融合抗氧化酶GS1XR对鼻咽上皮细胞的放射防护效应  

Radioprotective effect of fusion antioxidant enzyme GS1XR on nasopharyngeal epithelial cells

作　　者：何火聪[1,2] 韩亚南 张紫怡 潘剑茹 HE Huocong;HAN Yanan;ZHANG Ziyi;PAN Jianru(Laboratory of Radiation Biology,Clinical Oncology School of Fujian Medical University,Fujian Cancer Hospital,Fuzhou 350014,Fujian,China;College of Chemistry,Fuzhou University,Fuzhou 350108,Fujian,China;College of Biological Science and Engineering,Fuzhou University,Fuzhou 350108,Fujian,China;Key Laboratory of Marine Enzyme Engineering of Fujian Province,Fuzhou University,Fuzhou 350108,Fujian,China)

机构地区：[1]福建医科大学肿瘤临床医学院/福建省肿瘤医院放射生物学研究室,福建福州350014 [2]福州大学化学学院,福建福州350108 [3]福州大学生物科学与工程学院,福建福州350108 [4]福州大学福建省海洋酶工程重点实验室,福建福州350108

出　　处：《中国肿瘤生物治疗杂志》2024年第11期1116-1122,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金(No.81974482);福建省科技创新联合资金项目(No.2021Y9191);福建省自然科学基金(No.2023J011238)。

摘　　要：目的:本研究旨在探讨融合抗氧化酶GST-SOD1-X-R9(GS1XR)对正常鼻咽上皮细胞NP69的放射防护作用及其可能的机制。方法:培养NP69细胞,分为空白对照(Untr)组、EGFP-GS1组、EGFP-GS1R组和EGFP-GS1XR组,检测0.5 mg/mL不同融合抗氧化酶的跨膜效应。用CCK-8法测定3种融合抗氧化酶在0~1 mg/mL质量浓度范围内的细胞毒性。以DCFH-DA荧光探针测定0~6 Gy X射线和不同剂量(0~1 mg/mL)GS1XR对NP69细胞内ROS水平的影响。进一步实验将NP69细胞分为Untr组、4 Gy X线单纯照射Ctrl组和照射前分别预处理GS1、GS1R、GS1XR及阿米福汀(AMFT,4μg/mL)组,检测细胞内ROS水平,流式细胞术检测细胞的凋亡率,用WB法检测Nrf2入核量、抗氧化基因GCLC以及抗凋亡因子Bcl-2和凋亡因子BAX的表达。结果:实验结果显示,EGFP-GS1不具备跨膜能力,而EGFP-GS1R和EGFP-GS1XR能够有效跨膜进入NP69细胞(P<0.0001)。经24 h处理后,3种融合抗氧化酶均使细胞活力保持在80%以上,其中GS1XR处理组的细胞活力维持在100%以上。4 Gy X射线照射显著增加细胞内ROS水平(P<0.01),GS1XR以剂量依赖方式清除辐射诱导的ROS。与Ctrl组相比,GS1XR显著降低受照细胞内的ROS水平(P<0.05),促进Nrf2的入核(P<0.01),上调抗氧化基因GCLC(P<0.0001),降低细胞的凋亡率(P<0.0001)和抗凋亡因子Bcl-2(P<0.001)的表达,并下调促凋亡因子BAX的表达(P<0.05)。GS1XR的整体保护作用与GS1R相似,且与阿米福汀效果相当。结论:融合抗氧化酶GS1XR对NP69细胞具有显著的放射防护效应,其机制可能与其可进入细胞清除受照细胞内ROS,激活Nrf2信号通路,并调节Bcl-2和BAX的表达有关,GS1XR有望成为一种新型的放射防护剂。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,4μg/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<0.0001).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<0.0001),and reduced the apoptosis rate(P<0.0001).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 Amifost

关 键 词：放射防护 融合抗氧化酶 鼻咽上皮细胞 

分 类 号：R735.7[医药卫生—肿瘤]