| Background and ObjectsDiabetic encephalopathy(DE)is a chronic central nervous system complication caused by diabetes mellitus,and its clinical manifestations are acquired cognitive and behavioral dysfunction.The two diagnostic pathological changes of AD are neuritic plaques composed mainly of theβ-amyloid(Aβ)peptide and neurofibrillary tangles composed mainly of tau hyperphosphorylation which are characteristic pathological changes in DE as well.Due to the toxic effect of AP and prion protein-like characteristics of tau hyperphosphorylation,the two abnormal proteins have been considered as critical pathogenic factors of DE which will accelerate the process of cognitive impairment in diabetes.But the potential mechanism are still unclear.p38 mitogen activated protein kinase(p38MAPK)is an important signal transductive molecule in the cell,which is closely related to the increase of Aβ production and the hyperrphosphorylation of tau protein.The up-regulation of p38MAPK phosphorylation can not only increase the abnormal phosphorylation of tau protein and the expression of p53,but also have the inhibitory effect on Sirt1.Sirt1,as a longevity gene,was a pivotal regulatory factor to attenuate Aβ deposition and improve cognitive function by altering Sirt1-mediated Aβ metabolic pathway,including the α-secretase pathway,β-secretase pathway and autophagy.However,it is still necessary to further confirm that whether chronic high glucose can induce cognitive function and AD-like pathological changes through stimulating those signaling pathways mentioned above.Toll like receptor 9(TLR9)is an important immunomodulatory receptor in innate immunity.Previous studies suggested that TLR9 was just expressed in immune cells and tumor cells.However,recent studies have confirmed that parenchymal cells,such as neurons and cardiomyocytes,can also express TLR9 under stress which will initiate its non-inflammatory signaling pathway to regulate intracellular metabolic activity.Because p38MAPK/p53 is involved in the non-inflammatory signaling pathway of TLR9,the activation of TLR9/p38MAPK/p53 on neurons is likely to be one of the molecular mechanisms of DE.The purpose of this study is to observe the regulatory effects of TLR9 and Sirt1 on cognitive function and Aβ deposition of diabetic mice by the way of establishing a model of type 1 diabetes in the wild type and TLR9 gene knockout mice,and further silencing Sirtl gene.And in vivo experiments we detect the expression and phosphorylation of essential proteins in a-secretase pathway,β-secretase pathway and autophagy.Invitro study we perform experimients to suppress expression and activity of TLR9 and p38MAPK via selective depressant,and to up-regulate the expression of p53 in order to investigate the potential mechanism of high glucose on cognitive damage and biological effect of Aβ and tau hyperphosphorylation.Our aim is to explicit the relationship between high glucose and cognitive dysfunction,to explore the TLR9 mediated AD-like molecular signal mechanism.It might update the public understanding of DE and emphasize the importance of blood glucose management in diabetics.Method1.In vivo experimentsThe affects of TLR9 gene knockout and Sirtl gene silencing on the general condition and cognitive function of diabetic mice:(1)The wild-type C57BL/6J mice and the TLR9 knockout mice with the same genetic background(C57BL/6J-Tlr9M7Btlr/Mmjax,TLR9-/-type)were intervened via single i.p STZ injection(wild-type,180mg/kg;TLR9-/-,130mg/kg)to establish the type 1 diabetes model.Average body weight and plasma glucose were recorded per week.(2)Aecording to grouping,the bilateral hippocampus of diabetic mice were injected with saline,the vector(HBAAV2/9-GFP,2μL,the titer 1012 v.g./mL)and AAV-virus(HBAAV2/9-U6-sirt1 gRNA8-cas9,2μL,and the titer 1012 v.g./mL).(3)Morris’ water maze(MWM)was used to evaluate the spatial learning and memory ability of mice in each group,and to exclude the effect of diabetes on muscle strength and eye sight in mice.The effects of TLR9 gene knockout and Sirtl gene silencing on the expression of TLR9 and key proteins in a-secretase,β-secretase,and autophagy pathway in the hippocampus of diabetic mice:(1)After finishing the MWM cognitive assessment,WB was used to detect the expressions of p53,Sirt1 and Aβ in the hippocampus of mice.(2)WB was used to detect the expression of the key proteins in the Sirt1-medited α secretase,βsecretase and autophagy pathway.2.In vitro experimentsThe effects of selective inhibition for TLR9(ODN2088)and overexpression of p53 gene on the expression of the key proteins in the Sirt1-medited a secretase,β secretase and autophagy pathway in cultured HT22 cells under high glucose:(1)According to the interventions,the cells were divided into 8 groups.The interventions were listed above:High glucosse(50mM),ODN2088(5μM),ODN2088 Control(5μM),and sh-p53(rLV-mP53-ZsGreen-Puro,titer 108 TU/mL).(2)3 days later,WB detected TLR9 and its downstream-related genes,Sirt1,Aβ expression.The effects of TLR9(ODN2088)and p38MAPK(SB203580)on the expressions of key proteins in TLR9 signaling pathway and the tau hyperphosphorylation in cultured hippocampal neurons under high glucose and the possible molecular mechanism:(1)The primary cultured hippocampal neurons were incubated for 7 days.Further MAP2 immunofluorescence staining were used for neuronal identification.