抵抗素调节人冠状动脉内皮细胞MMP-9表达机制探讨

Effect of resistin on MMP-9 in human coronary artery endothelial cells

摘要:

目的：探讨抵抗素调节冠状动脉内皮细胞中基质金属蛋白酶（matrixmetalloproteinases，MMP）－9的表达及其机制。方法：用含1%胎牛血清（fetal bovine serum，FBS）的DMEM/F12培养基培养人冠状动脉内皮细胞（human coronary artery endothelial cells，HCAECs）16 h（血清饥饿培养），之后随机分为6组：（1）空白对照（Con）组：无抵抗素干预；（2）Res20组：抵抗素20 ng/ml干预；（3）Res40组：抵抗素40 ng/ml干预；（4）Res100组：抵抗素100 ng/ml干预；（5）Res40+U0126组：细胞中加入细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）抑制剂U0126（20 mmol/L）预处理1 h，再加入抵抗素40 ng/ml干预；（6）U0126组：细胞中仅加入ERK抑制剂U0126（20 mmol/L），细胞各组干预24 h后，用RT-PCR法检测MMP-9和蛋白酶抑制剂（tissue inhibitor of metalloproteinase，TIMP）－1 mRNA表达，ELISA法检测MMP-9和TIMP-1蛋白水平，Western blot检测HCAECs p-ERK1/2及ERK1/2的蛋白表达。结果：3种浓度抵抗素干预HCAECs 24 h后，MMP-9 mRNA与蛋白水平均较空白组明显升高（P＜0.05）；TIMP-1 mRNA与蛋白水平均较空白组明显降低（P＜0.05）。40 ng/ml抵抗素干预组加入ERK抑制剂U0126后，MMP-9 mRNA与蛋白水平明显下降（P＜0.05），TIMP-1 mRNA与蛋白无明显变化。与Con组、U0126组及Res40+U0126组相比，Res40组p-ERK1/2蛋白表达明显增高（P＜0.05）。结论：抵抗素可诱导HCAECs MMP-9的生成，可能通过下调TIMP-1的表达及激活ERK通路参与这一过程。

Abstract:

Objective：To investigate the expression of matrixmetalloproteinases-9（MMP-9） in human coronary artery endothelial cells （HCAECs） and to explore its underlying mechanism. Methods：HCAECs were incubated by 1% fetal bovine serum（FBS） in DMEM/F12 culture medium in six different group：（1）blank control（Con）group：without resistin；（2）Res20 group：with 20 ng/ml resistin；（3）Res40 group：with 40 ng/ml resistin；（4）Res100 group：with 100 ng/ml resistin；（5）Res40+U0126 group：incubating HCAECs with ERK inhibitor U0126 （20 mmol/L） pretreatment for 1 h ，then adding 40 ng/ml resistin；（6）U0126 group：with ERK inhibitor U0126 （20 mmol/L）. After 24 h，MMP-9 and tissue inhibitor of metalloproteinase-1（TIMP-1） mRNA and protein levels were measured by RT-PCR and ELISA. HCAECs p-ERK1/2 and ERK1/2 protein levels were measured by Western blot. Results：Compared with those in Con group，MMP-9 mRNA and protein levels were increased in Res20，Res40，Res100 groups（P＜0.05） and TIMP-1 mRNA and protein levels were decreased（P＜0.05）. MMP-9 mRNA and protein levels in Res 40+U0126 group were lower than those in Res40 group（P＜0.05） while there is no obvious change of TIMP-1 mRNA and protein levels. Compared with those in Con group，U0126 and Res40+U0126 group，p-ERK1/2 protein levels were higher in Res40 group（P＜0.05）. Conclusions：Expression of resistin can down-regulate the TIMP-1 and induce MMP-9 synthesis. The ERK1/2 is involved in MMP-9 expression in HCAECs exposed to re－sistin. These results suggest an active role of resistin in the pa－thophysiology of resistin-induced cardiovascular disease.