Exploring The Neuroprotective Effect Of Chaihu Shugan San On Alzheimer's Disease Based On Apoptosis And Autophagy Pathway

Objective:Based on the principle of prescription syndrome corresponding,exploration the molecular pathological basis of Alzheimer’s disease（AD）liver depression and qi stagnation syndrome.To investigate the pharmacodynamic activity,target and molecular mechanism of"Shugan Jieyu"representative formula Chaihu Shugan San（CSS）in degrading AD core pathological products and protecting neurons.To provide scientific experimental basis for the clinical treatment of CSS in AD,and expand new ideas for the research on the substance basis,mechanism of action and prescription syndrome corresponding of traditional Chinese medicine compound.Methods:（1）Chemical analysis of CSSAccording to the formula of CSS in jingyuequanshu,the CSS extracts were extracted by water,and then obtained by vacuum freeze-drying and analyzed and identified by high-performance liquid chromatography（HPLC）.（2）verified the effect of CSS on cognitive function and neuropathological in SAMP8 mice.5-month-old male SMRP8 mice were randomly divided into the model group,the high-dose group（CSSH,4.2g/kg）,the low-dose group（CSSL,2.1g/kg）,and the donepezil group（1mg/kg）.SAMR1 male mice of the same age served as the normal group.All mice were given intragastric administration once a day for 8 weeks.The Morris water maze experiment was conducted to observe the improvement effect of CSS on the learning and memory ability of SAMP8 mice starting from the 7th week of the experiment.At the end of the Morris,sections of brain tissues were taken and neuronal damage was detected by Nissler staining,andβ-amyloid（Aβ）expression in the hippocampal region and cortex of mice was detected by immunohistochemistry;the reagent kit was used to detect acetylcholinesterase（ACh E）andβ-secretase（BACE1）activities in mice brain tissue.（3）the exploration of CSS on neuron apoptosis and autophagy of AD cellsUsing Aβ_1-40（0.25μM）induced rat pheochromocytoma PC12 cells and established AD cell model.Cell viability and apoptosis were detected by MTT assay or Annexin V-FITC/PI staining separately.The lysosomes were investigated using MDC and Lyso Tracker staining.The expression of proteins relating to cell apoptosis and autophagy was measured by western blotting.（4）Network pharmacological analysis methods were used to comprehensively explain and predict the possible mechanisms of CSS and its compound-related targets in AD.Exploring the molecular pathological basis of AD liver depression and Qi stagnation syndrome,based on the principle of accordance of prescription and syndrome.Three public databases including TCMSP,Swiss Target Prediction,and STITCH were employed to identify the compounds in CSS and its compound-related targets.The acknowledged AD-related targets were collected from Therapeutic Target Database（TTD）,Gene Cards,and Mala Cards databases.Gene Ontology（GO）analysis and KEGG pathway enrichment analysis of selected targets were performed using the online functional annotation and enrichment database DAVID.Results:（1）Pharmacochemical analysisThe extract of CSS detected five components,including,Saikosaponin B1,Paeoniflorin,Glycyrrhizin,Ferulic acid,and Hesperidin.（2）Cognitive function and neuropathology in SAMP8 models of AD1)Behavioral research about learning and spatial memory functionCSS could improve the learning and spatial memory ability of SAMP8 mice after 8 weeks.On the 2nd-5th day of learning ability training,compared with the model group,the escape latency of mice in the CSSH group was significantly shortened（P<0.05 or P<0.01）,and the efficacy was equal to Donepezil.In terms of spatial memory ability,compared with the model group,the time of first finding the original platform was significantly shortened in the CSSH group（P<0.05）,and the frequency of mice crossing the platform was increased,which was equivalent to Donepezil.In terms of learning ability and spatial memory ability,there was no significant statistical