山柰酚-3-O-芸香糖苷对血管平滑肌细胞增殖、迁移及TGFBR1信号通路活化的影响

目的:探索山柰酚-3-O-芸香糖苷(KR)对血管平滑肌细胞(VSMC)增殖、迁移及转化生长因子β受体1(TGFBR1)信号通路活化的影响。方法:将KR(10、20和40μmol/L)孵育大鼠VSMC细胞系A7R5 24 h,或将40μmol/L KR孵育A7 R5细胞24、48和72 h后,MTT法检测细胞活力,Ed U染色检测细胞增殖情况,Transwell小室实验检测细胞迁移能力的变化,Western blot检测细胞迁移相关蛋白基质金属蛋白酶2(MMP2)及基质金属蛋白酶9(MMP9)的表达;分子对接技术探索KR与TGFBR1之间的相互关系,Western blot检测TGFBR1及其下游的Smad2和Smad3的激活情况。结果 :KR剂量和时间依赖性地降低细胞活力,剂量依赖性地减少Ed U染色阳性细胞数量,降低迁移细胞数量,减少迁移相关蛋白MMP2和MMP9的表达(P0.05)。KR与TGFBR1发生分子对接的结合力为-9.804 kcal/mol,与TGBFR1的SER-280、ARG-215、ASP-290和LYS-335氨基酸残基形成氢键连接。KR剂量依赖性地降低TGFBR1及其下游Smad2和Smad3的激活(P0.05)。结论:KR能抑制VSMC的增殖和迁移,其机制可能与抑制TGFBR1信号通路相关。

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TGFBR1 signaling pathway activation in vascular smooth muscle cells

AIM:To investigate the effects of kaempferol-3-O-rutinoside (KR) on the proliferation, migration of vascular smooth muscle cells (VSMC) and the activation of transforming growth factor β receptor 1 (TGFBR1) signaling pathway in the cells. METHODS:The viability of VSMC was detected by MTT assay. The proliferation of VSMC was measured by EdU staining. The migration ability of VSMC was examined by Transwell assay. The protein levels of the migration-associated proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were detected by Western blot. Molecular docking study was conducted to explore the interaction between KR and TGFBR1. The protein le-vels of the phosphorylated TGFBR1, Smad2 and Smad3 were determined by Western blot. RESULTS:KR inhibited the viability of VSMC in a dose-and time-dependent manner. KR reduced the ratio of EdU-positive cells in a dose-dependent manner. KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP2 and MMP9 (P<0.05). KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280, ARG-215, ASP-290 and LYS-335 of TGBFR1. KR dose-dependently suppressed the activation of TGFBR1 and its downstream proteins Smad2 and Smad3 (P<0.05). CONCLUSION:KR inhibits the proliferation and migration of VSMC, possibly via blocking the TGFBR1 signaling pathway.