融合抗氧化酶GS1XR对巨噬细胞极化的双向调节效应

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

摘要:

放射治疗可重塑肿瘤微环境，既能促进肿瘤相关巨噬细胞向抗肿瘤表型(M1样)极化，也可诱导免疫抑制表型(M2样)促进肿瘤进展。为评估融合抗氧化酶GS1XR在放疗相关条件下对巨噬细胞极化的干预作用，本研究建立了过氧化氢(hydrogen peroxide, H₂O₂)对巨噬细胞RAW264.7的氧化应激模型，分析比较了GS1、GS1R和GS1XR对该细胞的细胞毒性、胞内ROS清除能力与细胞摄取能力，之后通过巨噬细胞M1样和M2样极化报告细胞(RAW-PNos2-P和RAW-PArg1-P细胞)检测了3种融合抗氧化酶与N-乙酰半胱氨酸(N-acetylcysteine, NAC)对H₂O₂、转化生长因子β1及受照鼻咽癌细胞的条件培养基诱导的巨噬细胞极化的干预效果。结果表明，200 μmol/L H₂O₂显著升高胞内活性氧(reactive oxygen species, ROS)，而对细胞活力影响较小；GS1R、GS1XR跨膜进入巨噬细胞的能力强于GS1，三者在使用剂量下均未显示明显毒性；不同抗氧化预处理在3种巨噬细胞极化诱导模型中均呈现出“增强/维持M1样、抑制M2样”的一致趋势，且GS1R、GS1XR与NAC效果整体优于GS1，其中兼具跨膜与酶切响应设计的GS1XR具备作为放疗辅助分子的应用前景。本研究揭示了抗氧化处理对巨噬细胞极化的双向调节作用，为靶向巨噬细胞极化的放疗辅助策略提供了新的思路与策略。

Abstract:

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.