[Bidirectional regulatory effects of the fusion antioxidant enzyme GS1XR on macrophage polarization]

Radiotherapy can reshape the tumor microenvironment by promoting the polarization of tumor-associated macrophages (TAMs) toward either an antitumor (M1-like) phenotype or an immunosuppressive (M2-like) phenotype that facilitates tumor progression. To evaluate the modulatory effects of the fusion antioxidant enzyme GS1XR on macrophage polarization under radiotherapy-associated conditions, we used hydrogen peroxide (H₂O₂) to establish an oxidative stress model in RAW264.7 macrophages. The cytotoxicity, intracellular reactive oxygen species (ROS) scavenging capacity, and cellular uptake efficiency were compared among GS1, GS1R, and GS1XR. Subsequently, two macrophage polarization reporter cell lines-RAW-PNos2-P (for M1-like polarization) and RAW-PArg1-P (for M2-like polarization)-were employed to assess the effects of the three fusion antioxidant enzymes, as well as N-acetylcysteine (NAC), on macrophage polarization induced by H₂O₂, transforming growth factor beta 1 and conditioned medium from irradiated nasopharyngeal carcinoma cells. The results showed that 200 μmol/L H₂O₂ significantly increased intracellular ROS levels with minimal impact on cell viability. GS1R and GS1XR exhibited stronger membrane permeability than GS1, and none of the three enzymes showed notable cytotoxicity at the working concentrations. Across all the three polarization-inducing models, antioxidant pretreatment consistently demonstrated a dual regulatory effect of enhancing or maintaining M1-like polarization while suppressing M2-like polarization. GS1R, GS1XR, and NAC were more effective overall than GS1, with GS1XR-featuring both membrane-penetrating and enzyme-responsive functionalities-showing particular promise as a radiotherapy adjuvant. This study reveals the bidirectional regulatory role of antioxidant treatment on macrophage polarization and provides a novel strategy for radiotherapy targeting macrophage plasticity.