大黄素通过调控TLR4/NF-κB通路对脂多糖诱导血管内皮细胞氧化损伤的保护作用研究

Protective effect of emodin on lipopolysaccharide induced oxidative damage of vascular endothelial cells by regulating TLR4/NF-κB pathway

目的研究大黄素对脂多糖(LPS)诱导人脐静脉血管内皮细胞(HUVEC)氧化损伤的保护作用及机制。方法运用CCK-8细胞活力检测法筛选LPS体外诱导HUVEC细胞氧化损伤模型浓度及大黄素给药浓度。取HUVEC细胞,分为对照组、模型组(1μg/L LPS)、吡咯烷二硫代氨基甲酸铵(PDTC,阳性药,10μmol/L)组和大黄素低、高剂量(40、80μmol/L)组,置于37℃、5%CO2培养箱中培养24 h。ELISA法测定各组细胞上清液中一氧化氮(NO)、丙二醛(MDA)、活性氧(ROS)、肿瘤坏死因子-α(TNF-α)的含量;采用Western Blotting法检测各组细胞中Toll样受体(TLR4)、核因子κB(NF-κB p65)、TNF-α蛋白的表达。结果LPS浓度为1μg/L,作用24、48 h,HUVEC细胞存活率分别为64.8%、51.2%;40、80μmol/L大黄素作用24、48 h对HUVEC细胞存活率无显著影响,选择1μg/L作为LPS造模浓度,40、80μmol/L作用24 h作为大黄素给药条件。与对照组比较,模型组HUVEC细胞增殖活力显著下降,NO、MDA、ROS、TNF-α含量显著升高,TLR4、NF-κB p65、TNF-α蛋白表达升高(P<0.01);与模型组比较,40、80μmol/L大黄素给药后HUVEC细胞生存率显著升高,NO、MDA、ROS、TNF-α含量显著降低,TLR4、NF-κB p65、TNF-α蛋白表达显著降低(P<0.05、0.01)。结论大黄素对LPS诱导的HUVEC细胞氧化损伤具有显著保护作用,其作用机制可能与调控TLR4/NF-κB通路、抑制炎症有关。

Objective To study the protective effect and mechanism of emodin(EM)on oxidative damage of human umbilical vein endothelial cells(HUVEC)induced by lipopolysaccharide(LPS).Methods CCK-8 cell viability test was used to screen the concentration of LPS induced oxidative damage model and emodin.HUVEC cells were divided into control group,model group(1μg/L LPS),pyrrolidine dithiocarbamate(PDTC,positive drug,10μmol/L)group and emodin low and high dose(40,80μmol/L)group,and cultured in 37℃and 5%CO2 incubator for 24 h.ELISA was used to determine the contents of nitric oxide(NO),malondialdehyde(MDA),reactive oxygen species(ROS)and tumor necrosis factor(TNF-α)in the supernatant of each group and the western blotting was used to detect the expression of Toll-like receptor(TLR4),nuclear factor-κB(NF-κB p65),TNF-αprotein expression in each group of HUVEC cells.Results Compared with control group,the proliferative activity of HUVEC cells in LPSinduced model group decreased significantly,the contents of NO,MDA,ROS and TNF-αincreased significantly,and the expressions of TLR4,NF-kappa B p65,TNF-alpha and IL-6 increased significantly(P<0.01).After different concentrations of EM administration,the proliferation activity of HUVEC cells increased,the contents of NO,MDA,ROS and TNF-a decreased significantly,and the expressions of TLR4,NF-kappa B p65,TNF-a decreased significantly(P<0.05 and 0.01).Conclusion EM has a protective effect on LPS-induced oxidative damage in HUVEC endothelial cells,the mechanism may be related to the regulation of TLR4/NF-κB pathway,inhibit inflammation.