| Objective:To explore the relationship betweenα7 nicotinic acetylcholinergic receptor(α7n AchR)and septic associated encephalopathy(SAE),and to explore the protective mechanism ofα7n AchR on SAE via the JAK2/STAT3 pathway,so as to raise a academic foundation for the protective effect of cholinergic anti-inflammatory pathway on SAE rats.Methods:Forty healthy male SD rats were stochastically divided into four groups:sham operation group(Sham group),sepsis encephalopathy group(SAE group),SAE+α7n AchR agonist group(SAE+PNU-282987 group),SAE+α7n AchR agonist+STAT3inhibitor group(SAE+PNU-282987+Stattic group),10 rats in each group.SAE models were built by cecal ligation and puncture method(CLP).α7n AchR agonist group rats were intraperitoneally injected with 3.0mg/kg PNU-282987 30 minutes before surgery,α7n AchR agonist+STAT3 inhibitor group rats were intraperitoneally injected with 5.0 mg/kg Stattic or 3.0 mg/kg PNU-282987 respectively 30 minutes or 15 minutes before surgery,the remaining steps like SAE group.The rats in Sham group were only exposed to cecum without ligation and puncture.Performed neurobehavioral scores of each groups 24h after operation,then collected the hippocampal tissue and venous blood of rats.HE staining was applied to observe the pathological changes of hippocampus.TUNEL assay was used to detect the cell apoptosis in the hippocampus.Immunohistochemistry was used to detect the Arg-1~+and the Iba-1~+microglia cells in the hippocampal tissues of rats.WB was performed to detect the expression levels of JAK2,p-JAK2,NF-κB p65,STAT3,p-STAT3 in the hippocampus.Levels of IL-4,IL-1β,TNF-α,IL-6,S100βand NSE were detected by ELISA.Results:1.Compared with the Sham group,rats in the SAE group had lower neurobehavioral scores,the brain damage was significantly increased in the SAE group,the number of neurons in the hippocampus of rats was decreased and the level of neurons was unclear,the apoptotic cells in the hippocampus of rats were significantly increased,the levels of NSE,S100β,IL-1β,TNF-α,IL-6 in the serum were increased(P<0.001),the level of IL-4was decreased;the number of Iba-1~+M1 microglia was dominant in the CA1 area of hippocampus(P<0.001),the levels of IL-1β,TNF-α,IL-6 in the hippocampus were significantly increased(P<0.001),and the level of IL-4 was significantly decreased;the JAK2/STAT3 pathway were activated and the ratio of the expression of p-STAT3 and total STAT3(p-STAT3/STAT3)and NF-κB p65 were significantly increased(P<0.001).2.Compared with SAE group,the neurobehavioral score of rats in the SAE+PNU-282987 group recovered,the brain damage was significantly relieved in SAE+PNU-282987 group,the number of neurons in the hippocampus was increased,the apoptotic cells in the hippocampus of rats were significantly decreased,the levels of NSE,S100β,IL-1β,TNF-α,IL-6 in the serum were significantly decreased(P<0.001),the level of IL-4 was increased;the number of Arg-1~+M2 microglia in the hippocampus CA1region was significantly increased(P<0.001);the level of IL-1β,TNF-α,IL-6 in the hippocampus were significantly decreased,while the level of IL-4 was significantly increased(P<0.001);the expression of p-JAK2/JAK2 and p-STAT3/STAT3 was significantly increased,and the expression of NF-κB p65 was significantly decreased(P<0.001).3.Compared with SAE+PNU 282987 group,the neurobehavioral score and pathological damage in the hippocampus in SAE+PNU 282987+Stattic group were aggravating,the apoptotic cells in the hippocampus of rats were significantly increased,the levels of NSE,S100βin the serum were significantly increased(P<0.001);the numbers of Arg-1~+M2 microglia in the hippocampus CA1 region were decreased(P<0.001);the level of IL-1β,TNF-α,IL-6 in the hippocampus were significantly increased,while the level of IL-4 was decreased(P<0.001);the JAK2/STAT3 pathway were inhibited,the expression of p-STAT3/STAT3 was significantly decreased and the expression of NF-κB p65 was significantly increased(P<0.001).Conclusion:1.Septic encephalopathy rats showed sluggish and lost reflex,had nerve hippocampal injury and cell apoptosis,the pro-inflammatory Iba-1~+M1 was dominant in the CA1 area of hippocampus,the levels of IL-1β,TNF-α,IL-6 were increased,and levels of IL-4 were decreased,and the JAK2/STAT3 pathway was activated in the hippocampus.2.Activation ofα7n AchR receptor could promote the transformation of pro-inflammatory Iba-1~+M1 microglia to anti-inflammatory Arg-1~+M2 microglia in the hippocampus,significantly reduce the levels of pro-inflammatory cytokines IL-1β,TNF-αand IL-6 in the hippocampus and serum,and increase the level of anti-inflammatory cytokines IL-4,which can significantly reduce the brain injury and apoptosis and improve nerve reflex of septic encephalopathy rats.3.Activation ofα7n AchR plays a protective role in septic encephalopathy by promoting the phosphorylation of the JAK2/STAT3 pathway and inhibiting the expression of NF-κB,which can be reversed by STAT3 inhibitors. |
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