The inhibitory effect of homocysteine on the tube formation of endothelial cells can be reversed by the Danshen (Radix Salviae miltiorrhizae ) extract

Abstract

When endothelial cells are permitted to become superconfluent without change of the culture medium, they will assemble into capillary or vessel-like tubes. (Grant et al 1994) This phenomenon forms the basis of the functional assay for endothelial cells, the in vitro tube formation assay. On the other hand, hyperhomocysteinemia is believed to be responsible for the development of vascular disease via mechanisms that cause endothelial injury and dysfunction. (Clarke et al 1991) We studied the effect of homocysteine on the ability of human umbilical vein endothelial cells (HUVEC) to form capillary-like structure with the tube formation assay, and assessed the ability of Radix Salviae Miltiorrhizae (Danshen) to protect the endothelial cells from the damaging effect of homocysteine. Incubation of HUVEC culture with 5.0 mM homocysteine induced reduction of the number of branch points (mean = 13.2), which served as a quantitative measurement of tube formation as compared to the plain medium control (mean = 21.4). Incubation of cells with 5.0 mM and different concentrations (1, 10, 50, 100, 200, 400 μg/mL respectively) of total aqueous Danshen extracts led to a significant increase of the number of branch points. The response was dose-related, being optimal at 10μg/mL of Danshen extract. The results suggest that homocysteine at pharmacological level can induce endothelial injury with respect to tube formation in-vitro and that Danshen can reverse the adverse effect of homocysteine. Further investigations are being planned to look into the mechanism of the effects.

在不轉換培養環境的情況下，內皮細胞會出現超融合化的生長，並匯聚成管狀的結構 (Grant et al 1994)，此一現象可被利用來測定內皮細胞在實驗室的條件下組織管狀結構的能力，另一方面，高同型半胱氨酸血症被認為會通過損傷血管內壁而引發血管性病變 (Clarke et al 1991)。我們利用人臍帶靜脈的內皮細胞 (HUVEC)，研究同型半胱氨酸 (Hcy) 對內皮細胞組管能力的抑制，並且加入丹參的總水提物，觀察丹參保護HUVEC，令其免受Hcy損害的能力。我們用含5.0mM Hcy的培養液培養HUVEC，發現用以反映HUVEC組管能力的交叉點與生長於不含Hcy的HUVEC比較，數目明顯減少 (中位數由21.4減至13.2)，當加入不同濃度 (分別為1, 10, 50, 100, 200, 400 μg/mL) 的丹參總水提物於含Hcy的培養液後，交叉點的數目明顯增加 (中位數分別為26.8, 35.2, 31.6, 29.8, 23.8和22.2)，結果且顯示交叉點數目的增加與丹參水提物的濃度相關，而以10μg/mL表現最明顯的效應。我們認為內皮細胞於接觸到葯效濃度的Hcy後，短時間內即受到損傷，影響其組管的能力，而丹參可令內皮細胞免受損害。我們將進一步探討當中涉及的機制。