氟伐他汀对高糖环境中培养的原代人脐静脉内皮细胞损伤改善作用及其机制

Effect of fluvastatin on injury of primary human umbilical vein endothelial cells cultured in high glucose environment and its mechanism

目的 观察氟伐他汀(Flu)对高糖环境中培养的原代人脐静脉内皮细胞损伤的改善作用,并探讨其作用机制。方法 以原代人脐静脉内皮细胞为实验对象,并将其随机分为Flu+高糖组、Flu组、高糖组、对照组。Flu+高糖组在高糖培养基(含30.5 mmol/L葡萄糖的ECM培养基)中加入0.01、0.1、0.125、0.25、0.5、1.0μmol/L Flu处理12 h;Flu组在常规培养基中加入0.01、0.1、0.125、0.25、0.5、1.0μmol/L Flu处理12 h;高糖组于高糖培养基中培养24 h;对照组常规培养。采用MTS法检测各组细胞活力,根据细胞活力值,后续实验中Flu+高糖组中Flu的浓度选定0.25、0.5、1.0μmol/L。流式细胞术检测各组细胞内活性氧(ROS);化学比色法检测各组细胞内丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH);RT-PCR法检测各组细胞内Ⅲ型组蛋白去乙酰化酶(SIRT1)、叉头框转录因子O1(FOXO1)、SOD2 mRNA。结果 经24 h贴壁培养,与对照组比较,高糖组细胞活力降低(P均<0.05);与高糖组比较,Flu+高糖组(0.25、0.5、1.0μmol/L)细胞活力提高(P均<0.05)。与对照组比较,高糖组细胞内ROS和MDA水平高,SOD和CAT水平低,GSH水平高,SIRT1、SOD2 mRNA相对表达量低(P均<0.05);与高糖组比较,高糖+1.0μmol/L Flu组细胞内ROS、MDA水平降低,SOD、CAT水平升高,GSH水平降低,SIRT1 mRNA、SOD2 mRNA相对表达量高(P均<0.05)。各组FOXO1 mRNA相对表达量比较,P均>0.05。结论0.25、0.5、1.0μmol/L Flu可改善高糖诱导的原代人脐静脉内皮细胞损伤,其改善作用机制可能是提高细胞中抗氧化酶水平,上调SIRT1基因表达,抵抗高糖诱导的氧化损伤。

Objective lial cells(HUVECs)cultured in high glucose(HG)environment and its mechanism.Methods the experimental objects,and were randomly divided into the control group,HG group,Flu group,HG+Flu group. HUVECs in the control group were cultured routinely,and HUVECs in the HG group were cultured in high glucose medium(ECM medium containing 30. 5 mmol/L glucose)for 24 h. HUVECs in the Flu group were treated with 0. 01,0. 1,0. 125,0. 25,0. 5,and 1. 0 μmol/L Flu in the conventional medium for 12 h. HUVECs in the HG+Flu group were treated with 0. 01,0. 1,0. 125,0. 25,0. 5,and 1. 0 μmol/L Flu in the high glucose medium for 12 h. MTS was used to detect cell viability. In the subsequent experiments,the concentrations of Flu in the Flu+high glucose group were selected as0. 25,0. 5,and 1. 0 μmol/L. Flow cytometry was used to detect the level of intracellular reactive oxygen species(ROS).Chemical colorimetry was used to detect malonic dialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),and glutathione(GSH)in HUVECs. RT-PCR was used to detect the gene expression of Sirtuins 1(SIRT1),forkhead box transcription factor O1(FOXO1),and SOD2 mRNA.Results with the control group,the HG group had lower cell viability(P<0. 05). Compared with the HG group,the cell viability of the HG(0. 25,0. 5,and 1. 0 μmol/L)+Flu group was significantly improved(all P<0. 05). Compared with the control group,the HG group had higher ROS and MDA,lower SOD and CAT,higher GSH,lower relative expression levels of SIRT1 mRNA and SOD2 mRNA(all P<0. 05). Compared with the HG group,HG+1. 0 μmol/L Flu group had lower ROS and MDA,higher SOD and CAT,lower GSH,and higher relative expression levels of SIRT1 mRNA and SOD2 mRNA(all P<0. 05). There was no significant difference in the relative expression of FOXO1 mRNA among these groups(all P>0. 05).Conclusion nism may be that Flu increases the level of antioxidant enzymes in HUVECs,up-regulates the expression of SIRT1 gene,and resists the oxidative damage induced by high glucose.