| Background: Treatment of acute myocardial infarction (AMI) remains muchchallenge, though there have been enormous progresses in ischemic heart disease.Cell-based repair is emerging as a potential novel therapy for AMI. However, theefficacy of cell-based therapy is hindered by the deleterious local milieu of themyocardium.Objective: The study was conducted to evaluate whether Rehmannia GlutinosaOligosaccharides (RGOs) treatment can increase the efficacy of allogenic adiposetissue-derived stem cells (ASCs) transplantation in Chinese miniswine with AMIand to explore the potential mechanisms.Methods and results:The first part: Crude extract RGOs was extracted by boiled water fromRehmannia root and was further separated by cation exchange resin and anionexchange resin eluting and by charcoal column chromatography. The stachyose,the main component in the RGOs, was determined by High Performance LiquidChromatography (HPLC). The outputs of crude extract RGOs and purifiedproduct from raw material were41.64%and35.14%, respectively; and purifiedRGOs contained31.15%stachyose.The second part: The ASCs of miniswine were isolated and cultured from adiposetissue harvested from inguinal regions by enzyme digestion and adherent. Themolecular 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 acertain range of concentration (0.01mg/ml~1mg/ml), and the best proliferative effect was observed at concentration of0.1mg/ml.The third part: To determine whether RGOs (0.1mg/ml) pretreatment for12hourscauses increase of vascular endothelial growth factor (VEGF), hepatocyte growthfactor (HGF), insulin-like growth factor-1(IGF-1), basic fibroblast growth factor(bFGF) and stromal cell derived factor-lα (SDF-lα) release from human ASCs(hASCs) in vitro. The levels of VEGF, HGF, IGF-1, bFGF, SDF-1α in the hASCssupernatant were determined by enzyme-linked immunosorbent assay (ELISA).The results indicated that RGOs could promote the secretion of VEGF, HGF,IGF-1and SDF-1α in a time-dependent manner, but the secretion of bFGF did notincrease significantly.The fourth part: To investigate whether RGOs (0.01mg/ml,0.1mg/ml,1mg/ml,10mg/ml) pretreatment for12hours and continued presence could protect ASCsagainst apoptosis in a model of oxidative stress consisting of hydrogen peroxide-and serum deprivation-induced in vitro. Apoptosis of ASCs was assessed using anAnnexin V-FITC/PI apoptosis kit. Cell activity was determined by CCK-8assay.Caspase-3activity was detected by applying a caspase-3assay kit. Expression ofBax and Bcl-2was investigated using Western blot analysis. We found that RGOssignificantly attenuated hydrogen peroxide-and serum deprivation-induced ASCsapoptosis, showing a decreased apoptosis rate, an increase in cell activity, adecreased caspase-3activity, a down-regulated Bax expression and anup-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 extractalone from the3rd day prior to AMI to one month post AMI (4g, two times perday); ASCs transplantation alone (group A, n=4) and RGOs+ASCs (group RA,n=4). AMI models were created by occlusion of the left anterior descendingcoronary artery for90minutes. One week later (the7th to10th day post AMI),allogenic ASCs (0.8×106/kg) were injected into left anterior descending coronaryartery. At8weeks post transplantation, magnetic resonance imaging (MRI) showed that the left ventricular ejection fraction and wall thickness of infarctedregions were increased while the left ventricular mass index, the infarct size weredecreased only in group RA compared with group C (P＜0.05). ASCs survivalwas significantly better in group RA than in group A (P＜0.01). Microvasculardensities both in the infracted zone and the peri-infarct zone also increasedsignificantly in group RA but not in other three groups (P＜0.01). Massontrichrome stain showed that there was less fibrosis with more survivingmyocardium in group A and group RA than that in group C (P＜0.05and P＜0.01). TUNEL assay indicated that RGOs administration significantly decreasedcell apoptosis in peri-infarct myocardium in group R and group RA (P＜0.05andP＜0.01). Western blot analysis indicated that the expression levels of Bax ingroup R, group A and group RA were significantly decreased (P＜0.01,versusgroup C). However, the expression levels of Bcl-2in group R and group RA weresignificantly increased (P＜0.01,versus group C).Conclusion: RGOs treatment improves the therapeutic efficacy of ASCstransplantation by improving the local milieu of the ischemic myocardium,promoting ASCs proliferation and survival, enhancing paracrine function and byanti-apoptosis.
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