| Chronic Cerebral Hypoperfusion(CCH)is a long-term cerebral hypoperfusion state caused by various reasons such as hypertension,diabetes,heart disease,cerebral arteriosclerosis and smoking,which is a common cause of cognitive dysfunction.CCH is an important risk factor for cognitive dysfunction,and there are Alheimer-like pathological changes after CCH,such as causing oxidative stress,accelerating Aβ deposition,promoting tau protein hyperphosphorylation,and aggravating nerves cell damage,leading to synaptic protein imbalance,causing brain leukodystrophy and neuroimmune inflammation.The treatment of cognitive impairment and a series of pathophysiological changes caused by CCH has important clinical significance.For a long period of time,the brain was considered an organ insensitive to insulin.With the discovery of widespread distribution of insulin receptors in the brain,the role of the insulin pathway in the central nervous system was gradually revealed.In recent years,researches on the function of the insulin pathway in the central nervous system have been continuously improved.So far,the insulin receptor has been found to be an important regulator of processes such as synapse formation and remodeling,neuron survival/neuroprotection,and synaptic plasticity.Changes in the insulin pathway are found in neurodegenerative diseases such as Alzheimer’s disease,Huntington’s disease and Parkinson’s disease;diseases caused by abnormal brain development,such as autism,tumors,major depression and neurotoxicity.A more thorough understanding of the insulin pathway is helpful for Alzheimer’s disease,vascular dementia,Parkinson’s disease and other neurological diseases,and can provide new treatment approaches in future clinical practice.Intranasal insulin is a non-invasive approach that selectively activates central insulin pathway.Intranasal insulin enters the nasal cavity in the form of a nasal spray,first enters the nasal mucosa,and then bypasses the blood-brain barrier through the olfactory nerve and trigeminal nerve and is ultimately transported to the central nervous system.Intranasal insulin has been successfully used in the researches of neurodegenerative diseases and many other neurological conditions,and it has been proved to have a positive effect on patients with mild cognitive impairment and the early stages of Alzheimer’s disease.Intranasal insulin can improve memory function of rats with Alzheimer’s disease,as well as promote cerebral blood flow and glucose metabolism,inhibit hyperphosphorylation of tau protein,glial cell activation,neuron loss,neuroinflammation and amyloid expression.And the insulin pathway may play a vital role in the underneath mechanisms.This study used the right common carotid artery ligation method to establish a mouse model of CCH,and then explored the effect of intranasal insulin therapy on the cognitive function changes,neuronal apoptosis and tau protein phosphorylation in the hippocampus of mice with CCH,and the role of the insulin pathway.Part Ⅰ: The improvement effect of intranasal insulin on the cognitive functions of CCH mouseObjective: To investigate the effect of intranasal insulin on the cognitive function changes in mice with CCH after ligation of the right common carotid artery.Methods: Sixty healthy C57BL/6 male mice aged 16-20 weeks were selected and randomly divided into sham group,sham + intranasal insulin group,CCH group and CCH +intranasal insulin group.Each group had 15 animals.A mouse model of CCH was established by the classic right common carotid artery ligation.Six weeks after the operation,17.5μl of insulin or saline was administered nasally on a daily basis for a total of 4 weeks.After the intranasal process,behavior tests began,including open field test,elevated plus maze,new object recognition test and Morris water maze,to evaluate the behavioral changes of CCH mice.Results: The metabolic level of the CCH mice decreased,which was manifested as an increase in body weight compared with the other three groups,but there was no significant difference in food intake.Intranasal insulin can improve this defection.There was no significant difference among the four groups of mice in the open field test and elevated plus maze,suggesting that CCH and intranasal insulin did not have a significant effect on the general exploratory behavior and anxiety state of the mice.In the new object recognition test,the recent object recognition ability of the mice in CCH group decreased,and intranasal insulin could significantly improve it,and there was a statistical difference.In the learning phase of Morris water maze,the spatial learning ability of CCH mice decreases,and intranasal insulin can improve the spatial learning ability of the mice(P<0.05).In the testing phase,the results showed that the spatial memory ability of mice with