尾加压素Ⅱ诱导巨噬细胞分泌巨噬细胞集落刺激因子

Urotensin Ⅱ induces macrophages to produce macrophage colony-stimulating factor

目的探索尾加压素Ⅱ(UⅡ)能否促进小鼠单核巨噬细胞系RAW264.7细胞分泌巨噬细胞集落刺激因子(M-CSF),并探讨相关的信号机制。方法培养小鼠单核巨噬细胞系RAW264.7细胞,以Real-time PCR法和ELISA法分别检测细胞的M-CSF mRNA和蛋白表达水平,用细胞免疫印迹法检测p38MAPK和ERK的磷酸化水平。结果 UⅡ呈浓度依赖性和时间依赖性促进RAW264.7细胞中M-CSF mRNA和蛋白水平的表达,最大效应时间均为刺激12 h,M-CSF mRNA水平是对照组水平的2.73倍,蛋白表达水平显著高于对照组水平[(50.04±4.28)pg/ml比(20.39±2.75)pg/ml,P<0.01],是对照组水平的2.45倍;最大效应浓度均为10-8mol/L和10-7mol/L,以10-8mol/L UⅡ刺激12 h,M-CSF的mRNA和蛋白表达水平较对照组分别增加了97.3%和80.8%。采用UⅡ受体(UT)阻断剂Urantide、ERK阻断剂PD98059和p38MAPK阻断剂SB203580分别预处理细胞后,M-CSF蛋白水平较UⅡ组显著降低,分别为[(45±4.04)pg/ml比(57.47±2.93)pg/ml]、[(42.13±4.28)pg/ml比(57.47±2.93)pg/ml]和[(44±5.34)pg/ml比(57.47±2.93)pg/ml],差异均有统计学意义(均P<0.05)。10-8mol/L UⅡ刺激细胞3 min显著促进ERK和p38MAPK蛋白磷酸化,5 min时ERK磷酸化水平达到峰值,至15 min仍持续高值,而p38MAPK在15 min时磷酸化达到峰值;30 min时,两者的磷酸化水平均减弱。结论UⅡ可通过与受体结合活化ERK和p38MAPK信号通路促进RAW264.7细胞中M-CSF的表达。

Objective To investigate the role of urotensin ]I in M-CSF secretion from RAW264. 7 macrophages and the relevant signal mechanisms. Methods The RAW264.7 cell line was cultured. The mRNA expression and protein level of M-CSF were determined by real time polymerase chain reaction and Enzyme-linked immunosorbent assay, respectively. Western blot was applied to assay the phosphorylation status of ERK and p38MAPK. Results U Ⅱ increased the expression of M-CSF mRNA and protein in a concentration- and time-dependent manner with the peak at 12 h of treatment （P 〈 0. 01 ）, when the gene and protein level of M-CSF was about 1.73 and 1.45 times more than the baseline level, respectively. Themaximal effect was reached at 10^-8 mol/L and 10^-7 mol/L （ both P 〈 0. 01 ）. Compared with the control group, the mRNA and protein expression of M-CSF increased by 97.3% and 80. 8% after 12 h of U Ⅱ stimulation. The U 11 effects were significantly inhibited by its receptor （UT） antagonist urantide, the SB203580 p38MAPK inhibitor and the PD98059 ERK inhibitor. 10^-8 mol/L U Ⅱ also enhanced ERK and p38MAPK phosphorylation in RAW264. 7 macrophages. Conclusions Urotensin Ⅱ promotes M-CSF release in RAW264. 7 macrophages via p38MAPK and ERK signaling pathways.