摘要
目的研究内脏脂肪素(visfatin)对巨噬细胞基质金属蛋白酶-9(MMP-9)的作用及其机制。方法体外诱导THP-1单核细胞转化为巨噬细胞。为明确visfatin对MMP-9的作用,细胞分为两组:①巨噬细胞+visfatin12h组;②巨噬细胞+visfatin24h组,两组的visfatin的质量浓度均为:0(对照组)、50、100、200、400ng/mL。采用RT-PCR和Westernblotting测定MMP-9基因和蛋白表达,明胶酶谱法检测MMP-9的活性。为明确visfatin对MMP-9的作用机制,细胞分为五组:①巨噬细胞未加刺激组(对照组);②巨噬细胞+MAPKp38、ERK1/2、JNK信号通路抑制剂预处理1h后加visfatin(200ng/mL)24h组;③巨噬细胞+过氧化物酶体增殖剂活化受体(PPARγ)天然及人工配体/RXR配体预处理1h后加visfatin(200ng/mL)24h组;④巨噬细胞+visfatin(200ng/mL)24h组(Vis200组);⑤巨噬细胞+visfatin(200ng/mL)刺激不同时间组(5、10、15、30、60min)。Western blotting检测MMP-9蛋白和PPARγ蛋白表达及visfatin刺激下巨噬细胞p38、ERK1/2、JNKMAPK磷酸化水平。结果Visfatin能促进MMP-9基因及蛋白表达(P<0.05,P<0.01),同时增强了MMP-9的活性(P<0.01)。p38MAPK、ERK1/2MAPK通路抑制剂及RXR配体抑制visfatin对MMP-9表达具有上调作用;visfatin能促进p38MAPK和ERK1/2MAPK的磷酸化,但不影响PPARγ蛋白的表达。结论Visfatin增加了巨噬细胞炎症因子的表达,该作用与p38MAPK和ERK1/2MAPK信号通路有关;RXR可能参与了该过程。
Objective To investigate the effects and mechanism of visfatin on matrix metalloproteinases-9（MMP-9）expression and invasive activity in macrophages.Methods THP-1 monocytes were induced into macrophages.To investigate the effects of visfatin on MMP-9,cells were divided into 2 groups：①macrophages＋visfatin 12 h;②macrophages＋visfatin 24 h.The concentrations of visfatin in each group were：0（control）,50,100,200,400 ng/mL.MMP-9 mRNA and protein expression were analysed by RT-PCR and Western blotting,and MMP-9 invasive activity was assayed by gelatin zymography.To investigate the mechanism of visfatin on MMP-9,cells were divided into 5 groups：①macrophages without stimulation（control）;②macrophages pretreated with MAPK p38,ERK1/2,JNK pathway inhibitor for 1 h,then stimulated with visfatin（200 ng/mL）for 24 h;③macrophages pretreated with retinoid X receptors（RXR）nature ligand or peroxisome proliferators-activated receptor γ（PPARγ）natural/synthetic ligand for 1 h,then stimulated with visfatin（200 ng/mL）for 24 h;④macrophages stimulated with visfatin（200 ng/mL）for 24 h;⑤macophages＋visfatin（200 ng/mL）for different time（5,10,15,30,60 min）.MMP-9 expression,PPARγ expression,and the effect of visfatin on MAPK phosphorylation were analysed by Western blotting.Results Visfatin not only significantly enhanced MMP-9 mRNA and protein expression in macrophages（P〈0.05,P〈0.01）,but also up-regulated MMP-9 invasive activity（P〈0.01）.p38 MAPK inhibitor,ERK1/2 MAPK inhibitor and RXR ligand significantly blocked visfatin activity.Visfatin activated the phosphorylation of p38 MAPK and ERK1/2 MAPK,while had no effect on expression of PPARγ protein.ConclusionVisfatin enhances inflammatory factors expression and activity in macrophages which is related with ERK1/2 MAPK and p38 MAPK,and RXR may mediate visfatin activity.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2009年第9期1057-1061,1065,共6页
Journal of Shanghai Jiao tong University:Medical Science
