UⅡ／UT 系统对 LPS 刺激枯否细胞炎症信号通路分子 p38 MAPK 和 NF-κB 的影响

Effects of UⅡ/UT system on the expression of inflammatory signal molecules p38 MAPK and NF-κB in LPS-stimulated Kupffer cells

目的探讨urotensinⅡ（ UⅡ）/urotensinⅡ特异性受体（ UT ）系统对脂多糖（ lipopo-lysaccharide， LPS）刺激肝枯否细胞（Kupffer cell， KC）炎症信号通路分子p38丝裂原活化蛋白激酶（ p38 mitogen-activated protein kinase ， p38 MAPK）和核因子-κB（ nuclear factor-κB， NF-κB）的影响。方法采用胶原酶灌注消化和密度梯度离心分离大鼠枯否细胞。将细胞随机分成6组，各组细胞处置方法如下：1组：UⅡ（-）urantide（-）LPS（-）；2组：UⅡ（＋）urantide（-）LPS（-）；3组：UⅡ（-）urantide （＋）LPS（-）；4组：UⅡ（-）urantide（-）LPS（＋）；5组：UⅡ（＋）urantide（-）LPS（＋）；6组：UⅡ（-）uran-tide（＋） LPS（＋）。 p38 MAPK/p-p38 MAPK蛋白、NF-κB p65亚基表达水平采用Western blot分析方法检测；NF-κB与DNA结合活性采用凝胶迁移阻滞（EMSA）分析检测。结果各组KC核内p38 MAPK蛋白的表达水平无明显差异（P均＞0．05）；LPS刺激KC（4～6组）核内p38 MAPK蛋白磷酸化和p65蛋白表达水平以及NF-κB与DNA分子结合活性较正常对照组（1组）均明显升高（P均＜0．01），UⅡ（2组）或UT（3组）处理KC核内上述蛋白质的表达和活性水平与1组相比无明显统计学差异（ P＞0．05），而UT预处理（6组）组则较4组显著降低（P＜0．01）。结论 UⅡ/UT系统参与了LPS刺激KC炎症信号通路分子p38 MAPK和NF-κB的激活。

Objective To investigate the effects of urotensin Ⅱ/urotensin Ⅱreceptor （ UⅡ/UT） system on the expression of inflammatory signal molecules p 38 mitogen-activated protein kinase （ p38 MAPK） and nuclear factor-κB （ NF-κB ） in lipopolysaccharide （ LPS ）-stimulated Kupffer cells （ KCs ） . Methods Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifuga-tion.The isolated cells were randomly divided into six treatment groups including group 1：UⅡ（-） urantide （-）LPS（-）, group 2：UⅡ（＋）urantide（-）LPS（-）, group 3： UⅡ（-）urantide（＋）LPS（-）, group 4：UⅡ（-）urantide（-）LPS（＋）, group 5：UⅡ（＋） urantide（-） LPS（＋） and group 6：UⅡ（-）urantide（＋） LPS（＋） .Western blot assay was performed to detect p 38 MAPK/p-p38 MAPK protein and NF-κB p65 sub-unit.The DNA-binding activity of NF-κB was tested by electrophoretic mobility shift assay （EMSA）.Re-sults There was no significant difference with the expression of p 38 MAPK protein in KCs among the six groups （P〉0.05）.The expression of p65 protein and p-p38 MAPK and the DNA-binding activity of NF-κB were significantly enhanced in LPS-stimulated KCs from groups 4, 5 and 6 in comparison with those in group 1 （P〈0.01）.No significant differences with the levels of p65 protein and phosphor-p38 MAPK and the DNA-binding activity of NF-κB were observed between UⅡ/urantide-treated cells （ group 2 or group 3） and untreated cells （group 1） （all P〉0.05）, but that were decreased in group 6 than those in group 4 （all P〈0.01）.Conclusion UⅡ/UT system participated in the activation of p38 MAPK and NF-κB signaling pathways in LPS-stimulated primary Kupffer cells .