Studies On The Effect And Action Mechanism Of LW-AFC On Glutamatergic System Exception In Senescence Accelerated Mouse

Alzheimer’s disease is a neurodegenerative disease that is a serious health hazard in today’s society.The pathogenesis is complicated,and the excitotoxicity is one of the pathogenesis.Vesicular glutamate transporters determine vesicular glutamate filling,which makes VGLUTS play an important role in the pathogenesis of excitotoxicity in AD.SAMP8 mice were a good model for studying the pathogenesis of sporadic AD.Previous studies have shown that abnormalities in neural signaling in the brain of SAMP8 mice include amino acids and monoaminergic nervous systems.Our early study results show that the LW-AFC can reduce β-amyloid in genetically-related APP/PS1 transgenic mouse,reduce the content of Aβ40 and Aβ 42,reduce the loss of neurons,and relieve the damage of learning and memory ability and can improve the distributing AD animal models of senescence-accelerated mouse prone 8 strain(SAMP8)learning memory ability,but the LW-AFC is whether by adjust the the glutamatergic function of the nervous system of mouse to improving the ability of learning and memory is not clear.In order to study whether LW-AFC can regulate glutamatergic system abnormal,we observe the change of glutamate and other neurotransmitter content in brain regions-the key to learning and memory in the hippocampus in SAMP8 after gavage administration of LW-AFC,and through observe the effect of LW-AFC to glutamatergic system postsynaptic receptor,the removal of Glu in the synaptic cleft,Glu synthesis and vesicle glutamate transporters,to study the mechanism of LW-AFC adjust the glutamatergic system abnormal.SAMP8 mouse,classic AD animal model,were employed,SAMR1 mice for comparison.In order to observe the glutamatergic system abnormal in SAMP8,we first using the microdialysis combined HPLC-ECD method,determine the amino acid and monoamine neurotransmitter circadian rhythm and aging change in hippocampal tissue fluid;Second,using Western blot method to detect the expression of VGLUTs and the enzyme clevrge it,and postsynaptic NMDA and AMPA receptors,and EAATs that remove Glu in the synaptic cleft,and GS and Glutaminase for Glu synthesis,and SN1 and SAT1 of Gln transporter in hippocampus;Finally,we study the regulating effect of LW-AFC to glutamatergic system abnormal through the observe the effect of LW-AFC to the content of Glu in SAMP8,and observe the various stages of neural transmission in glutamatergic system,revealing the mechanism how LW-AFC regulate glutamatergic system abnormal and improve the learning and memory function in SAMP8.PartⅠ.The observation of glutamatergic system abnormal in SAMP81.Circadian rhythm and aging changes of excitatory neurotransmitters in the hippocampus of SAM miceMicrodialysis combined HPLC-ECD method to detect neurotransmitter content in hippocampal tissue fluid in SAM mice,the results show that the content of Glu excitatory amino acid neurotransmitter in SAMR1 has obvious circadian rhythm,in the early stage of the elderly(3 and 9 months)the content of Glu is high during the day,but low at night,the circadian rhythm in the old age(12 to 18 months)is contrary to the early stage of the elderly.Circadian rhythm of Glu in SAMP8 is in disorder compared to SAMR1,characterized by Glu content is low in the evening in 3 months,but 6,12,15 months is high,9 and 18 months without circadian rhythm change.Circadian rhythm of Glu content in AUC showed that both SAMR1 and SAMP8 mice Glu content are reduced with aging in hippocampal tissue fluid,and compared to age-related SAMR1,3 and 9 months Glu content decreased in SAMP8,but 12 months were significantly elevated.In addition,the circadian rhythm of Ser,Gln and ASP content are disorder in the hippocampus interstitial fluid in SAMP8,Ser and ASP content decreased in more months in SAMP8 compared to age-related SAMR1,while the content of Gln in 6,9 and 15 months increased in SAMP8.2.The circadian rhythm and aging changes of inhibitory neurotransmitters in the hippocampus of SAM mice.For determine GABA and Gly content of inhibitory amino acid in the hippocampal tissue fluid,results showed that the content of GABA is higher during the day,low at night in SAMR1 in adulthood,and is low during the day,higher at night in old age,however,the presence of Gly content is higher during the day,low at night in just 3 months,then show GABA circadian rhythm in old age.The circadian rhythm of GABA and Gly in SAMP8 mice was disturbed.Circadian rhythm of AUC Content of GABA and Gly AUC decreases with aging both SAMR1 and SAMP8 mice in hippocampal tissue fluid,compared to age-related SAMR1,GABA is decreased in 6 and 15 months in hippocampal tissue fluid of SAMP8,6,9 and 12 months Gly levels are elevated,but 15 months is decreased.3.The circadian rhythm and aging changes of the neurotransmitters in the hippocampal tissues of SAM miceThe content of noradrenaline(NE),Dopamine(DA)and its metabolic product 3,4-dihydroxy phenylacetic acid(DOPAC)and Homovanillic acid(HVA),serotonin(5-HT)and its metabolite product 5-hydroxy indole acetic acid(5-HIAA)in hippocampal tissue fluid has obvious circadian rhythm in SAMR1,showed higher at night in the early stage of the elderly,reduced at night in old age,however circadian rhythm is