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Radioprotective effect of fusion antioxidant enzyme GS1XR on nasopharyngeal epithelial cells
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Abstract:
Objective: To investigate the radioprotective effects of the fusion antioxidant enzyme GST-SOD1-X-R9 (GS1XR) on normal nasopharyngeal epithelial cells (NP69) and its potential mechanisms. Methods: NP69 cells were cultured and divided into the following groups: untreated control (Untr) group, EGFP-GS1 group, EGFP-GS1R group, and EGFP-GS1XR group. The transmembrane effect of different fusion antioxidant enzymes was evaluated at a concentration of 0.5 mg/mL. The cytotoxicity of the three enzymes within a concentration range of 0 to 1 mg/mL was determined using the CCK-8 assay. ROS levels in NP69 cells were measured using a DCFH-DA fluorescent probe following exposure to 0～6 Gy X-ray and varying doses (0～1 mg/mL) of GS1XR. In further experiments, NP69 cells were divided into blank control (Untr) group, 4 Gy X-ray only group (Ctrl), and groups pre-treated with GS1, GS1R, GS1XR, or Amifostine (AMFT, 4 μg/mL) before X-ray exposure. ROS levels, apoptosis rate, Nrf2 nuclear translocation, and expression of antioxidant gene GCLC, anti-apoptotic factor Bcl-2, and pro-apoptotic factor BAX were evaluated using flow cytometry and WB analysis. Results: EGFP-GS1 lacked transmembrane ability, whereas EGFP-GS1R and EGFP-GS1XR efficiently crossed the NP69 cell membrane (P < 0.000 1). After 24 hours of treatment, all three fusion antioxidant enzymes maintained cell viability above 80%, with the GS1XR-treated group maintaining cell viability above 100%. Exposure to 4 Gy X-ray significantly increased intracellular ROS levels (P < 0.01), while GS1XR effectively reduced radiation-induced ROS in a dose-dependent manner. Compared to the Ctrl group, GS1XR significantly decreased intracellular ROS levels (P < 0.05), promoted Nrf2 nuclear translocation (P < 0.01), upregulated the expression of antioxidant gene GCLC (P < 0.000 1), and reduced the apoptosis rate (P < 0.000 1). Additionally, it increased the expression of anti-apoptotic factor Bcl-2 (P < 0.001) and downregulated pro-apoptotic factor BAX (P < 0.05). The overall protective effects of GS1XR were similar to those of GS1R and comparable to the effects of Amifostine. Conclusion: The fusion antioxidant enzyme GS1XR exhibits significant radioprotective effects on NP69 cells, likely through its ability to enter cells, eliminate radiation-induced ROS, activate the Nrf2 signaling pathway, and regulate the expression of Bcl-2 and BAX. GS1XR shows potential as a novel radioprotective agent.
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