[Astragali Radix enhances radiotherapy efficacy in lung cancer by modulating SPOP-mediated homologous recombination repair].

This study aimed to investigate the molecular mechanism by which Astragali Radix enhances radiotherapy efficacy in lung cancer through speckle-type POZ protein(SPOP)-mediated homologous recombination repair. For in vitro experiments, A549 and H1299 cells were randomly divided into a blank group, a radiation group, and the radiation combined with 5%, 10%, and 20% Astragali Radix groups. Except for the blank group, all groups were irradiated with 4 Gy X-rays to establish the radiation model. After modeling, the radiation combined with low-, medium-, and high-dose Astragali Radix groups were treated with complete culture medium containing 5%, 10%, and 20% Astragali Radix-medicated serum, respectively, and cultured for 24 hours. Cell proliferation was detected by colony formation assay, apoptosis was detected by flow cytometry, and the protein expression of SPOP and homologous recombination repair proteins was detected by Western blot. For in vivo experiments, 40 C57BL/6J mice were randomly divided into a blank group, a tumor group, a radiation group, and a radiation combined with Astragali Radix group. Except for the blank group, the mice in all other groups were inoculated subcutaneously with Lewis lung carcinoma cells into the axilla to establish a tumor-bearing mouse model. After one week of feeding, when rice grain-sized protrusions appeared in the axilla, the mice received a single dose of 4 Gy irradiation, followed by intragastric administration of Astragali Radix decoction for 1 consecutive week, after which they were sacrificed and the tumor tissues were collected for analysis. Hematoxylin-eosin(HE) staining was used to observe the histopathological changes in tumor tissues, TUNEL staining was performed to detect apoptosis, immunofluorescence was used to detect the expression of phosphorylated histone H2AX(γ-H2AX) to assess DNA damage, immunohistochemistry was used to detect the expression of breast cancer susceptibility gene 1(BRCA1), breast cancer susceptibility gene 2(BRCA2), RAD51 recombinase(RAD51), and replication protein A(RPA), and Western blot was used to detect the protein expression of SPOP, BRCA2, BRCA1, RAD51, and RPA. The in vitro results showed that compared with the blank group, the number of A549 and H1299 cells in the radiation group decreased, their proliferative capacity was weakened, the apoptosis rate increased, and the protein expression of SPOP, BRCA2, BRCA1, RAD51, and RPA increased. Compared with the radiation group, the number of A549 and H1299 cells in the radiation combined with Astragali Radix group significantly decreased, their proliferative capacity was significantly weakened, the apoptosis rate significantly increased, and the protein expression of SPOP, BRCA2, BRCA1, RAD51, and RPA significantly decreased. In vivo HE staining results showed that the tumor group exhibited high tumor cell density with regular cell morphology, uniform arrangement, and no obvious tumor necrosis. The radiation group showed reduced tumor cell density, uneven distribution, enlarged nuclei, deep staining, marked morphological variation, and varying degrees of tumor necrosis. The radiation combined with Astragali Radix group displayed looser cell arrangement, further reduced cell volume, and a further decrease in nuclear mitotic activity. The TUNEL staining results showed that compared with the tumor group, the number of apoptotic cells in the radiation group increased; compared with the radiation group, the number of apoptotic cells in the radiation combined with Astragali Radix group significantly increased. The immunohistochemistry results showed that compared with the tumor group, the expression of proliferating cell nuclear antigen(Ki67) decreased; compared with the radiation group, Ki67 expression in the radiation combined with Astragali Radix group decreased. Both immunohistochemistry and Western blot results demonstrated that compared with the tumor group, the expressions of SPOP, BRCA2, BRCA1, RAD51, and RPA proteins in the tumor tissues of the radiation group mice significantly increased; compared with the radiation group, the expressions of SPOP, BRCA2, BRCA1, RAD51, and RPA proteins in the radiation combined with Astragali Radix group significantly decreased. Astragali Radix-medicated serum or Astragali Radix decoction combined with radiation can inhibit SPOP-mediated homologous recombination repair, increase tumor cell apoptosis, and improve radiotherapy efficacy.