| Objectives: Observe the chronic sleep deprivation on protein expression and gene expression of α-7 nAChR,coexpression of α-7 nAChR with astrocytes and microglias in mouse hippocampus and then observe the protein expression of p-AKT,p-GSK,Nrf2,HO-1,gene expression level of inflammatory factors(TNF-α,IL-1β,IFN-γ,MCP-1)and anti-inflammatory factors(Arg-1、CD206、YM-1、TGF-β).Further explore the inflammatory mechanism of the PI3K/AKT pathway of glia cells in chronic sleep deprivation and the possible mechanism of cognitive impairment after sleep deprivation.Method: Adult C57BL/6J mouses aged 7-8 weeks were randomly divided into three groups,12 individuals per group: CC group(blank control): fed in suitable environment for 1 week;SD group(chronic sleep deprivation 7 days): fed in suitable environment for 1 week,and 7 days of chronic sleep deprivation with modified multiple platform method;SD + PHA-543613 groups(chronic sleep deprivation for 7 days + PHA-543613): fed in suitable environment for 1 week,6 hours after 7 days of chronic sleep deprivation using the modified multiple platform method,intraperitoneal injection α 7-nAChR agonists PHA-543613(6 mg/kg)for three days.The α 7-nAChR positive cells in mouse hippocampus were examined by immunofluorescence staining,the protein expression of α 7-nAChR in mouse hippocampus were examined by Western Blot,the gene expression of α 7-nAChR in mouse hippocampus were examined by Rt-PCR;the coexpression of α-7 nAChR with astrocytes and microglias in mouse hippocampus were assesed by double-labeled immunofluorescence,the protein expression of p-AKT、AKT、p-GSK、GSK、Nrf-2、HO-1 in mouse hippocampus were evaluaed by Western Blot,and the gene expression of TNF-α 、IL-1β 、IFN-γ 、MCP-1、Arg-1、CD206、TGF-β 、YM-1 in mouse hippocampus were examined by Rt-PCR.Results: 1.Compared with CC group,α-7 nAChR positive cells reduced significantly in SD group,the expression of α-7 nAChR protein and mRNA decreased significantly in group B(P < 0.01,P < 0.01,P = 0.012 respectively);Compared with SD group,α-7 nAChR positive cells increased significantly in SD+PHA-543613 group,the expression of α-7 nAChR protein and mRNA inceased significantly in group C(P < 0.01,P = 0.037,P = 0.037respectively);2.Double-labeled immunofluorescence showed the coexpression of α-7 nAChR with astrocytes and microglial cells;3.Compared with CC group,the coexpression of α-7 nAChR with astrocytes and microglial cells reduced significantly in SD group(P < 0.01,P = 0.639respectively),the expression of P-AKT protein decreased and the expression of p-GSK protein increased in SD group(P = 0.011,P < 0.01,respectively),the expression of Nrf-2 and Ho-1 protein decreased significantly(P = 0.02,P = 0.16,respectively)in SD group;the gene expression of inflammatory factors(TNF-α,MCP-1)increased(P < 0.01,P < 0.01,respectively)and gene expression of suppression of inflammation factors(CD206,TGF-β)decreased significantly in SD group(P < 0.01,P < 0.01,respectively);4.Compared with SD group,the coexpression of α-7 nAChR with astrocytes and microglial cells increased in SD+PHA-543613 group(P=0.027,P >0.05,respectively),the expression of p-AKT and p-GSK protein increased significantly in group C(P=0.047、P =0.038,respectively),the expression of Nrf-2 and Ho-1 protein increased significantly(P = 0.02,P = 0.16,respectively)in SD+PHA-543613 group;the gene expression of inflammatory factors(TNF-α,MCP-1)decreased(P < 0.01,P < 0.01,respectively)and gene expression of suppression of inflammation factors(CD206,TGF-β)increased significantly in SD+PHA-543613 group(P=0.011、P=0.012,respectively).Conclusions: 1,The hippocampal tissues of normal mice were expressed with astrocytes and microglial cells.Chronic sleep deprivation inhibited the expression of α-7 nAChR gene and protein in the hippocampus of mice,and reduced the α-7 nAChR positive cells;2,α-7 nAChR regulates the signaling pathway of PI3K/AKT/ GSK-3,chronic sleep deprivation inhibits the expression of α 7 nAChR on glia in the hippocampal tissue.,the expression of p-AKT protein is decreased,and the expression of p-GSK-3β protein is increased,chronic sleep deprivation inhibited the signaling pathway of glia α 7-nAChR/PI3K/AKT/GSK-3β;3,Chronic sleep deprivation inhibit mice hippocampus antioxidant enzymes Nrf-2,HO-1 expression,promote the release of the inflammatory cytokine TNF-α,MCP-1,IL – 1β,IFN – γ,reduce the expression of anti-inflammatory cytokines CD206,TGF-β,thus produce inflammatory injury.4,In chronic sleep deprivation,glia cellsα-7 nAChR/PI3K/AKT/GSK-3β not only regulates the expression of antioxidant enzyme Nrf-2 and HO-1,also affects the transcription and release of anti-inflammatory cytokines CD206,TGF-β and inflammatory cytokines TNF-α,MCP-1;In summary,α-7 nAChR /PI3K/AKT pathway may be the result of long-term sleep deprivation cognitie impairment of one of the inflammatory mechanisms,may be an intervention target for cognitive impairment caused by chronic sleep deprivation,providing a theoretical basis for clinical treatment of chronic insomnia patients with cognitive decline. |
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