丹红注射液对共培养体系中平滑肌细胞舒张的影响及其作用机制研究

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

目的研究丹红注射液在共培养体系中通过人脐静脉内皮细胞(Ea.Hy926)对大鼠胸主动脉平滑肌细胞(A7r5)舒张的影响及其作用机制。方法以A7r5、Ea.Hy926为细胞模型,用Western blotting法检测丹红注射液对单独培养的A7r5细胞中肌球蛋白轻链(MLC)和磷酸化MLC(P-MLC)蛋白表达的影响;检测其对A7r5细胞内钙离子浓度的影响;用RT-PCR法检测A7r5细胞钙调蛋白(Ca M)m RNA表达的变化,MTT法观察其对重组人血小板源性生长因子-BB(PDGF-BB)诱导的A7r5细胞增殖的影响;最后用Transwell小室建立共培养体系,分别用Western blotting法和ELISA法检测丹红注射液对共培养体系中A7r5细胞MLC、P-MLC的蛋白表达以及环磷酸腺苷(c AMP)分泌的影响。结果丹红注射液(10μL/m L)能降低单独培养的A7r5细胞中P-MLC/MLC的值(P<0.05)和Ca M m RNA表达(P<0.01),对细胞内钙离子浓度和MLC蛋白表达无显著影响,也可降低共培养体系中A7r5细胞P-MLC/MLC值(P<0.05),并增加c AMP的分泌(P<0.01),此外还能抑制PDGF-BB诱导的A7r5细胞增殖(P<0.01)。结论丹红注射液能通过降低P-MLC/MLC值和Ca M的m RNA表达直接使平滑肌舒张,也能通过促进内皮细胞释放舒血管因子间接增加共培养体系中平滑肌细胞c AMP的分泌,使平滑肌细胞舒张。

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 Ca M m RNA 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 c AMP generation in A7r5 cells. Results DHI（10 μL/m L） contributed to the decrease of the ratio of P-MLC/MLC（P〈0.05） and Ca M m RNA 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/m L） could still decrease the ratio of P-MLC/MLC（P〈0.05） and increase the secretion of c AMP（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 Ca M m RNA, meanwhile indirectly relaxing the vascular smooth muscle cells in co-culture system by promoting the secretion of c AMP which is increased by vasodilatory factors in endothelial cells.