Based On Network Pharmacology And Proteomics To Explore The Mechanism Of Puerarin On Aβ_1-42 Damage SH-SY5Y Cells

Purpose:For the prevention and treatment of dementia,China has accumulated thousands of years of experience.Pueraria is sweet and pungent in taste,cool in nature,and can return to the spleen and stomach channels.It has the functions of relieving muscle and fever,penetrating rash,generating fluid and relieving thirst,rising Yang and stopping diarrhea.Modern pharmacological studies have found that Pueraria and puerarin have significant protective effects on animal and cell models of Alzheimer’s disease.However,its material basis and related mechanisms still need to be clarified.Based on the integration of bioinformatics,molecular biology and database information,combined with data mining,literature research,bioinformatics and other methods,our study of network pharmacology studied the interaction between"drug-target-biological progress"and predicted and inferred its active ingredients,targets and mechanisms.Objective to establish an HPLC method for the determination of active ingredient of puerarin content in SH-SY5Y cells.The characteristics of drug uptake by cells was investigated combined with the AD cell model of SH-SY5Y cells induced by Aβ_1-42,then the concentration of puerarin was screened.Label-free Quantitative Proteomics technology was used at the protein level to detect differential proteins between SH-SY5Y cell groups.Bioinformatics analysis methods including GO annotation and enrichment analysis,KEGG pathway annotation and enrichment analysis and protein-protein interaction network analysis were used to further explore the key links and molecular of the core active ingredients in Pueraria lobata about anti-AD.Based on the results of bioinformatics analysis,immunofluorometric assay,Annexin V/FITC-PI flow cytometry analysis,Western blot and Real-time PCR were used to observe the effects of the core active ingredients in Pueraria lobata on the corresponding biological processes and molecules.Material and method:Based on TCMSP database,"OB>30%and DL≥0.18"were used as the screening conditions for the main constituents of Pueraria lobata.At the same time,relevant literatures were consulted for supplement,and the main constituents and targets of Pueraria lobata were determined.Search Genecards,OMIM,Pharm Gk,TTD and Grug Bank database to download and integrate all the disease targets related to"Alzheimer’s disease".Cytoscape and Venny2.1 were used to intersect the component targets with the disease targets to obtain the action targets of main component of puerarin on AD.The main component targets,protein interaction targets and AD targets of Pueraria lobata were introduced,then the"component-target-related target"network was constructed by using Cytoscape combined with DIP database to screen the core components and targets of Pueraria lobata against AD.GO/KEGG enrichment analysis was performed by Metascape,P value≤0.01 was used as the screening condition for significant differences,and bubble map was used for observability analysis.Cytoscape was used to construct the―component-target-biological process‖network.The components,biological processes,and pathways were assigned with the Degree value and the P value respectively through topological analysis.The drug concentration（0,20,40,60,80,100,200,300,400,500μg/mL,n=6）was screened by cytotoxicity test.After 24hours of incubation,20μL of CCK-8 solution was added into each well and incubated for 1 h.The absorbance（A value）was determined at 450 nm,and the cell survival rate was calculated.HPLC method was established for the determination of puerarin in SH-SY5Y cells.The standard curve was drawn and the method specificity,recovery and precision were tested.The chromatographic conditions were as follows:mobile phase:methanol:0.1%citric acid solution（25:75）,flow rate:0.8 mL/min;column temperature:40℃;detection wavelength:250 nm;injection volume:20μL.5 groups of 1,2,6,12 and 24 h were set to screen the administration time;5 groups of 20,40,60,80,100μg/mL were set to screen the administration concentration（n=3）.The cells were divided into 9 groups:the control group was routinely added with complete medium without puerarin;the blank group was not inoculated with cells or puerarin,but added with the same volume of culture medium;the Aβ_1-42 injury model group was added with 10μM oligomeric Aβ_1-42 on the basis of the control group;the puerarin intervention group was intervened with puerarin of different concentrations and time selected by the