Simultaneously its protective effect can also be reversed by the administration of 4-P-PDOT,which significantly increased brain infarction volume,footfault index and asymmetry index.Western blot analysis showed that after 24 hours of ischemia in pMCAO,the administration of ramelteon can significantly reduce the levels of autophagy marker proteins(SQSTM1,LC3-?)and the levels of signal pathways activation(p-AMPK,p-mTOR,p-p70S6),and this phenomenon can be reversed by 4-P-PDOT.Administration of rapamycin after surgery can significantly reverse the protective effect of rapamycin.At 15 days after ischemia in photochemically embolized mice,ramelteon can also reduce autophagy marker proteins(SQSTM1,LC3-?)and signaling pathway activation(p-AMPK,p-mTOR,p-p70S6).These effects can also be reversed by 4-P-PDOT.Conclusions:Ramelteon has a protective effect on ischemic brain injury in mice.The efficacy and the effective time window of ramelteon is comparable to edaravone.Ramelteon can be a potential therapeutic drug for stroke.The agonism of melatonin receptors and the inhibition of autophagy may be involved in the neuroprotective effect of ramelteon in ischemic brains.