disorder of the levels of these neurotransmitters in SAMP8,more performance for lower at night.For monamine neurotransmitter,total content of SAMP8 and SAMR1 reaching the highest in 6 months after decrease with aging,when compared to age-related SAMR1,concentration of 5-HIAA is increased in SAMP8,NE content rise in only 3 to 9 months.Pearson correlation analysis results indicate that the content of excitatory neurotransmitter Gln,Ser,ASP and the content of inhibitory neurotransmitter GABA,Gly and the content of monamine neurotransmitter NE,HVA,5 – HT,DA and its metabolic product DOPAC was a positive correlation with Glu,prompt in addition to Glu abnormalities,other systems also have synergy to it in AD animal models of SAMP8 mice.PartⅡ.Study on the mechanism of glutamatergic system abnormal in SAMP8 miceIn order to study the mechanism of SAMP8 brain glutamatergic system abnormal,we examined the expression in the hippocampus,including presynaptic VGLUTs and the enzyme which can cleavage it,NMDA and AMPA receptors in postsynaptic,EAATs which can remove the synaptic cleft Glu and GS and Glutaminase which can reglute Glu synthesis and the transporter SN1 and SAT1 of Gln,the results are as follows:The expression of VGLUT1 and VGLUT2 protein increased in 9 and 12 months in hippocampus in SAMR1,then decreased,but decreased with aging in SAMP8,compared to age-related SAMR1,the expression of VGLUT1 is decreased,and VGLUT2 were significantly lower after 12 months.Calpain and Caspase3 were enzymes that degrade VGLUTs,and the results showed that there was no significant change in the expression of Calpain in the hippocampus in SAMP8,and the expression of Caspase3 were significantly elevated.The results indicated that the expression of VGLUTs in SAMP8 was decreased,and the increase of Caspase3 was the probable cause.The expression of NMDAR1,NMDAR2 A,NMDAR2B,NMDAR2 C,NMDAR2D protein were all decreased,AMPA2 were significantly elevated,the protein expression of AMPA1 has no significant change compared to age-related SAMR1,the results indicated that postsynaptic receptor is reduced in glutamatergic system in hippocampus in SAMP8.The expression of EAAT1 and EAAT2 protein in the hippocampus of SAMP8 mice were decreased,and the expression of EAAT3 protein showed no significant change compared to age-related SAMR1.The result indicated that the transporter which can clear Glu in synaptic cleft were decreased.The expression of SN1,glutaminase and GS protein were decreased in hippocampus in SAMP8,and the expression of SAT1 protein was found to increased compared to age-related SAMR1,indicating that Glu resynthesis was decreased.The above results suggest that the Glu content was significantly elevated in hippocampus interstitial fluid in the AD animal models of SAMP8,may be due to the reduce of postsynaptic receptor and transporter which can clear the Glu in synaptic cleft,and the reduce of VGLUTs on the presynaptic vesicle membrane may be due to the increase in Caspase3,on the other hand,may be due to the decreased of Glu synthesis caused by feedback.PartⅢ.Study on the regulation effect of LW-AFC on glutamatergic system abnormality in SAMP8 mice7-month male SAMP8 mice were gastric gavage of LW-AFC(1.6 g/kg)for 198 days,VGLUT1 and Calpain protein expression in the hippocampus on SAMP8 were significantly elevated,Caspase3 was decreased,at the same time can up regulation the expression of postsynaptic receptor 1,NMDANR2 A,NMDANR2B,NMDANR2 C,NMDANR2D,AMPA1,AMPA2 protein,EAAT2 protein expression was elevated,also can up-regulation the expression of glutaminase,SN1,and SAT1.The results indicated that LW-AFC had a regulating effect on the glutamatergic system abnormality in the brain of AD model mice.We selected LWD-b,which is the active site of LW-AFC that can into the brain,the results showed that the gastric gavage of SAMP8 mice LWD-b(0.216 g/kg)can increase the expression of VGLUT1 and VGLUT2 protein in the hippocampus in SAMP8,decreased the expression of Caspase3,and down-regulating the expression of EAAT1 and EAAT2,up-regulation the expression of NMDANR1 protein.Through the above research,this study mainly draws the following conclusions:(1)glutamatergic system exceptions in SAMP8 of AD model mice,show circadian rhythm disorders of the excitatory amino acids Glu,Ser,Gln and ASP content in hippocampal tissue fluid,Glu content was significantly elevated,the exceptions of inhibitory neurotransmitter GABA,Gly,monoamine neurotransmitter NE,5-HT,DA and its metabolites DOPAC may have synergy with Glu for glutamatergic system exceptions.(2)the mechanism of glutamatergic system exceptions in AD animal models of SAMP8 mice,on the one hand is the postsynaptic glutamate receptors and remove the synaptic cleft Glu transporter reduce cause Glu levels rise in hippocampal tissue fluid,on the other hand is Caspase3 increase and Glu synthetic decrease caused by feedback caused by presynaptic vesicle membrane VGLUTs decreases.(3)LW-AFC has a regulation for glutamatergic system exceptions in AD model mice brain,through increase the expression of VGLUT1 on the synaptic vesicle membrane,reduce Caspase3 protein expression,up-regulation the expression of postsynaptic receptor and transporter which can remove Glu in the synaptic cleft and increase the expression of the molecules of Glu synthesis.