uptake experiment on the basis of the injury model group,including 5 puerarin intervention group with 6 concentrations of puerarin（10,20,40,80,100μg/ml）.CCK-8 method was used to detect the activity of the cells.The a value was measured at 450 nm by multi-functional enzyme labeling instrument,and the cell survival rate was calculated.Aβimmunofluorescence assay,Annexin V/FITC-PI flow cytometry and TUNEL were used to observe the Aβcontent and apoptosis of SH-SY5Y cells induced by Aβ_1-42.Three groups of cell samples were collected,the number of samples was 9 cases,which were group A（puerarin intervention group）,group B（AD model group）and group S（control group）.Firstly,the samples were prepared,and then analyzed by LC-MS/Ms.The mass spectrometry database retrieval software was used to search Maxquant 1.6.0.16 and Uni Prot database for protein identification and screening,combined with GO annotation and enrichment analysis,KEGG bioinformatics data analysis and protein-protein interaction network analysis,can be used to find cell changes and the source and mechanism of these changes from the huge and complex experimental data.Immunofluorescence staining,Annexin V/FITC-PI flow cytometry analysis,TUNEL detection,Western blot and Real-Time PCR detection were used to observe the effect of Aβ,autophagy and apoptosis of puerarin on SH-SY5Y cells inducing by Aβ_1-42,and the ERK1/2 pathway related protein and mRNA expression level.Results:1.TSMSP database combined with literature reports which reported compounds with anti-AD activity and the main component of Pueraria lobata were used,finallyβ-sitosterol,formononetin,3’-methoxydaidzein,daidzein-4’,7-diglucoside and puerarin were identified as the main components.TCMSP was used to predict the target points of the main components of Pueraria lobata and their related interaction targets.After integration,a total of 152component targets and 222 interaction targets were obtained.Genecards database,OMIM database,Pharm Gkb database,TTD database and Grug Bank database were used to mine AD-related targets,and 8756 AD targets were obtained after deduplication.Through topological analysis of the"component-target-related target"network,Betweenness≥0,Closeness≥0.010492173,and Degree≥4,77 core targets were obtained,including 25 common core targets,51 DIP-related core targets,and 1 drug non-public core target.Finally,25 public core targets were used as key targets for pueraria to improve AD,at the same time,β-sitosterol,formononetin,3’-methoxy daidzein and puerarin were the core active ingredients.The network of"component-core targets"was further constructed combined with topological analysis,then 25 Pueraria lobata anti-AD core targets and 4 core active ingredients（β-sitosterol,formononetin,3’-methoxy daidzein and puerarin）were screened,of which the last three ingredients are all Isoflavones.At the same time,targets of puerarin appeared 18 out of 25 core targets,and the comprehensive situation in the analysis of topology parameters such as Average Shortest Path Length,Betweenness Centrality,Closeness Centrality and Degree was the best.Based on the four core active ingredients and 25 key targets screened out by topological analysis,Metascape was used to carry out the enrichment analysis of GO and KEGG pathways.Using P<0.01 as the screening condition,it was found that these proteins were mainly enriched in 513 biological processes,12 There are three cellular components,34molecular functions,and 95 signaling pathways.There are many processes in the top ten results of GO enrichment analysis that are directly related to neuron death and apoptosis.Many processes were directly related to neuron death and apoptosis in the top ten results of go enrichment analysis.Through the construction of"component-target-biological process"network and topological analysis,it was found that AKT1（272/513）,SIRT1（233/513）,BCL2（220/513）,HIF1A（181/513）,BAX（174/513）and other targets participate in more GO-BP,AKT1（67/95）,RELA（60/95）,JUN（45/95）and other targets participate in more signal pathways.KEGG functional enrichment classification results found that 10 cell processes had significant differences,among them,PValue in the process of apoptosis and the number of targets enriched in it ranked first.2.CCK-8 method was used to detect the toxicity of puerarin on SH-SY5Y cells.The results showed that when the concentration of Puerarin was below 100μg/mL,the cell survival rate was above 90%.When the concentration of Puerarin was higher than 200μg/mL,the survival rate of sh-sh5y cells decreased slightly,and the cell