整合素β1-黏附斑激酶在心肌纤维化中的作用及机制

Role and mechanism of integrin β1-FAK on myocardial fibrosis

摘要:

初步探讨整合素β1-黏附斑激酶（integrin β1-FAK）在心肌细胞纤维化中的作用与机制。方法将培养的心肌细胞随机分为对照组、血清脂肪酶（LPS）处理组、integrin β1抑制组、黏附斑激酶（FAK）抑制组及integrinβ1阻断+FAK抑制组。免疫荧光双标法检测原代心肌细胞纯度，逆转录聚合酶链反应（RT-PCR）与实时荧光定量聚合酶链反应（qRT-RCR）检测基质金属蛋白酶9（MMP-9）、转化生长因子β1（TGF-β1）、Ⅰ型胶原（Col1A1）、Ⅲ型胶原（Col3A1）和整合素β1 （integrin β1）mRNA 水平的变化，Western blot 检测FAK 的表达。结果①与对照组比较，LPS 能够刺激心肌细胞上调MMP-9、TGF-β1、Col1A1、Col3A1 及integrin β1表达；②与LPS 组比较，integrin β1阻断可以抑制LPS 诱导的TGF-β1、MMP-9、Col1A1 及Col3A1 信使核糖核酸（mRNA）的上调（p <0.05），并阻断LPS 诱导的磷酸化FAK的激活（p <0.05）；③FAK 蛋白抑制剂PF-573228可抑制LPS诱导的TGF-β1和MMP-9 mRNA上调水平，同时加入integrin β1封闭性抗体显示相似的效果。结论Integrin β1阻断能抑制FAK 的磷酸化，下调TGF-β1、MMP-9、Col1A1 及Col3A1 的表达，从而改善在持续炎症刺激条件下心肌细胞的纤维化反应进程，为未来临床治疗拓展新思路。

Abstract:

To study the effects and mechanisms of integrin β1-FAK on myocardial fibrosis. Methods Cells were randomly divided into five groups: the blank control group, the LPS-stimulated group,integrin β1 blocking group, FAK blocking group, blocking integrin β1 and FAK group. Double-immunostaining was used to test the purity of myocardial cells. RT-PCR and q-PCR were used to detect the gene expression of MMP-9, TGF-β, Col1A1, Col3A1 and integrin β1. Western blot method was used to detect the expression of FAK protein. Results Compared with those in the control group, the LPS -treated cells exhibited a significant increase of MMP-9, TGF-β, Col1A1, Col3A1 and integrin β1, while blocking the integrin β1 prevented the increase in LPS-induced up-regulation of MMP-9, TGF-β, Col1A1, Col3A1 and integrin β1 and inhibited the FAK phosphorylation. Furthermore, when the cells were treated with the FAK agonist, the expression of MMP-9 and TGF-βdecreased, and it had similar effect with blocking integrin β1. Conclusions Our study suggests that inhibition of integrin β1 suppresses the FAK phosphorylation, and decreasesexpressons of MMP-9, TGF-β, Col1A1, Col3A1, which contributes to alleviate the development of myocardial fibrosis under the condition of continuous inflammation stimulation and provides new ideas for the future clinical treatment stimulated H9c2 cardiomyocytes.