Ligustilide Inhibiting A7r5 Cells Migration By C-Myc/MMPs Signaling Pathway

Objective:1) To identify the inhibitory of ligustilide on the migration of vascular smooth muscle A7r5 cells induced by AngⅡ.2) To investigate the molecular mechanism of LIG regulating VSMCs migration:To analyzes the relationship between LIG and c-Myc in vitro; and then analyzes the interaction between c-Myc and MMP.Methods:1) The cell cytotoxicity was assessed using MTT method.2) To using Angiotensin Ⅱ induced rat thoracic aortic vascular smooth muscle A7r5 cells migration to structure inflammation model.3) When A7r5 at 70%-80%confluency, cell migration was manufactured by wound healing and Transwell migration assay. Then, cell treatment with different concentrations of ligustilide 0,2.5,5,10μg/ml. A7r5 migration was observed under microscope with different time at 0,6,12,24h.4) Zymography and Western blot were used to measure MMP2, MMP9 and c-Myc expression of LIG treated with A7r5 by AngⅡ.5) RNA interference was used to silence expression of c-Myc. Real-time PCR, Western blot were used to analyze MMP2 and c-Myc expression.6) Co-IP technology were used to analyze whether c-Myc bound with MMP2.Results:1) Constructed the model of A7r5 cell migration by AngⅡ, AngⅡ of the same times at different concentration had different effect on cell migration of A7r5 cells. AngⅡ concentration of 1μg/ml at 12 h A7r5 cell migration rate reached (14.49 ± 0.239) higher than other concentration group (P< 0.05). The significantly simulated of AngⅡ on A7r5 cells migration was most obvious under final concentration of 1μg/ml at 12 h.2) ligustilide can prevent the vascular smooth muscle A7r5 cell migration induced by AngⅡ. The effects of LIG on prevent migration was concentration dependent at 2.5～10 g/ml. The inhibitory effect of ligustilide on A7r5 cells migration is most obvious under final concentration of 10μg/ml at 12h.3) The results showed that LIG reduce the MMP2 and c-Myc expression (P<0.05). However, MMP9 expression was not found to be statistically significant.4) Silence of c-Myc expression with transfection of c-Myc abolished the increased expression of MMP2 induced by AngⅡ.5) Co-IP assay results revealed that AngⅡ could increased c-Myc interact with the MMP2. In addition, silenced c-Myc led to decreased MMP2 cooperate with c-Myc.Conclusion:ligustilide inhibited A7r5 cell migration induced by AngⅡ, the effects of LIG on prevent migration was concentration dependent and its reducethe MMP2 and c-Myc expression. The inhibitory effect of ligustilide on A7r5 cells migration is most obvious under final concentration of 10μg/ml at 12 h. AngⅡ mediated regulation of c-Myc/MMPs signaling pathway to up-regulated the c-Myc and MMP2 expression. Ligustilide inhibiting A7r5 cells migration by wane c-Myc and MMPs activity.