摘要目的 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

abstractsObjective 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.