Melatonin Attenuate Kainic Acid-induced Neuronal Apoptosis By Alleviating ER Stress And Mitochondrial Disturbance

Background:Kainic acid(KA)exerts neuronal excitotoxicity by activating glutamate receptors.The excitotoxicity could cause apoptosis.It has been reported that over-activated glutamate receptors caused calcium influx in neuonrs.Calcium is one of the most critical molecules in cell signaling.The Calcium overloading can activate mitochondrial apoptotic pathway leading to cellular apoptosis.Melatonin has anti-apoptotic and neuronal protective functions.It has been reported melatonin could protect neurons from apoptosis in ischemia-reperfusion model.In our research,we first explored the mechanism of KA-induced apoptosis.Then we explored and analyzed the mechanism behind the protective effects of melatonin on KA-induced neuronal apoptosis.Object:Firstly,we established a model of KA-induced neuronal apoptosis in vitro.The inhibiting effects of melatonin on KA-induced apoptosis was observed and analyzed to investigate how melatonin inhibited KA-induced apoptosis.Then we confirmed the protective effects of melatonin on KA-induced neuronal apoptosis in vivo.Method:First,we detected the viability of cells and release of LDH under treatment of a series of dosage of KA and melatonin to figure out the optimal dosage of KA and melatonin in further study.Then we detected the expression of active cleavage Caspase-12,Caspase-9,Caspase-3 and cytochrome C under treatment of KA and melatonin by western blotting.In the meantime we confirmed the effects of KA and melatonin on neuronal apoptosis by tunnel assay.Then we detected the effects of KA and melatonin on intracellular level of Calcium and calpain activity.Finally we established the KA-induced apoptosis model in vivo.We detected the expression of active cleavage Caspase-12,Caspase-9,Caspase-3 and cytochrome C in hippocampal tissue under treatment of KA and melatonin by western blotting.We also confirmed the neuronal apoptosis under treatment of KA and melatonin in hippocampus by Nissil’s staining.Results:By MTT and crystal violet assay,we observed a decline in cells viability under treatment of KA.The decline in cellular viability induced by KA presented a dosage-dependent manner.KA also induced LDH release,indicating neuronal cytotoxicity of KA.Based on effect of KA on cell viability and LDH release,we found out the optimal dosage of KA is 50 μM under treatment of 8 hrs.Melatonin showed the inhibitory effect on apoptosis and LDH release induced by KA.We found the optimal dosage of melatonin is 50 μMunder treatment of 1 hr before KA insulting.KA caused an increase in the expression of active cleavage Caspase-12,Caspase-9,Caspase-3 and cytochrome C indicating activation of apoptosis caused by KA.Melatonin inhibited the activation of Caspase-12,Caspase-9,Caspase-3 and declined the release of cytochrome C,indicating a protective effect of melatonin on KA-induced apoptosis.By tunnel assay,we observed an increase in percentage of tunnel positive cells in KA-treated group,which could be attenuated by melatonin,confirming the anti-apoptotic effect of melatonin.By detecting the level of intracellular Calcium and activity of calpain,we found KA triggered an increase in content of intracellular calcium and activity of calpain while melatonin reduced the level of calciurm and calpain activity.Last,we observed the increase in expression of active cleavage Caspase-12,Caspase-9,Caspase-3 and cytochrome C in hippocampal tissue treated with KA while melatonin decreased the expression of Caspase-12,Caspase-9,Caspase-3 and cytochrome C.By Nissil’s staining,we found melatonin could attenuate the neuron loss indued by KA in hippocampus.Conclusion:KA could induce neuronal apoptosis while melatonin inhibited apoptosis caused by KA.Melatonin might inhibit the KA-induced apoptosis by attenuating calcium overloading and calpain activation induced by KA.Background:In the first part,we observed Kainic acid(KA)triggered calcium overloading and calpain activation.Endoplasmic reticulum(ER)is the main organelles responsible for various cellular activities including protein synthesis,maturation and folding,participating the regulation of various cellular activities.The abnormal calcium hike could trigger Endoplasmic reticulum stress(ERS).Although ERS considered as an adaptive action to maintain cellular homeostasis under stress,the over-prolonged or over activated ER stress leads to apoptosis.As it has been reported neuronal excitatoxicity could cause ERS,we assumed that KA triggered ERS and ERS-related apoptotic pathway by evoking calcium overloading.We have observed the inhibiting effect of melatonin on activation of Caspase-12 in first part of this article.We assumed melatonin could inhibit the KA-induced apoptosis by blocking ERS.Objective:Explore the mechanism behind KA-induced neuronal apoptosis by