The Effects Of Ligustilide On Angiotensin Ⅱ-Induced Vascular Smooth Muscle Cell Migration And Mechanisms Involved

Objective:1、To identify the inhibitory effect of ligustilide on the migration of vascular smooth muscle cells induced by Angiotensin-II.2、To investigate the molecular mechanism of LIG regulating VSMCs migration: To investigate the effect of ligustilide on c-Myc,MMP2 expression and the relationship between c-Myc and MMP2.To investigate the effect of ligustilide on the expression of ROCK and JNK and the relationship between ROCK and JNK.Methods:1、The toxicity of angiotensin-II and ligustilide on vascular smooth muscle cells was determined by CCK-8 assay.2、Construction of an angiotensin-II model for inducing excessive migration of vascular smooth muscle cells.3、The effect of ligustilide on angiotensin-II induced vascular smooth muscle cell migration was determined by cell scratch test and Transwell assay.4、qPCR and Western blot were used to detect the changes of c-Myc,MMP2,ROCK and JNK in gene and protein expression of ligustilide-induced vascular smooth muscle cells.5、c-Myc inhibitor and ROCK inhibitor were used to treat cells,and the migration ability of vascular smooth muscle cells was evaluated to detect changes in c-Myc,MMP2,ROCK and JNK protein expression.Results:1、An angiotensin-II-induced model of excessive migration of vascular smooth muscle cells was established,and the effect of inducing excessive cell migration was observed within 24 h of the concentration of 2 μg/mL(over-migration rate was 38 % to 45.4 %).Non-cytotoxic and stable,can be used as a model for constructing vascular smooth muscle cell migration.2、Ligustilide can effectively inhibit the migration of vascular smooth muscle cells induced by angiotensin-II,and the inhibition of cell migration in a concentration range of 2.5 ~ 10 μg/mL is concentration-dependent.When the concentration was 10 μg/mL for 12 h,the cell migration rate reached 49 %,which was the optimal concentration and the best time.3、Ligustilide down-regulates the expression of c-Myc and MMP2 proteins induced by vascular smooth muscle cells.After c-Myc inhibitor10058-F4 was applied to cells,the migration rate of vascular smoothmuscle cells decreased significantly,and the expression of c-Myc and MMP2 decreased.Ligustilide can be inhibited by regulation of c-Myc/MMP2 signaling pathway.Angiotensin-II induced vascular smooth muscle cell migration.4、Ligustilide can effectively down-regulate the protein expression levels of ROCK1/2 and JNK induced by vascular smooth muscle cells.After reducing its kinase activity by ROCK-specific inhibitor Y-27632,vascular smooth muscle cell migration decreased,and ROCK and JNK expression were down-regulated.Ligustilide inhibited angiotensin-II induction vascular smooth muscle cells migration by regulating ROCK/JNK signaling pathway.Conclusion:Ligustilide can effectively inhibit angiotensin-II-induced vascular smooth muscle cell migration,and the inhibitory effect is dose-dependent at 2.5 ~10 μg/mL.The inhibitory effect is most obvious when the concentration is 10 μg/mL for 12 h(inhibition rate was 49%).Ligustilide downregulates angiotensin-II-induced c-Myc and MMP2,ROCK and JNK protein expression,and ligustilide inhibits angiotensin-II induced vascular smooth muscle cell migration via c-Myc / MMP2,ROCK / JNK signaling pathway.