Li Xiaolei, Chen Hui, Wang Shuguang, Dai Jihang, Yan Lianqi, Wang Jingcheng, Sun Yu
Department of Orthopedics and Orthopedic Institute, Clinical Medical College of Yangzhou University, Subei People's Hospital of Jiangsu Province, Yangzhou 225001, China.
Department of Orthopedics and Orthopedic Institute, Clinical Medical College of Yangzhou University, Subei People's Hospital of Jiangsu Province, Yangzhou 225001, China.
Biochem Biophys Res Commun. 2017 Sep 2;490(4):1197-1204. doi: 10.1016/j.bbrc.2017.06.181. Epub 2017 Jun 29.
Tacrolimus (FK506) has been demonstrated to reduce epidural fibrosis. However, the detailed mechanism of action has not been elucidated. Aberrant miR-429 is involved in many diseases. The aim of this study was to describe the exact mechanism of FK506 induced apoptosis in fibroblasts and the prevention of epidural fibrosis. FK506 induced fibroblast apoptosis was evaluated using CCK-8 assays, flow cytometry, and western blotting. The expression of miR-429 in fibroblasts treated with FK506 was determined by RT-qPCR. Additionally, luciferase activity assays were used to determine the target relationship between miR-429 and RhoE. Flow cytometry and western blot analysis were used to determine the effects of FK506 and miR-429 on fibroblast apoptosis. The effects of FK506 and RhoE on fibroblast apoptosis were determined by CCK-8 assay, flow cytometry, and western blotting. We also evaluate the effects of FK506 and miR-429 on epidural fibrosis in rats by using histological analysis and TUNEL-staining. The results revealed FK506 induces fibroblast apoptosis and significantly downregulates miR-429 expression in fibroblasts. Additionally, miR-429 downregulation caused the apoptosis of fibroblasts. The luciferase activity assay confirmed that RhoE is a direct target of miR-429 and RhoE promotes fibroblast apoptosis. The rat model demonstrated miR-429 inhibition promotes fibroblast apoptosis and epidural fibrosis, which is consistent with the results of FK506 treatment. Our study demonstrates that FK506 induces fibroblast apoptosis and reduces epidural fibrosis by regulating miR-429 expression and its target of RhoE.
他克莫司（FK506）已被证明可减少硬膜外纤维化。然而，其详细作用机制尚未阐明。异常的miR-429参与多种疾病。本研究的目的是描述FK506诱导成纤维细胞凋亡及预防硬膜外纤维化的确切机制。使用CCK-8检测、流式细胞术和蛋白质印迹法评估FK506诱导的成纤维细胞凋亡。通过RT-qPCR测定FK506处理的成纤维细胞中miR-429的表达。此外，使用荧光素酶活性测定法确定miR-429与RhoE之间的靶标关系。采用流式细胞术和蛋白质印迹分析确定FK506和miR-429对成纤维细胞凋亡的影响。通过CCK-8检测、流式细胞术和蛋白质印迹法确定FK506和RhoE对成纤维细胞凋亡的影响。我们还通过组织学分析和TUNEL染色评估FK506和miR-429对大鼠硬膜外纤维化的影响。结果显示，FK506诱导成纤维细胞凋亡，并显著下调成纤维细胞中miR-429的表达。此外，miR-429表达下调导致成纤维细胞凋亡。荧光素酶活性测定证实RhoE是miR-429的直接靶标，且RhoE促进成纤维细胞凋亡。大鼠模型表明，抑制miR-429可促进成纤维细胞凋亡和硬膜外纤维化，这与FK506治疗的结果一致。我们的研究表明，FK506通过调节miR-429表达及其靶标RhoE诱导成纤维细胞凋亡并减少硬膜外纤维化。
