维生素E、维生素C、牛磺酸对苯并a芘致SH-SY5Y细胞能量代谢损伤的影响
Effects of vitamin E, vitamin C, and taurine on energy metabolism injury of SH-SY5Y cells induced by benzoapyrene
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摘要:
目的 观察维生素E、维生素C、牛磺酸对苯并a芘（BaP）暴露引起人神经母细胞瘤细胞（SH-SY5Y）能量代谢损伤的影响。
方法 分别用DMSO、BaP（1μmoL/L）、BaP（1μmoL/L）+维生素E（50μmoL/L）、BaP（1μmoL/L）+维生素C（100 μmoL/L）、BaP（1 μmoL/L）+牛磺酸（100 μmoL/L）处理SH-SY5Y细胞24 h后，用MTT法检测细胞存活率，ROS活性氧检测试剂盒检测细胞内ROS水平，细胞丙二醛（MDA）测定试剂盒检测细胞内MDA含量，超氧化物歧化酶（SOD）分型测试盒检测细胞内SOD活性，Seahorse XFp细胞能量分析仪检测细胞线粒体呼吸功能和糖酵解功能。
结果 BaP染毒组细胞存活率（84.2%±1.2%）、SOD活性（60.19±1.02）U/mg低于溶剂对照组100%±3.74%、（67.37±0.78）U/mg（P < 0.05），ROS水平（60.73%±3.15%）和MDA含量（0.837±0.104）μmol/g则高于溶剂对照组31.27%±1.40%、（0.382±0.083）μmol/g（P < 0.05）。BaP染毒组基础有氧呼吸速率（2.64±0.43）pmol/min、ATP偶联的有氧呼吸速率（2.09±0.32）pmol/min、有氧呼吸最大值（3.42±0.03）pmol/min、有氧呼吸储备值（0.78±0.44）pmol/min均低于溶剂对照组（4.89±0.43、4.16±0.24、7.06±0.11、2.18±0.35）pmol/min（P < 0.05），质子漏耗氧速率（0.55±0.16）pmol/min、糖酵解水平（3.54±1.02）mpH/min、糖酵解最大值（5.94±0.47）mpH/min、糖酵解储备值（2.40±0.61）mpH/min与溶剂对照组（0.73±0.27）pmol/min、（2.71±0.66）mpH/min、（5.75±0.65）mpH/min、（3.04±0.19）mpH/min相比，差异无统计学意义（P > 0.05）。维生素E、维生素C、牛磺酸干预组细胞存活率（90.2%±1.1%、91.9%±3.1%、98.2%±2.1%）、SOD活性（67.28±0.43）、（66.23±0.70）、（65.47±1.17）U/mg明显高于BaP染毒组84.2%±1.2%、（60.19±1.02）U/mg（P < 0.05），细胞内ROS水平（34.53%±1.96%、37.07%±2.42%、38.77%±2.31%）、MDA含量（0.477±0.095）、（0.544±1.09）、（0.558±0.152）μmol/g则低于BaP染毒组60.73%±3.15%、（0.837±0.104）μmol/g（P < 0.05）。维生素E、维生素C、牛磺酸干预组基础有氧呼吸速率（4.30±0.36）、（4.44±0.35）、（4.42±0.51）pmol/min，ATP偶联的有氧呼吸速率（3.45±0.20）、（3.70±0.19）、（3.60±0.34）pmol/min，有氧呼吸最大值（5.53±0.03）、（5.52±0.04）、（5.56±0.02）pmol/min均高于BaP染毒组（2.64±0.43）、（2.09±0.32）、（3.42±0.03）pmol/min（P < 0.05），有氧呼吸储备值（1.23±0.35）、（1.08±0.31）、（1.14±0.50）pmol/min与BaP染毒组（0.78±0.44）pmol/min相比，差异无统计学意义（P > 0.05）。糖酵解压力测试结果显示，5组间3项指标的差异均无统计学意义（P >0.05）。
结论 BaP暴露可导致SH-SY5Y细胞氧化损伤，降低线粒体呼吸功能，维生素E、维生素C、牛磺酸对BaP暴露引起的能量代谢损伤具有一定的保护作用。
Abstract:Objective To observe the effects of vitamin E, vitamin C, and taurine on energy metabolism damage of human neuroblastoma cells (SH-SY5Y) induced by benzoapyrene (BaP) exposure.
