安寐丹下调EphA4/EphrinA3信号通路蛋白表达改善老年睡眠剥夺模型认知功能的机制OA

Objective:To investigate whether Anmei Dan(AMD)improves cognitive function and alleviates circadian rhythm disorders in an aged mouse model of sleep deprivation by inhibiting the erythropoietin-producing hepatocellular receptor A4(EphA4)/Eph receptor-interacting protein A3(EphrinA3)signaling pathway and its downstream Ras homolog family member A(RhoA)/Rho-associated coiled-coil containing protein kinase(ROCK)signaling,and to analyze its multi-target regulatory characteristics.Methods:An aged C57 mouse model of sleep deprivation was established by the modified multiple platform water environment method.The mice were randomized into blank,model,rhynchophylline(Rhy,antagonist,50 mg·kg-1·d-1),and antagonist plus AMD(26.26 g·kg-1·d-1)groups.Spontaneous activity was assessed by the open field test,and circadian rhythm activity was monitored.Hematoxylin-eosin(HE)staining and Nissl staining were conducted to observe neuronal morphology in the hippocampal CA1/CA3 regions.Western blot and immunofluorescence assay were employed to determine the protein levels of EphA4,EphrinA3,Neuronal Nuclei Antigen(NEUN),glial fibrillary acidic protein(GFAP),phosphorylated(p)-EphA4,RhoA,Rho-associated coiled-coil containing protein kinase 1(ROCK1),Rho-associated coiled-coil containing protein kinase 2(ROCK2),brain-derived neurotrophic factor(BDNF),synaptophysin(SYN),postsynaptic density protein 95(PSD95),and growth-associated protein 43(GAP43).The mRNA levels of EphA4 and EphrinA3 were determined by real-time PCR.Results:Compared with the blank group,the model group showed decreased spontaneous activity(P<0.01),circadian rhythm disruption(P<0.01),hippocampal neuron disarrangement and reduced Nissl bodies,upregulated protein levels of EphA4,EphrinA3,NEUN,p-EphA4,RhoA,ROCK1,ROCK2,and GFAP(P<0.01),and downregulated protein levels of NEUN,BDNF,SYN,PSD95,and GAP43(P<0.01).Compared with the model group,both the antagonist group and the antagonist plus AMD group showed recovery in the above indicators(P<0.05,P<0.01),with the combination group outperforming the antagonist group in behavioral,pathological,and molecular expression aspects(P<0.05,P<0.01).Conclusion:AMD can alleviate cognitive and rhythm disorders caused by sleep deprivation by inhibiting the EphA4/EphrinA3 signaling pathway and its downstream RhoA/ROCK signaling,and upregulating the expression of synapse-related proteins such as BDNF.Its combination with an EphA4 antagonist exhibits a synergistic effect,which suggests that the mechanism involves multi-link synergistic intervention.