羟基红花黄色素A促犬胸主动脉内皮细胞增殖及其机制初探

摘要

Aim To study the proliferative effect of hydroxysafflor yellow A (HSYA) on cultured canine aortic endothelial cell (VEC) in normoxic (21% O2) or hypoxic (10% O2) culture and the underlying mechanism. Methods The endothelial cells were scratched from trypsined canine aorta endothelium. HSYA was added to the cells at final concentrations of 1×10-3, 1×10-4 and 1×10-5 mol·L-1, respectively. VEGF (2.6×10-7 mol·L-1)-treated cells were used as the positive control. The proliferative effect of HSYA on VEC was determined at 48, 72, 96, and 120 h in normoxic culture by MTT assay. Similarly, the proliferation of VEC was determined at 12,24,48,and 72 h in hypoxic culture by MTT assay. The effects of HSYA on VEC proliferation and VEGF secretion were investigated by MTT and ELISA assays at the presence of the antibodies to VEGF and VEGF receptors. Results Pretreatment with HSYA at concentrations of 1×10-3 and 1×10-4 mol·L-1 enhanced VEC proliferation in normoxic culture. The most significant enhancing effect of HSYA on VEC proliferation was achieved at 24, 48, and 72 h in hypoxic culture in concentration-dependent and time-dependent manner. HSYA at 1×10-3 mol·L-1 showed a potency similar to VEGF at 2.6×10-7 mol·L-1. Pretreatment with the antibodies of Flt-1, KDR or VEGF blocked the proliferative effect of HSYA with similar potencies. Antibodies of Flt-1 or VEGF antagonized the promoting effect of HSYA on VEGF secretion. Conclusion HSYA promotes VEC proliferation either in normoxic or hypoxic culture, especially in the latter condition. This effect of HSYA is at least partly mediated by VEGF and VEGF receptor.%目的研究羟基红花黄色素A(HSYA)对常氧/低氧两种条件下体外培养的犬胸主动脉内皮细胞增殖的影响,且探讨其增殖作用是否与血管内皮生长因子(VEGF)有关.方法采用内膜消化刮取法获取犬胸主动脉内皮细胞;分别在常氧(21%)/低氧(10%)两种条件下,以噻唑蓝(MTT)法观察HSYA对血管内皮细胞(VEC)增殖的影响,VEGF作为阳性对照;并且观察VEGF抗体及其两种酪氨酸受体(Flt-1和KDR)的抗体对HSYA促VEC增殖及分泌VEGF的影响,采用ELISA法检测VEGF水平.结果 HSYA 1×10-3 mol·L-1、 1×10-4 mol·L-1在常氧条件下孵育72 h、 96 h和120 h或在低氧条件下孵育24 h、 48 h、 72 h对VEC都有明显促增殖作用,并具有浓度和时间依赖性,HSYA 1×10-3 mol·L-1与VEGF 2.6×10-7 mol·L-1在同样条件下对VEC的促增殖作用强度相当;10 μg·mL-1的VEGF、Flt-1和KDR的抗体均能明显抑制1×10-3 mol·L-1 HSYA的促VEC增殖作用,尤其10 μg·mL-1的VEGF抗体和Flt-1抗体能明显抑制1×10-3 mol·L-1 HSYA的促VEC分泌VEGF作用.结论在常氧/低氧两种条件下,HSYA均具有明显促VEC增殖作用,尤其在低氧条件下更为明显;而且HSYA的促VEC增殖作用可能与VEGF或VEGF受体有关.