difference between the CSSL and the model group.2)Study on neuropathologyNissler staining showed that the neuron cells in the hippocampus and cortex of the model mice were significantly reduced,with loose arrangement,abnormal cell morphology,cytoplasmic wrinkling,dark staining of Nissler staining,and the cells were in a damaged state.After treatment with CSS（CSSH,CSSL）and donepezil,the number,morphology,and structure of neuron cells in the hippocampus and cortex of the mice were significantly improved,and the cell damage was significantly reduced.Immunohistochemical showed that no Aβwas expressed in normal mice,while the model mice had higher Aβexpression in hippocampal tissues and cortex,and Aβexpression was lower after treatment with CSS（CSSH,CSSL）and donepezil.In addition,ACh E and BACE1 were elevated in the brain tissues of model mice（all P<0.001）,and CSS（CSSH,CSSL）and donepezil could reduce ACh E and BACE1 activity in brain tissues（all P<0.001）.（3）PC12 AD cell modelMTT showed that Aβ_1-40（0.125-2μM）stimulated PC12 cells for 24h or 48h causing different degrees of decrease in cell viability（P<0.001）.Compared with Aβ_1-40（0.25μg/m L）stimulating alone,Aβ_1-40（0.25μg/m L）+CSS（250μg/m L or 500μg/m L）significantly enhanced cell viability（P<0.05）,increased neuronal cell number and improved cell morphology induced by Aβ_1-40.Annexin V-FITC/PI double staining method showed that stimulation of Aβ_1-40（0.25μM）24 h significantly promoted early and late apoptotic stages（P<0.001,P<0.01）.CSS（500μg/ml）inhibited early and late apoptotic stages induced by Aβ_1-40（P<0.01）.CSS（250μg/ml）also reduced early and late apoptotic cells induced by Aβ1-40（P<0.01,P<0.05）.Co-stimulation of Aβ_1-40 and CSS further increased MDC green fluorescence intensity and Lyso Tracker green fluorescence compared with the addition of Aβ_1-40 alone（P<0.05）,promoted the formulation of autophagy-lysosome.Western-Blot results showed that Aβ_1-40significantly inhibited the p-Akt expression and p-m TOR,upregulated Bax levels,and LC3-II/LC3-Ⅰ（a ll P<0.05）.CSS increased p-Akt and LC3-II/LC3-Ⅰlevels and down-regulated p-m TOR and Bax levels（all P<0.05）.（4）Network pharmacologyA total of 152 active compounds,520 predicted biological targets,and 160 AD-related targets were identified in CSS.Among them,60 key targets were highly responsible for the effect of CSS against AD and had 770 interactions.Further GO functional enrichment analysis showed that the 60 key targets were mainly found in the nucleus and cytoplasm,with protein binding functions,and were involved in various biological processes including transcriptional regulation,signal transduction,regulation of the apoptotic process,drug response,vascular neogenesis,gene expression regulation,protein phosphorylation,and positive regulation of cell proliferation,etc.The KEGG pathway enrichment analysis showed that 60 key targets could be enriched to 125 signaling pathways,including PI3K/Akt signaling pathway,MAPK signaling pathway,HIF-1signaling pathway,Estrogen signaling pathway,m TOR signaling pathway,et al.Conclusions:The findings of the current study support that CSS can improve the learning and memory function of SMAP8 mice,protect neuron cells,reduce Aβdeposition,and decrease the activity of ACh E and BACE1 in brain tissue.CSS has multi-link and multi-target synergistic effects with Donepezil,the first-line drug of AD.Besides,CSS can also protect neurons,delay brain pathology through inhibiting neuronal apoptosis and enhancing the autophagic-lysosome pathway then reduce AD pathological product Aβ.Moreover,this study systematically carries out a network of pharmacological research on the effect of CSS in AD and expands a new sight for further systematic research.The therapeutic effect of CSS on AD is realized by multiple active ingredients,multiple signal pathways and multiple effect targets.Meanwhile,this study is methodologically significant for the efficient screening of the substance basis,action targets,and related molecular pathways of Chinese herbal compounds.