| Objective Percutaneous coronary intervention (PCI) has been widely used as an effective means for the treatment of coronary artery diseases. However, post-PCI restenosis remains a primary limitation for long-term success after PCI. Although the molecular mechanisms of restenosis are far elucidated, many studies have shown that under exogenous multiple stimuli, the proliferation, transdifferentiation and migration from the vascular media into intima of vascular smooth muscle cells (VSMCs) play pivotal roles in the pathophysiology of post-PCI restenosis. Astragaloside IV (AS-IV), a purified principal extract from traditional Chinese herb medicine Astragali Radix, exhibits anti-inflammatory, antiviral, antioxidant, anti-myocardial fibrosis and cardioprotective effects. However, its effect on post-intervention restenosis has not been studied and reported. The aim of the present study is to investigate the AS-IV effect on proliferation and migration of VSMCs that were induced by tumor necrosis factor-a (TNF-a), and related biological mechanism. This study will provide experimental basis for the prevention and therapy of post-PCI restenosis.Methods (1) For next related researches, primary cultures of VSMCs were prepared from the thoracoabdominal aorta of rats using explant method. Morphology of cells was observed by inverted microscope, and identified by immunohistochemical methods with antibody against SM-α-actin. (2) The model of VSMCs proliferation and migration was established by TNF-a inducer in vitro, and divided randomly into the following groups:①control group; ②TNF-α group; ③ TNF-α+AS-Ⅳ 0.5 ug/ml; ④TNF-α+AS-Ⅳ 5ug/ml; ⑤TNF-α+AS-Ⅳ 25ug/ml; and ⑥ TNF-α+AS-Ⅳ (50ug/ml). (3) A WST-8 assay was used to evaluate cell proliferation activity. (4) The wound migration assay and transwell invasion assay were used to analyze cell migratory and invasive capability. (5) The Quantitative real-time polymerase chain reaction (real-time PCR) and western blotting assay were used to examine the AS-IV effect on the mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2) in VSMCs.Results (1) Ninety percent cultured explants survived, and the purity of the fifth passage VSMCs was more than 95%. The passaged cells showed the typical "valley and peak " morphological appearance under inverted microscope. Immunohistochemical staining with antibody against SM-a-actin demonstrated more than 98% cells were positive. (2) The proliferative activity, migratory distance and invasive capacity of VSMCs were obviously increased in the TNF-α group. There was a significant difference as compared with the control group (P<0.01). The results suggested that the model of VSMCs proliferation and migration established by TNF-α inducer in vitro was successfully established. (3) The group treated with TNF-α alone significantly increased cell proliferation(P<0.01 compared to the control). However, when pretreated with AS-Ⅳ ahead of time, we found that AS-Ⅳ significantly inhibited TNF-α-induced VSMCs proliferation in a dose- and time-dependent manner in comparison to the TNF-α group. (4) In the wound migration assay, the migration of VSMCs to the wound areas was significantly promoted by TNF-α treatment relative to control group. On the contrary, AS-Ⅳ treatment at the concentration range of 0.5-50 ug/ml strongly suppressed TNF-α-induced migration in a dose- and time-dependent manner. (5) In the chamber invasion assays, TNF-α significantly enhanced the number of VSMCs invading into the lower chamber during 24 h exposure (P<0.01 compared to the control). Quantitative analysis demonstrated that AS-Ⅳ reversed TNF-α-induced enhancement of VSMCs invasion in a concentration-dependent manner (P<0.01 compared to the TNF-α group). (6) Real-time PCR and western blotting assays indicated that AS-Ⅳ (0.5-50 ug/ml) down-regulated TNF-α-induced gene expression and protein levels of MMP-2 and MMP-9, and up-regulated TNF-α-induced gene expression and protein levels of TIMP-1 and TIMP-2 in VSMCs. These findings revealed that AS-Ⅳ could influence the balance between MMPs and TIMPs, which would be effective in the VSMCs proliferation and migration.Conclusions:(1) AS-Ⅳ exerts inhibitory effects on VSMCS proliferation, migration and invasion in a dose- and time-dependent manner. (2) AS-Ⅳ exerts inhibitory effects on VSMCs proliferation and migration by restoring the balance of MMPs and TIMPs. Therefore, it may be a potentially be a useful therapeutic agent to treat cardiovascular restenosis after PCI. |
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