蟛蜞菊内酯对过氧化氢诱导人冠状动脉内皮细胞氧化损伤的保护作用

Protective effects of Wedelolactone on oxidative damage induced by hydrogen peroxide in human coronary artery endothelial cells

目的研究蟛蜞菊内酯(WEL)对人冠状动脉内皮细胞(HCAECs)氧化应激损伤的保护作用及其机制。方法将HCAECs分成6组:空白组、溶剂对照组、模型组和低、中、高3个剂量实验组。在对数生长期的HCAECs细胞中,加入200μmol·L_(-1) H_2O_2孵育24 h制备细胞损伤模型;空白组细胞不做任何处理。造模前,实验组加入低、中、高3个剂量(10,20,40μmol·L_(-1)) WEL预处理30min;溶剂对照组加入0. 1%二甲基亚砜。24 h后,以MTT法检测细胞存活率,按试剂盒说明测定乳酸脱氢酶(LDH)和谷胱甘肽过氧化物酶(GSH-Px)含量,以免疫印迹法测定帕金森病蛋白7(DJ-1/PARK7)的表达水平。结果空白组、溶剂对照组、模型组和低、中、高3个剂量实验组的细胞活力分别为(100. 0±15. 2)%,(99. 5±14. 6)%,(53. 1±13. 8)%,(65. 3±12. 7)%,(76. 8±13. 9)%和(85. 6±14. 4)%;这6组LDH释放量分别为(17. 12±1. 18),(17. 38±1. 22),(51. 42±4. 57),(44. 11±4. 02),(36. 25±3. 43)和(30. 11±2. 89) U·L_(-1);这6组的GSH-Px释放量分别为(141. 27±12. 81),(140. 56±12. 31),(70. 27±8. 04),(89. 32±9. 47),(110. 51±11. 76)和(121. 52±12. 73)μmol·L_(-1),模型组与空白组相比,细胞活力和GSH-Px释放量均明显下降,而LDH释放量上升,差异均有统计学意义(均P <0. 01);低、中、高3个剂量实验组与模型组比较,上述指标的差异均有统计学意义(均P <0. 01;低剂量实验组的LDH:P <0. 05)。空白组、模型组和低、中、高3个剂量实验组的的DJ-1的蛋白表达水平分别为1. 97±0. 31,0. 61±0. 08,0. 80±0. 11,0. 94±0. 21和1. 22±0. 26,模型组与空白组相比,差异有统计学意义(P <0. 05);低、中、高3个剂量实验组与模型组相比,差异均有统计学意义(均P <0. 05)。结论 WEL可以减轻过氧化氢引起的血管内皮细胞氧化应激损伤,机制可能与其上调DJ-1的表达有关。

Objective To investigate the protective effect and mechanism of Wedelolactone(WEL)on oxidative damage induced by hydrogen peroxide in human coronary artery endothelial cells(HCAECs).Methods HCAECs were divided into 6 groups:blank group,solvent control group,model group,and low,medium and high dose experimental groups.HCAECs in logarithmic growth phase were incubated with 200μmol·L-1 H2O2 to prepare injury model.The cells in the blank group did not do any treatment.Before modeling,the experimental groups was pretreated with WEL of low,medium and high concentrations(10,20,40μmol·L-1)for 30 min.The solvent control group was added 0.1%dimethyl sulfoxide.After administration 24 h,MTT assay was used to detect cell viability;lactate dehydrogenase(LDH)and glutathione peroxidase(GSH-Px)were measured according to the kit instructions,and immunoblotting was used to detect the expression level of Parkinson protein 7(DJ-1/PARK7).Results The cells viability rates in blank group,solvent control group,model group and low,medium,high doses experimental groups were(100.0±15.2)%,(99.5±14.6)%,(53.1±13.8)%,(65.3±12.7)%,(76.8±13.9)%and(85.6±14.4)%,respectively;the LDH release in the six groups were(17.12±1.18),(17.38±1.22),(51.42±4.57),(44.11±4.02),(36.25±3.43)and(30.11±2.89)U·L-1 respectively;the release of GSH-Px in the six groups were(141.27±12.81),(140.56±12.31),(70.27±8.04),(89.32±9.47),(110.51±11.76)and(121.52±12.73)μmol·L-1.Comparison between model group and blank group,the differences of the cell viability,LDH and GSH-Px release were statistically significant(all P<0.01);comparison between three doses experimental groups and model group,the differences of the factors were statistically significant(all P<0.01;LDH in low doses experimental group:P<0.05).The relative expression of the DJ-1 protein in blank group,model group and low,medium,high doses experimental groups were 1.97±0.31,0.61±0.08,0.80±0.11,0.94±0.21 and 1.22±0.26.Comparison between model group and blank group,the difference was significantly(P<0.05);comparison between three doses experimental groups and model group,the difference were significantly(all P<0.05).Conclusion WEL can alleviate oxidative stress injury induced by hydrogen peroxide in vascular endothelial cells.All these results may due to promoting expression of DJ-1 protein.