Angiogenesis Effects Of Morinda Officinalis Oligosaccharides On Human Umbilical Endothelial Cells

Background and Objective:Ischemic cardiovascular disease is the leading cause of mortality and morbidity worldwide. The present therapies for myocardial infarction such as thrombolysis, percutaneous tranluminal coronary angioplasty, and coronary artery bypass graft et al, can make reperfusion in serious ischemic myocardium for some AIM patient, regain the blood of infarction area to an extent and reduce infarct size. However, lots of AIM patients still not adapt to the above therapies and these therapies can't retrieve the function of the necrosed myocardium. Imagining that molecular bypass can cure blood-leaking heart disease,which means add the functionary coronary artery ramus and side to recover the blood supply of the blood-leaking cardiac muscle, to improve the situation of the patient. Our research group is investigating the protective effect of Chinese herbal medicine Morinda Officinalis How Oligosaccharide (MOO) on hearts. Recent research initially confirmed that MOO can obviously promote the proliferation and differentiation of myoblasts, and can promote the angiogenesis in the Chicken Chorioallantoic Membrane (CAM). However, the mechanism of angiogenesis is not clear, whether ERK and p-ERK expression is detected or not after MOO induction is still not clear. Therefore, on the basis of previous study, we use human umbilical vein endothelial cells (HUVEC) culture and human angiogenesis model experimental system including In vivo Matrigel plug assay and Endothelial cell migration assay to explore the mechanism of angiogenesis after MOO induction. In addition, we detect the protein expression of ERK1/2 and p-ERK1/2 by the western-boltting to observe the signal transduction pathways in the process of angiogenesis. So we can provide clues on the protection of myocardial ischemia.Methods1. HUVECs are cultured normally to set up hypoxia-reoxygenation injury model. HUVECs in 50ml culture flasks by adding 10% FBS RPMI1640 containing high glucose medium, at 37℃,5% CO2, saturated humidity incubator 1-2d, cells gradually grow into a single fusion-like. When the culture bottles are covered with 80%, cells can be digested with trypsin passaged or cryopreserved.2. This experiment is divided into six groups:Normal cells group, Hypoxia-reoxygenation injury model group, Hypoxia-reoxygenation injury model plus positive control Shexiangbaoxin Pill (2 g·L-1), Hypoxia-reoxygenation injury model plus small dose of MOO group (0.05 g·L-1), Hypoxia-reoxygenation injury model plus medium-dose group of MOO group (0.15 g·L-1), Hypoxia-reoxygenation injury model plus high-dose group of MOO group (0.45 g·L-1).3. Observe the state of HUVEC with the inverted microscope. Flow Cytometry (FCM) analysis the cell cycle.4. Transwell chamber migration assays calculated the number of migrated cells:Endothelial cell migration in response to MOO was determined as described earlier. HUVEC (1×105) were plated in duplicate onto the upper transwell chamber. The bottom chamber contained 0.6 ml of medium (serum free) alone or medium containing different concentrations of MOO. The cells were incubated for 24 h at 37℃and cells that did not pass through the membrane pores were removed. Migrated cells were stained using Hema 3 kit as per the manufacturer's instructions and counted in eight random fields (200×). SBW (2 g·L-1) was used as a positive control.5. Matrigel tubule formation assays assessed the area of tube formation: Growth factor-reduced Matrigel (50μ1) was pipetted onto 96-well culture plates and polymerized for 30 min at 37℃. HUVECs were seeded onto the surface of the Matrigel, MOO was then added and the plates incubated for 18 h at 37℃. The morphological changes to the cells were observed under a microscope and photographed.6. Western-blot analysis:Cell lysates from MOO-treated and untreated HUVEC were prepared using lysis buffer containing 20 mM Tris-HCl pH 7.5,150 mM NaCl,1% Triton X-100 and complete protease inhibitor cocktail. The membranes were blocked with 5% BSA and probed with anti-ERK1/2 antibody, followed by HRP conjugated anti-rabbit antibody. For phosphorylation experiments, cell lysates from HUVEC treated for different durations with MOO were examined using anti-phospho-ERK antibodies. Protein bands were visualized by an enhanced chemiluminescent detection system according to the manufacturer's protocol, and analyzed using the Q-one software.7. Data were analyzed by the SPSS 12.0 statistical software package. Results were expressed as mean±standard deviation (x±s). Different groups were compared through one-way ANOVA, followed by LSD. P-values of less than 0.05 were considered significant.Result:1. Observed under inverted microscope, cell nucleus is round, membrane clear, multilaterally-shaped spindle-shaped cells, wich showing a typical paving stone-like.2. The MOO increased the proportion of (G2+S) phase cells.3. The MOO obviously increased the number of cell migration.4. The MOO significantly improved the area of Tube formation.5. Western-blot showed MOO can increase the expression of p-ERK1/2.Conclusion:MOO can improve the state of cells induced by H/R, and stimulate the angiogenesis of HUVEC probably by the activation of ERK signaling.