脂多糖诱导血管平滑肌细胞分泌白细胞介素-6及p38丝裂素活化蛋白激酶的调控作用

目的 探讨p38丝裂素活化蛋白激酶(MAPK)信号转导通路在脂多糖(LPS)作用下血管平滑肌细胞(VSMC)分泌白细胞介素-6(IL-6)中的调节作用.方法 将体外培养的大鼠胸主动脉VSMC分为LPS刺激组、p38MAPK抑制剂SB203580干预组、SB203580对照组和溶液对照组.LPS组以终浓度100μg/L的LPS与VSMC共同孵育;干预组VSMC以p38MAPK抑制剂SB203580 10μmol/L预处理2 h,再加入终浓度100 9g/L的LPS共同孵育;对照组仅以SB203580 10 μmol/L预处理2 h;溶液组仅加入去血清培养液培养.各组于培养0、3、6、12、24 h后采用实时聚合酶链反应(real-time PCR)和酶联免疫吸附法(ELISA)分别检测细胞IL-6 mRNA和上清液中IL-6蛋白表达.结果 LPS刺激3 h,VSMC中IL-6 mRNA和蛋白表达即出现明显增高CmRNA(21.3±3.2)×104,蛋白(296.2±19.6)ng/L],12 h达高峰CmRNA(131.4±11.2)×104,蛋白(897.7±34.0)ng/L],24 h有所降低mRNA(15.3±4.7)×104,蛋白(194.3±24.0)ng/L],但仍显著高于溶液组(mRNA(9.4±1.9)×104,蛋白(29.4±4.4)ng/L,均P<0.05].干预组3、6、12 h可明显抑制LPS诱导VSMC中IL-6的分泌mRNA(15.4±3.6)×104、(43.2±6.6)X 104、(56.2±5.5)×104,蛋白(180.3±23.6)、(432.2±56.8)、(546.2±57.9)ng/L,均P<0.05].结论 LPS诱导VSMC可明显增加IL-6的mRNA和蛋白表达,p38MAPK抑制剂SB203580可显著抑制IL-6转录和蛋白合成,表明p38MAPK信号转导通路可能通过直接或间接作用参与了IL-6的分泌调节作用.

The role of p38 mitogen-activated protein kinase in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells

Objective To investigate the regulation mechanism of p38 mitogen-activated protein kinase(p38MAPK) in interleukin-6 (IL-6) expression of vascular smooth muscle cell (VSMC) induced by lipopolysaecharide (LPS).Methods Rat VSMCs were divided into LPS group, SB203580+LPS group,SB203580 group and control group.LPS group was treated with 100μg/L LPS for 0, 3, 6, 12, 24 hours,SB203580+LPS group was first treated with 10 μmol/L SB203580 for 2 hours and then exposed to 100μg/LLPS for 0, 3, 6, 12, 24 hours, SB203580 group was pretreated with 10 μmol/L SB203580 for 2 hours.The level of IL-6 mRNA was determined by real-time polymerase chain reaction (PCR) and IL-6 secretion in the culture medium was measured by enzyme linked immunosorbent assay (ELISA) at different time points.Results The expression of IL-6 mRNA and the release of IL-6 were increased significantly in VSMC as early as 3 hours after being treated with LPS mRNA;(21.3±3.2) ×104, protein: (296.2±19.6) ng/L],peaked in 12 hours mRNA : (131.4 ±11.2)× 104, protein : (897.7 ±34.0) ng/L], and the elevation persisted up to 24 hours after treatment mRNA: (15.3 ± 4.7) × 104, protein: (194.3 ± 24.0) ng/L]compared with control group mRNA: (9.4±1.9)±104, protein.(29.4±4.4) ng/L, all P<0.05].On the other hand, the expression of IL-6 was significantly suppressed by p38MAPK inhibitor SB203580 at 3, 6,12 hours mRNA: (15.4±3.6)×104, (43.2±6.6)X104, (56.2±5.5)×104, protein: (180.3±23.6),(432.2±56.8), (546.2±57.9) ng/L, all P<0.05].Conclusion The release of IL-6 and the expression of IL-6 mRNA was increased significantly in LPS-challenged VSMC;however, the induction of IL-6 was significantly suppressed by p38MAPK inhibitor, p38MAPK may play an important role in the release of IL-6 induced by LPS.