内脂素经核因子κB途径诱导人单核细胞基质金属蛋白酶-2和9表达的实验研究

Visfatin upregulates MMP-2 and MMP-9 expressions in human monocytes through activating NF-κB

目的 探讨内脂素对核因子κB（NF-κB）通路的激活及其在调节人单核细胞基质金属蛋白酶（MMP）-2和MMP-9的表达中的作用,以期进一步了解内脂素与动脉粥样硬化的关系.方法 以健康人外周血单核细胞为研究对象.抽取健康志愿者静脉血,分离、纯化单核细胞后随机分为空白对照组;内脂素组（100、200、400 ng/ml,24 h）;Bay11-7082抑制剂组（Bay11-7082 40μmol/ml预孵育30 min,内脂素400 ng/ml刺激24 h）.荧光定量PCR检测各组细胞MMP-2和MMP-9 mRNA的表达;Western blot检测MMP-2和MMP-9蛋白的表达;明胶酶谱法检测细胞培养基质中MMP-2和MMP-9的酶活性.内脂素刺激细胞后的核转位分别用Western blot和ELISA进行检测.结果 人外周血单核细胞经不同浓度的内脂素干预培养后24 h单核细胞MMP-2和MMP-9 mRNA及蛋白的表达均明显高于空白对照组（P均〈0.05）,且呈浓度依赖性.内脂素还上调细胞分泌到细胞培养基中的MMP-2和MMP-9酶活性,内脂素100 ng/ml组MMP-9、MMP-2的酶活性分别为空白对照组的（1.139 ±0.055）和（1.102±0.011）倍（P均〈0.05）.加入NF-κB抑制剂Bay11-7082后MMP-2、MMP-9的mRNA、蛋白的表达及酶活性均明显低,与内脂素400ng/ml组比较差异均有统计学意义（P均〈0.001）.内脂素浓度越高,核蛋白NF-κBp65表达越高,内脂素100 ng/ml组相对灰度值为空白对照组的（1.334±0.148）倍（P均〈0.05）;内脂素浓度越高,NF-κBp65亚单位核内转移越多,内脂素100ng/ml组为0.763±0.056,内脂素400 ng/ml组为1.290±0.065,均大于对照组的0.467±0.046（P均〈0.05）.结论 内脂素可通过激活NF-κB途径上调人单核细胞中MMP-2和MMP-9表达及活性.这可能是内脂素致动脉粥样硬化及与急性冠状动脉综合征密切相关的机制之一.

Objective To investigate the effects of visfatin on the MMP-2 and MMP-9 expressions in human monocytes and related mechanisms. Methods Human monocytes were isolated from blood, the expressions of MMP-2 and MMP-9 at mRNA and protein levels were detected in visfatin stimulated monocytes （0, 100, 200, 400 ng/ml） in the absence and presence of NF-κB inhibitor specific Bay11-7082 by Realtime PCR or Western blot, the MMP-2 and MMP-9 enzyme activity in the culture media was also detected by Gelatin Zymography. The NF-κB protein level and NF-κBp65 expression in visfatin stimulated cells were measured by Western blot and ELISA, respectively. Results Visftain upregulated MMP-2 and MMP-9 expressions in human monocytes in a dose dependent manner. After treatment with visfatin 400 ng/ml for 24 h, comparing with the free visfatin treatment, the protein expressions of MMP-2 and MMP-9 were up-regulated to 1. 644 ± 0.052 and 3. 578 ± 0. 081 （all P 〈 0. 001）; the enzyme activities of MMP-2 and MMP-9 were enhanced by 1. 661 ± 0. 036 （ P 〈 0. 001） and 1. 662 ± 0. 100 （P 〈 0. 001 ）. NF-κB was also activated in these cells by visaftin and these effects could be significantly attenuated by Bay 11-7082. Visftain induced a dose-dependent （ 100 - 400 ng） increase of NF-kBp65 nuclear translocation from 0. 763 ±0. 056 to 1. 290 ± 0. 065 at 100 and 400 ng/ml, comparing with free visfatin treatment 0. 467 ± 0.046 （all P〈0.05）. Bay11-7082 decreased the protein expression of MMP-2 and MMP-9 to 1. 183 ± 0. 030 and 2. 024 ± 0. 056 （all P 〈 0. 001 comparing with 400 ng/ml visfatin treatment）. Conclusion Visfatin enhanced the expression and activity of MMP-2 and MMP-9 in human monocytes via activating NF-κB signaling pathway.