| Senile plaque(SP)formed by Aβ deposition and neurofibrillary tangles(NFTs)formed by tau hyperphosphorylation are important pathological features of Alzheimer’s disease(AD).Recent studies have indicated that Aβ and tau protein have neurotoxic interaction,which can mediate glutamate excitotoxicity and cause neuron injury and synaptic damage.Our previous studies have shown that Huannao Yicong Decoction(HYD)can significantly reduce the deposition of Aβ and the phosphorylation of tau protein.On this basis,we put forward the hypothesis that HYD can interfere with the neurotoxic interaction between Aβ and tau protein,destroy the stability of Fyn/NMDAR/PSD95 complex,inhibit glutamate excitotoxicity,and improve synaptic plasticity of nerve cells.ObjectiveBased on the "neurotoxic interaction of Aβ-tau protein",this study clarified the specific mechanism of HYD in interfering with the toxic interaction between Aβ and tau protein.Moreover,the study clarified the mechanism of HYD in reducing NMDAR excitatory neurotoxicity to improve cognitive and synaptic function of APP/PS1/tau mice.MethodIn this study,HYD was used as an intervention drug,APP/PS1/tau mice and SH-SY5Y model cells overexpressing Aβ and tau protein were used as intervention objects,and Memantine was used as positive control drug.The animal experiment was divided into 5 groups.The control group was 28 C57BL/6J mice with the same genetic background and the same age.According to the different intervention,APP/PS1/tau mice were divided into four groups:model group,memantine group(MJG),HYD low dose group(HYD-L)and HYD high dose group(HYD-H),with 28 mice in each group.The cell experiment is divided into three parts,namely,cell modeling,pharmacodynamic study and verification of the mechanism of toxic interaction between Aβ and tau protein.The pharmacodynamic study of cell experiment was divided into 5 groups with the same group as animal experiment.The mechanism verification part was divided into distant groups according to the different kinases GSK-3β,CDK-5 and PP2A intervention.Namely model group(model),optimum concentration of HYD group(HYD),GSK-3β inhibitor group(GSK3β-I),GSK-3β overexpression group(GSK3β-o),CDK-5 inhibitor group(CDK5-i),CDK-5 overexpression group(CDK-o),PP2A agonist group(PP2A-o),PP2A inhibitor group(PP2A-i),GSK-3P overexpression+HYD group(GSK3β-o+HYD),CDK-5 overexpression+HYD group(CDK-o+HYD),PP2A inhibitor+HYD group(PP2A-i+HYD).In animal experiment,Morris water maze and novel object recognition test were used to observe the effect of HYD on learning and memory ability of APP/PS1/tau mice;electrophysiological experiment was used to record the slope of field excitatory postsynaptic potential(fEPSP)in hippocampal CA3-CA1 region of mice,and the levels of synaptic related proteins synapsin I(SYN1)and synaptophysin(SYP)in hippocampus were detected to observe the effect of HYD on synaptic plasticity in APP/PS1/tau mice.The Aβ deposition in hippocampal CA3 region of mice was observed by immunohistochemical staining,the contents of Aβ1-40 and Aβ1-42 were detected by ELISA,the neurofibrillary tangles in hippocampal CA3 region of mice were observed by silver staining,the levels of tau protein and phosphorylated tau protein in hippocampus were detected by Western blot,the expression levels of kinase GSK-3β and CDK-5 mRNA were detected by qPCR method,and the effect of HYD on the toxic interaction between Aβ and tau protein was observed.In addition,the levels of NR2A,NR2B,GluR1,PSD95,and Fyn were detected by Western blot to determine the effect of HYD on glutamate excitotoxicity in APP/PS1/tau mice.In cell experiment,SH-SY5Y model cells overexpressing Aβ1-42 and tau protein were constructed,and the content of Aβ1-42 was detected by ELISA.The axis-dendritic distribution of tau protein in SH-SY5Y model cells was observed by immunofluorescence,and the levels of tau protein and phosphorylated tau protein in SH-SY5Y model cells were detected by Western blot.The expression levels of phosphorylated kinase GSK-3β and CDK-5 mRNA were detected by qPCR.At the same time,the levels of NR1,NR2B,Fyn,PSD95,P-Fyn,P-PSD95,and P-NR2B were detected by Western blot to further clarify the effect of HYD on the toxic interaction between Aβ and tau protein and glutamate excitotoxicity.Result1 Effect of HYD on learning and memory ability of APP/PS1/tau miceMorris water maze test:after 5 days of positioning navigation test(4 times per day),the escape latency of each group was significantly shortened on the second day(P<0.05).With the increase of training times,the escape latency of each group was shortened in varying degrees,indicating that each group of mice have a certain spatial learning and memory ability to the fixed platform position.Compared with control group,the escape latency of model group was significantly longer and the number of times of platform crossing was significantly decreased(P<0.05).Compared with model group,the 5-day escape latency of HYD-H group was significantly shorter than that of model group,and the escape latency of MJG group and HYD-L group on the 3rd day was significantly shorter than that of model group(P<0.05).In the space exploration experiment on the 6th day,compared with model group,the number of times of crossing the platform and the distance ratio of the original platform quadrant in HYD-L group and HYD-H group were significantly higher than those in