表没食子儿茶素没食子酸酯对砷诱导心肌H9c2细胞氧化损伤的影响
|
摘要
目的研究表没食子儿茶素没食子酸酯(EGCG)对砷诱导的心肌细胞H9c2氧化损伤的影响及其分子机制。方法将心肌细胞H9c2分为6组:对照组(DMSO)、模型组(5μg·L~(-1)氯化亚砷)、5,6-二氯-1-β-D呋喃核糖基苯并咪唑(DRB)组(75 g·L~(-1)DRB)、联合组(400μg·L~(-1)EGCG+75 g·L~(-1)DRB)和低、高2个剂量EGCG组(100,400μg·L~(-1)EGCG)。以噻唑蓝(MTT)实验检测细胞存活率;以实时荧光定量PCR法和蛋白免疫印迹法检测核转录因子2抑制剂(Nrf2)和血红素氧合酶-1(HO-1)表达情况;酶标仪检测丙二醛(MDA)含量。结果干预48 h后,模型组、联合组和低、高2个剂量EGCG组的细胞存活率分别为(56. 43±11. 32)%,(78. 32±16. 21)%,(68. 58±13. 24)%,(80. 06±17. 52)%;这4组的Nrf2蛋白相对表达量为0. 58±0. 12,0. 83±0. 21,0. 73±0. 19,0. 86±0. 23;这4组的HO-1蛋白相对表达量为0. 50±0. 11,0. 71±0. 17,0. 62±0. 15,0. 75±0. 19;这4组的MDA含量分别为(6. 12±1. 52),(3. 31±0. 75),(4. 86±1. 07),(3. 24±0. 72) nmol·mg~(-1),低、高2个剂量EGCG组与模型组比较,以上指标差异均有统计学意义(均P <0. 05);联合组与高剂量EGCG组比较,以上指标差异均有统计学意义(均P <0. 05)。结论 EGCG可通过激活Nrf2/HO-1通路,减轻砷诱导的心肌细胞H9c2氧化损伤。
Objective To investigate the effect of epigallocatechin gallate( EGCG) on oxidative damage induced by arsenic in H9c2 cells and the molecular mechanism. Methods H9c2 cells were divided into six groups: control group( DMSO),model group( 5 μg ·L~(-1) AsCl_3),low,high dose EGCG groups( 100,400 μg·L~(-1) EGCG),5,6-dichloro-1-β-D-ribofuranosyl benzimidazole( DRB) group( 75 g · L~(-1) DRB),and united group( 400 μg·L~(-1) EGCG + 75 g·L~(-1) DRB).The expression of nuelear factor 2( Nrf2) and haem oxygenase-1( HO-1) in H9c2 cells was detected by real-time fluorescence quantitative-polymerase chain reaction and Western blot. The survival rates of H9c2 cells was detected by thiazolyl blue tetrazolium bromide assay. The levels of reactive oxygen species( ROS) and malondialdehyde( MDA) in H9c2 cells were detected by microplate reader. Results After 48 h ofintervention,the cell survival rates in model group, united group,and low,high doses of EGCG groups were( 56. 43 ± 11. 32) %,( 78. 32 ± 16. 21) %,( 68. 58 ± 13. 24) %,( 80. 06 ± 17. 52) %,respectively; the relative expression levels of Nrf2 protein were 0. 58 ± 0. 12,0. 83 ± 0. 21,0. 73 ± 0. 19,0. 86 ± 0. 23; and the relative expression levels of HO-1 protein in the 4 groups were 0. 50 ± 0. 11,0. 75 ± 0. 19,0. 71 ± 0. 17,0. 62 ± 0. 15; the MDA contents in the 4 groups were( 6. 12 ± 1. 52),( 3. 31 ± 0. 75),( 4. 86 ± 1. 07),( 3. 24 ± 0. 72) nmol·mg~(-1). Comparison between two doses of EGCG groups with model group,the difference of the above indicators had statistically significant( all P < 0. 05). Comparison between united group with high dose EGCG group,the difference of the above indicators had statistically significant( all P < 0. 05). Conclusion EGCG can alleviate oxidative damage induced by arsenic in H9c2 cells by activating the Nrf2/HO-1 pathway.
Objective To investigate the effect of epigallocatechin gallate( EGCG) on oxidative damage induced by arsenic in H9c2 cells and the molecular mechanism. Methods H9c2 cells were divided into six groups: control group( DMSO),model group( 5 μg ·L~(-1) AsCl_3),low,high dose EGCG groups( 100,400 μg·L~(-1) EGCG),5,6-dichloro-1-β-D-ribofuranosyl benzimidazole( DRB) group( 75 g · L~(-1) DRB),and united group( 400 μg·L~(-1) EGCG + 75 g·L~(-1) DRB).The expression of nuelear factor 2( Nrf2) and haem oxygenase-1( HO-1) in H9c2 cells was detected by real-time fluorescence quantitative-polymerase chain reaction and Western blot. The survival rates of H9c2 cells was detected by thiazolyl blue tetrazolium bromide assay. The levels of reactive oxygen species( ROS) and malondialdehyde( MDA) in H9c2 cells were detected by microplate reader. Results After 48 h ofintervention,the cell survival rates in model group, united group,and low,high doses of EGCG groups were( 56. 43 ± 11. 32) %,( 78. 32 ± 16. 21) %,( 68. 58 ± 13. 24) %,( 80. 06 ± 17. 52) %,respectively; the relative expression levels of Nrf2 protein were 0. 58 ± 0. 12,0. 83 ± 0. 21,0. 73 ± 0. 19,0. 86 ± 0. 23; and the relative expression levels of HO-1 protein in the 4 groups were 0. 50 ± 0. 11,0. 75 ± 0. 19,0. 71 ± 0. 17,0. 62 ± 0. 15; the MDA contents in the 4 groups were( 6. 12 ± 1. 52),( 3. 31 ± 0. 75),( 4. 86 ± 1. 07),( 3. 24 ± 0. 72) nmol·mg~(-1). Comparison between two doses of EGCG groups with model group,the difference of the above indicators had statistically significant( all P < 0. 05). Comparison between united group with high dose EGCG group,the difference of the above indicators had statistically significant( all P < 0. 05). Conclusion EGCG can alleviate oxidative damage induced by arsenic in H9c2 cells by activating the Nrf2/HO-1 pathway.
