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Background: Ionizing radiation can induce bystander effects (RIBE), and Astragalus polysaccharides (APS) have a protective effect against RIBE. It has been proved that RIBE occurred and APS played the inhibitory effects on the RIBE when radiation was carried out only one time. However, whether RIBE happened by multiple irradiations and the effects of APS on the RIBE are unclear. This study aimed to investigate whether APS suppress RIBE damage induced by multiple irradiations. Materials and Methods: A549 cells were irradiated with 2 Gy X-rays to obtain a conditioned medium. Bone mesenchymal stem cells (BMSCs) was incubated with the conditioned medium 5 times every 3 days or APS. Cell proliferation was detected by CCK-8 and colony formation assay, and genomic instability and DNA damages were detected by the micronucleus and immunofluorescence assay. The protein associated with methylation and cancer-associated proteins in BMSCs were detected by western blot. Results: After five stimulations with a conditioned medium, the proliferative capacity of BMSCs decreased; APS pre-intervention promoted the proliferation of BMSCs. And conditional medium intervention increased the micronucleus rate and a number of 53BP1 foci; APS pre-intervention reduced the micronucleus rate and number of 53BP1 foci. In addition, the conditional medium intervention reduced the expression of methylation-related proteins and increased the expression of cancer-related proteins, while APS pre-intervention reversed this trend. Conclusion: Irradiated A549 conditioned medium can induce RIBE of BMSCs, which might be related to methylation and cancer-associated proteins and APS may block RIBE by multiple irradiations in BMSCs. [ABSTRACT FROM AUTHOR]