Experimental Study Of Ethanol Extract Of Yanhusuo And The Alkaloid Fraction Of Yanhusuo Inhibiting Angiogenesis In Vivo And The Regulation Of VECF

Objective:To investigate the effects of the ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo on anti-angiogenesis in vivo and the regulation of VEGF.Methods:1. MTT assay was used to observe the effects of ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo on the proliferation of human gastric cancer cell lines BGC-823, human lung adenocarcinoma cell lines A549, human colon cancer cell lines LOVO and human hepatoma cell lines HepG-2.And the most sensitive tumor cells to the ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo had been screened.2. The most sensitive tumor cell lines were used as a control, the inhibiting effects of ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo on the proliferation of human umbilical vein endothelial cells (HUVEC) were measured by MTT assay.3. Chick chorio allantoic membrane (CAM) model was used to observe the effects of ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo on the neovascularization in vivo.4. Western blot was used to measure the protein expression of VEGF in A549cells and HUVEC cells. The expression and phosphorylation of AKT, MAPK,NF κB and other signaling pathways were also examined after treating with ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo.Results:1. Both ethanol extract of yanhusuo and the alkaloid fraction of yanhusuo could inhibit proliferation in vitro of HepG2cells, A549cells, LOVO cells and BGC-823cells in a concentration dependent manner. Inhibitory Concentration50(IC50) of ethanol extract of yanhusuo above four tumor cells were167.845μg/ml,153.078μg/ml,158.894μg/ml,155.865μg/ml, respectively; Under the same conditions,the inhiting effects of Corydalis alkaloids was stronger, IC50was:89.971μg/ml,45.672μg/ml,109.187μg/ml and62.62μg/ml, respectively. Therefore, the most sensitive cell line is human lung adenocarcinoma A549cell line.2Thanol extract of yanhusuo and the alkaloid fraction of yanhusuo, in the range of3.125μg/ml～25μg/ml, whose proliferated effects of A549cells were significantly lower than that of HUVEC, and the difference was significant (P<0.05). And the inhibiting ability of Corydalis alkaloids on the proliferation of HUVEC was better than that of Thanol extract of yanhusuo.3. Thanol extract of yanhusuo and the alkaloid fraction of yanhusuo, in the range of 3.125μg/ml～25μg/ml, could suppress angiogenesis of CAM in vivo.The neovascularization inhibition rate of Thanol extract of yanhusuo was between8.68%and25.2%, The neovascularization inhibition rate of Thanol extract of yanhusuo was between19.32%and39.9%.4. Corydalis alkaloids could reduce the expression of VEGF in both A549cells and HUVEC cells. Under the same conditions, Thanol extract of yanhusuo did not have the effects. Corydalis alkaloids (12.5μg/ml～200μg/ml), showed no significant impaction on p50of IκB-a exerpession of IκB-a phosphorylation in A549cells, but could reduce the exerpession of Akt phosphorylation in200μg/ml, which was significantly lower than that of the control. Either Corydalis alkaloids had no obvious effects on the MAPK3s pathway. Corydalis alkaloids can significantly decrease the protein expression of VEGF in a does dependent manner.ConclusionAlkaloid fraction of yanhusuo had a strong inhibition of proliferation in HepG2cells, A549cells, LOVO cells and BGC-823cells,whereas the inhibition of the ethanol extract of yanhusuo in the four tumor cell lines proliferation is relatively weaker; the ethanol extract of yanhusu and Alkaloid fraction of yanhusuo both had a certain anti-angiogenic effects in vivo; Corydalis alkaloids could reduce the expression of VEGF in A549cells and HUVEC cells through downregulating phosphorylation of Akt.