CCH also decreased.Intranasal insulin can improve the spatial memory ability of CCH mice.Part Ⅱ: The effect of intranasal insulin on tau hyperphosphorylation and neuron apoptosis in CCH miceObjective: To investigate the effect of intranasal insulin on tau hyperphosphorylation and neuron apoptosis in the right hippocampus of CCH mice after ligation of the right common carotid artery.Methods: Sixty healthy C57BL/6 male mice aged 16-20 weeks were selected and randomly divided into sham group,sham + intranasal insulin group,CCH group and CCH +intranasal insulin group.Each group had 15 animals.A mouse model of CCH was established by the classic right common carotid artery ligation.Six weeks after the operation,17.5μl of insulin or saline was administered nasally on a daily basis for a total of 4 weeks.Then,protein chip,Western Blot,immunofluorescence,TUNEL staining and other methods were used to explore the expression and phosphorylation tau and the apoptosis of cells in the right hippocampus of CCH mice.Results: The protein chip results showed that the expression of tau in the hippocampus of CCH mice increased,and the phosphorylation levels of tau p S396 and tau p S235 also increased.Intranasal insulin antagonized the expression of tau in the hippocampus and the increase in phosphorylation.In Western Blot,we found that the total tau level,tau p S396,tau p S262,and tau p T181 phosphorylation levels in the CCH group were higher than those in the sham group,and there was a statistical difference between the total tau and tau p S396(p<0.05).After intranasal insulin,the total tau and phosphorylation levels of tau p S262,tau p S396 and tau p T181 in the CCH + intranasal insulin group were lower than those in the CCH group(P<0.05).The results of TUNEL staining in the hippocampus showed that the apoptosis of hippocampus neurons in the CCH group increased,and intranasal insulin can improve neuronal apoptosis in hippocampus.In the protein chip detection,we also found that the phosphorylation level of caspase 9 p S196 in the hippocampus increased after CCH,and there was a downward trend after intranasal insulin,but there was no statistical difference.Part III: The molecular mechanism of intranasal insulin to improve tau hyperphosphorylation.Objective: To investigate the molecular mechanism of intranasal insulin on tau hyperphosphorylation,O-Glc NAc and insulin pathway in the hippocampus.Methods: Sixty healthy C57BL/6 male mice aged 16-20 weeks were selected and randomly divided into sham group,sham + intranasal insulin group,CCH group and CCH +intranasal insulin group.Each group had 15 animals.A mouse model of CCH was established by the classic right common carotid artery ligation.Six weeks after the operation,17.5μl of insulin or saline was administered nasally on a daily basis for a total of 4 weeks.The protein chip,Western Blot,immunofluorescence and other methods were applied to explore the effect of intranasal insulin on glycogen synthase kinase-3β(GSK 3β),protein kinase B(AKT),3-phosphoinsitide-dependent protein kinase-1(PDK1),insulin receptor substrate-1(IRS-1)and other protein expression and phosphorylation levels in the insulin pathway.We also used immune-spot method to explore the effect of intranasal insulin on OGlc NAc.In order to further investigate the molecular mechanisms of the effect of intranasal insulin on CCH,we used protein chip enrichment analysis to explore other cross-over molecular mechanisms that may participate in the process.Results: The protein chip results showed that intranasal insulin increased the phosphorylation level of GSK 3β S9,AKT1 S473,AKT1 T308,and PDK1 S241 in the insulin pathway,and the increase of phosphorylation level of AKT1 was the most obvious.In Western Blot,we also found that intranasal insulin can increase the phosphorylation level of AKT S473 and GSK 3β S9.The immune-spot test showed that the level of O-Glc NAc in the hippocampus increased after intranasal insulin(p<0.05),suggesting that the intranasal insulin could activate the insulin pathway to increase O-Glc NAc level,thereby antagonizing the phosphorylation of tau.In addition,we found through protein chip enrichment analysis that Erb B signaling pathway,PI3K-AKT signaling pathway,MAPK pathway,and apoptosis pathway may have potential cross-effects with insulin pathway.Conclusions:(1)Intranasal insulin can improve the damage of recent memory,spatial learning and spatial memory in CCH mice;(2)Intranasal insulin can improve neuronal apoptosis,total tau and tau hyperphosphorylation in the right hippocampus of CCH mice;(3)Intranasal insulin may increase the phosphorylation level of GSK 3β S9,AKT1S473,AKT1 T308,and PDK1 S241 in the insulin pathway,thereby increasing the level OGlc NAc,and antagonizing tau hyperphosphorylation. |
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