苦参碱对脂多糖诱导的人脐静脉内皮细胞炎性反应及氧化应激的影响

Effect of matrine on lipopolysaccharide-induced inflammation and oxidative stress in human umbilical vein endothelial cells

目的 探讨苦参碱(MAT)对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)炎性反应及氧化应激的影响.方法 将HUVECs随机分为5组:对照组;LPS组;LPS+MAT低、中、高剂量处理组.LPS组用100 ng/ml的LPS刺激12 h建立炎性反应及氧化应激损伤模型,LPS+MAT低、中、高剂量处理组分别以不同浓度MAT(0.5、1.0、1.5 mmol/L)预处理HUVECs 24 h,然后加入LPS(100 ng/ml)在培养箱中继续培养12 h.CCK-8法检测HUVECs的存活率,实时荧光定量PCR检测IL-1β、IL-6、单核细胞趋化因子1(MCP-1)、TNF-α、细胞间黏附分子1(ICAM-1)和血管细胞粘附分子1(VCAM-1)的mRNA水平,酶标仪检测单核巨噬细胞(THP-1)与HUVECs的黏附强度,Western blotting检测ICAM-1、VCAM-1和NF-κB P65的蛋白表达,DCFH-DA探针联合酶标仪检测细胞内反应活性氧(ROS)含量,试剂盒检测细胞内丙二醛(MDA)含量、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的活性.结果 MAT浓度≥2.0 mmol/L时表现出细胞毒性作用,可浓度依赖性降低HUVECs的存活率(P<0.05).与对照组相比,LPS组的IL-1β、IL-6、MCP-1、TNF-α、ICAM-1和VCAM-1的mRNA水平增加,THP-1细胞与HUVECs的黏附率升高,ICAM-1、VCAM-1和P65的蛋白表达增加,ROS、MDA含量升高,GSH-Px和CAT活性降低.MAT预处理后可以抑制LPS诱导的内皮细胞炎性反应,降低黏附因子表达,改善氧化应激水平,且浓度越高抑制作用越强(P<0.05).结论 MAT对LPS诱导的HUVECs损伤具有保护作用,且其保护作用可能与减少炎性因子表达、抑制氧化应激有关.

Objective To investigate the effects of matrine(MAT)on lipopolysaccharide(LPS)-induced inflammation and oxidative stress in human umbilical vein endothelial cells(HUVECs). Methods HUVECs were randomly divided into 5 groups:control group,LPS group,LPS+MAT low,medium and high dose treatment groups. In the LPS group,the model of inflammation and oxidative stress was established by stimulation with 100 ng/ml LPS for 12 h. In the LPS+MAT low,medium and high-dose treatment groups, HUVECs were pretreated with different concentrations of MAT(0.5,1.0,and 1.5 mmol/L)for 24 h,and then incubated with addition of LPS(100 ng/ml)for 12 h in the incubator. The viability of HUVECs was detected by CCK-8 assay. The mRNA expression levels of IL-1β,IL-6,MCP-1,TNF-α,ICAM-1 and VCAM-1 were detected by real-time fluorescent quantitative PCR. The adhesion of THP-1 cells to HUVECs was detected by microplate reader. Western blotting was used to detect the protein expression of ICAM-1,VCAM-1 and NF- κB P65. The DCFH-DA probe combined with the microplate reader was used to detect the reactive oxygen species(ROS)content in the cells. Intracellular dialdehyde(MDA)content,activity of glutathione peroxidase (GSH-Px)and catalase(CAT)were measured with specific tesk kits. Results MAT was found to exhibit cytotoxicity at levels ≥ 2.0 mmol/L,and reduce HUVECs viability in a dose-dependent manner(P<0.05). Compared with the control group,the LPS group showed elevated mRNA levels of IL-1β,IL-6,MCP-1, TNF-α,ICAM-1 and VCAM-1,higher rate of THP-1 cell adhesion to HUVECs,increased protein expression of ICAM-1,VCAM-1 and P65 protein expression,higher contents of ROS and MDA,and lowered activities of GSH-Px and CAT. MAT pretreatment was shown to inhibit LPS-induced inflammatory response in endothelial cells,reduce THP-1 cell adhesion to HUVECs,and improve oxidative stress. These effects were more prominent with higher doses(P<0.05). Conclusion MAT exhibits protective effect against LPS-induced HUVECs injury,which may be related to reduction of inflammatory cytokines expression and inhibition of oxidative stress.