| Objective:Circadian rhythm disturbance is not only one of the risk factors for the development of Alzheimer’s disease(Alzheimer’s disease,AD),but also a common concomitant symptom.Improving sleep as another way to treat dementia is a new way to prevent and cure AD.On the basis of previous studies,ancient books of traditional Chinese medicine related to dementia and circadian rhythm were collected and sorted out,which were reinterpreted from the perspective of TCM theory.From the perspective of biological clock genes and autophagy,the relationship between circadian rhythm and Alzheimer’s disease and the regulation effect and possible mechanism of Root-Securing and Brain-Fortifying method on circadian rhythm disturbance and cognitive impairment in Alzheimer’s disease were discussed.Methods:1.Theoretical discussion:Conduct literature research,sort out,analyze and summarize the correlation between dementia and circadian rhythm in ancient and modern literature,and refine its connotation.Take autophagy as the starting point,analyze and discuss the regulatory effect of circadian rhythm on Alzheimer’s disease from the perspective of consolidating the root-securing and Brain-Fortifying.2.Experimental study:experiment 1:Thirty 6-month-old APP/PS1 mice were randomly divided into Model group(Model),melatonin group(MT)and Brain-Fortifying Liquid group(RSBFL),and 10 equal-month-old gene-negative C57BL/6J mice were used as blank Control group(Control).Morris water maze test was used to evaluate the learning and memory ability of mice.The morphology of hippocampal neurons of mice in each group was observed by HE staining and Nizhi staining.The synaptic function of mice was observed by Golgi staining.The synaptic structure-related proteins PSD95,SYN1 and GAP-43 were observed by immunofluorescence.Experiment 2:The sleep biometric analysis system VitalRecord 3.0 was used to record 24h EEG/EMG biological signals,and the animal sleep analysis software Sleepsign3.0 was used to analyze the data and distinguish the sleep phases.The expressions of Clock genes Per1,Per2,Cry1,clock,Bmall,Rora,Rorb,REV-ERBA and DBP in the hippocampus of mice in each group were detected by Real Time PCR.Experiment 3:9 6-month-old APP/PS1 mice were randomly divided into 3 groups:Model group(Model group),Root-Securing and Brain-Fortifying Liquid group(RSBFL)and melatonin group(MT),and blank Control group(Control group)were equal month-old gene-negative C57BL/6J mice with 3 mice in each group.The intervention groups were given the corresponding Root-Securing and Brain-Fortifying Liquid intragastric administration at the dose of 2ml/100g/d,and the Control group and Model group were given the same volume of pure water intragastric administration for 12 weeks.mRNA transcriptome sequencing was performed in each group.After the completion of quality inspection,the mRNA of mice in each experimental group was sequenced and compared to screen out the differential genes,and then GO enrichment analysis and KEGG enrichment analysis were carried out at the same time to dig out the genes and pathways related to Alzheimer’s disease and circadian rhythm.Experiment 4:Adopt Western Blot analysis was performed to detect the expression levels of Beclin-1,LC3I,LC3-Ⅱ,p62,LAMP1 and CTSB,as well as the expression levels of PI3K,Akt and mTOR,phosphorylated proteins and Aβ in the PI3K/Akt/mTOR signaling pathway Expression.Result:1.The results of experiment 1(1)Results of Water maze testCompared with the Control group,the escape latency and total swimming distance of mice in Model group were significantly increased(P<0.01),but the escape latency and total swimming distance of mice in RSBF group and MT group were decreased to varying degrees(P<0.01,P<0.05).Compared with Control group,The Times of crossing the original platform and the time of staying in the target quadrant were significantly reduced in Model group(P<0.01),and the time of first arrival on the platform was significantly extended in model group(P<0.01).Compared with Model group,The Times of crossing the original platform and the time of staying in the target quadrant were increased in RSBFL and MT groups(P<0.01,P<0.05),and the time of first reaching the platform was decreased(P<0.01,P<0.05).(2)The results of HE stainingCompared with the Control group,the neurons in the CA1 and CA3 regions of the Model group were arranged loosely and disordered,the distance increased,the nuclei were deeply stained and irregular,and the number decreased significantly.Compared with Model group,the margins of CA1 and CA3 hippocampal neurons in RSBFL and MT groups were clear and closely arranged,and the number of neurons increased significantly.(3)The results of Nissl staining:Compared with the Control group,the cell boundaries of CA1 and CA3 regions in Model group were unclear and disordered,the number of nenistidia in the cytoplasm was significantly reduced,the staining was darker and there was obvious shrinkage,and the number of neurons was significantly reduced(P<0.01).In RSBFL and MT groups,the edge of hippocampal neurons in CA1 and CA3 regions was clear,the number of intracytoplasmic nishi was significantly increased with less wrinkle reduction,and the number of neurons was increased to varying degrees(P<0.01,P<0.05).