| With population ageing,the incidence of neurodegenerative diseases has increased year by year.Glutamate-induced excitotoxicity mediated by NMDA receptors plays an important role in the development of neurodegenerative diseases.Overactivation of NMDA receptors can trigger high level of calcium influx,which activates a series of apoptotic pathway leading to neuronal cells injury.Recent studies showed that NR2A-and NR2B-containing NMDARs play different roles in neuronal cells injury.As few subunit-specific NMDAR antagonists show good efficacy in clinical trials,great considerations have been given in the search for NR2B-selective antagonists considering the potential to cure neurodegenerative diseases Furthermore,NR2B-selective antagonists possessing the ability to scavenge free radical are likely to be a new kind of effective neuroprotectants,as preventing the development of neuronal injury in multiple ways.Objective:This study is aimed to search for potential NR2B subunit-selective NMDAR antagonists from natural compounds by computational virtual screen methods.Further,the potential protective mechanisms of ethyl acetate extract of Arctium lappa L.roots(EAE)and 1,5-O-dicaffeoyl-3-O-(4-malic acid methyl ester)-quinic acid(MQA)against neuronal injury are explored.The present study may lay the foundation for looking for leading compounds with neuroprotective activityMethods:(1)The present study is designed to search for compounds with high affinity to NMDAR NR2B subunit from Traditional Chinese Medicine database containing 41491 natural compounds by computer aided drug design technologies.Then the original herbs containing these compounds with potential neuroprotective activity were analyzed.(2)Neuroprotective properties of ten herbs extract were measured by MTT method in excitotoxicity and oxidative stress neuronal injury models in vitro.(3)Protective effects of EAE and MQA were evaluated by LDH leakage,Hoechst 33342 and Annexin V-FITC/PI double staining,antioxidant capacity assay,Ca2+ influx,ROS and MMP.Also,Western Blot was applied to explore the underlying mechanisms of protective effects against excitotoxicity and oxidative stress by measuring protein expressions,such as Bax/Bcl-2,cleaved caspase-3,ERK,JNK,p38 and NR2BResults:(1)A total of 91 herbs containing 200 compounds with high affinity to NMDAR NR2B subunit were identified.Neuroprotective activities and targeting on NMDARs of compounds distributed in 19 herbs are supported by previous studies,while 39 herbs only neuroprotective activities information available without targets details.For another 33 herbs,their neuroprotective properties have not been reported.(2)Neuroprotective effects of Arctium lappa L.root,Lycium barbarum L.root,Nelumbo nucifera Gaertn,semen,Senecio scandens Buch.-Ham.ex D.Don herb were first reported in vitro activity tests.Among all these samples,the extract of Arctium lappa L.root exhibited the strongest protective activity.(3)EAE significantly increased cell viability and reduced LDH leakage in a dose-dependent manner in glutamate-induced PC 12 cells injury model.Also,EAE significantly increased activities of GSH-Px and SOD,and reduced ROS formation and the decrease of MMP.Furthermore.Western Blot results revealed that EAE decreased the Bax/Bcl-2 ratio,and inhibited glutamate-induced release of cytochrome c,up-regulation of cleaved caspase-3,phosphorylation of ERK,JNK and p38(p<0.05).(4)EAE markedly increased cell viability and reduced LDH leakage in H2O2-induced oxidative stress injury model in SH-SY5Y cells.EAE also showed strong radical scavenging ability and significantly elevated activities of antioxidant enzyme(GSH-Px and SOD),reduced lipid peroxidation production,and prevented ROS formation and the decrease of MMP.Moreover,EAE decreased the Bax/Bcl-2 ratio and inhibited cytochrome c release,cleaved caspase-3,cleaved caspase-9 activities and expressions(p<0.05).(5)Pretreatment with MQA attenuated the loss of cell viability,LDH leakage and early apoptosis in a dose-dependent manner in NMDA-induced excitotoxicity injury model in SH-SY5Y cells.Also,MQA decreased the Bax/Bcl-2 ratio and inhibited cytochrome c release,cleaved caspase-3,cleaved caspase-9 activities and expressions.Further,MQA inhibited NMDA-induced phosphorylation of ERK,p38 and JNK,and increased phosphorylation of CREB,AKT and GSK-3?.In addition,MQA inhibited NMDA-induced Ca2+influx and up-regulation of NR2B subunit expression(p<0.05).(6)MQA markedly increased cell viability and reduced LDH leakage in H2O2-induced oxidative stress injury model in SH-SY5Y cells.MQA also significantly elevated activities of SOD,reduced MDA production,and prevented ROS formation and apoptosis.Moreover,MQA decreased the Bax/Bcl-2 ratio and inhibited cytochrome c release,cleaved caspase-3 and cleaved caspase-9 expressions.Further,MQA inhibited H2O2-induced phosphorylation of ERK,and increased phosphorylation of AKT and GSK-3?(p<0.05).Conclusion:(1)Computer aided drug design technologies were helpful to quickly and accurately screen compounds with neuroprotective effects by targeting on NMDAR NR2B subunit.(2)The potential protective mechanisms of EAE against glutamate-induced excitotoxicity were related with inhibition of protein expressions involved in the mitochondrial apoptotic pathway and phosphorylation of ERK,p38 and JNK.(3)The potential protective mechanisms of EAE against H2O2-induced oxidative stress were related with inhibition of caspase-3,caspase-9 activities,and protein expressions involved in the mitochondrial apoptotic pathway.(4)The potential protective mechanisms of MQA against NMDA-induced excitotoxicity may be related with blocking NMDARs NR2B unit,inhibiting Ca2+ influx mediated by overactivation of NMDARs,expression of proteins involved in the mitochondrial apoptotic pathway,regulating MAPKs,AKT/GSK-3? and CREB signaling pathways.(5)The potential protective mechanisms of MQA against H2O2-induced oxidative stress were related with ERK,AKT/GSK-3? signaling pathways,inhibition of protein expressions involved in the mitochondrial apoptotic pathway. |
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