Study On Protective Effect Of Biatractylolide On MPTP Opening Against A?_25-35 Induced Cell Injury In PC12 And SH-SY5Y Cells

Objective.To study the protective effect of Biatractylolide,a compound of Baizhu on stress induced mitochondrial membrane potential?mPTP?opening by Amyloid beta(A?_25-35)and cell apoptosis in rat adrenal pheochromocytoma cells?PC12?and a human bone marrow neuroblastoma cell line?SH-SY5Y?,and to explore its mechanism and further provide a systems network pharmacology model based on pharmacokinetic and pharmacological data to identify candidate compounds and potential targets of Baizhu,a traditional Chinese medicine with a neuroprotective effect in Alzheimer disease.Methods.Cells PC12 and SH-SY5Y were cultured in vitro.The PC12 and SH-SY5Y cells were treated with 20?M A?_25-355-35 for 24 hrs,pretreatment with Biatractylolide?5,10,20?M?for 2 hrs.Experiment was as follows:?1?blank control group,?2?A?_25-355-35 insulted group,?3?drug control group?low,medium,high?.By MTT assay cell proliferation was assayed.MMP?Mitochondrial membrane potential?was detected by staining Rhodamine 123.ROS kit was used for the measurement of intracellular ROS.Western blot analysis used to detect CypD and CytC.Network pharmacology is a useful approach that targets biological networks and analyzes the links among drugs,targets,and diseases in those networks.Networks have been constructed by using Cytoscape 3.2.1software.We have been made 3 networks:?1?C-T?compound target? candidate compounds of Baizhu and their potential targets have been searched on database and analysis available on Traditional Chinese Medicine Systems Pharmacology?TCMSP?and the Swiss Target Prediction platform.?2?T-D?Target disease?network was constructed by relating relevant targets to their diseases.The potential diseases were obtained from the Therapeutic Targets Database?TTD?and Drug Bank,and then classified into systems according to MeSH Browser database.?3?T-P?target pathways?has been obtained by inputted target proteins into Kyoto Encyclopedia of Genes and Genomes pathway analysis?KEGG?,and Gene Ontology?GO?respectively.Result.Incubation of PC12 and SH-SY5Y cells with 20?M of A?_25-355-35 for 24 hrs resulted in significant decrease of cell proliferation,decreased in membrane potential?MP?,increased ROS,increased CypD and apoptotic factor CytC increased in cytosol and decreased in mitochondria.Compared with A?_25-355-35 in control group pretreatment with Biatractylolide 5,10 and 20?M concentrations for 2 hrs,the activity of cell proliferation was improved,number of injured cells decreased,mitochondrial membrane potential had been increased and it was concentration dependent,and ROS release activity was also decreased.Western blot analysis manifested that CypD protein expression level in cell decreased and the other protein CytC expression level decreased in cytosol and increased in mitochondria.The result of Network pharmacology showed 18 candidate compounds of Baizhu,and their 20 potential targets.These compound and targets has been shown in compound target?C-T?network.We have found78 related diseases with these targets and exhibited in target disease?T-D? network.T-D network shows that more diseases are related to CNS as compared to others systems?24?.And finally we have found 22 signaling pathways and made a target pathway?T-P?network.Conclusion.Biatractylolide has a protective effect on Amyloid beta(A?_25-35)induced mPTP opening in PC12 cells and SH-SY5Y cells in a dose dependent manner,and mechanism is through the inhibition of mPTP opening.C-T?compound target?,T-D?target disease?,T-P?target pathway?networks supplied the systematic evidence that a TCM?Baizhu?formula has extensive pharmacological activities and successfully applied into the treatment of neurodegenerative diseases especially in Alzheimer disease.