鼠尾草酚对人脐静脉内皮细胞黏附淋巴细胞能力的影响及其机制

Effects of carnosol on lymphocyte adhesion of human umbilical vein endothelial cells

目的探讨鼠尾草酚对人脐静脉血管内皮细胞(HUVEC)黏附淋巴细胞能力的影响及其机制。方法将体外培养的人脐静脉血管内皮细胞(HUVEC)分为鼠尾草酚高剂量组、鼠尾草酚低剂量组、TNF-α组和对照组。鼠尾草酚高剂量组、鼠尾草酚低剂量组分别加入10、5μmol/L鼠尾草酚培养18 h,然后加入10 ng/mL TNF-α培养6h;TNF-α组加入10 ng/mL TNF-α培养6h;对照组只加EGM-2培养液培养24 h。用淋巴细胞黏附试验观察各组HUVEC黏附淋巴细胞的能力(以相对荧光强度表示),用酶联免疫吸附法(ELISA)法检测各组HUVEC细胞培养液上清液中的趋化因子白细胞介素8(IL-8)和单核细胞趋化蛋白-1(MCP-1),用Western blotting法检测各组HUVEC细胞中的黏附分子血管细胞黏附分子1(VCAM-1)、细胞间黏附分子1(ICAM-1)。结果鼠尾草酚高剂量组、鼠尾草酚低剂量组、TNF-α组和对照组相对荧光强度分别为1.74±0.33、2.08±0.28、2.61±0.29、1.00±0.01,TNF-α组荧光强度高于对照组(P<0.05),鼠尾草酚低、高剂量组荧光强度低于TNF-α组(P均<0.05),且鼠尾草酚高剂量组低于鼠尾草酚低剂量组(P<0.05)。四组HUVEC细胞培养液上清液中IL-8、MCP-1水平分别为1 910.00±156.33、3 766.00±336.88、5 629.00±324.61、961.00±48.13和952.00±57.81、1 289.00±223.46、2 285.00±209.61、211.00±6.36。四组HUVEC细胞中VCAM-1、ICAM-1相对表达量分别为0.38±0.03、0.51±0.05、0.99±0.01、0.08±0.02和0.33±0.06、0.42±0.05、0.98±0.02、0.06±0.01。与对照组相比,TNF-α组HUVEC细胞培养液上清液中IL-8、MCP-1蛋白水平及HUVEC细胞中VCAM-1、ICAM-1相对表达量升高(P均<0.05);与TNF-α组比较,鼠尾草酚低、高剂量组HUVEC细胞培养液上清液中IL-8、MCP-1蛋白水平及HUVEC细胞中VCAM-1、ICAM-1相对表达量下降(P均<0.05),且鼠尾草酚高剂量组低于鼠尾草酚低剂量组(P<0.05)。结论鼠尾草酚能下调HUVEC黏附淋巴细胞的能力。其机制可能是鼠尾草酚能抑制TNF-α诱导的HUVEC趋化因子IL-8、MCP-1及细胞黏附分子VCAM-1、ICAM-1表达上调。

Objective To investigate the effects of carnosol on adhesion of human umbilical vein endothelial cells （HUVEC） to lymphocytes and the mechanism.Methods HUVEC cultured in vitro were divided into four groups： the high-dose, low-dose carnosol groups were pretreated with 10, 5 μmol/L carnosol for 18 h and then were induced by 10 ng/mL TNF-α for 6 h;the TNF-α group was stimulated by 10 ng/mL TNF-α for 6 h;the control group was cultured with EGM-2 medium for 24 h.The ability of lymphocyte adhesion in HUVEC was analyzed by lymphocyte adhesion test （measured by relative fluorescence intensity）.We examined interleukin-8 （IL-8） and monocyte chemoattractant protein-1 （MCP-1） expression in cell culture supernates of HUVEC by ELISA.Western blotting was used to detect the expression of vascular cell adhesion molecule-1 （VCAM-1） and intracellular adhesion molecule-1 （ICAM-1）.Results The relative fluorescence intensities were 1.74＆#177;0.33, 2.08＆#177;0.28, 2.61＆#177;0.29, and 1.00＆#177;0.01 in the high-dose carnosol group, low-dose carnosol group, TNF-α and control groups, and the TNF-α group had significantly higher relative fluorescence intensity than that in the control group （P〈0.05）, while the high-dose and low-dose groups had significantly lower relative fluorescence intensities than that in the TNF-α group （both P〈0.05）, and the high-dose carnosol group had significantly lower relative fluorescence intensity than that in the low-dose carnosol group （P〈0.05）.The expression levels of IL-8 and MCP-1 in the four groups were 1 910.00＆#177;156.33, 3 766.00＆#177;336.88, 5629.00＆#177;324.61, 961.00＆#177;48.13, and 952.00＆#177;57.81, 1 289.00＆#177;223.46, 2 285.00＆#177;209.61, 211.00＆#177;6.36;VCAM-1 and ICAM-1 expression levels were 0.38＆#177;0.03, 0.51＆#177;0.05, 0.99＆#177;0.01, 0.08＆#177;0.02, and 0.33＆#177;0.06, 0.42＆#177;0.05, 0.98＆#177;0.02, 0.06＆#177;0.01.We demonstrated that the TNF-α group had significantly higher IL-8 and MCP-1 levels as well as VCAM-1 and ICAM-1 expression levels as compared with those of the control group （all P〈0.05）, and the low-dose and high-dose carnosol groups had significantly lower IL-8 and MCP-1 as well as VCAM-1 and ICAM-1 expression levels as compared with those of the TNF-α group （all P〈0.05）, while the high-dose carnosol group had significantly lower IL-8 and MCP-1 as well as VCAM-1 and ICAM-1 expression levels than those in the low-dose carnosol group （all P〈0.05）.Conclusion Carnosol may inhibit lymphocyte adhesion of HUVEC, and its underlying mechanism may be associated with the inhibitory effect of carnosol on the expression of chemokines IL-8, MCP-1, and CAMs VCAM-1 and ICAM-1.