抗氧化剂对砷诱导人膀胱上皮细胞氧化应激相关通路的影响

Effects of antioxidants on arsenic-induced oxidative stress-related pathways in SV-HUC-1 cells

目的研究抗氧化剂对亚砷酸钠诱导的人膀胱上皮细胞系(SV-HUC-1)氧化应激相关通路的影响。方法取处于对数生长期的SV-HUC-1细胞,分别暴露于含终浓度为0(对照,F12K培养基)、4μmol/L亚砷酸钠及4μmol/L亚砷酸钠与丁硫氨酸亚砜亚胺(BSO,0.5 mmol/L)、褪黑素(0.5 mmol/L)或N-乙酰半胱氨酸(NAC,0.5 mmol/L)的培养基中,于培养16 h后,收集细胞进行转录因子NF-E2相关因子2(nuclear factor erythroid 2-related factor 2,NRF2)通路相关蛋白及其下游基因[血红素加氧酶1(heme oxygenase-1,HO1)、醌氧化还原酶(NAD(P)H-quinone oxidoreductase 1,NQO1)]蛋白表达水平的检测;于培养1 h后,收集细胞进行丝裂原活化蛋白激酶(mitogen-Activated Protein Kinase,MAPK)通路关键蛋白[p-细胞外信号调节激酶(p-ERK)、p-p38、p-c-Jun-N末端激酶(p-JNK)]表达水平的检测。结果与对照组比较,亚砷酸钠暴露SV-HUC-1细胞内NRF2、NQO1、HO1蛋白及p-ERK、p-p38、p-JNK蛋白的表达水平均较高;巯基耗竭剂BSO与亚砷酸钠联合暴露可加剧砷诱导的SV-HUC-1细胞内NRF2、NQO1、HO1和p-p38、p-JNK蛋白表达水平的升高,而抑制p-ERK的升高,差异均有统计学意义(P<0.05)。褪黑素与亚砷酸钠组及亚砷酸钠+NAC组SV-HUC-1细胞内NRF2蛋白的表达水平明显低于对照组和亚砷酸钠组;NQO1蛋白的表达水平仅明显高于对照组;HO1蛋白的表达水平明显高于对照组,而明显低于亚砷酸钠组,差异均有统计学意义(P<0.05)。褪黑素+亚砷酸钠组SV-HUC-1细胞内p-ERK、p-p38蛋白的表达水平明显低于对照组和亚砷酸钠组;p-JNK蛋白的表达水平明显高于对照组和亚砷酸钠组,差异均有统计学意义(P<0.05)。亚砷酸钠+NAC组SV-HUC-1细胞内p-p38蛋白的表达水平明显低于对照组和亚砷酸钠组;p-JNK蛋白的表达水平仅明显高于对照组;p-ERK蛋白的表达水平明显高于对照组,而明显低于砷暴露组,差异均有统计学意义(P<0.05)。结论抗氧化剂能够抑制亚砷酸钠急性暴露导致的SV-HUC-1细胞氧化应激相关通路的活化。

Objective To explore the effect of antioxidants on sodium arsenite（NaAsO_2） induced oxidative stress-related pathways in human uroepithelial cells（SV-HUC-1）. Methods The cells were incubated with NaAsO_2 at the concentrations of0 and 4 μmol/L, or NaAsO_2（4 μmol/L） co-treated with BSO（0.5 mmol/L）,melatonin（0.5 mmol/L） or NAC（0.5 mmol/L）. The protein levels of NRF2 and its downstream genes including HO1 and NQO1 were measured by Western blot analysis after 16 hours incubation. The protein phosphorylations of MAPK pathway（p-ERK, p-38 and p-JNK） were measured by Western blot analysis after one hour incubation. Results Compared with the control group,the protein levels of the NRF2 pathway（NRF2,NQO1 and HO1） and MAPK pathway（p-ERK,p-p38 and p-JNK） were increased after exposure to 4 μmol/L NaAsO_2（P〈0.05）. In BSO ＋NaAsO_2 group, BSO exacerbated NaAsO_2-induced activation of NRF2, NQO1, HO1, p-p38 and p-JNK but attenuated the activation of p-ERK（P〈0.05）. In melatonin＋NaAsO_2 group and NAC＋NaAsO_2 group, the protein levels of NRF2 were significantly lower than the control and NaAsO_2 groups; The protein levels of NQO1 were higher than the control group;The HO-1 protein levels were higher than control but lower than NaAsO_2 group（P〈0.05）. In melatonin ＋NaAsO_2 group, the protein levels of p-ERK and p-p38 were significantly lower than the control and NaAsO_2 groups; But p-JNK levels were higher than control and NaAsO_2 groups（P〈0.05）. In NAC＋NaAsO_2 group, the protein levels of p-p38 were significantly lower than the control and NaAsO_2 groups; p-JNK levels were higher than control group; p-ERK were higher than control group but lower than NaAsO_2 group（P〈0.05）. Conclusion Antioxidants may inhibit NaAsO_2-induced activation of oxidative stress-related pathways in SV-HUC-1 cells.