Methods SH-SY5Y cells were treated with DMSO, BaP (1 μmoL/L), BaP (1 μmoL/L) + vitamin E (50 μmol/L), BaP (1 μmol/L) + vitamin C (100 μmol/L), and BaP (1 μmol/L) + taurine (100 μmol/L), respectively. After 24 h of treatment, cell viability was measured by MTT assay, reactive oxygen species (ROS) level was detected with ROS detection kit, cellular malondialdehyde (MDA) content was assessed with MDA assay kit, superoxide dismutase (SOD) activity was evaluated with SOD typing test kit, and mitochondrial respiratory and glycolysis of the cells were detected with Seahorse XFp cell energy analyzer.
Results The cell viability (84.2%±1.2%) and SOD activity(60.19±1.02) U/mg were significantly lower, while the ROS level (60.73%±3.15%) and MDA content(0.837±0.104) μmol/g were significantly higher in the BaP-treated group than in the vehicle control group100%±3.74%, (67.37±0.78) U/mg, 31.27%±1.40%, and (0.382±0.083) μmol/g, respectively (P < 0.05). The basal oxygen consumption rate (OCR)(2.64±0.43) pmol/min, ATP-linked OCR (2.09±0.32) pmol/min, maximal respiration (3.42±0.03) pmol/min, and spare respiratory capacity (0.78±0.44) pmol/min in the BaP-treated group were lower than those in the vehicle control group (4.89±0.43, 4.16±0.24, 7.06±0.11, and 2.18±0.35) pmol/min, respectively (P < 0.05), while the proton leak OCR(0.55±0.16) pmol/min, glycolysis level (3.54±1.02) mpH/min, glycolytic capacity (5.94±0.47) mpH/min, and glycolytic reserve(2.40±0.61) mpH/min in the BaP-treated group were not statistically different from those in the vehicle control group (0.73±0.27) pmol/min, (2.71±0.66) mpH/min, (5.75±0.65) mpH/min, and (3.04±0.19) mpH/min, respectively (P > 0.05). The cell viabilities (90.2%±1.1%, 91.9%±3.1%, and 98.2%±2.1%) and SOD activities (67.28±0.43), (66.23±0.70), and (65.47±1.17) U/mg in the groups treated with vitamin E, vitamin C, and taurine in addition to BaP were significantly higher than those in the BaP-treated group84.2%±1.2% and (60.19±1.02) U/mg, while the ROS levels (34.53%±1.96%, 37.07%±2.42%, and 38.77%±2.31%) and MDA contents (0.477±0.095), (0.544±1.09), and (0.558±0.152) μmol/g were lower60.73%±3.15% and (0.837±0.104) μmol/g (P < 0.05). The basal OCR (4.30±0.36), (4.44±0.35), and (4.42±0.51) pmol/min, ATP-linked OCR (3.45±0.20), (3.70±0.19), and (3.60±0.34) pmol/min, and maximal respiration (5.53±0.03), (5.52±0.04), and (5.56±0.02) pmol/min in the groups treated with vitamin E, vitamin C, and taurine in addition to BaP were significantly higher than those in the BaP-treated group (2.64±0.43), (2.09±0.32), and (3.42±0.03) pmol/min, respectively (P < 0.05), and there was no significant difference in the spare respiratory capacity between the three groups (1.23±0.35), (1.08±0.31), and (1.14±0.50) pmol/min and the Bap-treated group (0.78±0.44) pmol/min (P > 0.05). The results of glycolysis stress test showed no differences in the three glycolysis function indicators among the five groups (P > 0.05).
Conclusion Exposure to BaP can cause oxidative damage in SH-SY5Y cells and reduce mitochondrial respiratory, which may be protected by vitamin E, vitamin C, and taurine.
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