Study On Anti-parkinson’s Mechanism Of Paeoniflorin Based On Network Pharmacology And Molecular Docking

Parkinson’s disease（PD）is a complex and multifactorial neurodegenerative disease.PD is the second most common neurodegenerative disease after Alzheimer’s disease.At present,drugs for the treatment of PD mainly include levodopa preparations,dopamine receptor agonists,monoamine oxidase B inhibitors,methyltransferase inhibitors and other drugs such as anticholinergic drugs,etc.,and multi-drug combination is required in the late stage of the disease.Although these drugs can relieve patients’symptoms,they require long-term use,are resistant,and some have serious adverse reactions.Traditional Chinese medicine has an ideal therapeutic effect in the treatment of many chronic diseases.Peony is one of the commonly used traditional Chinese medicines,which has been used in traditional medicine for more than 2,000 years.Paeoniflorin（PF）is the main active component of Paeoniae Radix Alba and Paeoniae Radix Rubra.It has been documented that PF can act on the nervous system,and animal experiments have shown that PF has neuroprotective effect,but the specific mechanism and target need to be further studied.Network pharmacology and molecular docking is one of the important means to find drug action targets.In this study,we first used the online pharmacology Disgenet database,TTD database and DRUGBANK database to search for PD related disease targets,combined the targets of the three databases,screened out the duplicates,and obtained1160 PD disease targets.TCMSP database was used to query PF chemical structure formula,and files saved in the format of Mol2 were downloaded.The number of 117 targets was obtained by using Swiss Target Predict database to predict PF action targets.The intersection of PD target and PF target was found,and 36 targets were obtained as the functional targets of PF in the treatment of PD.Protein-related proteins of 36 targets were found in the String database,and protein-protein interactions（PPI）were analyzed using Cytoscape 3.2.1 software.Score values greater than 0.7（with high confidence）were screened to obtain 23 core targets and their related proteins,and a PPI network was constructed.GO（Gene Ontology）annotation analysis and KEGG analysis were performed in the David database,and 84 biological processes,18 molecular functions,29 cellular components and 14 pathways were obtained.Then,molecular docking between PF and the sifting core targets was performed.Finally,a PD rat model was established by subcutaneous injection of rotenone to verify the network pharmacology and molecular docking prediction results.PD rat model was established by subcutaneous injection of rotenone.A total of 72 adult male SD rats were randomly divided into 6 groups with12 rats in each group.Normal group:subcutaneous injection of normal saline;vehicle control group:subcutaneously injected sunflower oil,1m L·kg^-1·d^-1;Model group:Rotenone（sunflower oil as solvent,containing2mg rotenone per m L）was injected subcutaneously,1m L·kg^-1·d^-1,and double steamed water(10 m L·kg^-1·d^-1)was given intragastric administration on the day of modeling;PF low-dose,medium-dose and high-dose groups:1m L·kg^-1·d^-1was injected subcutaneously with rotenone,and different concentrations of PF solutions(10,20 and 40 mg·kg^-1·d^-1for low-dose,medium-dose and high-dose paeoniflorin solutions were given intragastric administration on the modeling day,respectively).The experiment lasted for 28 days.The body weight and general health condition were monitored daily during the period of the administration of various drugs.Each group of rats after dosing 28 d,the general behavioral deficits was assessed.The limb motor coordination of rats was observed by hanging test.Muscle rigidity of rats was evaluated by swimming test and grid test.The degeneration of dopaminergic neuronwas observed using HE staining.TH-positive neurons in the substantia nigra pars compacta（SNpc）were estimated by immunohistochemical method.The expression levels of the SNCA,TH and Caspase-3 protein with ELISA experiment.And the enzyme activity of TH,Caspase-3 and Ach E was detected by spectrophotometry.The results showed that there were no statistical differences in the behavioral scores,the expression of the SNCA,TH and Caspase-3,and the enzyme activity of TH,Caspase-3 and Ach E between normal group and vehicle group.These results suggest that the solvent sunflower oil had no effect on the above indexes in rats（P>0.05）.Compared with the vehicle group,the hanging time on the wire and swimming time of rats in the model group were significantly reduced,and the time required from the static state to the movement of any paw was significantly prolonged in the grid experiment,with statistical differences（P<0.01）.The results of HE staining showed that the cells were sparse,nucleoli blurred and cytoplasm concentrated in SNpc.Immunohistochemical results showed that TH-positive neuronswere decreased in SNpc.ELISA results showed that SNCA aggregation increased,Caspase-3 protein expression increased,TH expression decreased（P<0.01）.Spectrophotometric experiment results showed that the activity of caspase-3 and Ach E was significantly increased,and the activity of TH was significantly decreased,with statistical differences.Compared with the model group,PF can inhibit the nerve damage of rotenone-induced Parkinson’s disease model rats,reduce the formation of SNCA aggregation,increase the expression of TH,decrease the expression of Caspase-3,enhance the activity of TH,and inhibit the activity of Ach E and Caspase-3.In this study,the anti-PD target of PF was predicted by network pharmacology and molecular docking,and the experimental verification was carried out.This study preliminarily revealed the anti-PD mechanism of PF,and provided a basis for further research on the mechanism of PF.