Astragalus small molecules protect BMSCs from radiation-induced bystander effect and enhance lung cancer radiosensitivity via the primary cilium/TGF-βR1/Smad3 pathway.

Radiotherapy is an important treatment for lung cancer. However, in the course of radiotherapy, treatment-related side effects and decreased radiosensitivity remain challenging issues. TGF-βR1 can induce radiation-induced bystander effect (RIBE) through the primary cilia; however, this mechanism needs to be further elucidated. At present, traditional Chinese medicine (TCM) shows great advantages in protecting against RIBE, in which Astragalus and its related formulations show good protective effects against radiation; however, the mechanisms by which Astragalus exerts these protective effects are unknown. Therefore, this study aims to investigate the molecular mechanisms by which TGF-βR1 exerts RIBE through the primary cilia, enhancing radiosensitivity, and to reveal the therapeutic effects of small molecules derived from Astragalus membranaceus via this pathway. A co-culture model of A549 cells and bone marrow mesenchymal stem cells (BMSCs) was established, and network pharmacology was employed to identify key proteins involved in the repair of radiation-induced DNA damage in BMSCs. The role of the primary cilium/TGF-βR1 pathway in the repair of radiation-induced DNA damage in adjacent BMSCs was investigated using immunofluorescence and Western blot techniques. Molecular docking technology was utilized to screen effective small molecules from Astragalus that target the primary cilium/TGF-βR1 pathway. The screened effective small molecules were then combined, and their effects on radiation-induced bystander effect in neighboring BMSCs were studied through the CCK-8 assay, colony formation assay, apoptosis assay, cell cycle analysis, immunofluorescence, and Western blot experiments. The core differentially expressed gene IFT88 was identified by bioinformatics analysis. In the co-culture model with BMSCs following A549 irradiation with 2 Gy of X-ray, BMSCs were inhibited. After irradiation, TGF-βR1, IFT88, and RAD51 were abnormally activated in the adjacent BMSCs. However, after knockdown of IFT88 (SiIFT88), the protein expressions of TGF-βR1 and RAD51 were significantly decreased. Based on molecular docking screening for TGF-βR1 and IFT88 using the Astragalus small molecule compounds vanillic acid and 3-hydroxy-9,10-dimethoxy rosewood, the expression of TGF-βR1 and RAD51 proteins and the number of primary cilia were decreased by the intervention of these two small molecules alone or in combination with radiation in paracellular and lung cancer cells, but the expression level of TGF-βR1 was not affected. Primary cilia play a key role in the repair of radiation-induced DNA damage in adjacent BMSCs and in enhancing the radiosensitivity of lung cancer. Vanillic acid and rosewood in A. membranaceus small molecules can regulate DNA damage in BMSCs through the TGF-βR1/primary cilia.