survival rate decreased significantly when the concentration was higher than 300μg/mL.Therefore,the drug concentration of 100μg/ml was selected as the highest uptake concentration.The method specificity experiment results showed that the impurities in blank cells had no interference on the determination of puerarin,and could be separated effectively.The regression equation was y=104684x+3818.1.Puerarin had good linearity in the concentration range of 0.2-100μg/mL（R²=0.9999）.The results of recovery and precision test showed that the recoveries of low,medium and high concentration groups were（104.39±3.86）%,（93.19±5.12）%,（88.29±7.12）%,RSD were 3.70%,5.50%,8.06%（n=6）,intra day RSD were 7.97%,3.04%,3.21%（n=6）,and inter day RSD were 8.96%,4.76%and 5.33%（n=6）,respectively.The results of drug administration time screening test showed that the drug uptake in SH-SY5Y cells increased gradually with the prolongation of uptake time,reached the maximum at 6 h,and gradually reached equilibrium with the extension of time,so 6 h was taken as the time for cell uptake.The results of drug concentration screening test showed that puerarin uptake was concentration dependent in the concentration range of 20-100μg/mL.The experimental results of puerarin on the cell viability of SH-SY5Y cells induced by Aβ_1-42 showed that compared with the control group,the cell viability of SH-SY5Y cells in model group was significantly decreased（P<0.01）,and the cell survival rate was 56%.The survival rate of SH-SY5Y cells in puerarin 20,40,80 and 100μg/mL groups were significantly increased,which were 71.10%,79.71%,83.41%and 84.77%,respectively（P<0.01）.It is suggested that puerarin can improve the inhibitory effect of Aβ_1-42 on SH-SY5Y cell proliferation in dose-dependent manner,and its effective range is 20-100μg/mL.3.In the proteomics test,9239 unique peptides,28947 identified peptides and 2283 proteins were identified in group A;10423 unique peptides,34267 identified peptides and 2402proteins were identified in group B;10752 unique peptides,35486 identified peptides and2400 proteins were identified in groups,11781 unique peptides and 98 unique peptides were identified in group S.A total of 11781 unique peptides,98700 identified peptides and 2511proteins were identified.The results showed that there were 753 significantly different proteins,311 differential proteins,446 up-regulated proteins and 307 down regulated proteins between group A and group S;there were 336 significantly different proteins in group B compared with group S,172 with or without difference proteins,172 up-regulated proteins and 164 down regulated proteins;compared with group B,there were a total of 753significantly different proteins,311 with or without differential proteins,446 up-regulated proteins and 307 down-regulated proteins;compared with group B,there were a total of 336significantly different proteins There were 104 significantly different proteins,131 with or without differential proteins,27 up-regulated proteins and 77 down regulated proteins.The results showed that there were 2186 common proteins in group A and group S,97 proteins in group A were not detected in group S,214 proteins in group S were not detected in group A,2306 common proteins were detected in group B and group S,96 proteins were not detected in group S,94 proteins were not detected in group B and group A In group B,153 proteins were not detected in group A,and 34 proteins in group A were not detected in group B.The results showed that most of the proteins predicted were related to cellular component organization or biogenesis,cellular component organization and cellular component Biogenesis correlation.In the classification of cell composition,all the proteins were mainly located in dendritic tree,cytosol and cytospray.It is related to protein binding,drug binding and binding in molecular function classification.KEGG enrichment analysis and pathway annotation showed that the P value in the top ten pathways are as follows:Pathogenic Escherichia coli infection,m TOR signaling pathway,Regulation of autophagy,Regulation of actin cytoskeleton,Spliceosome,Hepatocellular carcinoma,Tight junction,Non-small cell lung cancer,Apoptosis,Gap junction.Among them,in m TOR pathway,the expression of CAB39,IRS1,ATP6V1D,DVL2,MAP2K2（MEK）,PRKCA,RPS6KA3,MAPK1（ERK1/2）and other differential proteins were all down-regulated.In Regulation of autophagy pathway,the expression of IRS1,SH3GLB1,MAP2K2,MAPK1,EIF2S1 and other differential proteins were all down-regulated.In Apoptosis pathway,the expression of