Relaxation of Danhong Injection in vascular smooth muscle cells in co-culture system and study on its mechanism / 中草药

ABSTRACT

Objective: In this study, we examined whether Danhong Injection (DHI) exerted a relaxation effect in vascular smooth muscle cells (A7r5) in co-culture system by human umbilical vein endothelial cells (Ea.Hy926) and studied its mechanisms. Methods: First of all, Western blotting assay was carried out to quantify the protein expression of MLC and P-MLC in A7r5 cells. Secondly, calcium assay kit was used to detect the changes in intracellular calcium while the changes of CaM mRNA expression were subsequently detected by RT-PCR. MTT assay was used to detect the effect of DHI in proliferation of A7r5 cell, which is induced by PDGF-BB. Finally, the co-culture system of EA.Hy926 and A7r5 cells was constructed by a Transwell. After the treatment of Hy926 cells with DHI for co-culture system, the protein expression of MLC and P-MLC in A7r5 cells in co-culture system was also quantified by Western blotting. Cell-based ELISA was used to observe the cAMP generation in A7r5 cells. Results: DHI (10 μL/mL) contributed to the decrease of the ratio of P-MLC/MLC (P < 0.05) and CaM mRNA expression (P < 0.01) in A7r5 cells, which are cultured individually, but had no significant effect in intracellular calcium concentration and the expression of MLC proteins. In co-culture system, DHI (10 μL/mL) could still decrease the ratio of P-MLC/MLC (P < 0.05) and increase the secretion of cAMP (P < 0.01) in A7r5 cells. In addition, DHI contributed to suppress the proliferation of A7r5 cells which were induced by the PDGF-BB (P < 0.01). Conclusion: DHI can directly relax vascular smooth muscle cells by reducing the ratio of P-MLC/MLC and the expression of CaM mRNA, meanwhile indirectly relaxing the vascular smooth muscle cells in co-culture system by promoting the secretion of cAMP which is increased by vasodilatory factors in endothelial cells.