引文
[1]高艳芳,何云.砷暴露的心血管毒性效应及其机制研究进展[J].毒理学杂志,2016,30(6):469-471.
[2]代春美,宋雨泽,杨伟,等.EGCG对高糖诱导的HK-2细胞氧化应激损伤的保护作用[J].天然产物研究与开发,2016,28(5):673-679.
[3]脱红芳,孟惠彦,杨卫振,等.表没食子儿茶素没食子酸酯对雨蛙素导致的小鼠急性胰腺炎肺损伤的作用[J].中国临床药理学杂志,2018,29(4):425-427.
[4]霍魁媛,常宏,王勇,等.槲皮素保护H2O2诱导的H9C2心肌细胞氧化损伤的作用机制[J].中国实验方剂学杂志,2016,32(15):122-127.
[5]唐海淑,崔惠,甫尔哈提.吾守尔,等.脊髓灰质炎病毒实时荧光定量RT-PCR检测方法的建立及应用[J].现代预防医学,2015,42(15):2803-2805.
[6]文慧,王李昂,刘春颖,等.旋毛虫病人血清Western blot对成虫可溶性蛋白中早期诊断抗原的鉴定[J].中国病原生物学杂志,2017,30(2):132-135.
[7]乔乔,李静,张文强,等.MTT试验检测EV71 2A蛋白对宿主细胞损伤作用[J].中国人兽共患病学报,2016,32(6):525-528.
[8]丁雨,陈雪梅,吴思思.大鼠心肌细胞H9C2活性氧水平检测方法探究[J].中国现代医学杂志,2017,24(5):8-12.
[9]SITI H N,KAMISAH Y,KAMSIAH J.The role of oxidative stress,antioxidants and vascular inflammation in cardiovascular disease(a review)[J].Vasc Pharmacol,2015,71(1):40-56.
[10]FU X Y,YANG M F,CAO M Z,et al.Strategy to suppress oxidative damage-induced neurotoxicity in PC12 cells by curcumin:the role of ROS-mediated DNA damage and the MAPK and AKT pathways[J].Mol Neurobiol,2016,53(1):369-378.
[11]KUBBEN N,ZHANG W,WANG L,et al.Repression of the antioxidant NRF2 pathway in premature aging[J].Cell,2016,165(6):1361-1374.
[12]CHOI Y H.The cytoprotective effect of isorhamnetin against oxidative stress is mediated by the upregulation of the Nrf2-dependent HO-1 expression in C2C12 myoblasts through scavenging reactive oxygen species and ERK inactivation[J].General Physiol Biophys,2016,35(2):145-154.
[2]代春美,宋雨泽,杨伟,等.EGCG对高糖诱导的HK-2细胞氧化应激损伤的保护作用[J].天然产物研究与开发,2016,28(5):673-679.
[3]脱红芳,孟惠彦,杨卫振,等.表没食子儿茶素没食子酸酯对雨蛙素导致的小鼠急性胰腺炎肺损伤的作用[J].中国临床药理学杂志,2018,29(4):425-427.
[4]霍魁媛,常宏,王勇,等.槲皮素保护H2O2诱导的H9C2心肌细胞氧化损伤的作用机制[J].中国实验方剂学杂志,2016,32(15):122-127.
[5]唐海淑,崔惠,甫尔哈提.吾守尔,等.脊髓灰质炎病毒实时荧光定量RT-PCR检测方法的建立及应用[J].现代预防医学,2015,42(15):2803-2805.
[6]文慧,王李昂,刘春颖,等.旋毛虫病人血清Western blot对成虫可溶性蛋白中早期诊断抗原的鉴定[J].中国病原生物学杂志,2017,30(2):132-135.
[7]乔乔,李静,张文强,等.MTT试验检测EV71 2A蛋白对宿主细胞损伤作用[J].中国人兽共患病学报,2016,32(6):525-528.
[8]丁雨,陈雪梅,吴思思.大鼠心肌细胞H9C2活性氧水平检测方法探究[J].中国现代医学杂志,2017,24(5):8-12.
[9]SITI H N,KAMISAH Y,KAMSIAH J.The role of oxidative stress,antioxidants and vascular inflammation in cardiovascular disease(a review)[J].Vasc Pharmacol,2015,71(1):40-56.
[10]FU X Y,YANG M F,CAO M Z,et al.Strategy to suppress oxidative damage-induced neurotoxicity in PC12 cells by curcumin:the role of ROS-mediated DNA damage and the MAPK and AKT pathways[J].Mol Neurobiol,2016,53(1):369-378.
[11]KUBBEN N,ZHANG W,WANG L,et al.Repression of the antioxidant NRF2 pathway in premature aging[J].Cell,2016,165(6):1361-1374.
[12]CHOI Y H.The cytoprotective effect of isorhamnetin against oxidative stress is mediated by the upregulation of the Nrf2-dependent HO-1 expression in C2C12 myoblasts through scavenging reactive oxygen species and ERK inactivation[J].General Physiol Biophys,2016,35(2):145-154.