TLR4/NF-κB信号通路在同型半胱氨酸致内皮细胞损伤中的作用研究

An investigation on mechanism of the TLR4/NF-κB on Hcy to promote the damage of HUVECs

目的:Toll样受体4(Toll-like receptor-4,TLR4)/核因子-κB(Nuclear factor kappa B,NF-κB)在同型半胱氨酸(Homo-cysteine,Hcy)致动脉粥样硬化(Atherosclerosis,AS)过程中损伤内皮细胞的作用机制。方法:体外培养原代人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs),用0、50、100、200、500μmol/L Hcy和100μmol/L Hcy+30μmol/L(维生素B12+叶酸)分别与HUVECs培养72 h后,采用MTT法观察Hcy对HUVECs活性的影响;用分光光度比色法检测各组HUVECs中丙二醛(Malondialdehyde,MDA)和超氧化物歧化酶(Superoxide dismutase,SOD);酶免法检测NF-κB;荧光定量PCR和Westernblot分别测TLR4 mRNA和蛋白表达。结果:Hcy可抑制HUVECs的活性,各Hcy干预组与对照组比较细胞活性均降低,其中100、200、500μmol/L Hcy组与对照组比较差异有统计学意义(P<0.05),而其他组与对照组比较差异无统计学意义(P>0.05);随Hcy浓度增加MDA和NF-κB含量和TLR4 mRNA和蛋白表达明显增加,而各实验组SOD活性明显降低,其中,100、200、500μmol/L Hcy组与对照组比较差异有统计学意义(P<0.05),其他组与对照组比较差异无统计学意义(P>0.05)。结论:TLR4/NF-κB可能是Hcy致HUVECs损伤的重要机制之一,其中MDA和SOD起了重要的作用。

Objective：To investigate the mechanism of TLR4/NF-κB on Hcy to promote the damage of the vein endothelial cells on plaques of atherosclerosis.Methods：Cultured HUVECs were treated by 0,50,100,200 μmol/L and 500 μmol/L Hcy,100 μmol/L Hcy＋30 μmol/L（VitaminB12＋Folic acid） concentrations of Hcy for 72 h.The activity of HUVECs was examined by MTT assay.The MDA and NF-κB contents and SOD activity were measured by chromatometry and ELISA.The mRNA and protein expressions of TLR4 were detected by fluorescent quantitation PCR and Western blot.Results：MTT test showed that Hcy could restrain the activity of HUVECs after being treated by Hcy for 72 h.The activity of 100 to 500 μmol/L Hcy compared to control cells were significant（P0.05）,but other cells compared to control cells were not significant（P0.05）;the increasing concentration of Hcy increased MDA and NF-κB contents as well as the mRNA and protein expressions of TLR4,but it reduced the SOD contents.The activity of 100,200 μmol/L and 500 μmol/L Hcy compared to control cells was significant（P0.05）,but other cells compared to control cells were not significant（P0.05）.Conclusion：The TLR4/NF-κB may act as one of important mechanisms in Hcy to promote the damage of HUVECs,in which MDA and SOD may play an important role.