Protective Effect And Mechanism Of Isoimperatorin On Staurosporine Induced Neuronal Apoptosis

BackgroundStaurosporine（STS）is an alkaloid that can antifungally obtained in microorganisms to inhibit the activity of platelet aggregation and a variety of protein kinases.As a common apoptosis inducer,it is often used to study the apoptosis of dopaminergic neurons.Isoimperatorin（ISO）belongs to furan coumarin compounds.It is an effective component of Angelica dahurica,notopterygium notopterygium,North ginseng and other traditional Chinese medicine.It has the effects of anti-cancer,analgesia and inhibition of inflammation.Recent studies have found that isoimperatorin can inhibit oxidative stress response and apoptosis of nerve cells.However,whether isoimperatorin has a protective effect on STS induced neuronal apoptosis and its mechanism are not clear.Objectives1.To explore the protective effect of isoimperatorin on STS induced neurocytotoxicity;2.To clarify the molecular mechanism of isoimperatorin on the protection of neuronal apoptosis.Methods1.To detect the effect of isoimperatorin on the viability of neuroblastoma cell SH-SY5YAfter SH-SY5 Y cells were treated with different concentrations of isoimperatorin,the effect of different concentrations of isoimperatorin on cell viability was detected by CCK-8method.2.To investigate the effect of isoimperatorin on STS induced apoptosis of SH-SY5 Y cellsThe cells were treated with isoimperatorin for 24 h in advance,and then STS was added to the cell culture medium for 4 hours to induce apoptosis.The cell viability was detected by CCK-8 method,the expression levels of apoptosis related proteins cleaved PARP,Bad and anti-apoptosis Bcl-2 were detected by Western blot to determine the effect of isoimperatorin on the apoptosis of SH-SY5 Y cells.3.To analyze the effect of isoimperatorin on intracellular signal pathway induced by STSThe cells were pretreated with isoimperatorin for 1 h,and then the cells were cultured in the medium added with STS for 4 h.The phosphorylation levels of intracellular signal molecules JNK,ERK,p38 and Akt were detected by Western blot to reveal which signal pathways mediate the neuroprotective effect of isoimperatorin.4.Detection of specific inhibitors of related signal pathways was used to verify STS induced neuronal apoptosisAfter the cells were treated with various specific inhibitors for 1 hours,the STS treated cells were used to construct the apoptosis model in vitro.The expressions of apoptotic proteins cleaved PARP were detected by Western blot,The cell viability was detected by CCK-8 method,and the role of related signal pathways in isoimperatorin protecting nerve cells from STS induced apoptosis was analyzed.Results1.When the concentration of isoimperatorin increased to 40 μM,it still had no significant effect on the viability of SH-SY5 Y cells;2.Isoimperatorin has neuroprotective effect on SH-SY5 Y cytotoxicity induced by STS;3.Isoimperatorin can inhibit the activation of intracellular signal pathway MAPK pathway induced by STS;At the same time,it can promote the activation of AKT signal pathway induced by STS;4.In SH-SY5 Y cells,the protective effect of isoimperatorin on STS induced cytotoxicity can be simulated by using corresponding specific MAPK inhibitors;Treatment of SH-SY5 Y cells with Akt specific inhibitors promoted STS induced apoptosis of SH-SY5 Y cells.ConclusionBy studying the molecular mechanism of isoimperatorin’s neuroprotective effect,it was found that isoimperatorin synergistically protected SH-SY5 Y cells from STS induced apoptosis by inhibiting MAPK signal pathway and activating PI3K/Akt signal pathway.This suggests that isoimperatorin may play an important role in the treatment of neurodegenerative diseases and provide a new experimental basis for the development of new drugs for the treatment of neurodegenerative diseases.