HYD-L group and HYD-H group(P<0.05).The number of times of crossing the platform in MJG group was not significantly higher than that in model group,suggesting that the spatial learning and memory ability of APP/PS1/tau mice was impaired.HYD and memantine could improve the spatial learning and memory ability of APP/PS1/tau mice,and the effect of HYD was more obvious.Novel object recognition experiment:compared with control group,the recognition time of new object C in Model group was shorter than that in Model group.Compared with model group,the recognition time and discrimination coefficient of new object C in HYD-L group and HYD-H group increased significantly,while the discrimination coefficient of new object C in MJG group did not increase significantly(P>0.05).It is suggested that 12-month-old APP/PS1/tau mice have the ability to recognize short-term memory,and HYD can improve the short-term memory ability of APP/PS1/tau mice.2 Effect of HYD on synaptic plasticity in hippocampal CA3-CA1 region of APP/PS1/tau miceThe electrophysiological results showed that after TBS induction,the EPSP slope of hippocampus in model group was significantly lower than that in control group(P<0.05).Compared with model group,the EPSP slope of hippocampus in HYD-H group was significantly higher,and the EPSP slope in MJG group was significantly higher than that in MJG group,but there was no significant difference between the two groups(P>0.05).The results of immunohistochemical staining showed that the optical density(IOD)and area of Synapsin I and Synaptophysin in hippocampal CA1 and CA3 region of model group were lower than those of control group.Compared with model group,the IOD value of SYN1 protein in CA1 and CA3 region of hippocampus in MJG group and HYD-L group increased,and the SYN1 area in CA3 region increased(P<0.05).The IOD value of SYP protein in hippocampal CA1 region and the SYP area of CA1 and CA3 region in MJG group,HYD-L group and HYD-H group were significantly increased,and the IOD value of SYP protein in hippocampal CA3 region in HYD-L group and HYD-H group was significantly increased(P<0.05),indicating that HYD can increase LTP,increase the levels of Synapsin I and Synaptophysin,and improve synaptic plasticity in APP/PS1/tau mice.3 Effect of HYD on neurotoxic interaction between Aβ and tau proteinThis study found that Aβ deposition and NFTs in hippocampal CA3 region of model group were significantly higher than those of control group.Compared with model group,Aβ deposition and NFTs in hippocampal CA3 region of MJG group and HYD-H group were significantly decreased(P<0.05).ELISA detection showed that the contents of Aβ1-40 and Aβ1-42 in hippocampus of MJG group,HYD-L group and HYD-H group were significantly lower than those of model group.At the same time,the levels of tau protein phosphorylation sites(Ser396,Ser404,Ser202/Thr205)in the hippocampus of MJG group,HYD-L group and HYD-H group were significantly lower than those of model group,and the mRNA expression of kinase GSK-3β and CDK-5 was significantly decreased(P<0.05).The results of cell experiment also showed that the content of AP1-42,the level of tau protein and its phosphorylation sites(Ser396,Ser404,Ser202)in MJG group,HYD-L group and HYD-H group were significantly lower than those in model group,and the mRNA expression of kinase GSK-3P and CDK-5 was significantly lower than that in model group.In addition,in the cell mechanism verification experiment,it was found that HYD could directly inhibit the activity of kinase GSK-3β and CDK-5,increase the activity of kinase PP2A and decrease the level of tau protein phosphorylation.These results suggest that HYD can reduce Aβ deposition,inhibit the activity of kinase GSK-3β and CDK-5,increase the activity of kinase PP2A,inhibit the hyperphosphorylation of tau protein,and then interfere with the toxic interaction and synergistic toxicity between Aβ and tau protein.4 Effect of HYD on glutamate excitatory toxicityCompared with model group,the levels of NR2A and NR2B in hippocampus of MJG group,HYD-L group and HYD-H group were significantly decreased(P<0.05),while the levels of GluR1 and PSD95 were increased.The cell experiment results showed that the levels of NR1,NR2B,Fyn,P-Fyn,P-PSD95,and P-NR2B in MJG group and HYD-H group were lower than those in model group(P<0.05),and the levels of PSD95 were higher than those in model group(P<0.05).These results indicated that HYD could reduce the levels of NMDARs,increase the levels of GluR1 and PSD95,decrease the phosphorylation of Fyn,NR2B,and PSD95,ultimately inhibiting glutamate excitotoxicity and improving synapse function.Conclusion1 HYD can improve the spatial learning and memory ability of APP/PS1/tau mice.enhance LTP and improve synaptic plasticity.2 HYD can enhance LTP,increase SYN1 and SYP levels,and improve synaptic plasticity of mice.3 HYD can inhibit the neurotoxic interaction between Aβ and tau protein by reducing Aβ deposition,inhibiting the activities of GSK-3β and CDK-5 kinase,enhancing the activity of PP2A kinase,and inhibiting the hyperphosphorylation of tau protein and the formation of NFTs.3 HYD can decrease the overexpression of NMDARs,increase the levels of GluR1 and PSD95,reduce the phosphorylation of Fyn,NR2B,and PSD95,ultimately inhibiting glutamate excitotoxicity and improving synapse function. |
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