蕨麻多酚对糖氧剥夺损伤血管内皮细胞一氧化氮、一氧化氮合酶、内皮素及缺氧诱导因子的影响

Effects of Potentilla anserina L. Polyphenols on NO,NOS,ET-1 and HIF-1α in Human Umbilical Vein Endothelial Cells Injured by Glucose and Oxygen Deprivation

目的:研究蕨麻多酚对糖氧剥夺损伤的血管内皮细胞中一氧化氮(NO)、内皮素(ET-1)、内皮型一氧化氮(eNOS)、诱导型一氧化氮(iNOS)及缺氧诱导因子(HIF-1α)的影响。方法:利用混合气体培养箱建立糖氧剥夺损伤的血管内皮细胞模型,用高(2.4 mg·ml^(-1))、中(1.2 mg·ml^(-1))、低(0.6 mg·ml^(-1))浓度的蕨麻多酚处理细胞,以复方丹参滴丸为阳性药物(2.4 mg·ml^(-1)),UV法检测各组细胞培养介质中NO含量和NOS活性;放射免疫法检测ET-1含量; RT-PCR法检测各组细胞HIF-1αmRNA、eNOS mRNA、iNOS mRNA及ET-1 mRNA的表达; Western Blot法检测各组细胞HIF-1α和eNOS蛋白的表达。结果:与正常对照组比较,模型对照组细胞中ET-1含量和NOS活性显著升高,NO含量显著降低,HIF-1αmRNA、ET-1mRNA、iNOSmRNA和HIF-1α蛋白表达上调,eNOS mRNA和eNOS蛋白表达下调(P<0.01)。与模型对照组比较,除低浓度蕨麻多酚组ET-1含量、ET-1mRNA和eNOSmRNA无统计学差异外,蕨麻多酚各浓度组细胞NO分泌量均显著升高,ET-1含量和NOS活性显著下降,HIF-1αmRNA、ET-1mRNA、iNOSmRNA和HIF-1α蛋白表达下调,eNOSmRNA和eNOS蛋白表达上调(P<0.05或P<0.01)。与阳性药物组比较,蕨麻多酚各浓度组能够显著降低iNOSmRNA的表达,高、中浓度组能够显著增加eNOSmRNA的表达,降低NOS活性,高剂量组能够进一步增加NO分泌量,降低ET-1含量(P<0.05或P<0.01)。结论:蕨麻多酚可能在血管内皮细胞缺氧时通过下调HIF-1α,进而抑制ET-1合成和分泌;上调eNOS,下调iNOS,调控NO合成和分泌;调节NO和ET-1的动态平衡,发挥抗缺氧保护作用。

Objective: To study the effects of Potentilla anserina L. polyphenols on NO,ET-1,eNOS,iNOS and HIF-1α in human umbilical vein endothelial cells injured by glucose and oxygen deprivation. Methods: Human umbilical vein endothelial cells (HVECs) injured by glucose and oxygen deprivation were cultured in a mixed gas incubator. The cells were treated with polyphenols (2.4,1.2 and 0.6 mg·ml-1) and Danshen dropping pills (2.4 mg·ml-1) was used as the positive drug. The content of NO and the activity of NOS in the medium were detected by spectrophotometry. The content of ET-1 was detected by immunoassay. HIF-1α mRNA,eNOS mRNA,iNOS mRNA and ET-1 mRNA were detected by RT-PCR,and HIF-1α and eNOS protein were detected by Western blot.Results: Compared with the normal control group,the content of ET-1 and NOS activity increased,the content of NO decreased,the expression of HIF-1α mRNA,ET-1 mRNA,iNOSmRNA and HIF-1α protein increased and the expression of eNOS mRNA and eNOS protein decreased in the model control group (P<0.01).Compared with the model control group,there was no significant difference about ET-1,ET-1 mRNA and eNOS mRNA in the low concentration Potentilla anserina L. Polyphenols group. The secretion of NO increased significantly,the content of ET-1 and the activity of NOS decreased significantly,the expression of HIF-1α mRNA,ET-1 mRNA,iNOSmRNA and HIF-1α protein decreased,the expression of eNOS mRNA and eNOS protein increased in other concentration groups of Potentilla anserina L. Polyphenols (P < 0.05 or P < 0.01). Compared with the positive drug group,the expression of iNOSmRNA was significantly decreased in Potentilla anserina L. Polyphenols,the expression of eNOSmRNA and NOS activity were significantly increased in the high and medium concentration groups. The secretion of N0 increased and the secretion of ET-1 decreased in Potentilla anserina L. Polyphenols high concentration group (P<0.05 or P<0.01).Conclusion: Potentilla anserina L. polyphenols may inhibit the synthesis and secretion of ET-1 by down-regulating the expression of HIF-1α;regulate the synthesis and secretion of NO by up-regulating the expression of eNOS and down-regulating the expression of iNOS,which plays a role in anti-hypoxia protection by maintaining the dynamic balance of NO and ET-1.