The Effect Of Guifudihuang Drug-containing Serum Inhibiting The Proliferation Of Angiotension Ⅱ-induced Rat Vascular Artery Smooth Muscle Cells

ObjectiveAs we have established the model of cell proliferation and phenotype transformation of rat vascular artery smooth cells by using the AngⅡ. Then our studies will aim to observe the effect of Guifudihuang Drug-containing serum on preventing the proliferation of rat VSMCs in vitro. This study will investigate the possible mechanism and provide the theoretical basis for the prevention and treatment of UA diseases by using Guifudihuang in the cell level. Methods1. The original generation culture VSMCs by the methods of tissure adherent and cells from3-5generations were identified by immunohistochemistry specific staining of smooth muscle-specific alpha actin (a-SMA).2. Randomly, the rats were divided into drug-taking group and blank group.The drug was administered orally for one week. Then we obtained the blood and separate the serum from it. Prepare the blank serum and the different concerntration of Guifudihuang serum including5%,10%,20%.3. Use different dose of AngⅡ-treated (10-8-10-6M) to stimulate VSMCs. After24h, using the methods of CCK8assay to measure the OD values, which shows the degree of proliferation of VSMCs.4. Divide the VSMCs into different groups randomly, incluing control gr oup, AngⅡ treated group, AngⅡ with5%Guifudihuang-containing serum tre ated group, AngⅢ with10%Guifudihuang-containing serum treated group an d AngⅡ with20%Guifudihuang-containing serum treated group. The time of stimulation is24h.5. CCK8assay and Brdu-Elisa used to indirectly and directly detetmine the proliferation of VSMCs respectively.6.Statistical analysis:Data were expressed as (X±S). Comparisons between groups were analyzed with one-ways analysis of variance(ANOVA), The experimental data was analyzed by the software of SPSS17.0. Differences were considered statistically significant at p<0.05or p<0.01.Results1. Immunohistochemistry staining shew more than98%of cells positively expressed a-SMA.2. The results of CCK8showed a dose-dependent stimulation of VSMCs growth at the concentration of10-5to10-8mol/L compared with control group after24h Ang II treatment. The significant stimulation was observed from the dose of10^M in VSMCs (p<0.01).3. The results from CCK8assay and Brdu-Elisa showed that compared wit h Ang II treated the OD values was lower in the both10%and20%of Guifud ihuang-containing serum treated groups, the difference were statistically significant (p<0.05), in which the latter group had the more significant effect (p<0.01)Conclution1.The cultured cells express smooth muscle-specific a-actin. They are identified as vascular smooth muscle cells and the percentage of smooth muscle cells can be higher than98%.2. The result indicated that AngⅡ could lead to growth-stimulating effect on VSMCs. And we chrose the dose of10-6M as the one of Ang Ⅱ treatment in the follow steps.3. Guifudihuang drug-containing serum could inhibit the proliferation of angiotension Ⅱ-induced VSMCs, which is showed by the way of the number of cells and the DNA synthese.4. Both the groups of10%and20%of Guifudihuang-containing serum could inhibit the proliferation of AngiotensionII-induced VSMCs and high dose is much more effective.