A Study To Compare Angiogenic Effect Of Rhodiola Rosea And Its Compound And Study Its Angiogenic Mechanism

Coronary artery disease (CAD) is one of the leading causes of death these days. About 1/3 of People have sudden death whose first sign of CAD are acute myocardial infarction. Most of CAD people can get better after proper secondary preventive intervention. But some of them are still under bad condition after ordinary drug therapy. And more, they are not suitable to have percutaneous coronary intervention and bypass therapy. For these people, angiogenic therapy is one of the best choices. Chinese medicine is one of the important interventions of angiogenic therapy. Our research group has done much about screening angiogenic drug of Chinese medicine by using the chick embryo chorioallantoic membrane (CAM) model. Five of them have best angiogenic effect. Rhodiola Rosea is the top one among them. Though it is proved to be angiogenic on infarct myocardium and can improve the function of the heart after acute myocardial infarction, but how it does that is still under cover. To deeper study its mechanism and to compare its effect with its compound, we design following experiments.Part I a study to compare the angiogenic effect of Rhodiola Rosea and its compound , Wuwei fangObject: 1 to compare the angiogenic effect of Rhodiola Rosea and its compound , Wuwei fang on CAM2 to select the proper condition of drug serum to study its angiogenic effect;3 to compare the angiogenic effect of drug serum of Rhodiola Rosea and Wuwei fang.Methods: Using CAM as studying model, by adding Rhodiola Rosea, Wuwei fang, Siwei fang, which not contain Rhodiola Rosea, Chinese medicine A, Chinese medicine B, Chinese medicine C, Chinese medicine D, all drugs have two different way to distill except Wuwei fang and Siwei fang, to compare their angiogenic effect on CAM. Then, useWuwei fang and Shexiang Baoxinwan as the tool drug to select the proper condition of drug serum study. Three major factor, animal feeding days, feeding dose and time of exsanguinations, and four different level of each factor are set. Compare the angiogenic effect on CAM of drug serum under different condition and between different drug serum. After having the proper condition of drug serum to study its angiogenic effect, Rhodiola Rosea serum is made under the same condition and then compare its effect with Wuwei fang.Results: Rhodiola Rosea has the best angiogenic effect among these drugs and also better than its compound, Wuwei fang. The best condition of drug serum is feeding 4 days and 5 times as clinic dosage. Rhodiola Rosea serum has better angiogenic effect than Wuwei fang serum.Conclusions: Rhodiola Rosea has better angiogenic effect than its compound Wuweifang.Part II to study the effect of Rhodiola Rosea on angiogenic genes on HUVECObject: study the possible angiogenesis mechanism of Rhodiola Rosea on HUVECMethods: 1) choose the best dose of Rhodiola Rosea to have proper growth effect on HUVEC by MTT array; 2) study the influence of Rhodiola Rosea on angiogenic genes on HUVEC by using GEArray Q Series Human angiogenesis gene array; 3) to validate the results of gene array by using real-time PCR.Results: 1) 500ug/ml Rhodiola Rosea is the proper dose for HUVEC to grow.2) Among 96 angiogenic genes, Rhodiola Rosea influent 50 of them and up regulate their expression >2 times as control. They are within VEGF and its receptor family, FGF and its receptor family, TGFb and its receptor family, cytokines such as CSF,TNF, MDK, and matrix protein such as MMPs, transcription factors such as ETS1, SMAD1 and others as endoglin ets.3) By using real-time PCR, we validate Rhodiola Rosea has influence on CSF, TNF, VEGFR2, TGFbR1 and R2, ETS1, SMAD1.Conclusions: Rhodiola Rosea has influence on several different angiogenic genes besides VEGF and FGF. These genes comprise a complex net and go up or down to get equilibrium. Among the net VEGF, FGF and TGFb are the key genes influenced by Rhodiola Rosea.Part III to study the effect of Rhodiola Rosea on myocardium during angiogenesis period after acute myocardial infarctionObjects: to study the effect of Rhodiola Rosea on expression of HIF1a, VEGFR2, ETS1, GDF15 and ERK1/2 of infarcted area, border and noninfarcted area during 2 weeks after acute myocardial infarction.Methods: Rats have LADC as AMI model. After that Rats are randomized to Rhodiola Rosea group, which are fed Rhodiola Rosea everyday, and NS group which are fed NS instead of Rhodiola Rosea everyday. Two other groups are sham group and normal group. Within 24 hours after AMI, Rhodiola Rosea or NS are fed to rats. Then, lday, 4days, 7days and 14days after feeding, rats are sacrificed. There are 4 rats of Rhodiola Rosea group, 4 rats of NS group, 2 rats of sham group and 1 rat of normal group sacrificed each time. After get the heart, half of it is conserved in 10%formalin to do CD34 immmunohistochemistry test later and among another half, issue from infarcted, border and noninfarcted area are taken to do real-time PCR test of HIF1a, VEGFR2, ETS1, GDF15 and western blot test of ERK1/2. Rats after 4days are test heart function before sacrificed.Results: Both NS group and Rhodiola Rosea group have up regulated expression of MVD after AMI from dayl to day14. MVD are on the top on day 7 and day 14. Rhodiola Rosea group have greater MVD on day4, day7 and dayl4 as compared to NS group. The heart function is improved on day14 in both groups. And Rhodiola Rosea group has a better function on day14. Rhodiola Rosea group influent expression of HIF1a fP VEGFR2/KDR of myocardium more great on day7. The influence to HIF1a 和 VEGFR2/KDR by Rhodiola Rosea is consistent. Rhodiola Rosea group influent expressionof ETS1 of myocardium mostly on day4 and day7.on day4 its expression on border and infarcted area is higher than noninfarcted area while on day7 its expression on border and noninfarcted area are higher than infracted area. Rhodiola Rosea group influent expression of GDF15 mostly on day4 of border and on day7 of noninfarcted area, both is up regulated. But in NS group GDF 15 are most expressed on day 14 which is consistent with its function improve. The phosphate of erk1/2 are great on day1 and day4 following down regulating which are not consistent with expression of HIF1a, VEGFR2/KDR and ETS1. The influence of Rhodiola Rosea group on phosphate of erk1/2 is mostly on day4 and day7.Conclusions: HIF1a, VEGFR2/KDR and ETS1 have different expression on different area of myocardium of infarction and consistent with angiogenic changes. Rhodiola Rosea group influence these genes mostly during inflammation period. But phosphate of erk1/2 is not consistent with changes of these genes. Rhodiola Rosea group influent GDF15 expression earlier than its heart function improved.