Exploration Of The Function And Molecular Mechanism Of Atractylodin In Osteosarcoma Based On Network Pharmacology And Experiments In Vitro And Vivo

Object: In this study,we explored the inhibitor effect of atractylodin in osteosarcoma through cytological experiments,xenograft mouse model assay,network pharmacology and molecular experiment.Methods:1.In cytological experiments,CCK8 assay was used to detect the inhibitory effect of atractylodin to the proliferation of 143 B and U2 OS osteosarcoma cell lines.In addition,the wound healing and transwell matrigel assays were exerted to assess the inhibitory effect of atractylodin on the migration and invasion of osteosarcoma cells.2.Subcutaneous tumor model was used to clarify the anti-osteosarcoma effects in vivo,and analyzed the biosafety of atractylodin by HE staining further.3.Network pharmacology technology was used to predict the potential molecular mechanism.Firstly,we obtained the atractylodin targets,osteosarcoma targets and intersection targets of two.Then,protein-protein interaction(PPI)network and GO/KEGG enrichment analyses were taken to select the top targets and pathways.And molecular docking technology was applied to verify the binding affinity between atractylodin and targets.Finally,Kaplan-Meier Plotter and GEPIA databases were applied to predict the relationship between core targets with the prognosis of osteosarcoma patients.4.Finally,the molecular mechanism was verified by biomarkers in osteosarcoma cell lines and immunohistochemistry of nude mice tumors.Western blot was used to assess the expression level of proteins changed by atractylodin in the MAPK pathway.And immunohistochemical sections stain was applied to verify relevant marks of proliferation,invasion,apoptosis and pathway.Results:1.Atractylodin could inhibit the proliferation,migration and invasion of osteosarcoma cell lines 143 B and U2 OS in a concentration-dependent and time-dependent manner;2.Xenograft mouse model confirmed the anti-osteosarcoma effect and biosafety of atractylodin in vivo.3.Bioinformatics research showed that atractylodin caould exert characteristics of anti-osteosarcoma effects as "multi-target and multi-pathway",and mainly influence the MAPK pathway with MAPK1(ERK2)4.Western blot and IHC were verified that atractylodin might regulate MAPK signal pathway to anti-osteosarcoma.Conclusion:1.Atractylodin could inhibit the proliferation,migration and invasion of osteosarcoma cells in vitro,and inhibit the growth of osteosarcoma in vivo.2.Atractylodin might inhibit osteosarcoma by targeting MAPK1 and regulating the MAPK signal pathway...