(2)According to the interventions,the cells were divided into 6 groups.The interventions were listed above:High glucosse(50mM),ODN2088(5μM),ODN2088 high glucosse(50mM),ODN2088(5μM),ODN2088 Control(5μM)and SB203580(20 M).(3)3 days later,WB detected the expression of TLR9 and its downstream-related genes,Unc93b1,and the level of tau hyperphosphorylation.The effects of selective inhibition of TLR9 and p38MAPK,and silencing p53 gene on the apoptosis of hippocampal neurons under high glucose:(1)After the primary cultured hippocampal neurons were incubated for 7 days,we initiated the interventions with high glucose(50mM),ODN2088(5μM).ODN2088 Control(5μM),SB203580(20μM)for 3 days.Tunel test was used to label the apoptotic neurons.(2)The HT22 cells were intervened with high glucose(50mM),ODN2088(5μM)、ODN2088 Control(5μM)and sh-p53.And the apoptotic rate of the neurons was detected by the Flow Cytometry.ResultsThe effects of TLR9 gene knockout and Sirtl gene silencing on the general condition and cognitive function of diabetic mice:(1)The increase in the average weight of the diabetic mice was significantly slowed down and the blood glucose of diabetic mice was greatly increased compared with the non-diabetic mice.But the TLR9 gene knockout and the Sirtl gene silencing had no effect on the average weight and blood glucose among the mice.(2)MWM test showed that the spatial learning and memory ability of diabetic mice were impaired and TLR9 gene knockout could improve the cognitive impairment of diabetic mice.But the Sirtl gene silencing in the hippocampus would reverse the cognitive protection of TLR9 knockout on diabetic mice.In ascending platform test,there was no significant difference in escape latency and swimming speed among the groups.The molecular mechanism of TLR9 gene knockout and Sirtl gene silencing on the cognitive function of diabetic mice:(1)WB showed that the expression of Sirtl in the hippocampus of diabetic mice was significantly down-regulated,and the expression of p5 3 and Aβ was significantly increased.TLR9 gene knockout could reverse the trend of Sirtl,p53 and Aβ in diabetic mice.Once selective silencing Sirtl,the expression of p53 was affected,but the production of Aβ was obviously increased,suggesting the negative regulation of TLR9 on Sirtl which mediated excessive Aβ deposition in hippocampus of diabetic mice.(2)The changes in the expression of Sirtl and key proteins in a secretase,βsecretase pathway and the autophagy pathway in the hippocampus indicated that Sirtl was in accordance with the regulatory trend of ADAM 10,but was contrary to BACE1.Although TLR9 could activate autophagy,but down-regulation of Sirtl had no effect on autophagy,suggesting that Sirtl-mediated inhibition of a secretase pathway and stimulation of Aβ secretase pathway was a molecular mechanism of exacerbated Aβ deposition in diabetic hippocampus.Pharmacological inhibition of TLR9 could significantly improve the accumulation of Aβ in the neurons incubated in the high glucose condition,that would simultaneously inhibit p38MAPK,p53,and recover the expression of Sirtl.The over-expression of p53 gene has no effect on the expression of TLR9 and the level of phosphorylation of p38MAPK,but it could inhibit Sirtl and increase the content of Aβ in the primary cultured neurons.It suggested that the down-regulation of Sirtl expression mediated by TLR9/p38MAPK/p53 played a vital role in the aggravation of Aβ in the hippocampus of diabetic mice.Pharmacological inhibition of TLR9 expression and p38MAPK activation could significantly reduce the abnormal phosphorylation of tau protein in primary cultured hippocampal neurons.under high glucose in vitro.Pharmacological inhibition of TLR9 expression and p38MAPK activation could significantly reduce the apoptosis of neurons in the model of diabetic encephalopathy in vitro.Over-expression of p53 gene could reverse the neuroprotective effect of TLR9 and p38MAPK inhibitors.Chronic high-glucose stimulation could induce the expression of Unc93b1,which lead to activate the inflammatory signal pathway of TLR9/NF-κB.Futher studies supported that high glucose induced activation of TLR9/NF-κB was not affected by p38MAPK interference.ConclusionOur findings suggest that high-glucose mediated TLR9 non-inflammatory signaling pathway plays an critical role in cognitive impairment and formation of AD-like pathological changes in diabetic mice.TLR9 can activate the p38MAPK/p53 signaling pathway to modulate Sirtl and lead to its downstream abnormality of a-secretase and β-secretase pathways,which finally increases the formation of Aβ in the hippocampus and impairs the learning and memory ability of diabetic mice.Furthermore,TLR9/p38MAPK has the ability to up-regulate hyperphosphorylation of tau protein.The regulation is independent of the TLR9/NF-κB pathway.What’s more,the activation of TLR9/p38MAPK/p53 participates the apoptotic process of neurons under high glucose,these studies indicates that activation of TLR9/p38MAPK/p53 signaling pathway was associated with pathogenesis of DE,and was considered as a potential molecular target for diagnosis and treatment of DE.Further researches on DE might update the public understanding of diabetic central nervous system complication and provide new ideas to delay the progress of this disease. |
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