The Study Of Effect Of Danggui Shaoyao San On Insulin Signaling Pathway In PC12 Cells Injured From High Glucose Concentration

Objective:1.The aim of this study is to explore and investigate mechanisms of Danggui Shaoyao San for treatment of Alzheimer’ s disease(AD)by using a network pharmacology approach,to lay the foundation for follow-up research.2.Based on the results of network pharmacology,the possible molecular mechanism of Danggui Shaoyao San in the treatment of Alzheimer’ s disease was preliminarily discussed through cell experiments.3.The fingerprint of Decoction of compound Danggui Shaoyao San was established,which provided reference for the analysis of chemical composition and the establishment of quality control standard.Method:1.First,TCMSP database were used to screen the components of Danggui Shaoyao San and then predict the potential targets of alzheimer’ s disease and selected compounds.String database was selected to form a protein-protein interaction(PPI)networks for interselected targets of compounds.After that,Cytoscape 3.5.1 software was used to visualize the network.Finally,to investigate the biological function and pathway of potential target of traditional Chinese medicine(TCM),and to explore the main pathway of Danggui Shaoyao San in the treatment of AD,gene ontology(GO)and KEGG pathway analysis of active target genes,which was queried in the Unitprot database,were carried out by DAVID online analysis tool.2.This experiment takes the PC12 cell model as the research object,takes the compound Danggui Shaoyao San as the starting point,combined with the previous research results,tries to investigate the effect of Danggui Shaoyao San on the insulin signaling pathway PC12 cell injured from high Glucose Concentration,to explore whether the AD pathogenesis is related to insulin resistance,reveals the compatibility regularity of this compound.It provides reference for the analysis of Chinese herbal compound and the study of the principle of traditional Chinese medicine.1)According to the results obtained in step 1,combined with the research progress in recent years,this experiment uses PC12 cells as the research object,intervene with Danggui Shaoyao San decoction,insulin freeze-dried powder solution and GSK 1838705A(IGF-1 inhibitor)respectively,using Methyl Thiazolyl Tetrazolium(MTT)assay to detect cell survival rate,the effect of drugs on the activity of PC12 cells and the range of concentrations which do not influence their growth;2)The PC12 cells were cultured in culture medium with different glucose concentration,and the concentrations that could cause significant damage were screened and selected for the next test.3)The PC12 cells were cultured in the medium with high glucose concentration selected in step 2,to establish the model of high glucose injury,and the experiment was divided into 4 groups:blank control group,model control group,GSK1838705A(IGF-1 inhibitor)group,Danggui Shaoyao San decoction group.After culturing 24h,Western blot was used to detect the effects of Danggui Shaoyaosan Decoction on the expression of APP protein and insulin signaling pathway proteins,such as IGF-1,mTOR/p-mTOR and P70S6K/p-P70S6K in PC12 cells.3.Using HPLC-DAD method,gradient Elution method was used to explore the chromatographic conditions of compound Danggui Shaoyao San decoction to ensure that the composition of water decoction could be detected as much as possible in vitro and determined.Then the representative standard product is selected to calibrate the characteristic peak,and the HPLC-DAD fingerprint of compound Danggui Shaoyao San is established by this method.Results:1.In total of 38 screened compounds and 9 potential targets were obtained for the treatment of Alzheimer’ s disease.Results indicated that Danggui Shaoyao San might have a role in treating AD by pathway,nervous system development,signal transduction,etc.,and some molecular functions,such as G protein coupled acetylcholine receptor activity,peroxidase activity,Acetylcholine binding.The mechanisms might be concerned with cholinergic synaptic pathway,neural ligand-receptor interaction pathway,calcium signaling pathway,nicotine addiction-related signal pathway and PI3K-Akt signaling pathway.At present,some of the drugs confirmed by the study act on the signaling pathway of AD,and there are relatively many researches on the PI3/AKT signaling pathway.2.The results of MTT experiment showed that there were significant differences between PC12 cell activity and control group when the glucose concentration of medium was greater than or equal to 100 mmol/1.Therefore,the high sugar culture medium of 100 mmol/1 was selected in the late experiment to culture the PC12 cells,and the scatterwater decoction of compound Danggui Shaoyao San had no obstruction to the growth of PC12 cells in the concentration 1-10 mg/ml of raw medicines.The insulin dry powder solution promoted the growth of PC12 cells in the concentration range of 0-20 μg/ml.We selected 5 μg/ml as experimental concentration;GSK1838705A had no effect on the activity of PC12 cells in the concentration range of 50-70 nmol/ml,and the 60 nmol/ml was selected as the experimental concentration.The results of Western blot show that:Danggui Shaoyaosan could reduce the expression of APP and IGF-1 protein in PC12 cells damaged by high glucose,while the levels of mTOR,p-mTOR,P70S6K and p-P70S6K protein were significantly increased.3.In this experiment,the HPLC-DAD fingerprint of compound Danggui Shaoyao San Decoction was established,22 common peaks were determined,and the results of fingerprint similarity calculation are more than 0.91.Conckution:1.This part of the study reflects the characteristics of multi-component,multi-target and multi-pathway of traditional Chinese medicine,and analyses the mechanism of Danggui Shaoyao San in the treatment of Alzheimer’s disease.It provides ideas and methodological theoretical basis for further research on the mechanism of Danggui Shaoyao San in the treatment of Alzheimer’s disease.2.Danggui Shaoyao San can inhibit the expression of APP protein and IGF-1 protein in PC12 cells damaged by high glucose through insulin signal transduction pathway,which may have the effect of reversing insulin resistance at the molecular level.The results showed that the levels of mTOR,p-mTOR,P70S6K and p-P70S6K protein were significantly increased,suggesting that there was an effect of activating the m-TOR/P70S6K pathway downstream of insulin signal transduction pathway.3.The fingerprint of Danggui Shaoyao Powder was established in this experiment,and a comprehensive and low detection limit method was established to ensure that the components contained in the decoction were fully detected in vitro,which could provide reference for the accurate and controllable use of drugs in later pharmacological experiments.