BAX,TUBA1C,MAP2K2,CAPN1,TUBA1B,MAPK1,EIF2S1,TUBA1A and other differential proteins were all down-regulated.Protein interaction network analysis showed that Cap1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.In addition,in the interaction network,ERK1/2 pathway is closely related to apoptosis and autophagy.4.The result of Aβimmunofluorescence showed that the Aβof the puerarin intervention group was significantly less than that of the AD model group.Annexin V/FITC-PI flow cytometry analysis showed that compared with the AD model group,the apoptosis rate of the puerarin intervention group was significantly reduced[（10.98±0.45）%vs（21.93±0.22）%,P<0.01].TUNEL detection showed that the apoptosis rate of puerarin intervention group was significantly lower than that of AD model group[（10.30±4.16）%vs（18.80±4.10）%,P<0.01].Western blot detection of the expression of autophagy-related proteins LC3Ⅱand LC3Ⅰshowed that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after inducing by Aβ,and the degree of this up-regulation was further enhanced in puerarin intervention group.The protein expression level of LC3Ⅰamong the groups was no significant difference.The trend of the ratio of LC3Ⅱ/LC3Ⅰwas the same as the protein expression level related LC3Ⅱamong each group.The expression level of autophagy-related protein p62 decreased in the control group,AD model group,and puerarin intervention group.In addition,the protein expression level of p-ERK1/2 in the AD model group was significantly higher than that in the control group（P<0.01）,and the expression of p-ERK1/2was significantly down-regulated after the intervention of puerarin（P<0.01）.There was no significant difference in ERK1/2 in each group.Compared with the control group,the expression level of the pro-apoptotic protein BAX in the AD model group was significantly up-regulated（P<0.01）.Compared with the AD model group,the protein expression of BAX was significantly down-regulated after puerarin intervention（P<0.01）,and there was no significant difference from the control group.The protein expression level of Bcl-2 in the AD model group was significantly lower than that in the control group（P<0.01）,the expression was significantly up-regulated after puerarin intervention（P<0.01）.The protein expression level of CAP1 was significantly higher in the AD model group than in the control group（P<0.01）,and the expression was significantly down-regulated after puerarin intervention（P<0.01）.Real-Time PCR showed that there was no significant difference in the mRNA expression of ERK1/2 among the groups.Compared with the control group,the expression of Bcl-2 mRNA in the AD model group was significantly down-regulated（P<0.01）,and the expression was significantly up-regulated after puerarin intervention（P<0.01）.The mRNA expression of Bax in the AD model group was significantly higher than the other two groups（P<0.01）,and the expression was significantly down-regulated after puerarin intervention（P<0.01）.The expression of CAP1 mRNA in each group was basically the same as that of Bax.The AD model group was significantly higher than the other two groups（P<0.01）,and the expression was significantly down-regulated after puerarin intervention（P<0.01）.Conclusion:1.With the help of TCMSP,Genecards,OMIM,Pharm Gkb,TTD,Grug Bank and other databases,the network of"drug-target-biological process"was constructed and topological analysis was carried out.The result showed that puerarin was an important material basis for pueraria to improve AD,and its potential pharmacodynamic mechanism may be closely related to neuronal apoptosis and autophagy.2.For SH-SY5Y cells,the optimal administration condition of puerarin was 100μg/mL for 6h.In addition,puerarin can improve the inhibitory effect of Aβ_1-42 on SH-SY5Y cells in a dose-dependent manner.The effective range of puerarin is 20-100μg mL.3.Through label With the method of free proteomics,104 significantly different proteins and131 with or without different proteins were found in SH-SY5Y cells of Aβ_1-42-induced AD model group and puerarin intervention group.Among these protein rich pathways,P values of apoptosis and autophagy pathway are significantly different,and they are closely related to the pathogenesis of AD,and ERK1/2 pathway may play an important role in this process.4.The mechanism of puerarin improving AD may be to accelerate the clearance of Aβby inducing autophagy and reduce its cytotoxicity on the one hand,and to promote the survival of neurons by inhibiting apoptosis on the other hand.ERK1/2 may be an important target of puerarin.