红景天苷抑制动脉平滑肌细胞增殖、迁移、分泌表型的分子机制研究

Mechanism of salidroside in inhibiting proliferation,migration and promoting phenotypic switching of arterial smooth muscle cells

探讨红景天苷(salidroside, SAL)抗血小板源性生长因子-BB(platelet-derived growth factor-BB,PDGF-BB)诱导人主动脉平滑肌细胞(human aortic smooth muscle cells, HASMC)表型转化,并对其药理机制进行探析。应用乳酸脱氢酶释放实验筛选SAL的安全有效浓度。应用PDGF-BB诱导HASMC建立细胞表型转化体外模型。分为对照组、模型组、SAL组。应用CCK-8法检测细胞增殖率,Transwell检测细胞迁移,荧光标记的鬼笔环肽对F-肌动蛋白(fibrous actin, F-actin)染色观察细胞骨架结构,Western blot检测增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、迁移相关蛋白基质金属蛋白酶9(matrix metalloprotein 9,MMP-9)、纤维粘连蛋白(Fibronectin)、表型转换标志物α-肌动蛋白(α-smooth muscle actin,α-SMA)、骨桥蛋白(osteopontin, OPN)。由结果可知造模使HASMC增殖明显、迁移增加并促使HASMC由收缩表型转变成分泌表型,使其细胞骨架结构排列紊乱。加入SAL后HASMC增殖、迁移能力减弱,“收缩表型标志物”α-SMA表达增加,“分泌表型标志物”OPN表达减少、细胞骨架结构排列紊乱改善。机制研究细胞信号相关通路Akt、mTOR,以及Akt/mTOR信号通路上下游相关蛋白磷酸酶和张力蛋白同源物(phosphatase and tensin homologue, PTEN)、血小板源性生长因子受体β(platelet-derived growth factor receptor β,PDGFR-β)及缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的表达。模型组HASMC信号通路Akt、mTOR蛋白磷酸增加,PTEN、HIF-1α、PDGFR-β相对表达量显著增高。SAL组磷酸化Akt、mTOR表达减少,PTEN、PDGFR-β、HIF-1α蛋白的表达降低。综上所述,SAL对PDGF-BB诱导的HASMC具有保护作用,可能与HASMC的PDGFR-β/Akt/mTOR/HIF-1α信号通路相关。

This study aims to examine the effect of salidroside(SAL)on the phenotypic switching of human aortic smooth muscle cells(HASMC)induced by the platelet-derived growth factor-BB(PDGF-BB)and investigate the pharmacological mechanism.Firstly,the safe concentration of SAL was screened by the lactate dehydrogenase release assay.HASMC were divided into control,model,and SAL groups,and the cells in other groups except the control group were treated with PDGF-BB for the modeling of phenotypic switching.Cell proliferation and migration were detected by the cell-counting kit(CCK-8)assay and Transwell assay,respectively.The cytoskeletal structure was observed by F-actin staining with fluorescently labeled phalloidine.The protein levels of proliferating cell nuclear antigen(PCNA),migration-related protein matrix metalloprotein 9(MMP-9),fibronectin,α-smooth muscle actin(α-SMA),and osteopontin(OPN)were determined by Western blot.To further investigate the pharmacological mechanism of SAL,this study determined the expression of protein kinase B(Akt)and mammalian target of rapamycin(mTOR),as well as the upstream proteins phosphatase and tensin homologue(PTEN)and platelet-derived growth factor receptorβ(PDGFR-β)and the downstream protein hypoxia-inducible factor-1α(HIF-1α)of the Akt/mTOR signaling pathway.The results showed that the HASMCs in the model group presented significantly increased proliferation and migration,the switching from a contractile phenotype to a secretory phenotype,and cytoskeletal disarrangement.Compared with the model group,SAL weakened the proliferation and migration of HASMC,promoted the expression ofα-SMA(a contractile phenotype marker),inhibited the expression of OPN(a secretory phenotype marker),and repaired the cytoskeletal disarrangement.Furthermore,compared with the control group,the modeling upregulated the levels of phosphorylated Akt and mTOR and the relative expression of PTEN,HIF-1α,and PDGFR-β.Compared with the model group,SAL down-regulated the protein levels of phosphorylated Akt and mTOR,PTEN,PDGFR-β,and HIF-1α.In conclusion,SAL exerts a protective effect on the HASMCs exposed to PDGF-BB by regulating the PDGFR-β/Akt/mTOR/HIF-1αsignaling pathway.