(4)The results of Golgi stainingCompared with Control group,the amount of black in Golgi staining of brain tissue of mice in each group was significantly reduced,and the neuronal dendritic spines were also correspondingly decreased,especially in Model group(P<0.01).Compared with Model group,the number of black in RSBFL group and MT group was increased in different degrees,and the neuronal dendritic spines in hippocampal area were increased in different degrees,the difference was statistically significant(P<0.01,P<0.05).(5)The results of PSD95/SYN1/GAP43 immunofluorescenceCompared with Control group,the red fluorescence distribution area of PSD95 in CA3 and DG region was smaller in Model group,and the fluorescence intensity was significantly decreased,especially in DG region(P<0.01).Compared with Model group,the red fluorescence distribution area and fluorescence intensity of PSD95 in CA3 and DG regions of RSBFL group and MT group were significantly increased(P<0.01).Compared with Control group,the green fluorescence intensity of SYN1 in CA3 and DG regions of hippocampal tissue in Model group was significantly decreased(P<0.01).Compared with Model group,the green fluorescence distribution area and fluorescence intensity of SYN1 in CA3 and DG regions of RSBFL and MT groups were significantly increased(P<0.01).Compared with Control group,the pink fluorescence intensity of GAP43 in CA3 and DG regions of hippocampal tissue in Model group was significantly decreased(P<0.01).Compared with Model group,the pink fluorescence distribution area and fluorescence intensity of GAP43 in CA3 and DG regions of RSBFL and MT groups were significantly increased(P<0.01,P<0.05).(6)The results of TEMCompared with the Control group,the Model group showed different sizes of hippocampal nuclei,significantly reduced mitochondria,less endometrium and matrix,significantly reduced ribosomes,and irregular shape of rough endoplasmic reticulum.The morphology and structure of hippocampal neuron nuclei in RSBFL and MT groups were relatively normal,some mitochondria had clear structure,the rough endoplasmic reticulum had mild flat sac expansion,and the number of ribosomes increased.2.Results of experiment 2(1)The results of EEG/EMGCompared with the Control group,the Wake time of mice in the Model group increased,the NREM sleep duration decreased,and the REM sleep duration decreased correspondingly,especially in the daytime(P<0.01,P<0.05).Compared with Model group,Wake duration decreased and NREM sleep duration increased significantly in each administration group,especially during the day(P<0.01,P<0.05),while REM sleep duration showed no statistical difference at night(P>0.05).(2)The results of Real Time-PCRCompared with Control group,the expressions of Perl,Cry2,Bmal1,Rev-erba,Npas2,Per2,Rora,Cry1,Clock and Dbp in Model group were significantly decreased(P<0.01).Compared with Model group,the expressions of Perl,Cry2,Bmal1,Rev-erba,Npas2,Per2,Rora,Cry1,Clock and Dbp in RSBFL group and MT group were increased to varying degrees(P<0.01,P<0.05).3.Results of experiment 3In this study,a total of 216 differential genes were screened,which significantly affected 27 signaling pathways,including PI3K-Akt,mTOR,Wnt,AMPK,P53,JAK-STAT NF-kappa B and other classical pathways.signaling pathway PI3K/AKT/mTOR is significantly enriched,and the expression of its related genes has significantly changed.It is speculated that the PI3K/AKT/mTOR signaling pathway plays an important regulatory role in the occurrence of Alzheimer’s disease and circadian rhythm disturbance.The effect of,Root-Securing and Brain-Fortifying Liquid may be closely related to the intervention of this pathway.4.Results of experiment 4(1)The results of autophagy signature protein detectionCompared with Control group,the ratio of LC3-Ⅱ to LC3-Ⅰ in hippocampus of Model group was increased,and the protein expression level of P62 was also increased significantly(P<0.01).The expression levels of Beclin-1,Lamp1 and CTSB proteins were significantly decreased(P<0.01).Compared with Model group,the ratio of LC3-Ⅱto LC3-Ⅰ in RSBFL group and MT group was increased(P<0.01),and the protein expression level of P62 was decreased(P<0.01,P<0.05).The expression levels of Beclin-1,Lamp1 and CTSB proteins were increased in different degrees(P<0.01,P<0.05).(2)The results of Western BlotCompared with Control group,the expression levels of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR in hippocampus of Model group were significantly decreased(P<0.01).Compared with Model group,the expression levels of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR in RSBFL group and MT group were significantly increased(P<0.01).(3)The results of Aβ protein expressionCompared with Control group,the expression level of Aβ protein in hippocampus of Model group was significantly increased(P<0.01).Compared with Model group,the expression level of Aβ protein in RSBFL group and MT group was significantly decreased(P<0.01).Conclusion:(1)RSBFL has conducted in-depth analysis of AD and circadian rhythm disturbance from the perspective of Yin-Yang,conversion of Yingwei,the camp and the WuZang Shen,The two cause and effect of each other,can be used to consolidate the brain of the day,and the Five internal Organs to the brain to prevent and cure.(2)RSBFL can improve the learning and memory ability of APP/PS1 mice,can reducing Aβ deposition,improve neuron-synaptic damage in APP/PS1 mice.(3)RSBFL can improve sleep-wake circadian rhythm disturbance in APP/PS1 mice.(4)RSBFL can improve the learning and memory ability and circadian rhythm disturbance of APP/PS1 mice,which may be achieved by regulating of autophagy signaling pathway PI3K/Akt/mTOR. |
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