Effects of Rehmannia Glutinosa Oligosaccharides on Allogenic Adipose Tissue-derived Stem Cells Transplantation in Miniswine with Acute Myocardial Infarction
|Keywords||Acute myocardial infarction Rehmannia Glutinosa Oligosaccharides Adipose tissue-derived stem cells Paracrine Apoptosis|
Background:Treatment of acute myocardial infarction (AMI) remains much challenge, though there have been enormous progresses in ischemic heart disease. Cell-based repair is emerging as a potential novel therapy for AMI. However, the efficacy of cell-based.therapy is hindered by the deleterious local milieu of the myocardium.Objective:The study was conducted to evaluate whether Rehmannia Glutinosa Oligosaccharides (RGOs) treatment can increase the efficacy of allogenic adipose tissue-derived stem cells (ASCs) transplantation in Chinese miniswine with AMI and to explore the potential mechanisms.Methods and results:The first part:Crude extract RGOs was extracted by boiled water from Rehmannia root and was further separated by cation exchange resin and anion exchange resin eluting and by charcoal column chromatography. The stachyose, the main component in the RGOs, was determined by High Performance Liquid Chromatography (HPLC). The outputs of crude extract RGOs and purified product from raw material were41.64%and35.14%, respectively; and purified RGOs contained31.15%stachyose.The second part:The ASCs of miniswine were isolated and cultured from adipose tissue harvested from inguinal regions by enzyme digestion and adherent. The molecular phenotypes of ASCs at passages4were examined by flow cytometry, and results showed that ASCs were positive for CD29, CD44, CD90and CD105, but negative for CD31, CD34, CD45and HLA-DR. According to CCK-8colorimetry’s results, RGOs could accelerate proliferation of ASCs in vitro in a certain range of concentration (0.01mg/ml-lmg/ml), and the best proliferative effect was observed at concentration of0.1mg/ml.The third part:To determine whether RGOs (0.1mg/ml) pretreatment for12hours causes increase of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor-1(IGF-1), basic fibroblast growth factor (bFGF) and stromal cell derived factor-la (SDF-la) release from human ASCs (hASCs) in vitro. The levels of VEGF, HGF, IGF-1, bFGF, SDF-la in the hASCs supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The results indicated that RGOs could promote the secretion of VEGF, HGF, IGF-1and SDF-1a in a time-dependent manner, but the secretion of bFGF did not increase significantly.The fourth part:To investigate whether RGOs (0.01mg/ml,0.1mg/ml, lmg/ml,10mg/ml) pretreatment for12hours and continued presence could protect ASCs against apoptosis in a model of oxidative stress consisting of hydrogen peroxide-and serum deprivation-induced in vitro. Apoptosis of ASCs was assessed using an Annexin V-FITC/PI apoptosis kit. Cell activity was determined by CCK-8assay. Caspase-3activity was detected by applying a caspase-3assay kit. Expression of Bax and Bcl-2was investigated using Western blot analysis. We found that RGOs significantly attenuated hydrogen peroxide-and serum deprivation-induced ASCs apoptosis, showing a decreased apoptosis rate, an increase in cell activity, a decreased caspase-3activity, a down-regulated Bax expression and an up-regulated Bcl-2expression.The fifth part:Seventeen Chinese miniswine were divided into four groups: control (group C, n=5);RGOs alone (group R, n=4), administration crude extract alone from the3rd day prior to AMI to one month post AMI (4g, two times per day); ASCs transplantation alone (group A, n=4) and RGOs+ASCs (group RA, n=4). AMI models were created by occlusion of the left anterior descending coronary artery for90minutes. One week later (the7th to10th day post AMI), allogenic ASCs (0.8×106/kg) were injected into left anterior descending coronary artery. At8weeks post transplantation, magnetic resonance imaging (MRI) showed that the left ventricular ejection fraction and wall thickness of infarcted regions were increased while the left ventricular mass index, the infarct size were decreased only in group RA compared with group C (P<0.05). ASCs survival was significantly better in group RA than in group A (P<0.01). Microvascular densities both in the infracted zone and the peri-infarct zone also increased significantly in group RA but not in other three groups (P<0.01). Masson trichrome stain showed that there was less fibrosis with more surviving myocardium in group A and group RA than that in group C (P<0.05and P<0.01). TUNEL assay indicated that RGOs administration significantly decreased cell apoptosis in peri-infarct myocardium in group R and group RA (P<0.05and P<0.01). Western blot analysis indicated that the expression levels of Bax in group R, group A and group RA were significantly decreased (P<0.01,versus group C). However, the expression levels of Bcl-2in group R and group RA were significantly increased (P<0.01,versus group C).Conclusion:RGOs treatment improves the therapeutic efficacy of ASCs transplantation by improving the local milieu of the ischemic myocardium, promoting ASCs proliferation and survival, enhancing paracrine function and by anti-apoptosis.