The Experimental Research Of The Stimulating Angiogenesis Effect Of Morinda Officinalis How Oligosaccharides

Background:Coronary artery disease(CAD), which severely affects health, is a type of cardiovascular diseases with high incidence. Therapeutic angiogenesis has become a promising research in medical domain of the world. Stimulating revascularization in ischemic myocardium is a novel strategy, especially in CAD. It has been proved that various blood vessel growth factors and transgenic therapies can promote angiogenesis. However, they also include effective and safe problems in clinical applications. Recently, angiogenic research of traditional Chinese medicine has attained lots of achievements. It has been demonstrated that the tradional Chinese medicine that invigorate the kidney can promote angiogenesis. Radix morinda officinalis (RMO) is commonly used as a type of tradional Chinese medicine that can invigorate kidney and strengthen yang. Morinda officinalis How oligosaccharides (MOO) is the effective component of water fraction of the ethanolic extract of RMO. Our previous research has shown that MOO can obviously resist hypoxia /reoxygenation injury and protect myocardium. However, it has not been reported whether MOO can stimulate angiogenesis, whether its myocardial protective effect is related to the establishment of collateral circulation of coronary artery in ischemic myocardium.Objective:On the basis of our previous studies, through preparing models of chick chorioallantoic membrane(CAM) and acute myocardial infarction (AMI) of rats, our aim is to observe the effect of MOO on angiogenesis of ischemic myocardium in the models of CAM and AMI of rats. From point of view of therapeutic angiogenesis, we aim to investigate the effect of MOO on cardiovascular disease and study the mechanisms, in order to provide more proofs for the potential protective effect of RMO on cardiovacular diseases.Methods:1. Preparation of serum containing MOO 16 Wistar rats were randomly divided into small, middle and large doses of MOO groups and vacuity serum group. Each group included 4 rats. Adopting the method of four-day intragastric administration, rats in each group were treated respectively with water fraction of the ethanolic extract of RMO at doses of 0.7g/kg/day, 1.4g/kg/day, 2.8g/kg/day and normal saline(NS) at equal volume each day. Blood was drawn from the abdominal aorta 1h after the last intragastric administration. Then small, middle and large doses of serum containing MOO and vacuity serum were prepared respectively.2. The experimental research of the stimulating angiogenesis effect of MOO on the CAM model 60 chick embryoes were randomly divide into NS group, vacuity serum group, bFGF group, small, middle and large doses of MOO groups. Each group included 10 embryoes. CAM models were prepared after 7 days incubation. Then NS, vacuity serum, bFGF(2500U/ml), small, middle and large doses of serum containing MOO were added respectively onto the carriers on the CAM. CAM sample was prepared after 3 days incubation. The state of angiogenesis was observed and the amounts of first and second grade blood vessels were counted under stereomicroscope.3.The experimental reserch of the stimulating angiogenesis effect of MOO in the ischemic myocardium of the rats after AMI 40 male Wistar rats, after ligation of left coronary artery, were randomly divided into 5 groups: model group, Shexiangbaoxin Pill(Shexiang) group, small, middle and large doses of MOO groups. Each group included 8 rats. Another 10 rats were set as sham-operated group(sham group). The Shexiang group was treated with suspension of Shexiangbaoxin Pills 30mg/kg/day. The small, middle and large doses of MOO groups were treated respectively with the water fraction of the ethanolic extract of RMO at doses of 0.7g/kg/day, 1.4g/kg/day, 2.8g/kg/day. The model group was treated with NS at equal volume. The sham group was treated with nothing, raised commonly. Each exprimental group was treated with drug during 24h after model preparation. After sacrificed 6 weeks later, left ventricles of each rat were removed, imbedded with paraffin and then sliced. Then the histomorphology of myocardial slice was observed. Microvessel density(MVD), vascular endothelial growth factor(VEGF), basic fibroblast growth factor(bFGF) in ischemic myocardium of rats in each group were detected by immunohistochemistry assay. The optical density(OD) values of protein expression of VEGF and bFGF in ischemic myocardium were calculated and analyzed by image analysis system.Results:1. The state of angiogenesis in CAM model There was no specificity on angiogenesis in NS group and vacuity serum group. However, in each bFGF group and MOO group, it was observed that the bole blood vessels grew closely to carrier, and the small and middle vessels proliferated obviously, especially small vessels grew radiatly surrounding the center of carrier.2. Effects of serum containing MOO on angiogenesis of the first and second grade vessels in CAM model Compared with vacuity serum group, the amounts of first and second grade vessels in each MOO group increased significantly(P < 0.05). But the drug action was lower than bFGF(P < 0.05). Compared with small dose group, the amounts of first and second grade vessels increased significantly in middle and large doses groups(P < 0.05). But there was no significance between the latter two groups(P > 0.05). There was no significance of the amounts of first and second grade vessels between NS group and vacuity serum group(P > 0.05).3. Observation of histomorphology of myocardial slice Neoformative microvessels could be observed in all groups(sham, model, Shexiang, small, middle, large doses of MOO). There were no obvious proliferative microvessels in sham group, some in model group, temperate in small and middle doses of MOO groups, more in Shexiang and large doses of MOO groups.4. Effects of MOO on MVD in the ischemic myocardium of the rats after AMI Compared with sham group, MVD was significantly increased in model group(P < 0.05). Compared with model group, MVD was signicantly increased in middle and large doses of MOO groups(P < 0.05), but still lower than Shexiang group(P > 0.05). There was no significant difference of MVD between small dose of MOO group and model group(P > 0.05). But MVD among 3 doses of MOO groups showed significant differences (P < 0.05).5.Effects of MOO on expression of VEGF and bFGF protein in the ischemic myocardium of the rats after AMI Compared with sham group, the OD values of VEGF and bFGF were significantly increased in model group, Shexiang group, small, middle and large doses of MOO groups(P < 0.05); there was significant difference between Shexiang group and other groups(P < 0.05). Compared with model group, OD values of VEGF and bFGF were significantly increased in middle and large doses of MOO groups(P < 0.05), but still lower than Shexiang group(P > 0.05). There was no significant difference of OD values of VEGF and bFGF between small dose of MOO group and model group(P > 0.05). There was significant differences of OD value of bFGF between small and middle doses of MOO groups, also between small and large doses of groups(P < 0.05). But the OD values of bFGF showed no significant difference between middle and large doses of groups. There were significant differences of OD values of VEGF among 3 doses of MOO groups.Conclusion:1. MOO can obviously stimulate angiogenesis of chick chorioallantoic membrane (CAM).2. MOO can obviously stimulate angiogenesis in the ischemic myocardium of the rats after AMI, and improve collateral circulation of coronary artery in ischemic myocardium.3. MOO can obviously increase the expression of VEGF and bFGF protein in the ischemic myocardium of the rats after AMI. It may act as one of the mechanisms of MOO's stimulating angiogenesis effect.