小檗碱减轻DSS诱导的结肠上皮细胞损伤的研究

Effects of berberine in alleviating DSS induced colonic epithelial cell injury

目的探讨小檗碱保护葡聚糖硫酸钠(DSS)诱导的结肠上皮细胞损伤的作用机制。方法将NCM-460细胞随机分为空白组(常规培养)、模型组(40mg·mL^(-1)DSS)、低剂量组(5μmol·L^(-1)小檗碱)、高剂量组(10μmol·L^(-1)小檗碱)、siRNA组(10μmol·L^(-1)小檗碱+转染siRNA质粒)、si-SelS组(10μmol·L^(-1)小檗碱+转染si-SelS质粒)。用蛋白质印迹法检测硒蛋白S(SelS)表达水平,用细胞计数试剂盒-8(CCK-8)实验和5-乙炔基-2′-脱氧尿苷(EdU)实验检测细胞增殖情况,用Milli-cellERS仪检测细胞转移上皮电阻(TEER)水平,用试剂盒法检测超氧化物歧化酶(SOD)水平,用流式细胞术检测细胞凋亡情况。结果空白组、模型组、高剂量组、siRNA组、si-SelS组SelS蛋白相对表达水平分别为1.02±0.13、0.42±0.05、0.90±0.08、0.89±0.10和0.30±0.03,48h细胞光密度值分别为0.85±0.05、0.48±0.04、0.70±0.08、0.68±0.05和0.51±0.05,EdU阳性细胞率分别为(33.78±2.72)%、(11.90±2.00)%、(25.74±1.94)%、(24.29±1.96)%和(15.17±1.16)%,TEER值分别为(100.00±3.64)%、(43.47±4.19)%、(73.28±7.38)%、(76.25±7.68)%和(53.49±4.42)%,SOD活性分别为(13.32±0.73)、(5.33±0.55)、(9.63±1.13)、(9.69±0.88)和(6.40±0.57)U·mL^(-1),细胞凋亡率分别为(3.21±0.02)%、(24.59±2.35)%、(10.90±1.09)%、(11.11±1.24)%和(16.73±1.56)%。模型组的上述指标与空白组比较,高剂量组的上述指标与模型组比较,si-SelS组的上述指标与siRNA组比较,在统计学上差异均有统计学意义(均P<0.05)。结论小檗碱可通过上调SelS抑制氧化应激,改善DSS诱导的结肠上皮细胞屏障损伤。

Objective To investigate the protective effect of berberine on the injury of colon epithelial cells induced by dextran sulfate sodium(DSS).Methods NCM-460 cells were randomly divided into blank group(conventional culture),model group(40 mg·mL^(-1)DSS)and low-dose group(5μmol·L-1berberine),high-dose group(10μmol·L^(-1)berberine),siRNA group(10μmol·L^(-1)berberine+transfected siRNA plasmid),si-SelS group(10μmol·L^(-1)berberine+transfected si-SelS plasmid).The expressions of selenioprotein S(SelS)were detected by Western blot;cell proliferation was detected by cell counting kit-8(CCK-8)and 5-ethynyl-2'-deoxyuridine(EdU)tests;trans epithellal electric resistance(TEER)levels were detected by Milli-cell ERS;superoxide dismutase(SOD)was detected by kit method;apoptosis was detected by flow cytometry.Results The relative expression levels of SelS protein in blank group,model group,high-dose group,siRNA group and si-SelS group were 1.02±0.13,0.42±0.05,0.90±0.08,0.89±0.10 and 0.30±0.03,respectively;the cell optical density at 48 h were 0.85±0.05,0.48±0.04,0.70±0.08,0.68±0.05 and0.51±0.05,respectively;the EdU positive cell rates were(33.78±2.72)%,(11.90±2.00)%,(25.74±1.94)%,(24.29±1.96)%and(15.17±1.16)%,respectively;TEER values were(100.00±3.64)%,(43.47±4.19)%,(73.28±7.38)%,(76.25±7.68)%and(53.49±4.42)%,respectively;SOD activities were(13.32±0.73),(5.33±0.55),(9.63±1.13),(9.69±0.88)and(6.40±0.57)U·mL-1,respectively;the apoptosis rates were(3.21±0.02)%,(24.59±2.35)%,(10.90±1.09)%,(11.11±1.24)%and(16.73±1.56)%,respectively.The above indexes in the model group were compared with those in the blank group,and those in the high-dose group were compared with those in the model group.The above indexes of si-SelS group were statistically significant compared with those of siRNA group(all P<0.05).Conclusion Berberine can inhibit oxidative stress and improve DSS induced colon epithelial cell barrier damage by up-regulating SelS.