内脏脂肪素对巨噬细胞MMP-9的作用及其机制探讨

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+visfatin 12 h组；②巨噬细胞+visfatin24 h组，两组的visfatin的质量浓度均为：0（对照组）、50、100、200、400 ng/mL。采用RT-PCR和Western blotting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+ MAPK p38、ERK1/2、JNK信号通路抑制剂预处理1 h后加visfatin（200 ng/mL）24 h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1 h后加visfatin（200 ng/mL）24 h组；④巨噬细胞+visfatin（200 ng/mL）24 h组（Vis200组）；⑤巨噬细胞+visfatin（200 ng/mL）刺激不同时间组（5、10、15、30、60 min）。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、JNK MAPK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）。p38 MAPK、ERK1/2 MAPK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p38 MAPK和ERK1/2 MAPK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p38 MAPK和ERK1/2MAPK信号通路有关；RXR可能参与了该过程。

Effects and mechanism of visfatin on MMP-9 in macrophages

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. Conclusion Visfatin enhances inflammatory factors expression and activity in macrophages which is related with ERK1/2 MAPK and p38 MAPK, and RXR may mediate visfatin activity.