白藜芦醇对IEC-6细胞辐射损伤的防护作用及机制研究

The protective effect and mechanism of resveratrol against radiation-induced IEC-6 cell injury

目的探讨白藜芦醇对大鼠小肠隐窝细胞(IEC-6)辐射损伤的防护作用及机制。方法采用不同剂量(2、4、6、8、10 Gy)的6 MeV高能X射线对大鼠IEC-6细胞进行照射,应用克隆形成实验检测细胞增殖,建立IEC-6细胞辐射损伤模型。应用CCK-8检测不同浓度白藜芦醇(0.1、0.5、1、5、10、20、40μmol/L)对IEC-6细胞活力的影响,以及不同浓度白藜芦醇(0.1、1、2、4、6、8μmol/L)对照射后细胞活力的影响。将细胞分为对照组、模型组(10 Gy)和白藜芦醇组(10 Gy,1μmol/L),对照组接受伪照射并予等量载体孵育;模型组接受6 MeV高能X射线照射,予等量载体孵育;白藜芦醇组在照射前2 h予相应浓度的白藜芦醇预处理,接受与模型组等剂量的高能X射线照射。采用Annexin V-PI染色检测细胞凋亡;DCFH-DA荧光探针检测活性氧;β-Gal染色检测细胞衰老;Real-time qPCR、Western Blot检测p16、p21、CAT和SOD2的mRNA水平及相应蛋白质表达情况。多组间数据比较采用单因素方差分析,应用Tukey’s HSD法进行事后两两比较。结果4~10 Gy电离辐射明显抑制IEC-6细胞增殖,呈剂量依赖性(均P<0.05);浓度为0.1、0.5、1、5μmol/L的白藜芦醇对细胞活力无明显影响(均P>0.05);浓度为0.1μmol/L、1μmol/L的白藜芦醇可增加电离辐射后细胞活力(均P<0.05)。白藜芦醇组细胞凋亡率、DCFH-DA荧光强度、β-Gal阳性率均低于模型组(均P<0.05);白藜芦醇组细胞中CAT、SOD2的mRNA水平及相应蛋白质表达较模型组升高(均P<0.05),但p16、p21的mRNA水平及相应蛋白质表达较模型组降低(均P<0.05)。结论白藜芦醇可以降低活性氧水平,抑制细胞衰老和凋亡,从而减轻IEC-6细胞辐射损伤。

Objective To investigate the protective effect and mechanism of resveratrol against radiation-induced rat small intestinal crypt cells(IEC-6)injury.Methods For establishment of radiation injury model,cells were exposed to different doses(2,4,6,8,and 10 Gy)of 6 MeV high-energy X-rays,and clone formatting assay was used to detect cell proliferation.CCK-8 assay was used to detect the effect of resveratrol(0.1,0.5,1,5,10,20 and 40μmol/L)on IEC-6 cell viability and the effect of resveratrol(0.1,1,2,4,6 and 8μmol/L)on cell viability post irradiation.The cells were divided into control group,model group(10 Gy)and resveratrol group(10 Gy,1μmol/L).The control group received sham-irradiation and was incubated with equivalent vehicle.The model group received 6 MeV high energy X-ray irradiation and was incubated with equivalent vehicle.The resveratrol groups were pre-pretreated with resveratrol 2 hours before irradiation and received the same dose of irradiation as model group.Annexin V-PI staining was used to detect apoptosis;DCFH-DA was used to detect total reactive oxygen species.β-galactosidase(β-Gal)staining was used to detect cellular senescence.Realtime qPCR and Western Blot were used to detect the levels of mRNA and the expressions of p16,p21,CAT,and SOD2.Comparisons between the 3 groups were performed using One-way analysis of variance followed by a Tukey’s post hoc test.Results Ionizing radiation inhibits IEC-6 cell proliferation at doses of 4,6,8,and 10 Gy,and in a dose-dependent manner(all P<0.05).Resveratrol at concentration of 0.1,0.5,1,and 5μmol/L had no significant effect on cell viability(all P>0.05)while concentration of 0.1 and 1μmol/L increased the cell viability post irradiation(all P<0.05).The apoptosis rate,DCFH-DA fluorescence intensity,andβ-Gal positive rate of the resveratrol group were lower than that of the model group(all P<0.05).The mRNA levels and protein expressions of CAT and SOD2 in the resveratrol group were higher than that of the model group(all P<0.05),while the mRNA levels and protein expressions of p16 and p21 were all lower than that of the model group(all P<0.05).Conclusion Resveratrol protects IEC-6 cells against radiation-induced damage,which may be related with the decreasing of ROS and the inhibition of senescence and apoptosis.