triggering ERS.Then detect the inhibiting effect of melatonin on apoptosis induced by KA-triggered ERS.Method:First,we investigated the inhibiting effect of PBA on ERS and ERS mediated apoptosis by detecting the expression of C-Caspase-12,GRP78,CHOP and calpain.Then we investigated how calpain involved in ERS mediated-apoptosis by detecting the expression of ATF-4 and calpain under treatment of Salubrinal and KA.Last,we confirmed the inhibiting effect of melatonin on KA-induced ERS and apoptosis by detecting the expression of GRP78.CHOP and calpain.We also detected the reduction of calpain activity under melatonin adminstration in vivo.Conclusion:KA could trigger activation of calpain by inducing FERS,finally leading to an ERS mediated apoptosis.Melatonin could inhibit ERS mediated apoptosis caused by KA via blocking ERS and activation of calpain.Background:In the first part of this article,we have confirmed Kainic acid(KA)could induce neuronal apoptosis.We have also confirmed the inhibiting effect of melatonin on apoptosis induced by KA.The mitochondria-related apoptotic pathway has been proved involved in KA-induced apoptosis.Mitochondria are highly dynamic organelles that undergoing continuous mitochondrial dynamics fission/fusion.Mitochondrial dysfunction could trigger apoptosis.The imbalance of mitochondrial fission and fusion trigger mitochondrial impairment leading to mitochondrial dysfunction eventually inducing apoptosis.In this case,we assumed KA could trigger the mitochondrial dynamics disturbance which activates mitochondria related apoptotic pathway.In the first part of this article,we have observed melatonin could attenuate the cytochrome C release and Caspase-9 activation induced by KA.Based on that,we assumed that melatonin might reduce mitochondria related apoptosis by inhibiting the mitochondrial fragmentation and alleviating mitochondrial dysfunction.Objective:First,explore the relationship between ER stress and mitochondrial fragmentation by detecting the effect of PBA on KA induced mitochondrial dysfunction,morphological impairment and dynamic disturbance.Then explore the mechanism behind KA induced mitochondria related apoptosis by detecting the effect of BAPTA-AM and calpeptin on mitochondrial morphology,function,expression of dynamic protein Mitofusin-2(Mfn-2)and active cleavage caspase-3.Further,analyze the mechanism behind the protective effect of melatonin on KA induced neuronal apoptosis by observing the effect of KA and melatonin on mitochondrial structure,morphology,amount,length,function and expression of dynamics related protein.Method:First,detect the effect of KA and PBA on mitochondrial length,amount,expression of Mfn-2 and C-Caspase-3,content of ATP and ROS and the mitochondrial membrane potential.Then detect the effect of BAPTA-AM and calpeptin on mitochondrial length,amount,expression of Mfn-2 and C-Caspase-3,content of ATP and ROS and the mitochondrial membrane potential.Meanwhile,detect the expression of Mfn-1,Mfn-2.OPA-1 and Drp-1 on level of protein and mRNA under treatment with KA and melatonin.Furthermore,detect the effect of KA and melatonin on mitochondrial morphology and average length by transmission electron microscope and mito-tracker assay.Last,detect the effect of KA and melatonin on mitochondrial membrane potential,content of ATP and ROS.Result:PBA could maintain mitochondrial membrane potential,increase the amount and average length of mitochondria,up-regulate the Mfn-2 level and ATP synthesis;decrease the content of ROS and expression of C-Caspase-3.BAPTA-AM and calpeptin could maintain mitochondrial membrane potential,increase the amount and average length of mitochondria,up-regulate the Mfn-2 level and ATP synthesis,decrease the content of ROS and expression of C-Caspase-3.KA could reduce the expression of Mfn-2 while the expression of Mfn-1,OPA-1 and Drp-1 on protein level and the expression of all these four genes on mRNA level remained uneffected.Melatonin could attenuate the reduction of Mfn-2 induced by KA.By transmission electron microscope,we observed a mitochondrial impairment induced by KA.Then we observed mitochondrial fragmentation by using mito-trakcer probe.Melatonin could restore the amount and average length of mitochondria.We also observed melatonin restored mitochondrial membrane potential and ATP synthesis while KA induced a drop in mitochondria membrane potential and intracellular ATP level.Melatonin could also reduce the level of ROS in N2a cells while KA caused a significant increase in level of ROS in cells.Conclusion:KA could trigger calcium overloading,calpain activation and mitochondria fragmentation,leading to mitochondrial impairment and dysfunction eventually causing apoptosis.Melatonin could inhibit mitochondrial fragmentation by blocking calcium overloading and calpain activation,presenting an inhibiting effect on KA induced apoptosis.