摘要
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 .
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2014年第7期503-508,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金项目(81070357,30660066)