安寐丹调控CX3CL1/CX3CR1信号通路介导海马炎症改善老年睡眠剥夺小鼠学习记忆的作用机制OA

Objective:To investigate the effects of Anmei Dan(AMD)on the expression of proteins in the C-X3-C motif chemokine ligand 1(CX3CL1)/C-X3-C chemokine receptor 1(CX3CR1)signaling pathway,hippocampal inflammation,and learning and memory in an aged sleep-deprived model.Methods:Sixty aged C57 mice were randomly assigned into a control group,a model group,a melatonin group(1.3 mg·kg-1·d-1),and high-,medium-,and low-dose AMD groups(26.26,13.13,6.565 g·kg-1·d-1,respectively),with 10 mice per group.Continuous sleep deprivation was administered for 4 weeks through a custom-built sleep deprivation chamber.The cognitive function of mice was assessed via the Y-maze test.Histomorphological alterations in pyramidal cells of the hippocampal CA1 and DG regions were examined by hematoxylin-eosin(HE)and Nissl staining.Synaptic morphology in the hippocampus was visualized with Golgi staining.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the levels of inflammatory cytokines interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in the hippocampal tissue as well as the level of interleukin-10(IL-10)in the prefrontal cortex.Real-time PCR was performed to detect markers of M1-type[cluster of differentiation(CD)86 and inducible nitric oxide synthase(iNOS)]and M2-type[CD206 and arginase 1(Arg1)]macrophages.Western blot was employed to quantify the protein levels of CX3CL1,CX3CR1,phosphorylated nuclear factor κB p65 subunit(p-NF-κB p65),and phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK)in the hippocampus.Immunofluorescence double staining was performed to co-label CX3CR1 with ionized calcium-binding adapter molecule 1(Iba-1)for observation of the co-localization of target proteins with microglia.Results:Compared with the control group,the model group exhibited disrupted daily activity patterns with reduced spontaneous alternation rates(P<0.01),disorganized morphology and arrangement of hippocampal neurons,accompanied by reduced numbers of Nissl bodies and dendritic spines(P<0.01),upregulated protein levels of IL-6,IL-10,IL-1β,TNF-α,CX3CL1,CX3CR1,p-NF-κB p65,p-p38 MAPK,and Iba-1 and mRNA levels of CD86 and iNOS(P<0.01),and downregulated mRNA levels of CD206 and Arg1(P<0.05,P<0.01).Compared with the model group,the melatonin group and medium-and high-dose AMD groups showed increased spontaneous alternation rates(P<0.01),improved neuronal morphology,number,and spine density in the hippocampal CA1 and DG regions(P<0.01),declined levels of IL-6,IL-10,IL-1β,and TNF-α(P<0.05,P<0.01),downregulated protein levels of CX3CL1,CX3CR1,p-NF-κB p65,p-p38 MAPK,and Iba-1 and mRNA levels of CD86 and iNOS,and upregulated mRNA levels of CD206 and Arg1(P<0.01).Conclusion:AMD may improve the learning and memory in aged sleep-deprived mice by mediating neuroinflammation through the CX3CL1/CX3CR1 signaling pathway.